For instance, the site of gravity perception and signal transduction (endodermal cells) overlaps wit...

For instance, the site of gravity perception and signal transduction (endodermal cells) overlaps with the site of curvature response in shoots, which has been proposed to occur simultane[r]

Edited by

SIMON GILROY

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First edition, 2008

Library of Congress Cataloging-in-Publication Data

Plant Tropisms/edited by Simon Gilroy, Patrick H Masson.-1st ed p cm

Includes bibliographical references ISBN 13:978-0-8138-2323-2 (alk paper) ISBN 10:0-8138-2323-4

1 Tropisms Growth (Plants) I Gilroy, Simon II Masson, Patrick H

QK745.P59 2007 571.8⬘2—dc22

v

List of Contributors ix

Preface xiii

Chapter 1: Mechanisms of Gravity Perception in Higher Plants 3 ALINE H VALSTER AND ELISON B BLANCAFLOR

1.1 Introduction

1.2 Identification and characterization of gravity perception sites in plant organs 1.2.1 Roots

1.2.2 Hypocotyls and inflorescence stems (dicotyledons) 1.2.3 Cereal pulvini (monocotyledons)

1.3 The starch-statolith hypothesis

1.3.1 A variety of plant organs utilize sedimenting amyloplasts to sense gravity 1.3.2 Amyloplast sedimentation is influenced by the environment and developmental

stage of the plant 11

1.4 The gravitational pressure model for gravity sensing 11 1.5 The cytoskeleton in gravity perception 12

1.6 Concluding remarks and future prospects 14 1.7 Acknowledgment 15

1.8 Literature cited 15

Chapter 2: Signal Transduction in Gravitropism 21

BENJAMIN R HARRISON, MIYO T MORITA, PATRICK H MASSON, AND MASAO TASAKA

2.1 Introduction 21

2.2 Gravity signal transduction in roots and aboveground organs 22

2.2.1 Do mechano-sensitive ion channels function as gravity receptors? 24

2.2.2 Inositol 1,4,5-trisphosphate seems to function in gravity signal transduction 26 2.2.3 Do pH changes contribute to gravity signal transduction? 27

2.2.4 Proteins implicated in gravity signal transduction 28

2.2.5 Global ‘-omic’ approaches to the study of root gravitropism 32 2.2.6 Relocalization of auxin transport facilitators or activity regulation? 37 2.2.7 Could cytokinin also contribute to the gravitropic signal? 38

2.3 Gravity signal transduction in organs that not grow vertically 39 2.4 Acknowledgments 40

2.5 Literature cited 40

Chapter 3: Auxin Transport and the Integration of Gravitropic Growth 47 GLORIA K MUDAY AND ABIDUR RAHMAN

3.1 Introduction to auxins 47

3.2 Auxin transport and its role in plant gravity response 47 3.3 Approaches to identify proteins that mediate IAA efflux 51 3.4 Proteins that mediate IAA efflux 51

3.6 Regulation of IAA efflux protein location and activity during gravity response 55 3.6.1 Mechanisms that may control localization of IAA efflux carriers 56 3.6.2 Regulation of IAA efflux by synthesis and degradation of efflux carriers 58 3.6.3 Regulation of auxin transport by reversible protein phosphorylation 59 3.6.4 Regulation of auxin transport by flavonoids 61

3.6.5 Regulation of auxin transport by other signaling pathways 61 3.6.6 Regulation of gravity response by ethylene 64

3.7 Overview of the mechanisms of auxin-induced growth 65 3.8 Conclusions 67

3.9 Acknowledgements 68 3.10 Literature cited 68

Chapter 4: Phototropism and Its Relationship to Gravitropism 79 JACK L MULLEN AND JOHN Z KISS

4.1 Phototropism: general description and distribution 79 4.2 Light perception 80

4.3 Signal transduction and growth response 82 4.4 Interactions with gravitropism 83

4.5 Importance to plant form and function 84 4.6 Conclusions and outlook 85

4.7 Literature cited 86

Chapter 5: Touch Sensing and Thigmotropism 91

GABRIELE B MONSHAUSEN, SARAH J SWANSON, AND SIMON GILROY 5.1 Introduction 91

5.2 Plant mechanoresponses 91

5.2.1 Specialized touch responses 92

5.2.2 Thigmomorphogenesis and thigmotropism 94 5.3 General principles of touch perception 95

5.3.1 Gating through membrane tension: the mechanoreceptor for hypo-osmotic stress in bacteria, MscL 98

5.3.2 Gating through tethers: the mechanoreceptor for gentle touch in Caenorhabditis

elegans 99

5.3.3 Evidence for mechanically gated ion channels in plants 101 5.4 Signal transduction in touch and gravity perception 103

5.4.1 Ionic signaling 103

5.4.2 Ca2+signaling in the touch and gravity response 103

5.5 Insights from transcriptional profiling 107

5.6 Interaction of touch and gravity signaling/response 110 5.7 Conclusion and Perspectives 113

5.8 Acknowledgements 114 5.9 Literature cited 14

Chapter 6: Other Tropisms and their Relationship to Gravitropism 123 GLADYS I CASSAB

6.1 Introduction 123 6.2 Hydrotropism 123

6.2.1 Early studies of hydrotoprism 124 6.2.2 Genetic analysis of hydrotropism 125

6.2.4 ABA and the hydrotropic response 128 6.2.5 Future experiments 129

6.3 Electrotropism 129 6.4 Chemotropism 131

6.5 Thermotropism and oxytropism 132 6.6 Traumatropism 134

6.7 Overview 135 6.8 Acknowledgments 135 6.9 Literature cited 135

Chapter 7: Single-Cell Gravitropism and Gravitaxis 141

MARKUS BRAUN AND RUTH HEMMERSBACH

7.1 Introduction 141

7.2 Definitions of responses to environmental stimuli that optimize the ecological fitness of single-cell organisms 141

7.3 Occurrence and significance of gravitaxis in single-cell systems 142 7.4 Significance of gravitropism in single-cell systems 143

7.5 What makes a cell a biological gravity sensor? 144

7.6 Gravity susception—the initial physical step of gravity sensing 145 7.7 Susception in the statolith-based systems of Chara 145

7.8 Susception in the statolith-based system Loxodes 149

7.9 Susception in the protoplast-based systems of Euglena and Paramecium 150 7.10 Graviperception in the statolith-based systems of Chara 150

7.11 Graviperception in the statolith-based system Loxodes 151

7.12 Graviperception in the protoplast-based systems Paramecium and Euglena 151 7.13 Signal transduction pathways and graviresponse mechanisms in the statolith-based

systems of Chara 153

7.14 Signal transduction pathways and graviresponse mechanisms in Euglena and Paramecium 154 7.15 Conclusions 155

7.16 Acknowledgements 156 7.17 Literature cited 156

Color Section

Chapter 8: Space-Based Research on Plant Tropisms 161

MELANIE J CORRELL AND JOHN Z KISS

8.1 Introduction—the variety of plant movements 161 8.2 The microgravity environment 162

8.3 Ground-based studies: mitigating the effects of gravity 165 8.4 Gravitropism 166

8.4.1 Gravitropism: gravity perception 166 8.4.2 Gravitropism: signal transduction 168 8.4.3 Gravitropism: the curving response 169 8.5 Phototropism 171

8.6 Hydrotropism, autotropism, and oxytropism 172 8.7 Studies of other plant movements in microgravity 174 8.8 Space flight hardware used to study tropisms 175 8.9 Future outlook and prospects 177

Chapter 9: Plan(t)s for Space Exploration 183 CHRISTOPHER S BROWN, HEIKE WINTER SEDEROFF, ERIC DAVIES,

ROBERT J FERL, AND BRATISLAV STANKOVIC

9.1 Introduction 183

9.2 Human missions to space 184 9.3 Life support 184

9.4 Genomics and space exploration 185 9.5 Nanotechnology 187

9.6 Sensors, biosensors, and intelligent machines 187 9.7 Plan(t)s for space exploration 188

9.8 Imagine 192 9.9 Literature cited 192

ix Elison B Blancaflor

Plant Biology Division

The Samuel Roberts Noble Foundation 2510 Sam Noble Parkway

Ardmore, OK 73401 USA

Tel: (580) 224-6687 Fax: (580) 224-6692

E-mail: eblancaflor@noble.org Markus Braun

Gravitationsbiologie

Institut für Molekulare Physiologie und Biotechnologie der Pflanzen Universität Bonn

53115 Bonn Germany

Tel: (49) 228-73-2686 Fax: (49) 228-732677 E-mail: mbraun@uni-bonn.de Christopher S Brown

Kenan Institute for Engineering, Technology & Science

North Carolina State University Raleigh, NC 27695

USA

Tel: (919) 513-2457 Fax: (919) 515-5831

E-mail: cbrown@gw.fis.ncsu.edu

Gladys I Cassab

Department of Plant Molecular Biology Institute of Biotechnology

National Autonomous University of Mexico P.O Box 510-3

Cuernavaca, Mor 62250 Mexico

Tel: (52) 5556-22-7660 Fax (52) 7773-13-9988 E-mail: gladys@ibt.unam.mx Melanie J Correll

Department of Agricultural and Biological Engineering

University of Florida 209 Frazier Rogers Hall P.O Box 110570

Gainesville, FL 32611-0570 USA

Tel: (352) 392-1864 Fax: (352) 392-4092 E-mail: Correllm@ufl.edu Eric Davies

Department of Plant Biology North Carolina State University 1231 Gardner Hall

Box 7612

Raleigh, NC 27695 USA

Tel: (919) 513-1901 Fax: (919) 515-3436

Robert J Ferl

Department of Horticulture University of Florida Gainesville, FL 32611 USA

Tel: (352) 392-1928 Fax: (352) 392-4072 E-mail: robferl@ufl.edu Simon Gilroy

Biology Department

The Pennsylvania State University 208 Mueller Laboratory

University Park, PA 16802 USA

Tel: (814) 863-9626 Fax: (814) 865-9131 E-mail: sxg12@psu.edu Benjamin R Harrison

Laboratory of Genetics (Room 3262) University of Wisconsin–Madison 425G Henry Mall

Madison, WI 53706 USA

Tel: (608) 265-8632 Fax: (608) 262-2976

E-mail: brharrison@wisc.edu Ruth Hemmersbach

Institute of Aerospace Medicine DLR (German Aerospace Research

Establishment) 51140 Köln Under Höhe Germany

Email: ruth.hemmersbach@dlr.de John Z Kiss

Department of Botany Pearson Hall

Miami University Oxford, OH 45056 USA

Phone: (513) 529-5428 Fax: (513) 529-4243 E-mail: kissjz@muohio.edu

Patrick H Masson

Laboratory of Genetics (Room 3262) University of Wisconsin–Madison 425G Henry Mall

Madison, WI 53706 USA

Tel: (608) 265-2312 Fax: (608) 262-2976

E-mail: phmasson@wisc.edu Gabriele B Monshausen Biology Department

The Pennsylvania State University 208 Mueller Laboratory

University Park, PA 16802 USA

Tel: (814) 863-9625 Fax: (814) 865-9131 E-mail: gbm10@psu.edu Miyo T Morita

Graduate School of Biological Sciences Nara Institute of Science and Technology 8916-5 Takayama

Ikoma, Nara 630-0101 Japan

Phone: (81) 743-72-5487 Fax: (81) 743-72-5487 E-mail: mimorita@bs.naist.jp Gloria K Muday

Department of Biology Wake Forest University

Winston-Salem, NC 27109-7325 USA

Tel: (336) 758-5316 Fax: (336) 758-6008 E-mail: muday@wfu.edu Jack L Mullen

Department of Bioagricultural Sciences and Pest Management

Plant Science Building, Room C 129 Colorado State University

Fort Collins, CO 80523-1177 USA

Tel: (970) 491-5261

Abidur Rahman Biology Department University of Massachusetts 611 North Pleasant St 106 Morrill

Amherst, MA 01003 USA

Phone: (413) 545-2776 Fax: (413) 545-3243

E-mail: abidur@bio.umass.edu Heike Winter Sederoff Department of Plant Biology North Carolina State University Raleigh, NC 27695

USA

Phone: (919) 513-0076 Fax: (919) 515-3436

E-mail: heike_winter@ncsu.edu Bratislav Stankovic

Brinks Hofer Gilson & Lione

455 N Cityfront Plaza Drive, Suite 3600 Chicago, IL 60611-5599

USA

Sarah J Swanson Biology Department

The Pennsylvania State University 208 Mueller Laboratory

University Park, PA 16802 USA

Tel: (814) 863-9625 Fax: (814) 865-9131 E-mail: sjs31@psu.edu

Masao Tasaka

Graduate School of Biological Sciences Nara Institute of Science and Technology 8916-5 Takayama

Ikoma, Nara 630-0101 Japan

Phone: (81) 793-72-5480 Fax: (81) 793-72-5489 E-mail m-tasaka@bs.naist.jp Aline H Valster

Plant Biology Division

The Samuel Roberts Noble Foundation 2510 Sam Noble Parkway

Ardmore, OK 73401 USA

Tel (580) 224-6756 Fax (580) 224-6692

xiii

As sessile organisms, plants spend their entire lives at the site of seed germination Consequently, they require a suite of strategies to survive very diverse environmental stresses Part of this plasticity relies on the ability of most plant organs to grow in direc-tions that are dictated by specific cues from the environment, seeking out better condi-tions to fulfill their primary funccondi-tions Typical guidance for the growth of plant organs is provided by gravity, light, touch, gradients in humidity, ions, oxygen, and temperature Such directional growth, defined by vectorial stimuli, is called a tropism and is believed to significantly contribute to plant survival

The concept of tropism was introduced 200 years ago, when Knight (1806) postulated that a plant’s perception of gravity might modulate its ability to direct shoots to grow up-ward and guide roots downup-ward Eighty years later, Darwin (1880) made seminal contri-butions to the field by documenting a wide array of tropic responses and identifying re-gions of the root and shoot specialized for the perception of light and gravity He also predicted the existence of auxin by proposing the presence of a plant growth regulator (hormone) whose gravity-induced redistribution across the tip of an organ might signal differential growth

Since these discoveries, our analysis of tropic growth has expanded to include meas-urements of responses to light, touch, and gradients in humidity, ions, chemicals, and oxygen However, only recently have the data converged to provide a picture of the phys-iological, molecular, and cell biological processes that underlie plant tropisms Thus, the last few years have witnessed a true renaissance in the analysis of tropic response, mainly driven by the marrying of modern tools and strategies in the fields of forward and reverse genetics, biochemistry, cell biology, expression profiling, and proteomics, to a very care-ful analysis of the growth process itself

When such analyses have been coupled with the utilization of model systems such as

Arabidopsis thaliana and rice, where their entire genome has been sequenced, these

strategies have provided an unprecedented power of resolution in our analysis of growth behaviors Consequently, our conception of tropisms has evolved from their being con-sidered as simple laboratory curiosities to becoming important tools/phenotypes with which to decipher basic cell biological processes that are essential to plant growth and development Thus, current insight into tropisms is intimately involved in our understand-ing of auxin transport and response; cytoskeleton organization and its involvement in the control of anisotropic cell expansion; the perception and transduction of stimuli such as light, touch, humidity, ions, or oxygen; the biogenesis and function of organelles such as plastids and vacuoles; and even the control of vesicular trafficking, to name but a few (Blancaflor and Masson 2003)

Chapter 1, Valster and Blancaflor describe our current models of gravitropic sensing in plants, a theme further developed in Chapter 2, where Harrison and colleagues discuss the molecular mechanisms behind transduction of the gravity signal In Chapter 7, Braun and Hemmersbach further explore sensing and signaling in plants by comparison to the wealth of data on how single-celled organisms detect and respond to gravity Similarly, in Chapter 4, Mullen and Kiss describe the remarkably detailed knowledge we now have of the mechanisms whereby plants perceive light and translate that cue into a phototropic growth

Despite Darwin’s prediction of the action of auxin in tropic response as early as 1880, only recently have the mechanisms behind auxin transport and action been defined to the molecular level For example, we now understand that the relocalization of auxin trans-porters is a central component regulating tropic response pathways and critical compo-nents of the auxin transport pathway have been defined with molecular precision In Chapter 3, Muday and Rahman provide an overview of this extremely rapidly evolving field

Although individual tropic stimuli are often studied in a controlled laboratory setting, nature provides a harsh environment where multiple vectorial stimuli often signal con-flicting information for a plant organ An important step in our conceptualization of plant responses to such a complex environment has been the realization that organs not only perceive and respond to each one of these parameters, but they also have to integrate and interpret the corresponding environmental information into global “decisions” that man-ifest themselves into complex growth behaviors

The integration of other tropic stimuli with the gravitropic response has recently re-ceived intense analysis and, in Chapters and 6, Monshausen and colleagues and Gladys Cassab describe the wealth of tropic responses in plants and specifically how responses to touch and moisture alter gravitropic response Such integrated responses to combined environmental cues appear to involve complex intra- and intercellular communications Recent analyses have uncovered some of these fascinating signaling events (Fasano et al 2002), opening the possibility of, one day, being able to engineer plants that are capable of using a defined set of directional cues for growth guidance while being oblivious to other cues Such engineering accomplishments could find applications in agriculture and in more futuristic endeavors such as space exploration

Indeed, spaceflight has offered researchers a unique opportunity to dissect tropic re-sponse in the absence of the effects of gravity However, in space, in addition to exposure to microgravity, organisms also suffer from a lack of convection, growth-space limita-tions, lower light exposures, and increased radiation levels Hence, the spaceflight envi-ronment appears quite unfavorable to plant success, and tropic responses are likely to be altered accordingly Because plants have been identified as an ideal choice for utilization in bioregenerative life-support systems during long-term space exploration missions, there is a definite need for a better understanding of their growth behavior and sustain-ability during long-term exploration travels in order to prevent or overcome potential cat-astrophic system breakdowns in the midst of a mission

experiments have added new information on plant growth responses to directional cues such as gravity, light, or oxygen gradients and, in Chapter 8, Correll and Kiss describe the opportunities that spaceflight has provided to understand how a range of such tropic responses operate

However, spaceflight experimentation has also been plagued by a variety of constraints that have diminished their potential scientific value Hence, a combined approach, in-cluding both ground- and orbit-based research, is necessary to gain a better understand-ing of the behavior of plants and their organs under micro- or hypergravity environments in the hope of being able to, one day, engineer cultivars that are better-adapted to the con-ditions likely to be encountered during space exploration missions

Thus, the field of plant tropisms has received considerable attention in the last few years for its impact on both basic understanding of plant growth and development and applied aspects, such as crop response or application to spaceflight We hope this book will pro-vide a comprehensive yet integrated coverage of our current state of knowledge on the mo-lecular and cell biological processes that govern plant tropisms, with major emphasis on gravitropism (one of the most extensively studied plant tropisms) Our understanding of tropic responses is rapidly increasing and, with each new insight, the potential to engineer new traits into plants moves closer Therefore, for the last chapter of the book we asked Chris Brown and colleagues to present a vision for how our increasingly detailed under-standing of these plant growth responses might translate into designing plants to sustain human endeavors in perhaps the most inhospitable environment for life imaginable— space

Simon Gilroy Patrick H Masson

Literature Cited

Blancaflor EB and Masson P 2003 Update on Plant gravitropism Unraveling the ups and downs of a complex process Plant Physiology 133: 1677–1690.

Darwin C 1880 The Power of Movement in Plants London: John Murray.

Fasano JM, Massa GD and Gilroy S 2002 Ionic signaling in plant responses to gravity and touch

Journal of Plant Growth Regulation 21: 71–88.

Knight T 1806 On the direction of the radicle and germen during the vegetation of seeds

Aline H Valster and Elison B Blancaflor*

3

1.1 Introduction

Plant growth and development is influenced by a multitude of exogenous and endogenous signals Among the signals a plant encounters during its lifetime, gravity is one that re-mains constant throughout development Since the plant needs to orient its organs to po-sition itself within available environmental resources such as light and soil nutrients, the gravity stimulus is significant for its survival From the moment the seed germinates, the seedling orients its emerging root such that it grows downward, toward the gravity vec-tor, whereas it directs its shoot to grow upward, opposite the gravity vector This phenom-enon, referred to as gravitropism (geotropism in the older literature) requires the coordi-nated response and interaction of different cell types Furthermore, an array of cellular structures and endogenous molecules, which in turn are modulated by a variety of envi-ronmental stimuli including light, moisture, oxygen, and touch, eventually determine the final manifestation of the gravity response (Blancaflor and Masson 2003; Morita and Tasaka 2004; Perrin et al 2005; Esmon et al 2005)

Gravitropism has traditionally been divided into a series of events: gravity perception, signal transduction, and the growth response (Sack 1991; Kiss 2000) In higher plants, these events appear to take place in spatially distinct regions of the organ, in contrast to tip-growing cells such as rhizoids of the green algae Chara and protonemata of moss and

Chara where, as discussed in Chapter 7, all phases of gravitropism occur within the same

cell (Sievers et al 1996; Schwuchow et al 2002) Since gravity must ultimately work on a mass to exert its effect on a given biological system, it has been widely accepted that plants sense gravity through falling organelles (statoliths) within specialized cells (stato-cytes) Through the years, this model of plant gravity perception has been refined and al-ternative hypotheses have been proposed, including the possibility that the settling of the whole cell protoplast rather than sedimenting organelles is responsible for gravity sens-ing (Staves 1997) A number of excellent articles which provide a historical perspective on gravity perception in plants include Sack (1991, 1997) and Kiss (2000) The reader is referred to these articles for an in-depth discussion and critical analysis of the experimen-tal data that have led to current models on how plants sense gravity

In this chapter, we revisit the topic of gravity perception mechanisms, focusing prima-rily on roots and shoots of higher plants Although we occasionally refer to some of the older literature, this chapter will highlight recent findings that are leading to new, testable models explaining how plants sense gravity

1.2 Identification and Characterization of Gravity Perception Sites in Plant Organs

Gravity has been shown to regulate the orientation of different plant organs such as roots, shoots (Fukaki et al 1998; Morita and Tasaka 2004; Perrin et al 2005), leaves (Mano et al 2006), inflorescence stems (Weise et al 2000), cereal pulvini (Perera et al 2001), and peanut gynophores (Moctezuma and Feldman 1999) The response to gravity in the ma-jority of these plant organs is manifested as differential cell growth between opposite flanks of the organ, leading to upward or downward bending Since not all cells within the organ undergo differential growth (Sack et al 1990), an important question in grav-itropism research is how the different cell and tissue types within the organ contribute to the gravity response A more specific question is whether the machinery for sensing grav-ity occurs in the same sites as the responding tissues

To address these questions, research spanning two centuries has focused on elucidat-ing the spatial regulation of gravitropism For example, work that began with Charles Darwin in the late 1800s and followed-up by several other investigators in the 1900s iden-tified the cap as the major gravity perception site in roots (reviewed by Konings 1995; Boonsirichai et al 2002) These early experiments showed that surgical removal of the root cap tissue inhibited the gravitropic curvature without affecting overall root growth In this section, we briefly review experimental evidence that has further reinforced the existence of specific gravity-sensing sites, distinct from the responding tissues, in the best-studied multicellular plant organs, namely roots, dicot stems, and grass pulvini

1.2.1 Roots

As noted earlier, gravity must work on a mass to elicit a specific biological response Therefore, cells that would be prime candidates for perceiving gravity are those which ex-hibit a distinct structural polarity with respect to the gravity vector Indeed, detailed ultra-structural studies of the cap of vertically growing roots in a variety of plant species re-vealed that the central region of the cap (called the columella) contains cells with organelles that are consistently positioned at the bottom of the cell (reviewed in Sack 1991, 1997) These organelles, later identified as starch-containing plastids called amyloplasts (Figure 1.1A and Color Section), would rapidly change position (i.e., sediment) when the root was reoriented The sedimentation of amyloplasts is the most widely accepted expla-nation for how plant organs sense gravity, a model currently known as the starch-statolith hypothesis (refer to The Starch-Statolith Hypothesis section later in this chapter)

(Figure 1.2), had the strongest inhibitory effect on the gravity response—identifying those specific cells of the cap as the most important for gravity sensing Destroying the lower part of the cap in horizontally positioned roots with heavy-ion microbeams also in-hibited gravitropism, possibly by interfering with the cap tissue responsible for transmis-sion of the gravity signal from the columella (Tanaka et al 2002)

Another set of studies implicating the root cap in the gravitropic response employed a genetic approach to remove root cap cells A protein synthesis inhibitor (diphtheria toxin A) was expressed under a root cap specific promoter in Arabidopsis, killing the express-ing cells (Tsugeki and Fedoroff 1999) In addition to havexpress-ing altered morphology, the re-Figure 1.1 (also see Color Section). A Longitudinal section of the root cap of Medicago truncatula show-ing the centrally located columella cells (c) containshow-ing starch-filled plastids (a, amyloplasts) Note that the columella cells are highly polarized with the nucleus (n) located at the upper side of the cell and amyloplasts (a) sedimented on the bottom side B Hypocotyl of a Medicago truncatula seedling bends upward when po-sitioned horizontally Longitudinal section of the reoriented hypocotyl shows amyloplasts (a) sedimented to the new bottom side of the cell White arrow indicates the direction of gravity

sulting roots were agravitropic, providing further evidence that the cap is the primary site of gravity perception in roots

Despite overwhelming evidence supporting the cap as a major gravity-sensing site in roots, there are sparse reports demonstrating that the root cap might not be the only tis-sue that is able to perceive gravity Early experiments employing centrifugation methods suggested that the elongation zone might also be involved (reviewed in Boonsirichai et al 2002) However, these experiments are difficult to interpret because the centrifugation technique itself possibly introduces mechanical effects that could contribute to the bend-ing response of the root More recently, Wolverton et al (2002a) devised a method (named ROTATO) that allowed different regions of the root outside the cap to be main-tained at a defined angle to the vertical (continuously gravistimulated) If a section of the elongation zone of the root was kept at a defined angle, curvature of the root persisted even after the root cap had reached its normal vertical position From these experiments it was concluded that the elongation zone can contribute to gravitropic sensing, although to a lesser extent than the root cap It was estimated that 20% of the total rate of curva-ture originates from the distal elongation zone or the apical portion of the central elonga-tion zone

The finding that the elongation zone contributes to root gravitropic sensing might ex-plain why roots sometimes curve past the vertical and why starchless mutants of

Arabidopsis still have a residual gravitropic response (Wolverton et al 2002a, b) In

sup-port of the notion that other tissues outside the cap can sense gravity was the recent ob-servation that gravitropic curvature in decapped roots of maize can be restored by myosin and actin inhibitors This indicates the existence of a mechanism for gravity sensing out-side the cap that relies on a dynamic cytoskeleton (Mancuso et al 2006; see The Cytoskeleton in Gravity Perception section) Although these new findings continue to support the conclusion made more than a century ago that the root cap is a major site for gravity perception, it appears that it may not be the sole site The availability of techniques such as ROTATO should allow more detailed investigations into alternative gravity-sensing sites in roots

1.2.2 Hypocotyls and Inflorescence Stems (Dicotyledons)

to encode transcription factors necessary for the normal development of the endodermis in roots and shoots Both mutants failed to develop an endodermal cell layer and, as a re-sult, lacked sedimentable amyloplasts in their shoots However, both mutants had normal columella cells containing sedimentable amyloplasts, indicating that amyloplast forma-tion or sedimentaforma-tion itself was not impaired Importantly, although shoot gravitropism was defective in these mutants, root gravitropism was not Thus, it was concluded from these studies that the presence of a normal endodermal layer is important in shoot grav-itropism (Fukaki et al 1998)

The importance of the endodermis for gravitropism was also recently demonstrated in other plant species For example, an agravitropic mutant in the Japanese morning glory (Pharbitis nil) called weeping was shown to be defective in the formation of the endoder-mal layer, similar to the sgr1 mutant in Arabidopsis (Hatakeda et al 2003) Interestingly, the disrupted gene in weeping was recently shown to be an ortholog of Arabidopsis scr. When the wild-type scr from morning glory (PnSCR) was introduced into the

Arabidopsis scr mutants for complementation, the agravitropic phenotype of Arabidopsis

was rescued (Kitazawa et al 2005)

Results from the above studies are also beginning to shed light on the relationship be-tween gravitropism and circumnutation (i.e., oscillatory plant movements) It has long been debated whether gravitropism and circumnutation are causally related to each other (Kiss 2006) Interestingly, it appears that circumnutation and winding movement defects in weeping could be attributed to loss of endodermal function since the circumnutation phenotype in Arabidopsis scr1 could be rescued by scr from wild-type morning glory. Although this provided compelling evidence that other types of plant movements (such as circumnutation) require gravity-sensing cells, recent studies in rice coleoptiles indicate that the relationship between gravitropism and circumnutation may be more complex For example, Yoshihara and Iino (2005) demonstrated that red light abolished circumnutation in rice coleoptiles without affecting the gravitropic response Furthermore, coleoptiles of the rice mutant lazy not circumnutate but contain sedimentable amyloplasts, which suggests that mechanisms independent of gravitropism might operate in plant circumnu-tations (Yoshihara and Iino 2006)

In addition to proving directly that the endodermis is a major gravity-sensing cell layer, the screen for shoot gravitropic mutants has helped uncover additional features of the en-dodermis that might be important for gravity perception Although much attention has been given to sedimenting amyloplasts, other cellular compartments in the gravisensitive cells are likely important For example, in the above-mentioned screen performed by the group of Tasaka, several other shoot gravitropic mutants were isolated Of these, the sgr2,

sgr3, and sgr4/zigzag mutants are notable because they are implicated in vacuolar

Further evidence for the role of vacuolar membrane dynamics in shoot gravitropism comes from the analysis of another mutant in Arabidopsis, namely the sgr3 mutant (Yano et al 2003) The SGR3 gene encodes a syntaxin (AtVAM3) that also has been implicated in vacuolar transport The SGR3 protein is localized to the prevacuolar compartment and the vacuole itself The sgr3 mutant shows abnormal appearance of vacuoles in endoder-mal cells with irregular curves and aberrant membranous structures This mutant also dis-plays abnormal sedimentation of amyloplasts in the shoot endodermis, coupled with al-tered gravitropism in inflorescence stems (Yano et al 2003) This study provides additional evidence for a role of vacuolar membrane dynamics and vacuolar biogenesis in proper amyloplast sedimentation and, by extension, graviperception

Further evidence for a role of membrane dynamics comes from yet another mutant The gravitropism defective mutant (grv2) in Arabidopsis exhibits a defect in shoot and hypocotyl gravitropism The responsible gene, GRV2, appears to code for a protein that is similar to the RME-8 protein in Caenorhabditis elegans, which is important for endo-cytosis (Silady et al 2004) Since the endodermal cell layers in shoots of grv2 mutant dis-play defects in amyloplast sedimentation, endocytosis might be involved in the initial gravity perception steps as well as the membrane dynamics necessary for targeting of auxin efflux carriers such as PIN3 to their correct position within the columella cell (Friml et al 2002; Chapter in this book)

1.2.3 Cereal Pulvini (Monocotyledons)

In contrast to roots and shoots in dicotyledons, the shoots of grasses have a specialized tissue that mediates gravitropism, namely the pulvinus The pulvinus is a cushion-like swelling at the base of each internode and the vascular bundles within it are surrounded by bundle sheath cells that contain sedimenting amyloplasts Upon reorientation of the monocotyledon plant within the gravity field, the amyloplasts sediment to the bottom cell wall in the same way as described for columella and endodermal cells (Allen et al 2003) Also, in pulvini it is this sedimentation that is thought to set off the signal transduction cascade that ultimately regulates the reorientation of the stem of the plant However, in contrast to the primary root, the processes of gravity perception, signal transduction, and growth response (i.e., the establishment of a gradient of cell elongation) all occur in the same tissue (Collings et al 1998) In order for the cells in the pulvini to be gravi-competent, it appears that they must delay maturation Maturation of the surrounding cells in the tissue of the stems involves lignification of the cell wall and rearrangement of microtubules from transverse to oblique after elongation is completed Once these cel-lular processes have taken place, the pulvinus is no longer capable of responding to grav-istimulation During the onset of the gravitropic curvature, maturation occurs only on the upper side of the pulvinus, whereas the elongating cells in the lower side mature only after maximum bending capacity of the stem has been reached (about 30 degrees upward bending) (Collings et al 1998)

within seconds to minutes after gravistimulation As discussed in Chapter 2, the role for InsP3 as a likely universal signaling molecule in the gravity response was recently demonstrated by constitutively overexpressing a human type I polyphosphate 5-phosphatase in Arabidopsis This enzyme specifically hydrolyzes InsP3 This experi-ment resulted in a 90% reduction in InsP3 levels and a 30% decline in the gravity re-sponse of roots, hypocotyls, and inflorescence stems (Perera et al 2006) Genetic and biochemical studies such as these are yielding important information regarding the sig-nal transduction pathways activated upon gravistimulation However, demonstrating how the cells that sense gravity via sedimenting plastids convert this directional infor-mation into biochemical signals that include InsP3transients remains a major challenge in gravitropism research

1.3 The Starch-Statolith Hypothesis

It is clear from the previous section that a common denominator of gravisensitive plant organs is the presence of statoliths/amyloplasts which sediment to the bottom of the graviperceptive cells and that, upon reorientation of these cells within the gravity field, the amyloplasts rapidly resediment to the new bottom of the cell The starch-statolith hy-pothesis poses that the sedimentation of the statoliths within the gravisensitive cells is the event that translates the gravity-driven mechanical stimulus into a chemical signal that further regulates differential growth of the flanks of the reorienting organ How the “falling” of the statoliths exactly converts into a chemical signal is not clear yet, but it is thought that possible interactions of amyloplasts with other cell components such as the endoplasmic reticulum, cytoskeleton, or vacuole are important The starch-statolith hy-pothesis is now widely accepted as the major gravity-sensing mechanism despite the lack of knowledge about some details

1.3.1 A Variety of Plant Organs Utilize Sedimenting Amyloplasts to Sense Gravity

Evidence in support of a role for the starch-filled amyloplasts in the gravitropic response comes from several Arabidopsis mutants that are impaired in amyloplast formation For example, the phosphoglucomutase mutant (pgm) is impaired in starch synthesis and as a result shows reduced gravitropic responses in both roots and shoots (Kiss et al 1989; Kiss et al 1997) Some controversy about the starch-statolith hypothesis came from a paper published in 1989 by Caspar and Pickard (1989) showing that an Arabidopsis mu-tant lacking plastid phosphoglucomutase activity still was able to respond to gravistimu-lation, albeit in a reduced manner From this study it was concluded that starch is unnec-essary for gravitropism However, upon careful ultrastructural examination of this mutant, it appeared that not all starch formation was inhibited Apparently, some starch grains were formed in a subset of the amyloplasts, explaining the reduced but not com-pletely abolished gravitropic response (Saether and Iversen 1991)

support for the starch-statolith hypothesis comes from the observation that gravitropism is impaired in shoots of the eal1 mutant but not in roots Further compelling support comes from the laser ablation experiments mentioned earlier, where it was shown that a strong correlation exists between the maximum amyloplast sedimentation rates in the different cell layers of the root cap (S1, S2, and S3: Figure 1.2) and their involvement in the gravitropic response (Blancaflor et al 1998) Moreover, a series of elegant experi-ments employing a high-gradient magnetic field to displace amyloplasts statoliths mim-icked a gravitropic response (i.e., induced curvature) in roots and shoots, providing fur-ther strong support for the starch-statolith hypothesis (Kuznetsov and Hasenstein 1996; Weise et al 2000)

Recently, other less-studied plant organs have been shown to exploit sedimenting amy-loplast for the purpose of gravity sensing For example, Arabidopsis plants shift their leaves upward against the gravity vector when kept in the dark This movement was shown to be a combination of nastic and gravitropic movement (Mano et al 2006) Cells with sedimenting amyloplasts were observed in several cell layers around the vasculature of the proximal region of the petiole Two mutants in amyloplast formation (pgm and

sgr2-1) showed that abnormal distribution or absence of amyloplasts in the petioles

re-sulted in reduced upward bending of Arabidopsis leaves, indicating that sedimenting amyloplasts are in part responsible for this process (Mano et al 2006)

An unusual example of gravitropism can be found in peanut gynophores (Moctezuma and Feldman 1999) The gynophore is a specialized organ which ensures that developing fruits are buried in the soil This is a step that is essential to the life cycle of the peanut plant Moctezuma and Feldman (1999) explored gravitropism in the peanut gynophore and found that sedimenting amyloplasts are present in the starch sheath cells that sur-round the vasculature in the gynophore De-starching the plastids by incubation with gib-berellic acid and kinetin in the dark did not affect overall growth but abolished 82% of the gravitropic response Gravitropism in the peanut gynophore is interesting because the gravity-sensing mechanism seems similar to gravity sensing in shoots, yet the induced growth response is opposite (i.e., the gynophore grows down rather than upward) Thus, studies on the peanut gynophore might prove extremely helpful in addressing the ques-tion on how the positive versus negative gravitropic response is determined in roots and shoots

Another example where sedimenting plastids appear to mediate a particular gravi-tropic response is in peg formation in cucumber hypocotyls Cucumber seedlings form a specialized protuberance called a peg on the concave side of the bending site between the hypocotyl and the root, which assists in shedding of the seed coat upon germination The formation of the peg is gravity-dependent and the amyloplast-containing sheath cells of the vascular bundles in this area are responsible for gravity sensing (Saito et al 2004) In this case, gravity is responsible for the inhibition of peg formation on the convex side of the bending zone as, under microgravity conditions, two pegs (one on either side of the bending site) will form (Takahashi et al 2000)

1.3.2 Amyloplast Sedimentation Is Influenced by the Environment and Developmental Stage of the Plant

To optimize exposure to the available environmental resources, it would be beneficial for the plant to regulate the gravitropic response of its organs to a certain degree One can imagine that under certain circumstances it is in the plant’s best interest to suppress the gravitropic response Indeed, evidence for such regulatory mechanisms does exist For example, a downward-growing root (i.e., positively responding to the gravity stimulus) encountering a rock in the soil may benefit from temporarily suppressing its gravitropic response so that it can grow around the encountered obstacle As discussed in Chapter 5, Massa and Gilroy (2003) have shown that the gravitropic response in Arabidopsis roots is suppressed after tactile stimulation of the peripheral cap cells Interestingly, it seems that this suppression is correlated with the inhibition of normal amyloplast sedimentation in the statocytes This finding indicates a complex and direct interaction between mech-anisms of thigmo- and gravitropism allowing the roots to navigate the soil and grow around rocks and other obstacles

Another example of down-regulation of the gravitropic response comes from experi-ments in which it was shown that roots of Arabidopsis and radish show greater hydrotrop-ism and reduced gravitrophydrotrop-ism after columella amyloplasts are degraded (Iino 2006, and references therein) The interaction between these two tropisms allows the root to inhibit the gravitropic response (by disintegrating amyloplasts) in favor of a search for water (re-viewed in Chapter 6) Regulated gravitropism can also be found in lateral roots Lateral roots need to grow out horizontally before becoming plagio-gravitropic (i.e., assume an oblique orientation relative to the gravity vector) It appears that lateral root gravitropism is delayed until amyloplasts mature and accumulate numerous starch grains in the lateral root columella cells (Kiss et al 2002)

In line with these observations is a recent study by Ma and Hasenstein (2006) that ad-dresses the question of when the embryonic root is capable of sensing gravity To this end, flax seeds were gravistimulated and allowed to germinate during clinorotation The onset of gravisensing was determined as the time after which 50% of the emerging roots bent in the direction of the gravity vector during gravistimulation It was found that the onset of graviperception was established 11 hours before root emergence at the time of germi-nation (and hours after imbibition) and, interestingly, coincided with the development of mature amyloplasts in embryonic columella cells (Ma and Hasenstein 2006)

Taken together, the role of starch-statoliths in gravity perception seems undeniable Almost all graviperceptive tissues and organs in higher plants (stems, leaves, roots, gynophores, and pegs) display sedimenting amyloplasts in their gravisensitive cells As mentioned above, in some reported cases the development of mature amyloplasts coin-cides with the ability of cells to perceive gravity and the plant seems to be able to down-regulate the gravity-sensing ability by disintegration of amyloplasts

1.4 The Gravitational Pressure Model for Gravity Sensing

proposal, called the gravitational pressure model, originated with studies in single intern-odal cells of Chara Chara internodes display a gravity-dependent polarity of cytoplas-mic streaming such that the downward velocity of streaming is 10% faster than the up-ward stream However, when positioned horizontally, left and right streaming velocity of the internodes occur at the same rate A series of studies in the 1990s suggest that this change in the polarity of streaming is a physiological response to gravity and thus reflec-tive of gravity sensing (Wayne and Staves 1996) Because of the lack of sedimentable or-ganelles in the internodes, the gravitational pressure model proposed that the cell per-ceives gravity by sensing the weight of the entire protoplast Differential tension between the upper and lower part of the cell then activates receptors in the plasma membrane, ini-tiating events relevant to the gravity response (reviewed in Staves 1997)

Although the gravitational pressure model seems to adequately explain the gravity-induced changes in cytoplasmic streaming in giant internodal cells of Chara, the debate continues as to its applicability to gravity sensing in higher plants One interpretation made by proponents of the gravitational pressure model is that amyloplasts contribute to gravity sensing by simply adding to the weight of the protoplast They argue that if grav-ity sensing was dependent on the mass of the protoplast, then media of higher densgrav-ity should inhibit gravitropism Indeed, when rice roots were grown in aqueous media of higher density due to the addition of various solutes, gravitropism was inhibited without affecting amyloplast sedimentation (Staves et al 1997) However, these data have yet to be confirmed in other plant species Instead, one report on protonemata of the moss

Ceratodon purpureus contradicts the earlier studies with rice In plastid-containing

pro-tonemata of Ceratodon, robust upward bending was shown to proceed despite growth in high-density media (Schwuchow et al 2002)

It has also been suggested that both plastid and protoplast pressure-based sensing may operate in regulating gravitropism Indeed, although sedimenting plastids appear to accel-erate a gravity response in Arabidopsis inflorescence stems, they are not required to elicit this response (Weise and Kiss 1999) There are earlier examples in the literature that sup-port the gravitational pressure model for gravity sensing (Kiss 2000), but it seems that most of the cell biological and genetic studies in the last five years continue to point to sedimenting plastids as a primary mechanism to explain gravity perception in higher plants (Blancaflor and Masson 2003; Morita and Tasaka 2004)

1.5 The Cytoskeleton in Gravity Perception

The plant cytoskeleton is a network of dynamic filamentous proteins that consist of microtubules, actin filaments (F-actin), and a number of accessory proteins (Blancaflor et al 2006) In the mid- to late 1980s, interest in the cytoskeleton as a mediator of plant gravity sensing was ignited when Wolfgang Hensel published a series of papers describ-ing the organization of the cytoskeleton in the root columella in an attempt to explain the highly polarized nature of this particular cell type (Hensel 1985, 1988, 1989) This was followed by a series of studies on the structure of the cytoskeleton in gravity-sensing cells of roots and shoots, which continued for several years (e.g., White and Sack 1990; Baluˇska et al 1997; Collings et al 1998; Collings et al 2001; Driss-Ecole et al 2000); (Figure 1.2)

The widespread interest in describing the nature of the cytoskeleton in statocytes was likely triggered by the proposal of Andreas Sievers and colleagues in the late 1980s that the cytoskeleton may not only be involved in maintaining statocyte polarity but could also be a direct cellular target of sedimenting amyloplasts, raising the possibility that it might function as a gravity receptor Sievers et al (1991) suggested that sedimenting amylo-plasts could pull on the cytoskeleton, particularly on F-actin networks, triggering a cas-cade of signaling events leading to differential organ growth Indeed, this proposal has been supported by several studies demonstrating that amyloplast movement is dependent on the actin cytoskeleton since drugs that disrupt F-actin alter the dynamics and sedimen-tation of amyloplasts in space (Volkmann et al 1999) and on the ground (Hou et al 2004; Saito et al 2005; Palmieri and Kiss 2005) In shoot endodermal cells, for example, amy-loplast position is restricted by the large, centrally located vacuole, and amyamy-loplasts have to traverse the thin cytoplasmic strands within the cell to sediment (Kato et al 2002; Saito et al 2005)

As noted earlier, mutants impaired in shoot gravitropism have defects in vacuolar membrane dynamics, and it is possible that the cytoskeleton may function in shoot grav-ity sensing by regulating the trafficking of vesicles to the vacuole and indirectly influenc-ing amyloplast sedimentation (Morita et al 2002; Yano et al 2003) Indeed, shoots treated with latrunculin B, a drug that disrupts F-actin, showed reduced amyloplast settling upon gravistimulation (Friedman et al 2003; Palmieri and Kiss 2005; Saito et al 2005) One would predict that if amyloplast sedimentation was impeded by F-actin disruption, the ability of shoots to bend after a gravity stimulus would be inhibited due to altered grav-ity sensing Although one study in snapdragon inflorescence stems has demonstrated that this is indeed the case (Friedman et al 2003), a number of other reports surprisingly show that altering the actin cytoskeleton with latrunculin B can actually promote gravitropism in shoots (Yamamoto and Kiss 2002; Palmieri and Kiss 2005; Saito et al 2005)

gen-erated by these “rogue” amyloplasts could be amplified several-fold, leading to a stronger gravitropic response Alternatively, extended contact between amyloplasts and the vacuo-lar membrane surface in endodermal cells with a disrupted actin network could be re-sponsible for the enhanced gravity response in shoots (Palmieri and Kiss 2005)

Similar to shoots, latrunculin B has a promotive effect on root gravitropism The en-hanced gravity response in roots is clearly manifested as persistent curvature on a slowly rotating clinostat after a short gravistimulus is provided (Hou et al 2003) In contrast to endodermal cells of shoots, actin disruption has consistently induced the enhancement of amyloplast sedimentation in the root columella upon gravistimulation (Baluˇska et al 1997; Yoder et al 2001; Hou et al 2004) This could be explained by smaller vacuoles in the columella being less of an impediment to amyloplast movement Nonetheless, like in shoots, the increased sensitivity of roots to gravity may result from diminished system noise and the amplified signals generated by rapidly falling amyloplasts (Hou et al 2004) Although the above proposal is purely speculative at this point, the ability to ma-nipulate cytoskeletal dynamics specifically in the gravity-sensing cells should provide a significant step toward further testing the relationship between the cytoskeleton and plastid-based gravity sensing in plants

There have been other explanations as to how the cytoskeleton and amyloplasts in the columella interact to generate a gravity signal, including a proposal that localized disrup-tion of the actin network can produce a direcdisrup-tional signal by altering the balance of forces acting on plasma membrane receptors (Yoder et al 2001) Moreover, a recent report showing that actin disruption of decapped maize roots can partially restore gravitropism, which points to the intriguing possibility that actin-dependent gravity sensing may occur outside the root cap (Mancuso et al 2006) Although it will require additional work to tease apart the different possibilities, results from recent cellular and pharmacological ap-proaches are leading to new, testable hypotheses on how the cytoskeleton mediates in gravity perception in higher plants

1.6 Concluding Remarks and Future Prospects

over-whelming Both processes working in tandem might be part of a redundant mechanism to ensure the plant’s best chances for survival in its environment

In conclusion, although our view about gravity perception in higher plants continues to revolve around the idea of sedimenting plastids, one of the more pressing issues is how the gravity-induced mechanical stimulus of falling statoliths or, alternatively, the pressure exerted by the entire protoplast, is converted to a chemical signal that subsequently reg-ulates a physiological response As discussed in Chapter 2, studies involving the interac-tions between amyloplasts and other cell organelles such as the vacuole, the cytoskeleton, and the endoplasmic reticulum are fruitful avenues for research that will help address this issue In addition, studies of gravitropism in other plant organs might provide useful ad-ditional information about the gravitropic response For example, the peanut gynophore could be developed into a useful model to discern how the gravity signal is translated into a positive (i.e., downward) or negative (i.e., upward) response Moreover, transcript and protein profiling are helping to identify additional molecular players in the gravitropic sponse (Moseyko et al 2002; Kimbrough et al 2004) An exciting avenue for future re-search will be to observe changes in transcript, protein, and metabolite levels specifically in the cells that are presumed to sense gravity

1.7 Acknowledgment

Funding from the Samuel Roberts Noble Foundation Inc and the National Science Foundation (DBI-0400580) is gratefully acknowledged

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Benjamin R Harrison, Miyo T Morita, Patrick H Masson*, and

Masao Tasaka

21

2.1 Introduction

As discussed in Chapter 1, most plant organs use gravity as a growth guide However, dif-ferent organs will interpret that information in difdif-ferent ways Shoots grow upward toward light, aboveground environments in order to optimize photosynthesis, exchange gases, and perform their reproductive functions Most roots, on the other hand, grow downward, into the soil, where they anchor the plant and take up water and nutrients necessary for plant growth, development, and reproduction To understand how these organs interpret differ-ently the information provided by gravity, we first need to understand the molecular mech-anisms that govern gravity signal transduction in gravity-sensing cells, termed statocytes Before we describe the current state of our knowledge on the mechanisms that gov-ern gravity signal transduction in plants, it is important to understand that the gravity-responding organs of higher plants are diversified in their tissue structure and develop-mental origin In cereal grasses, the graviresponsive coleoptile of seedlings is a hollow cylindrical sheath, whereas the pulvini of adult plants are swellings at the base of each internode In dicots, hypocotyls, and epicotyls of young seedlings and leaf petioles and stems of adult plants are all graviresponsive Similarly, both primary and lateral roots are graviresponsive in monocots and dicots, even though the site of root gravicurvature does not contain obviously differentiated statocytes Hence, the morphology and cytol-ogy of different plant organs may affect the machinery that modulates their gravitropic responses In spite of such diversity, all graviresponsive organs share two common fea-tures: they contain graviperceptive cells with sedimentable amyloplasts (Sack 1997), and they develop asymmetry in auxin concentration between their upper (lower concen-tration) and lower (higher concenconcen-tration) flanks upon gravistimulation (Philippar et al 1999; Muday and DeLong 2001; Friml et al 2002; Long et al 2002) Thus, within these organs, a gravitational signal perceived through the relocalization of amyloplasts within differentiated statocytes is converted into biochemical signal(s) that is (are) transmitted to adjacent cells, leading to the formation of a lateral gradient of auxin at the elongation zone, responsible for the gravitropic curvature (see also Chapter 3)

Although seemingly similar in global terms, the physiological and biochemical events that accompany gravitropism in aboveground organs and roots differ substantially in the details For instance, the site of gravity perception and signal transduction (endodermal cells) overlaps with the site of curvature response in shoots, which has been proposed to occur simultaneously and uniformly along the organs (Firn and Digby 1980) Roots, on the other hand, show a physical separation between the primary site of gravity perception and signal transduction (the root cap columella) and the site of curvature response (the

elongation zones) (Masson et al 2002) Hence, in roots, the gravity-induced lateral auxin gradient generated across the cap has to be transmitted basipetally, from the site of per-ception and signal transduction to the site of response in order for a gravitropic curvature to develop (Figure 2.1A and Color Section)

In aboveground organs, the signal has only to be transmitted laterally, across the organ In the Arabidopsis shoot elongation zone, the epidermis, cortex, gravity-perceiving endo-dermis, and stele are arranged radially, in successive layers, and Arabidopsis stem seg-ments dissected from any part of the elongation zone are gravitropic (Fukaki et al 1996) Thus, the gravity signal perceived in the endodermal cell layer may be transmitted in an inner-to-outer fashion (laterally), leading to a unilateral, asymmetric auxin distribution between the lower and upper flanks that results in differential cell elongation and conse-quent gravitropic curvature (Fukaki et al 1998; Tasaka et al 1999) This centrifugal as-pect of gravitropic signaling between cell types seems general in aboveground organs However, we not know whether all cells in the endodermal layer play equal roles in gravity perception and signaling (Figure 2.1B and Color Section)

Recent physiological and molecular genetic studies have provided insights into the mechanisms that govern gravity signal transduction in roots, hypocotyls, shoots, and ce-real pulvini This information, summarized in this chapter, will provide the foundation for future research aimed at better understanding how this machinery might be modu-lated by endogenous and environmental cues

2.2 Gravity Signal Transduction in Roots and Aboveground Organs

Roots constitute an excellent system for analysis of gravity signal transduction in plants The physical separation existing between the primary site of gravity perception (the root cap columella) and the site of differential cellular elongation that drives the correspon-ding curvature response (the elongation zones) allows for careful dissection of the mo-lecular mechanisms involved in each phase of the process Hence, it is not surprising that several teams have focused their research on this system to decipher some of the molec-ular mechanisms that govern gravity signal transduction The gravity receptor remains unknown However, several potential gravity signal transducers have recently been un-covered They include cytoplasmic cations, inositol 1,4,5-trisphosphate (InsP3), and

sev-eral proteins

23 seedling (left) showing the cap, distal and central elongation zones (DEZ and CEZ), and the mature zone (MZ) The inset (right) represents a confocal image of a propidium iodide-stained root tip showing the root cap (with its L1, L2, and L3 layers of columella cells, and lateral cap cells, LRC), the promeristem (with its quiescent center cells, QC, surrounded by initials) The root proper is composed of several cell layers, includ-ing the epidermis (Ep); the cortex (C); the endodermis (En); and the stele (St) The primary site of gravity perception in roots is the root cap, with the statocytes located in the first two layers of columella cells, whereas the curvature response initiates in the DEZ Therefore, a gravitropic signal has to be transmitted basipetally upon gravistimulation B Top of an inflorescence stem showing the stem ended by a shoot apical meristem (SAM), flowers, and siliques The entire stem region located below the SAM in this picture is part of the elongation zone (EZ) The middle drawing corresponds to a region of shoot stem Microscopical image of a longitudinal section of this region (indicated by the rectangle in the drawing) is represented on the right showing the epidermis (Ep), three layers of cortical cells (C), one layer of endodermis (En), and the stele (St) Sedimenting amyloplasts, located in the endodermal statocytes, are indicated by black arrowheads Hence, the sites of gravity sensing and curvature response overlap in stems, and the gravitropic signal has to be trans-mitted outward, rather than basipetally, in order for a curvature response to develop

The same second messengers have been implicated in the modulation of gravity sig-nal transduction in aboveground organs as in roots, including cytoplasmic pH (Johannes et al 2001) and InsP3(Perera et al 1998, 1999, 2001) Furthermore, several proteins have

been identified as potential gravity signal transducers in shoots (Yamauchi et al 1997; Wyatt et al 2002; Morita et al 2006) Yet, there has been no indication of relocalization of auxin transporters within the statocytes in aboveground organs, even though evidence for asymmetric downward transport of auxin across gravistimulated organs also exists in these systems (Li et al 1991; Kaufman et al 1995; Philippar et al 1999; Friml et al 2002; Long et al 2002; Abas et al 2006) Below, we briefly discuss how these pieces of the gravity signal transduction puzzle might fit together to promote pathways that ulti-mately lead to the curvature of roots and aboveground organs

2.2.1 Do Mechano-Sensitive Ion Channels Function as Gravity Receptors?

As discussed in Chapter 1, several researchers have proposed that membrane-associated mechano-sensitive ion channels might function as gravity receptors in plant statocytes The sedimentation of, and/or pressure/tension exerted by amyloplasts would trigger the opening of such channels at sensitive membranes This would allow for a flux of Ca2+ ions within the statocytes, serving as second messengers to trigger a cascade of events that would ultimately lead to the lateral polarization of statocytes, as discussed above (Sievers et al 1984; Sievers et al 1989; Pickard and Ding 1993; Volkmann and Baluˇska 1999; Yoder et al 2001)

As discussed in Chapter 5, a number of pharmacological studies support a role for Ca2+in gravity signal transduction Agents that inactivate mechano-sensitive ion chan-nels (i.e., Gd3+or La3+) alter the function of Ca2+regulatory proteins (calmodulin and

Ca2+-ATPases), and Ca2+ chelators all inhibit gravitropism (reviewed in Sinclair and Trewavas 1997; Fasano et al 2002)

If Ca2+-selective, mechano-sensitive ion channels contribute to gravity signal trans-duction in the statocytes, one should be able to detect changes in cytosolic Ca2+levels

Unfortunately, the aequorin-derived signal could only be detected if hundreds of

Arabidopsis seedlings were examined in bulk, and the luminescence was too weak for

identification of its source In the future, it will be necessary to use a similar detection strategy to analyze gravity-induced Ca2+ transients in seedlings that express aequorin specifically within the root and hypocotyl statocytes to determine whether the signal de-rives from these cells It will also be important to determine whether such a signal disap-pears in mutants that are defective in early phases of gravity signal transduction, such as the starch-deficient and signal-transduction mutants described below

In separate experiments using either the system described above or Ca2+-sensitive fluorophores, other researchers were unable to detect changes in cytosolic Ca2+levels in the statocytes upon gravistimulation (Sedbrook et al 1996; Legue et al 1997; Fasano et al 2002; Massa and Gilroy 2003) Although these negative results cast doubt on the pos-sible involvement of Ca2+ in gravity signal transduction within the statocytes, they may reflect a lack of sensitivity of the Ca2+ detection approaches used, or a highly localized, yet functionally significant, Ca2+pulse undetectable by these sensors It is interesting to

note that even small and/or highly localized changes in cytosolic Ca2+levels within the root statocytes might be functionally relevant because these cells express high levels of calmodulin (Sinclair et al 1996; see also Chapter 5)

Additional complexity arises in studies that investigate the role of cytosolic Ca2+in gravity signal transduction within the root statocytes As emphasized in Chapter 1, touch stimulation of the root tip promotes a fast increase in cytosolic Ca2+ levels within the stimulated peripheral cap cells, which propagates to surrounding cells to eventually reach the columella region There, the corresponding Ca2+wave appears to inhibit gravitropism by interfering with amyloplast sedimentation (Massa and Gilroy 2003) Hence, if a Ca2+ wave signals an inhibition of gravitropic sensitivity in the statocytes in response to a touch stimulus at the cap, a role for gravity-induced Ca2+flux in gravity signal transduc-tion within the statocytes would require a corresponding Ca2+signal that displays a

dis-tinctive signature (Massa and Gilroy 2003)

the plastids’ osmotic pressure during division through mechanisms related to those of mechano-sensitive ion channels (Haswell and Meyerowitz 2006)

As of now, there has been no direct evidence for an involvement of these two potential mechano-sensitive ion channels in gravity signal transduction (Haswell and Meyerowitz 2006) However, some of the eight remaining Arabidopsis MSL genes are predicted to en-code proteins targeted to other cellular compartments Even more strikingly, some of these are highly expressed in the root statocytes and are transcriptionally responsive to gravis-timulation (Neal and Masson, unpublished data; Nawy et al 2005) Careful functional analysis of this outstanding set of genes, along with that of other potential plant ion chan-nels with as-yet poorly defined properties and functions (Schulz et al 2006), may soon yield important new insights into the gravitropic response

It remains quite possible that gravity reception involves mechanisms that not rely upon mechano-sensitive ion channels It is quite exciting to note recent developments in the study of model systems that involve single-cell gravitropic responses As discussed by Braun and Hemmersbach in Chapter of this book, Chara rhizoids sense gravity through the sedimentation of BaSO4-containing statoliths Elegant experiments with this system have demonstrated that the statoliths must sediment onto sensitive membranes at a subapi-cal region of the cell for the signal to be perceived and transduced into a curvature response (Braun 2002) Interestingly, experiments involving short-term exposure to hypo- and hyper-gravity have indicated that simple contact of statoliths with the sensitive membrane is suf-ficient for gravity perception to occur; differential pressure or tension is not needed (Braun 2002; Limbach et al 2005) This result led the authors to postulate that gravity signal trans-duction might be triggered by molecular interaction between ligands carried by the sedi-menting statoliths and receptors located at the sensitive membranes (Limbach et al 2005)

A similar ligand-receptor model of gravity reception should not be excluded in higher plants In fact, experiments involving starch-deficient mutants of Arabidopsis (which have lighter amyloplasts that not appear to sediment), or seedlings exposed to drugs that destabilize the actin filaments in shoot statocytes—thereby disabling amyloplast sed-imentation (see Chapter 1)—have suggested that a few “rogue” sedimenting amyloplasts might be responsible for the remaining gravitropic capability associated these systems (Kiss et al 1997; Palmieri and Kiss 2005; Saito et al 2005) This interesting model could be tested by subjecting higher plants to short-term hyper- and hypogravity treatments similar to those performed on Chara (Limbach et al 2005).

2.2.2 Inositol 1,4,5-Trisphosphate Seems to Function in Gravity Signal Transduction

A potential role for inositol 1,4,5-trisphosphate (InsP3) in gravity signal transduction was

recently suggested from elegant physiological, biochemical, and genetic studies utilizing both aboveground and root model systems These experiments demonstrated both the ex-istence of gravity-induced changes in InsP3 levels in stimulated organs and a need for wild-type levels of InsP3for full graviresponsiveness

occurred only in the lower pulvinus halves and correlated with the bending response These changes in InsP3levels were functionally relevant because treatments with an in-hibitor of phospholipase C, an enzyme that contributes to InsP3synthesis, blocked the in-crease in InsP3levels and inhibited the bending response in oat (Perera et al 2001)

Recently, the same investigating team detected similar biphasic changes of InsP3 lev-els upon gravistimulation in Arabidopsis inflorescence stems (Perera et al 2006) In both cereal pulvini and dicot inflorescence stems, the second increase in InsP3levels preceded

visible bending responses, suggesting its involvement in early phases of the pathway To further investigate the potential role of InsP3in gravity signal transduction,

trans-genic Arabidopsis thaliana plants expressing human type I inositol polyphosphate 5-phosphatase, an enzyme that specifically hydrolyzes InsP3, were generated These

plants grew like wild type despite containing very low levels of InsP3(10% of wild-type levels) However, they exhibited reduction in the kinetics of inflorescence-stem, hypocotyl, and root gravitropism, enhanced root gravitropic sensitivity to extracellular Ca2+, decrease in basipetal auxin transport along the root, and delay in the development of lateral auxin gradients upon gravistimulation, as deduced from expression analyses of auxin-sensitive reporter constructs (Perera et al 2006) Because InsP3is a soluble second

messenger that propagates localized Ca2+fluxes through the cell and to neighboring cells, and the levels of both molecules display parallel, biphasic increases upon gravistimula-tion, it appears likely that both Ca2+and InsP3contribute to gravity signal transduction in most or all organs of the plant, possibly by modulating auxin transport (Plieth and Trewavas 2002; Perera et al 2006) Determination of the tissue(s) within responding or-gans where both InsP3and Ca2+changes occur upon gravistimulation should provide

im-portant insights into their mode of action

2.2.3 Do pH Changes Contribute to Gravity Signal Transduction?

Although an involvement of cytosolic Ca2+as a second messenger in gravity signal trans-duction remains speculative, better evidence exists for a contribution of cytoplasmic pH in this phase of the response

If cytosolic pH contributes to gravity signal transduction, its level in the statocytes should change upon gravistimulation, and interference with such changes should affect the response Indeed, both assumptions were recently validated Rapid cytoplasmic pH changes upon gravistimulation were observed in the statocytes of both maize pulvini and

Arabidopsis roots (Scott and Allen 1999; Fasano et al 2001; Johannes et al 2001;

Boonsirichai et al 2003; Hou et al 2004; Young et al 2006) By using longitudinal maize stem sections loaded with a pH indicator, Johannes and collaborators were able to moni-tor the cytoplasmic pH of both gravity-sensing bundle-sheath and parenchyma cells upon gravistimulation They found that gravistimulation promotes a fast alkalinization of the bundle-sheath statocytes without altering the pH of parenchyma cells They also found that the gravity-induced cytoplasmic alkalinization in the pulvinus statocytes occurs only in a restricted region of the cytoplasm where the sedimenting amyloplasts accumulated

baseline levels within to 10 (Scott and Allen 1999; Fasano et al 2001; Boonsirichai et al 2003; Young et al 2006) Although Scott and Allen suggested distinct kinetics in the cytoplasmic alkalinization of layer-2 cells (layers of columella cells are represented in Figure 2.1A) between upper and lower statocytes of gravistimulated roots, and an acid-ification of layer-3 cells, Fasano and collaborators described similar alkalinization in all statocytes within both columella tiers (Fasano et al 2001) Gravity-induced alkalinization of the root statocytes was dramatically attenuated in pgm, a mutant that contains starch-less plastids and displays altered gravisensitivity (Fasano et al 2001) It was also accom-panied by an acidification of the apoplast in wild-type roots, suggesting it might result from the activation of plasma membrane or vacuolar H+transporters (Fasano et al 2001; Li et al 2005)

Interestingly, altering the cytoplasmic pH of root statocytes by releasing preloaded caged protons resulted in a delay in the gravitropic response (Fasano et al 2001) Furthermore, treating root caps with agents that acidify the apoplast and the cytoplasm at concentrations that not alter the overall rate of root growth resulted in an enhancement of the gravitropic response, whereas treatments with alkalinizing agents delayed it (Scott and Allen 1999) Likewise, treatments with agents that disrupt actin filaments resulted in both sustained cytoplasmic alkalinization upon short periods of gravistimulation and en-hanced gravicurvature, as discussed in Chapter (Hou et al 2004) Even though the ini-tial studies differed in the details of their observations, current data converge to suggest an important role for cytosolic pH in gravity signal transduction in both coleoptiles and roots It should be cautioned that the data obtained so far not demonstrate an essential role for pH in this pathway, as none of the cytosolic pH manipulations performed so far have led to a complete elimination of gravitropism

In conclusion, cytoplasmic pH changes may have a universal role in the early signaling phases of gravitropism What might they be doing in this process? We currently have no definite answer to this important question, partly because we have only a rudimentary un-derstanding of the molecular mechanisms that govern it We also have a limited knowl-edge of the locale of these gravity-induced pH changes within individual statocytes It has been proposed that gravity-induced pH changes in the statocytes might facilitate auxin transport (Fasano et al 2002) Indeed, such pH changes may be related to the asymmetric pH responses that were observed at the surface of gravistimulated roots by proton-selective microelectrodes These asymmetric surface-pH changes originated at the root cap and progressed along the root tip at a rate comparable with polar auxin transport (Monshausen and Sievers 2002) It is possible that surface-pH changes and polar auxin transport are related, and that the gravity-induced pH changes in columella cells regulate the activity or cellular distribution of auxin transporters in the statocytes (Fasano et al 2002) In agreement with this model, mutant and transgenic Arabidopsis plants with altered expression of a H+-pyrophosphatase (AVP1) display altered auxin transport along with al-tered expression and mislocalization of the PIN1 auxin efflux facilitator (Li et al 2005)

2.2.4 Proteins Implicated in Gravity Signal Transduction

as-pects of polar auxin transport or auxin response, a few have been obtained that affect ear-lier phases of gravity signal transduction that occur in the statocytes For instance, muta-tions that affect starch biosynthesis, such as pgm, have been shown to affect gravitropism. As discussed in Chapter 1, this is not surprising since starch is a dense material that in-creases the weight of amyloplasts, enabling their sedimentation in the favorable environ-ment presented by the statocytes’ cytoplasm (Kiss et al 1989)

Other mutations that affect gravitropism without altering phototropism, starch synthe-sis, amyloplast sedimentation, or growth responses to auxin, other phytohormones, or polar auxin transport inhibitors have also been identified This class of mutants likely af-fects genes involved specifically in gravity signal transduction The first such mutants isolated in Arabidopsis thaliana affected the ARG1 locus The rhg/arg1 mutants dis-played altered root and hypocotyl gravitropism while maintaining wild-type root-growth responses to phytohormones and polar auxin transport inhibitors, as well as normal pho-totropism (Fukaki et al 1997; Sedbrook et al 1999) The latter observation was particu-larly revealing because it indicated that the mutant organs retained their ability to curve in response to other directional cues Hence, the defect was likely to lie in the early phases of gravity signal transduction

The ARG1 gene encodes a J-domain protein that is conserved between plants and the worm Caenorhabditis elegans, but is absent in yeast or other animals (Sedbrook et al. 1999) This protein was found to contain a J-domain at its N-terminus, a central hy-drophobic region and a C-terminal domain predicted to form a coiled coil structure (typ-ically involved in protein–protein interactions) The C-terminal region shares sequence similarity with proteins that interact with the cytoskeleton, although strong evidence for cytoskeleton interaction is currently lacking (Sedbrook et al 1999; Boonsirichai et al 2003)

In other, better-characterized, J-domain proteins, the highly conserved J-domain di-rectly interacts with the HSP70 chaperone, modulating its ATPase activity The residues needed for this interaction are conserved in ARG1, suggesting that this protein might also function in association with HSP70 in the folding, trafficking, localization, and/or regu-lation of gravity signal transducers in the statocyte (Sedbrook et al 1999)

A combination of biochemical fractionation and functional GFP-fusion localization studies demonstrated that ARG1 is a peripheral membrane protein that associates with multiple components of the vesicle trafficking pathway in all plant cells Targeting its ex-pression to the root or hypocotyl statocytes of an arg1-2 null-mutant rescued the gravi-tropic phenotype of the corresponding organ (root or hypocotyls, respectively), demon-strating ARG1’s role in early phases of gravity signal transduction

Although the Arabidopsis thaliana genome contains more than 90 J-domain genes (Miernyk 2001), only two encode proteins that are similar to ARG1 throughout their lengths: ARG1-Like (ARL1) and ARL2 A reverse genetic approach was used to demonstrate that ARL2 also contributes to early phases of gravity signal transduction, whereas ARL1 does not In fact, physiological and molecular studies of arl2 mutant seedlings also showed defects in lateral auxin transport and PIN3 protein relocalization within the root statocytes upon gravistimulation (Guan et al 2003; Harrison and Masson 2006) Hence, ARG1 and ARL2 appear to function in the same pathway In agreement with this conclusion, arg1-2 arl2-1 double mutants show intermediate gravitropic defects that are similar to those of the corresponding single mutants (Guan et al 2003)

Analysis of double mutants between arg1-2, arl2-1, and pgm-1 also led to surprising results Remember that pgm-1 affects phosphoglucomutase, an enzyme that contributes to starch biosynthesis As discussed above, both pgm-1 and arg1-2 or arl2-1 display al-tered kinetics of gravitropism However, their gravitropic defects are not complete and their organs still develop reasonably strong gravitropic responses, though with slower ki-netics If PGM and ARG1/ARL2 contribute to a linear gravity signal transduction path-way in the statocytes, double mutants should display a phenotype similar to that of sin-gle mutants

When arg1-2 pgm-1 and arl2-1 pgm-1 double mutants were analyzed, a surprisingly strong enhancement of the gravitropic defect was observed relative to that of single mu-tants (Boonsirichai et al., unpublished data; Guan et al 2003) This result can be ex-plained in several ways First, it is possible that ARG1 and ARL2 function in a pathway that is distinct from the PGM pathway In fact, as discussed in Chapter 1, several exper-iments have suggested the existence of more than one mechanism of gravity sensing in roots Second, it is possible that these mutations affect partially their respective steps in a linear pathway Indeed, ARG1 has been postulated to function as part of a chaperone complex that might modulate, but not be required for, the targeting or activity of membrane-associated proteins in the statocytes, whereas pgm-1 mutants are not com-pletely defective in amyloplast sedimentation (Saether and Iversen 1991)

In addition to revealing the possible topology of the gravisensing network, these double-mutant studies suggest alternative genetic strategies to search for novel gravity signal transducers For instance, a screen for genetic enhancers of arg1-2/arl2-3 may lead to the identification of new gravity signal transducers in the “PGM genetic pathway,” whereas searching for enhancers of pgm will likely lead to the discovery of genes that function in the “ARG1/ARL2 genetic pathway.” The recent isolation and initial character-ization of mutations falling in the first group (genetic enhancers of arg1-2) allowed the identification of an outer plastid membrane-associated protein as a possible gravity sig-nal transducer, boding well for the success of this approach (Stanga et al 2006)

Genetic studies have also been quite effective at uncovering gravity signal transducers in aboveground organs through careful investigations of Arabidopsis mutants with de-fects in shoot gravitropism As discussed in Chapter 1, a number of shoot gravitropism

(sgr) mutants have been identified in Arabidopsis which exhibit reduced gravitropic

sedi-mentation in gravity susception, and uncovered an important role for vacuolar biogene-sis and function in gravity perception or signal transduction in shoots (Saito et al 2005) In addition, other sgr mutants also identified new gravity signal transducers in

Arabi-dopsis shoots.

sgr5 and sgr6 are among the shoot gravitropism mutants that are most likely to be

de-fective in early phases of gravity signal transduction within the shoot statocytes Indeed, amyloplasts in mutant shoot endodermal cells sediment almost like wild type, indicat-ing that gravity susception is not affected (Morita et al 2006; Yano et al., unpublished results) SGR5 is a zinc-finger protein that is localized in the nucleus and is mainly ex-pressed in the endodermis In addition, endodermis-specific expression of wild-type

SGR5 in the sgr5-1 mutant restored shoot gravitropism to wild-type levels Hence,

SGR5 is probably a transcription factor that contributes to early events of gravity per-ception and/or signaling in the statocytes (Morita et al 2006) Further analyses, such as exploration of downstream target genes of SGR5, should clarify its function in shoot gravitropism

A similar analysis of sgr6 mutants suggested that the SGR6 protein is also involved in an early step of gravity signal transduction in stem statocytes, subsequent to amyloplast sedimentation Unfortunately, the molecular function of SGR6 remains unknown, consid-ering that its amino acid sequence is conserved with predicted orthologous proteins of unknown function in higher eukaryotes (Yano et al., unpublished results)

An alternative screening approach was also developed to isolate additional shoot grav-itropism mutants with defects in gravity perception or early phases of signal transduction (Wyatt et al 2002) Arabidopsis inflorescence stems show no response to gravistimula-tion at 4°C However, stems that are gravistimulated by horizontal placement at 4°C can execute a bending response to the cold gravistimulus if returned to the vertical position at room temperature within the next hour (Fukaki et al 1996) It has been demonstrated that basipetal auxin transport is abolished in the inflorescence stems of wild-type plants at 4°C (Nadella et al 2006)

Taking advantage of this unusual behavior, Wyatt and collaborators (2002) isolated several gravity persistence signal (gps) mutants that exhibit normal shoot gravitropism at room temperature but display abnormal bending responses to stimuli provided in the cold gps1 does not bend, gps2 bends in the wrong direction, and gps3 over-responds when returned to room temperature after cold gravistimulation (Wyatt et al 2002) Amyloplasts sediment in the direction of gravity in all gps mutants during cold gravis-timulation, indicating that gravity susception is not affected

To investigate a possible effect of the gps mutations on the ability of inflorescence stems to “remember” a cold stimulus by developing a lateral auxin gradient upon return to vertical position at room temperature, Wyatt and her collaborators studied expression of the auxdependent pIAA2::GUS gene in cold gravistimulated wild-type and gps in-florescence stems As expected, wild-type stems showed asymmetrical activation of GUS expression on the lower side of a section of its stem elongation zone On the other hand,

gps mutant plants displayed patterns of GUS expression that were consistent with the

displayed increased GUS expression on the lower side in an extended region along the elongation zone of its stems (Nadella et al 2006) These results suggest that the gps mu-tants fail to properly establish a lateral auxin gradient across their inflorescence stems after cold gravistimulation, supporting a role for the corresponding genes in early phases of gravity signal transduction

Initial results in the molecular genetic analysis of three GPS genes appear to support their involvement in gravity signal transduction (Sarah Wyatt, personal communication)

GPS1 encodes a cytochrome P450 of unknown function Although GPS1 is not

func-tional in the roots, a root-specific family member has been identified that is up-regulated in response to gravistimulation Initial experiments indicate that these P450s may be in-volved in synthesis of flavonoids and, thus, the regulation of auxin transport through that pathway (Buer and Muday 2004; Withers and Wyatt, unpublished data) GPS2 encodes a hypothetical synaptobrevin/vesicle-associated membrane protein, v-SNARE (McCallister and Wyatt, unpublished data) GPS2 protein may be involved in transport of the PIN ef-flux carriers or other regulatory molecules in the inflorescence stem (see Chapter 5) Finally, GPS3 encodes a transcription factor with a B3 DNA-binding domain similar to auxin response factors (ARFs) However, GPS3 protein lacks the C-terminal dimerization domain common among ARF proteins (see Chapter 5) Initial subcellular localizations of GPS3 using a GFP fusion support nuclear localization for the protein, but its role in grav-itropic signal transduction is as yet unknown (Nadella and Wyatt, unpublished data) Hence, these GPS genes hold great promise to further our understanding of the molecu-lar mechanisms that govern gravity signal transduction in shoots

2.2.5 Global ‘-omic’ Approaches to the Study of Root Gravitropism

Although the genetic approach has been successful at identifying new gravity signal transducers, it also has limitations due to functional redundancy associated with frequent gene duplications in plants and from pleiotropy, both of which mask function in gravi-tropism Trying to bypass such difficulties, several groups have recently used techniques derived from genomics and proteomics to identify genes or proteins whose expression varies early in response to gravistimulation Their hope is that some of these candidates will contribute to gravitropism

con-tained short, conserved sequences in their promoters, suggesting a potential role for these DNA motifs as cis-elements in the regulation of gene expression by gravistimulation (Moseyko et al 2002)

Although this initial study provided exciting new information on transcriptional re-sponses to gravistimulation in Arabidopsis seedlings, it suffered from the fact that entire seedlings were analyzed, including organs that respond in opposite ways to gravistimula-tion (hypocotyls and roots), and from probing only a fracgravistimula-tion of the Arabidopsis genome. A second study used similar strategies to monitor transient changes in gene expression in primary root tips of Arabidopsis thaliana seedlings in a time course during the first hour of gravity- and/or mechano-stimulation The whole-genome Affymetrix ATH1 microar-ray was used in this analysis (Kimbrough et al 2004)

This study helped uncover clusters of genes that show similar kinetics of expression change in the root tip upon gravistimulation A vast majority of the differentially ex-pressed genes (1,665 genes, or 96% of the regulated genes) were regulated by both gravity- and mechano-stimulation Only 65 differentially expressed genes showed spe-cific up-regulation in response to gravistimulation Five were up-regulated by a factor of three or more within of gravistimulation, and remained high during the first 30 of the response (Kimbrough et al 2004) These fast graviresponding genes did not change their expression in response to gravistimulation in arg1-2 and arl2-3 mutant back-grounds, confirming the key role played by ARG1 and ARL2 in early phases of gravity signal transduction in Arabidopsis root tips, upstream of the transcriptional responses (Yester et al 2006)

Most of the genes found to be regulated by gravity- or mechano-stimulation again fell into only a few functional classes: Transcription (258 genes); Metabolism (144 genes); Protein fate (114 genes); and Signal transduction (97 genes) Only a minority of the dif-ferentially expressed genes fell in the Defense (31) and Stress (14) functional categories, which were the most highly represented classes in Moseyko et al (2002) Furthermore, only three genes were found to be regulated by gravity and/or touch in both studies (Moseyko et al 2002; Kimbrough et al 2004) The difference in results between these two studies probably reflects differences in experimental procedures, as discussed in Kimbrough et al (2004)

Now that multiple genes have been identified whose expression varies in response to gravistimulation, they can be tested for a contribution to gravity signal transduction by reverse genetics (Kimbrough et al 2004)

Although global expression profiling is useful for identifying clusters of genes with similar expression profiles under defined conditions, it cannot uncover post-transcriptional regulatory processes Hence, attempts have been made at identifying proteins whose abundance, localization, and/or post-translational modifications are altered by gravistim-ulation

samples, and separated by two-dimensional gel electrophoresis (2D-GE) This form of electrophoresis separates proteins based on their isoelectric point in the first dimension, and on their molecular weight in the second dimension After electrophoresis the gels were autoradiographed, leading to protein-spot profiles for the extracts under investiga-tion When protein profiles from control and gravistimulated seedlings were compared, out of approximately 600 detectable spots increased in intensity in the gravistimulated samples relative to control A similar experiment testing the effect of continuous stimu-lation by rocking the dishes over a period of 24 hours led to the identification of 10 grav-ity up-regulated and gravistimulation-specific protein spots (Sakamoto et al 1993)

To detect potential differences in protein phosphorylation upon stimulation, control and continuously rocked samples were labeled with 32P-orthophosphate during the stim-ulus Proteins that were phosphorylated during the period of treatment should appear as radioactive spots on the 2D-GE gels By comparing radioactive protein spots between control and continuously rocked samples, Sakamoto et al (1993) were able to demon-strate that continuous rocking enhances the phosphorylation of two protein spots Hence, an important conclusion of these studies is that gravity- and/or mechano-stimulation pro-mote changes in 2D-GE protein spot intensity, reflective of changes in protein abundance and/or post-translational modification, and differential phosphorylation of specific pro-teins in Arabidopsis thaliana seedlings.

Another attempt at establishing a role for protein phosphorylation in gravity signal transduction sought phosphoproteins with differential levels of expression between upper and lower flanks of gravistimulated oat pulvini (Chang and Kaufman 2000; Chang et al 2003) These investigations uncovered two soluble and two membrane-associated pro-teins that are differentially phosphorylated in lower versus upper pulvinus halves in re-sponse to gravistimulation Subsequent work defined more thoroughly the gravity-induced phosphorylation of one of the soluble oat proteins, demonstrating that it occurs as early as after initiation of gravistimulation and requires a newly synthesized pro-tein This time of initial phosphorylation correlates well with the minimal gravistimula-tion time needed to activate a productive transducgravistimula-tion pathway leading to curvature re-sponse (presentation time), which is 5.2 in oat pulvini The differentially phosphorylated 50kD oat protein is itself a kinase, as demonstrated in autophosphoryla-tion experiments Altogether, these data indicate that the differential phosphorylaautophosphoryla-tion of this 50kD protein in graviresponding oat pulvini might contribute to gravity signal trans-duction in this system (Chang et al 2003)

In these early proteomic experiments, no attempts were made to identify the proteins present in the differentially represented 2D-GE protein spots (Sakamoto et al 1993; Chang et al 2003) However, the last decade witnessed amazing developments in mass spectrometry that truly revolutionized our ability to identify proteins based on their mass, on the mass of their proteolytic products, and on their amino acid content (Li and Assmann 2000) Taking advantage of this technological revolution, researchers are now able to identify differentially represented proteins as long as they are working with an or-ganism whose genome has been completely sequenced It is not surprising that recent proteomic studies have identified a number of Arabidopsis proteins whose abundance or modification varies in response to gravistimulation

Ca2+ signal transduction pathway as being differentially represented in the root tip of

Arabidopsis thaliana seedlings upon 0.5 and hours of gravistimulation Three other

pro-teins were found to change transiently their molecular weight, but not their pI, upon gravistimulation, suggesting post-translational modification (Kamada et al 2005) The stimulation times used in this study allowed significant gravitropic curvature, implying that the identified proteins have the potential of functioning in any phase of gravitropism, from gravity perception or signal transduction to the curvature response

In an attempt to focus on early phases of gravity perception and signal transduction, another study analyzed the protein profiles of 12-min gravistimulated Arabidopsis root-tip samples (Murthy, Young, Sabat, and Masson, in preparation) This time point was cho-sen because it is sufficient to promote productive gravity signal transduction (as deter-mined by the ability of 12-min gravistimulated root tips to develop tip curvatures after subsequent clinorotation; see Chapter 1), but insufficient for curvature initiation

Control and 12-min gravistimulated root tips were dissected, and proteins were ex-tracted using a three-step fractionation procedure Protein fractions were subjected to 2D-GE, followed by silver-staining of the corresponding gels The protein profiles of control and gravistimulated samples were compared Fifty-seven protein spots were uncovered whose staining intensity was altered after 12 of gravistimulation relative to unstim-ulated controls, and the corresponding proteins were identified by mass spectrometry An additional control was included in this experiment, in which Arabidopsis seedlings were gently rotated to the horizontal, then immediately returned to the vertical for an addi-tional 12 min, as a way to control for the mechano-stimulus that accompanies gravistim-ulation Only of the 57 graviresponding proteins also showed differential regulation in response to the mechano-stimulus control Hence, a vast majority of these proteins re-sponded specifically to gravistimulation

Most of the proteins identified in the latter study fell into the following functional cat-egories: Unknown function (24%); Metabolism (17%); Stress and detoxification (13%); Defense (10%); and Energy (10%) (Murthy et al., in preparation) It is striking that only one of these differentially represented proteins is encoded by a gene also found to be tran-scriptionally regulated by gravistimulation (Kimbrough et al 2004) This difference be-tween root-tip transcriptional- and proteomic-response profiles may again reflect differ-ences in the experimental procedures Indeed, transcriptional profiling was carried out on dark-grown seedlings, whereas analysis by Murthy et al involved light-grown material (Kimbrough et al 2004; Murthy et al 2007) On the other hand, of the 16 proteins iden-tified by Sakamoto et al (2005), or their paralogs, were also ideniden-tified as differentially represented by Murthy and collaborators, indicating some consistency between independ-ent proteomic studies

Among the 53 gravity-responding root-tip proteins identified by Murthy and collabo-rators, function in the S-adenosylmethionine (AdoMet) methyl-donor pathway This pathway generates precursors for ethylene and polyamine synthesis, and provides methyl groups for transmethylation reactions that target a number of plant regulatory molecules such as auxin, cytokinin, jasmonate, salicylate, etc This result suggested an involvement of the AdoMet cycle in gravity signal transduction (Young et al 2006)

A reverse genetic approach was used to investigate this possibility One of two

gravitropism A mutation in this gene resulted in plants displaying altered kinetics of root graviresponse and longer presentation time relative to wild type Root cap morphology was also altered, probably as a consequence of altered auxin accumulation in the root cap Upon gravistimulation, adk1-1 mutant seedlings failed to relocalize the auxin efflux fa-cilitator PIN3 to the lower membrane of their root statocytes, confirming a role for this gene in early phases of gravity signal transduction (Young et al 2006)

Surprisingly, adk1-1 roots displayed wild-type cytoplasmic alkalinization of their sta-tocytes in response to gravistimulation Furthermore, when expression of three of the five fast gravity-responsive genes described above was analyzed, one (At4g23670) still in-creased in expression in adk1-1 root tips upon gravistimulation as it did in wild type, whereas the two other genes (At5g38020 and At5g48010) did not respond to gravistimu-lation in adk1-1 (Yester et al 2006) Hence, adk1-1 affected differently the expression response to gravistimulation of three fast gravity-responsive genes, even though arg1-2 and arl2-1 obliterated completely the response of all three, as discussed above (Yester et al 2006) It is interesting to note here that At5g38020, whose expression response to gravistimulation is obliterated by adk1-1, encodes an enzyme whose predicted biochem-ical function (AdoMet-dependent methyltransferase activity) is directly associated with the AdoMet cycle (Kimbrough et al 2004; Schoor and Moffatt 2004; Yester et al 2006)

Together, these fascinating results can be interpreted in several ways For instance, gravity signal transduction could involve a single linear pathway in which ADK1 func-tions downstream of ARG1 to mediate gravity-induced PIN3 relocalization and differen-tial expression of At5g38020 and At5g48010 in the statocytes, with gravity-induced cy-toplasmic alkalinization and differential expression of At4g23670 requiring only the presence of functional ARG1 (Figure 2.2A) Alternatively, it is possible that the gravity signal transduction pathway is bifurcated, with the ADK1-dependent branch of the path-way leading to PIN3 relocalization and up-regulation of At5g38020 and At5g48010 whereas the other branch would lead to cytoplasmic alkalinization and activation of

At4g23670 In this case, ARG1 would function upstream of ADK1, before the point of

pathway bifurcation (Figure 2.2B) Further genetic analysis of double and multiple mu-tants should help resolve this ambiguity

The data discussed above support a role for ADK1 in early phases of gravity signal transduction in roots However, the molecular mechanisms underlying its contribution re-main uncharacterized Does adenosine, the re-main substrate of ADK that feedback inhibits the AdoMet cycle (Schoor and Moffatt 2004), function in cellular signaling like it does in animal systems (Nishizaki 2004)? Or other AdoMet cycle-derived regulatory com-pounds modulate gravity signal transduction? A systematic study on the contribution of distinct biochemical branches derived from the AdoMet pathway in different phases of the root gravitropic response will undoubtedly yield new insights into these long-ranging questions Preliminary results suggest a role for spermine, an AdoMet-derived polyamine, in the signal-transmission or curvature-response phases of root gravitropism (Young et al 2006)

They also constitute an outstanding list of markers that can be used to better define the pathways involved in gravity signal transduction, as illustrated by the expression analy-sis of fast gravity-responsive genes in adk1-1, arg1-2, and arl2-1 mutant root tips It is likely that a combination of reverse genetics and marker-gene-expression analysis will yield important insights into the molecular mechanisms that govern gravity signal trans-duction in plants

2.2.6 Relocalization of Auxin Transport Facilitators or Activity Regulation?

As reviewed in Chapter 3, activation of the gravity signal transduction pathway in roots results in a relocalization of the PIN3 auxin efflux facilitator to the bottom membrane of the statocytes (Friml et al 2002) PIN3, a member of the PIN family of transmembrane auxin efflux facilitators, is expressed in the statocytes of both roots (columella cells) and shoots (endodermal cells) It localizes to the plasma membrane and to vesicles that cycle between plasma membrane and endosome Mutations in the PIN3 gene result in altered root and hypocotyl gravitropism, supporting a role for this protein in gravity signal trans-duction (Friml et al 2002) In statocytes of vertical roots, PIN3 is positioned symmetri-cally at the plasma membrane It rapidly relocalizes laterally, to the bottom membrane, upon gravity stimulation (Friml et al 2002) PIN3 relocalization initiates within of gravistimulation, therefore preceding establishment of a lateral auxin gradient across the root tip As discussed above, PIN3 relocalization requires the presence of fully functional

ARG1, ARL2, and ADK1 (Harrison and Masson, unpublished data; Young et al 2006),

suggesting that it functions downstream of these proteins in the gravity signal transduc-tion pathway in roots

associated molecules within the root statocytes (Boonsirichai et al 2003) It is interesting to note that several of the molecules suggested to function as second messengers in grav-ity signal transduction, including Ca2+, proton flux, and phosphoinositides, have been shown to function in the regulation of vesicular trafficking in other cell types, thus argu-ing for a direct connection between gravity signal transduction and vesicular traffickargu-ing (reviewed in Blancaflor and Masson 2003) However, we have not yet eliminated the pos-sibility that gravity-induced PIN3 relocalization in the root statocytes is a consequence of transporter activity regulation right at the plasma membrane Indeed, changes in auxin level have been shown to result in changes in the cycling and polar localization of PIN pro-teins in other cell types (Blilou et al 2005; Paciorek et al 2005) More work is needed to resolve this ambiguity

The situation is even more complex if one considers potential effects of gravity signal transduction on the polarity of vesicular trafficking and on the localization of auxin trans-porters in shoot statocytes Indeed, PIN3 is also an ideal candidate for molecular linkage between gravity perception and asymmetric auxin distribution in shoots Here, PIN3 is localized to the plasma membrane of the inner longitudinal and bottom sides of endoder-mal cells in vertically oriented organs (Friml et al 2002), and no data currently exist to support or contradict a possible intracellular relocalization of PIN3 upon gravistimula-tion Careful immunolocalization or GFP-PIN fusion expression studies are needed to de-termine whether such a relocalization also occurs in shoot statocytes, and to establish whether it involves other auxin transporters It is essential to establish whether gravistim-ulation regulates the activity of auxin transporters on the outer side of lower flank stato-cytes, or directly regulates the vesicular trafficking of auxin transporters As reviewed in Chapter 3, the transduction pathway could also lead to differential phosphorylation of auxin transport facilitators, or it could regulate the levels of small-molecule effectors of auxin transporters, thereby contributing to the regulation of lateral auxin transport and lateral gradient formation in the absence of transporter relocalization These possibilities will have to be investigated carefully in order to gain a better understanding of the mo-lecular mechanisms that govern gravity signal transduction in both roots and shoots

2.2.7 Could Cytokinin Also Contribute to the Gravitropic Signal?

Recent studies provide potential auxin-related explanations for the surprising character-istics of the initial phases of root gravicurvature, including early gravity-induced increases in the levels of flavonoids at the root tip, which inhibit general auxin transport (Buer and Muday 2004); and an indirect effect of decreased auxin levels on the vesicular trafficking and degradation of the AGR1/EIR1/PIN2/WAV6 auxin efflux facilitator at the top flank (Abas et al 2006) However, independent observations suggest that cytokinin might also contribute to at least some aspects of root gravicurvature Using a cytokinin-sensitive ARR5-GUS reporter construct to indirectly follow changes in cytokinin levels within the root tip, investigators were able to demonstrate that gravistimulation promotes a fast asym-metrical increase in reporter expression within the bottom lateral cap flank, potentially re-flecting differential increases in cytokinin levels at that flank (Aloni et al 2004) The same authors also showed that application of exogenous cytokinin to one side of the elongation zone of vertically oriented roots results in curvature development in the direction of cy-tokinin application Hence, they suggested that gravistimulation might promote a lateral transport of cytokinin across the cap, with accumulation on the lower flank and consequent differential cellular elongation resulting in initial curvature response (Aloni et al 2004)

Although this model is attractive at first glance, it should be cautioned that genetic sup-port for it is currently lacking The cytokinin-deficient mutants analyzed so far have not shown dramatic changes in root gravitropism However, it is true that none of these mu-tants showed complete obliteration of cytokinin sensitivity (Aloni et al 2004) Further-more, expression of one of the genes that contribute mainly to cytokinin biosynthesis in the Arabidopsis root cap, isopentenyl transferase (IPT5), is auxin-sensitive (Miyawaki et al 2004), suggesting the possibility that the asymmetrical activation of ARR5-GUS ex-pression on the lower flank of gravistimulated root caps might simply be a consequence of lateral auxin transport, rather than reflecting an effect of gravity signal transduction on the lateral transport of cytokinins across the cap The kinetics of differential ARR5-GUS ex-pression across gravistimulated root tips appear to precede the asymmetrical activation of the auxin-sensitive DR5-GUS reporter (Aloni et al 2004) However, these two genes are indirect reporters of cytokinin and auxin levels, respectively, functioning at the end of their respective pathways Hence, data derived from comparative analyses of their expression kinetics upon gravitimulation should be interpreted with great caution

Future work will be needed to test asymmetrical activation of ARR5-GUS expression across the root tip in different mutants, such as arg1-2, arl2-1, adk1-1, or pgm, in order to test whether this response lies in one of the known gravity signal transduction pathways Investigation of this response in a variety of mutants affected in diverse aspects of cy-tokinin synthesis and response is also crucial For instance, an analysis of the simple, dou-ble, and multiple mutants carrying defects in the three cytokinin receptor genes found in

Arabidopsis thaliana (Higuchi et al 2004) should be carried out to evaluate their relative

contributions to the initial phases of the root gravitropic curvature

2.3 Gravity Signal Transduction in Organs that Do Not Grow Vertically

organs will tend to grow at a defined angle from the vertical (called gravity set point angle or GSA) after emerging from the primary organ This distinct growth behavior, termed plagiogravitropism, is pushed to its extreme in organs that grow horizontally (di-agravitropism) For instance, stolons and rhizomes grow horizontally, exploring a plant’s neighborhood and colonizing it through vegetative reproduction at their nodes

Interestingly, GSA can also vary depending on the environmental status of a growing organ As discussed in Chapters and 6, touch or lateral humidity gradients can tem-porarily inhibit gravitropism, thereby allowing a root to change its growth pattern to reach better environments despite the influence of gravity (Massa and Gilroy 2003; Takahashi et al 2003) Similarly, light can modulate gravitropism in addition to promoting distinct tropic responses called phototropism (Kiss et al 2002; Kiss et al 2003)

The developmental stage of a plant organ will also influence its GSA For instance, the peanut gynophore will switch from negative (upward) to positive (downward) gravitro-pism upon fertilization, driving the developing fruit into the sand where it has to be lo-cated in order complete its developmental and maturation program (Moctezuma and Feldman 1998)

Hence, not only is it necessary for a plant organ to perceive gravity, it is also impor-tant for that organ to transduce the corresponding vectorial information into a defined growth pattern that will ultimately allow it to reach environments that are better suited for plant growth and development How is this accomplished? Although new information hints at some of the molecular mechanisms that allow distinct directional stimuli (such as touch and humidity gradients) to affect gravitropism in roots (discussed in Chapters and 6), very little is known about how a specific GSA is actually set for a defined organ based on its developmental program or surrounding environment Yet, the tools used in the study of organ growth behavior in plants are becoming increasingly sophisticated, such that we can anticipate a future that will shed light on the regulatory mechanisms that tune gravity signal transduction to the characteristics of an organ’s endogenous and external environment, thereby modulating overall growth and morphogenesis

2.4 Acknowledgments

We thank John Stanga, Laura Vaughn, Jessica Will, Elison Blancaflor, and Simon Gilroy for critical comments on this manuscript This work was supported by grants from NSF, NASA, UW College of Agriculture and Life Sciences Hatch funds, and UW Graduate School Grant-in-Aid to PHM

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Gloria K Muday* and Abidur Rahman

47

3.1 Introduction to Auxins

Auxins have been implicated in controlling elongation, branching, and development of plant organs (as reviewed in Woodward and Bartel 2005), as well as the asymmetric growth known as tropisms, which is the focus of this chapter Of the plant hormones, auxin best resembles the canonical concept of a messenger, being synthesized in one place and acting in another Indole-3-acetic acid (IAA) is the most studied and abundant natural auxin, yet in some plants indole-3-butyric acid (IBA) is at almost equivalent lev-els (as reviewed in Ludwig-Muller et al 1993) Auxins also include synthetic compounds such as 1-naphthaleneacetic acid (1-NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D), which have been used in many studies because they are not susceptible to photolysis from blue and ultraviolet lights, like the native auxin IAA (Stasinopoulos and Hangarter 1990) The synthetic auxins have differences in activity (as reviewed in Woodward and Bartel 2005) and in transport properties (Delbarre et al 1996), which make them useful for de-signing experiments to test specific aspects of auxin function

3.2 Auxin Transport and Its Role in Plant Gravity Response

Auxin moves through plants by a unique cell-to-cell polar transport mechanism, from the shoot meristem and young leaves (Ljung et al 2002) toward the base of stems (as re-viewed in Blakeslee et al 2005) Figure 3.1 contains a diagram summarizing the move-ments of IAA in a seedling Polar auxin transport results in an auxin gradient down the length of the stem or hypocotyl, with the highest auxin concentrations found in the re-gions of greatest elongation (Ortuno et al 1990) Auxin is also synthesized in the root tip (Ljung et al 2005), where auxin transport is more complex, with two distinct polarities Shoot-derived IAA moves acropetally (toward the root apex) through the central cylinder, and basipetally (from the apex toward the base) through the outer layers of root cells (Tsurumi and Ohwaki 1978) Arabidopsis roots also have a tip-focused IAA gradient (Casimiro et al 2001) and basipetal transport of radiolabeled auxin applied to the root tip moves only within the apical centimeter of the root tip (Rashotte et al 2000; Geisler et al 2005) It is this basipetal IAA transport movement that is specifically linked to root gravitropism (Rashotte et al 2000)

In addition to polar transport down the length of plant tissues, the Cholodny-Went hy-pothesis suggests that the lateral transport of auxin across gravity-stimulated plant tissues drives differential gravitropic growth, as indicated in Figure 3.1B (as reviewed in Evans 1991; Trewavas 1992; Muday 2001; see also Chapters and 2) Such a lateral

ution of auxin also appears to be responsible for the curvature response to lateral light stimulation (phototropism; see Chapter 4)

Gravity perception in stems occurs in the starch sheath parenchyma tissues that run the length of the hypocotyl (Fukaki et al 1998) Lateral auxin transport then is believed to occur in multiple cells along the hypocotyl, and the elevated levels of auxin on the lower flank of the hypocotyl stimulate cellular elongation and allow upward growth (Blancaflor and Masson 2003) In contrast, roots sense gravity very locally in the columella cells in the root cap (Blancaflor et al 1998), and auxin is redistributed from the root tip to the lower side of the root after gravity stimulation, rather than being laterally transported across the root (as reviewed by Blancaflor and Masson 2003; see Chapters and 2) As elevations in auxin concentration generally inhibit root growth, this redistribution would result in slower growth on the lower side relative to the upper side, resulting in downward root growth

Asymmetric redistribution of radiolabeled IAA has been measured in both shoots (Parker and Briggs 1990) and roots (Young et al 1990), preceding differential gravi-tropic growth (Parker and Briggs 1990) Additionally, gradients in endogenous free IAA have been observed across gravity-stimulated oat and maize pulvini and maize coleop-tiles (Kaufman et al 1995; Philippar et al 1999; Long et al 2002) Growth of seedlings on IAA efflux inhibitors (as reviewed in Rubery 1990) leads to a rapid inhibition of the gravity response in a number of plant species under conditions where growth still occurs Figure 3.1. Auxin transport is polar in Arabidopsis and other plants A In an upright hypocotyl, inflores-cence, and other stem tissues, auxin moves in single direction, from the shoot apex toward the base (basipetal) In roots, movement from the shoot into the root is from the base toward the root apex (acropetal) through cells in the central cylinder In roots, auxin also moves from the root tip toward the base in a basipetal direction through cells of the cortex and/or epidermis B In a plant reoriented 90 degrees relative to gravity, although auxin transport continues in a polar fashion, lateral auxin transport also occurs In shoot tissues, this transport may occur across the hypocotyl, whereas in roots, redirection of auxin transport is believed to be controlled from the root cap The regions in which gravitropic bending will occur are also indicated

(Katekar and Geissler 1980; Muday and Haworth 1994; Rashotte et al 2000) Recently, synthetic and naturally occurring inhibitors of auxin influx have been identified and these compounds also inhibit gravitropic bending (Rahman et al 2001a; Parry et al 2001a)

Although the validity of the Cholodny-Went hypothesis has been debated (Trewavas 1992), molecular and genetic evidence has provided significant support to this hypothe-sis (as reviewed in Blancaflor and Masson 2003) As discussed in Chapters and 2, one powerful test of this hypothesis has been the examination of auxin-induced gene expres-sion across gravity-stimulated plants Transgenic plants carrying several different auxin-responsive promoters driving the expression of the gene encoding ß-glucuronidase (GUS) or green fluorescent protein (GFP) have now been used to show asymmetric auxin-induced gene expression across gravity-stimulated shoots (McClure and Guilfoyle 1989; Li et al 1991; Li et al 1999) or roots (Larkin et al 1996; Luschnig et al 1998; Rashotte et al 2001; Ottenschläger et al 2003) Although GUS and GFP reporters indi-rectly measure changes in auxin accumulation, the ability to easily observe the expres-sion with high spatial resolution makes this a powerful approach to explore the role of auxin in tropisms

The asymmetric expression of the DR5::GUS reporter in Arabidopsis roots that are vertical and roots reoriented 90 degrees relative to gravity is shown in Figure 3.2 (also see Color Section The inhibition of gravitropic bending and differential auxin-regulated gene expression by IAA efflux inhibitors, as shown in Figure 3.2C (Li et al 1991; Rashotte et al 2001; Paciorek et al 2005), indicates that lateral auxin transport is re-quired for differential gene expression It is likely that this asymmetric gene expression also requires a change in auxin sensitivity (Salisbury et al 1988), perhaps through acti-vation of transcription factors necessary for auxin-induced gene expression (as reviewed in Leyser 2006 and discussed below)

50

pression was visualized in 7-day-old seedlings homozygous for the DR5-GUS reporter construct (D) or for a DR5-GFP reporter Root tips are shown for (A), a vertically grown seedling on control media, and (B and C) seedlings hours after gravity stimulation on (B) control media, (C) µM NPA, or (D) hours after grav-ity stimulation on control media The scale bar is equal to 100 microns A model showing the mechanism of IAA redistribution and gradient formation at the root tip is shown in E and F To simplify the model, both PIN proteins, which are part of the IAA efflux protein complex, and AUX1, an auxin influx protein, have been shown to be asymmetrically localized in the diagram, although the localization of AUX1 was reported to be axial For gradients in IAA to form across a horizontal root, there are likely to be mechanisms to in-crease IAA transport to lower side of the root and dein-crease IAA transport to the upper side that act to change the activity and abundance of IAA efflux proteins

3.3 Approaches to Identify Proteins that Mediate IAA Efflux

Biochemical and genetic approaches have identified a number of proteins associated with auxin transport Auxin transport inhibitors have been used in both of these approaches Auxin efflux inhibitors reduce polar IAA transport, efflux of auxin from membranes and cells, and inhibit physiological processes dependent upon auxin transport, such as gravi-tropic curvature (as reviewed in Rubery 1990) In particular, the inhibitor, naphthylph-thalamic acid (NPA), has been used in biochemical experiments, since a tritiated form of this molecule can be used to follow the activity of one class of auxin transport proteins, termed NPA binding proteins (Muday et al 1993) These proteins have been biochemi-cally characterized using their NPA binding activity (Dixon et al 1996; Butler et al 1998; Hu et al 2000, as reviewed in Muday 2000)

Recently, NPA affinity chromatography has been used to identify a number of proteins that bind NPA (Murphy et al 2002) with some proteins identified by both this method and genetic approaches (Noh et al 2001), as described below More recently, auxin in-flux inhibitors have also been identified (Imhoff et al 2000; Rahman et al 2001a; Parry et al 2001a), but as yet these inhibitors have been used to characterize previously identi-fied proteins, not to identify new proteins

Genetic approaches have been the most productive in identifying candidates for auxin transporters and associated regulatory proteins The screens that have identified these proteins have included altered responses to auxin (Maher and Martindale 1980) or to auxin transport inhibitors (Ruegger et al 1997), altered growth and developmental processes that are dependent upon auxin transport, including gravity response (Chen et al 1998), and a number of developmental processes (Okada et al 1991; Noh et al 2001) Many of the mutant genes have been identified and the functions of the encoded proteins have been linked to IAA influx or efflux, as described below

3.4 Proteins that Mediate IAA Efflux

ex-pressed in roots and is localized asymmetrically in the plasma membrane, consistent with a role in mediating basipetal IAA transport (Müller et al 1998)

A recent report suggests that PIN function is redundant, using an eir1/pin2 mutant line transformed with a PIN1::GFP variant that has localization consistent with PIN2 but op-posite to PIN1 (Wisniewska et al 2006) This construct restored gravitropic bending and lateral IAA transport to eir1/pin2, whereas wild-type PIN1::GFP did not, indicating that the polar localization of PIN proteins is sufficient to direct IAA movement with appro-priate polarity (Wisniewska et al 2006) Finally, the overexpression of PIN7 in tobacco cell cultures significantly enhanced IAA, 1-NAA, and 2,4-D efflux, but not the trypto-phan movement (Petrasek et al 2006), conclusively demonstrating that PIN proteins can mediate IAA efflux

A second family of proteins has also been suggested to participate in IAA efflux by both genetic and biochemical approaches The multidrug resistance/P-glycoprotein (MDR/PGP) gene family encodes proteins with sequence similarity to genes in the ATP Binding Cassette (ABC) transporter superfamily, which transport a variety of molecules in addition to the cytotoxic compounds for which they were initially isolated (Geisler and Murphy 2006) AtMDR1/PGP19, AtPGP1, and AtPGP2 proteins have been shown to bind to an NPA affinity column and expression of AtMDR1 in yeast increases NPA bind-ing activity (Noh et al 2001), consistent with the possibility that these proteins are the target for IAA efflux inhibitors The mdr1/pgp19 mutant has reduced basipetal IAA transport in inflorescence (Noh et al 2001) and roots (Geisler et al 2005), and has phe-notypes consistent with altered auxin transport (Noh et al 2001; Lin and Wang 2005; Geisler et al 2005), including enhanced gravitropic responses in both inflorescences (Noh et al 2003) and roots (Lin and Wang 2005) The pgp1 mutant has a weak pheno-type that enhances the mdr1/pgp19 mutant phenopheno-type (Noh et al 2001; Noh et al 2003; Lin and Wang 2005), suggesting partially redundant functions for these two proteins The

pgp4/mdr4 mutant also has reduced root basipetal IAA transport and gravitropic response

(Terasaka et al 2005)

The ability of PGP1 and PGP4 to mediate IAA and NAA efflux in heterologous sys-tems (Geisler et al 2005; Terasaka et al 2005) further links these proteins to the process of IAA transport (Blakeslee et al 2005) Surprisingly, current evidence supports a role of PGP4 in control of IAA influx, not efflux (Terasaka et al 2005) Additionally, PGP19/ MDR1 has also been shown to mediate IAA efflux from plant tissue culture (Petrasek et al 2006), although it has been difficult to demonstrate its function in heterologous sys-tems (Geisler and Murphy 2006) PGP1, PGP4, and MDR1/PGP19 all have membrane localizations that are asymmetrically distributed across auxin transporting cells, suggest-ing that their localization could convey directional control of auxin transport (Geisler et al 2005; Terasaka et al 2005; Geisler and Murphy 2006) Additionally, AtPGP1 and 19 have been shown to participate in protein complexes with TWISTED DWARF1, a unique plasma membrane-anchored, immunophilin-like protein, which is required for maximal auxin transport and appropriate plant development (Geisler et al 2003)

com-plex formation (as reviewed in Geisler and Murphy 2006) The altered localization of PIN1 in the mdr1/pgp19 mutant is consistent with a protein complex (Noh et al 2003) that is altered in the absence of MDR/PGP proteins The possibility of cooperativity be-tween PIN1 and PGP1 and MDR1/PGP19 was examined in pgp1/pgp19 double mutants that overexpressed PIN1 under the control of an estradiol response promoter (Petrasek et al 2006) PIN1 overexpression in both wild-type and pgp1/pgp19 mutants was sufficient to induce agravitropic root growth in both cases, suggesting that PIN1 action did not re-quire PGP1 and PGP19 protein (Petrasek et al 2006) The recent demonstration that the potassium carrier, TRH1, can also mediate IAA efflux in vivo and in vitro further sug-gests the complexity of proteins that mediate IAA efflux (Vicente-Agullo et al 2004) A complete understanding of the complex assembly of auxin efflux carriers awaits further experimentation

The specificity of IAA transport machinery for naturally occurring auxins other than IAA has also been examined IBA transport has been examined in several species (Ludwig-Muller 2000; Rashotte et al 2003) In Arabidopsis, plants that have either the

aux1 or pin2/eir1/agr1 mutation exhibit wild-type levels of IBA transport (Rashotte et al.

2003) Similarly, IBA transport is not affected by auxin efflux inhibitors such as NPA (Rashotte et al 2003) The Arabidopsis rib1 mutant has altered IBA, but not IAA, trans-port, suggesting that the RIB1 protein may participate in IBA specific transport (Poupart et al 2005) Similarly, in rice roots, the arm2 mutant has altered IBA uptake but wild-type levels of IAA uptake (Chhun et al 2005) These results suggest that there may be additional as-yet unknown mechanisms that mediate transport of other auxins

3.5 IAA Influx Carriers and Their Role in Gravitropism

For many years the existence of an IAA influx carrier was questioned based on the chemiosmotic model of IAA transport (as reviewed in Goldsmith 1977) This model posits that because of the low pH of the extracellular space and a pKa for IAA of 4.8,

some extracellular IAA should be protonated and the hydrophobicity of uncharged IAA should allow it to passively enter plant cells (as reviewed in Goldsmith 1977) Yet, the ma-jority of IAA will be at pH above the pKa, so carrier-mediated uptake of the IAA anion would increase IAA accumulation The demonstrations that IAA uptake was saturable in suspension cells (Rubery and Sheldrake 1974) and that there is substrate-specific uptake of auxins, with IAA and 2,4-D but not 1-NAA, moving into plant cells by carrier-mediated uptake (Delbarre et al 1996) further supported the concept of protein-carrier-mediated IAA uptake Our understanding of auxin influx has been extensively improved through the molecular and functional characterization of AUX1’s activity as an IAA influx pro-tein (as reviewed by Parry et al 2001b; Blakeslee et al 2005) and identification of com-pounds which function as auxin influx inhibitors (Imhoff et al 2000; Rahman et al 2001a)

root branching and root hair formation (Maher and Martindale 1980; Marchant et al 2002; Rahman et al 2002) Consistent with a role in mediating IAA influx, roots of aux1 show a selective resistance to the auxins whose uptake appears to be carrier-mediated, IAA and 2,4-D, but not to the membrane-diffusible auxin, 1-NAA (Delbarre et al 1996; Marchant et al 1999; Yamamoto and Yamamoto 1998) The uptake and transport of IAA are also reduced in aux1 mutants (Rahman et al 2001a; Rashotte et al 2003).

Molecular cloning of AUX1 supported the idea that this protein mediates IAA trans-port as the gene is similar in sequence to the ATF (amino acid transtrans-porter family) of pro-teins (Bennett et al 1996; Young et al 1999; Ortiz-Lopez et al 2000) In the Arabidopsis genome, three other genes showed a high degree of sequence similarity to AUX1 and are termed the LAX (Like AUX1) gene family (Parry et al 2001b) However, the function of the other family members has not yet been reported AUX1 encodes a membrane protein of 48 KD composed of 11 transmembrane (TM)-spanning domains, with a cytoplasmic facing N-terminal domain (Swarup et al 2004) The functional characterization of aux1 alleles revealed that the central region of AUX1 appears particularly important for pro-tein function as nine of the missense mutations cluster between TM VI and VII On the other hand, studies of the sole conditional allele aux1-7 mutant suggest that the C-terminal region of AUX1 may perform a regulatory function (Rahman et al 2001a; Swarup et al 2004) Finally, a recent report provided direct evidence that AUX1 indeed functions as an auxin influx career (Yang et al 2006) Expression of wild-type AUX1 protein increased auxin uptake in Xenopus oocytes In contrast, expression of three inde-pendent point mutants of AUX1, which abrogate the AUX1 function in planta, did not me-diate auxin influx in oocytes The substrate specificity of AUX1 in heterologous expres-sion system is similar to the auxin specificity in plants, showing movement of IAA and 2,4-D, but not NAA and IBA (Yang et al 2006)

The tissue-specific expression pattern of AUX1 provides insight into its developmen-tal role in planta In the lateral root cap cells, AUX1 is localized without polarity, whereas in epidermal cells it is mainly axial, localized at both upper and lower sides (Marchant et al 1999; Swarup et al 2005) This expression pattern has been proposed to facilitate the basipetal transport of auxin between the sensing columella cells and the gravity-responsive cells of the distal elongation zone, but suggests that AUX1 does not specify the basipetal polarity (Swarup et al 2005) Consistent with this model, aux1 mutations disrupt basipetal auxin transport and lead to agravitropic growth (Rashotte et al 2003; Swarup et al 2005) This idea is further confirmed by the restoration of gravitropic re-sponse of aux1 by tissue-specific expression of AUX1 (Swarup et al 2005) in plants transformed with constructs expressed in cells of the lateral root cap and the epidermal cells of the elongation zone (Swarup et al 2005)

to the ␣/ß hydrolyase super-family and the protein localizes to the ER membrane, consis-tent with the accumulation of AUX1 protein in the ER of the axr4 mutant (Dharmasiri et al 2006) One additional study suggested that brefeldin A (BFA), a molecule that blocks vesicle transport (described in detail below), may prevent the localization of AUX1 to the appropriate membrane in protophloem cells (Grebe et al 2002)

Identification of IAA influx inhibitors has also enhanced our understanding of the process of IAA influx Imhoff et al (2000) screened a large number of aryl and ary-loxyalkylcarboxylic acids for their ability to block IAA influx in suspension-cultured tobacco cells Two compounds, 1-napthoxyacetic acid (1-NOA) and 3-chloro-4-hydroxyphenylacetic acid were found to inhibit auxin influx at micromolar concentra-tions (Imhoff et al 2000) and to inhibit polar IAA transport in plants (Parry et al 2001a) These influx inhibitors phenocopy the differential auxin resistance as well as agravitropic phenotypes of aux1, with 1-NOA having a specific effect on IAA influx (Parry et al. 2001a; Rahman et al 2002) The ability of 1-NOA to specifically inhibit AUX1-mediated IAA influx was confirmed when AUX1 was expressed in Xenopus oocytes, as described above (Yang et al 2006)

A naturally occurring plant secondary metabolite, chromosaponin I (CSI), reduces IAA influx and phenocopies the agravitropic root growth of the aux1 mutant (Rahman et al 2001a) In most alleles of aux1, where the mutation lies in the central domain or in the N-terminal domain (to date, 13 alleles tested), CSI either completely inhibited the gravi-tropic response in weak alleles or did not have any effect on the already agravigravi-tropic root growth in strong alleles (Swarup et al 2004) Interestingly, CSI had a very different ef-fect in aux1-7, which carries a mutation in the C-terminal domain CSI rescued the gravi-tropic response and auxin uptake defects in aux1-7, indicating that CSI may directly in-teract with AUX1 protein (Rahman et al 2001a) via the C-terminal domain (Swarup et al 2004) The restoration of gravitropic response by CSI in an engineered transgenic line expressing HA-aux1-7 in a null allele (aux1-22) background confirmed this direct inter-action (Swarup et al 2004)

The finding that chromosaponin acts as a naturally occurring influx inhibitor parallels the identification of flavonoids as regulators of auxin efflux, described below The iden-tification of these two molecules suggests that regulation of auxin movements by endoge-nous small molecules may be an important and general mechanism to control auxin trans-port and dependent physiological processes

3.6 Regulation of IAA Efflux Protein Location and Activity during Gravity Response

For a better understanding of changes in auxin transport, two important mechanisms need to be clarified First, the initial establishment of polarity of auxin transport, and second, how this polarity is changed in response to gravity stimulation The presence of multiple

PIN and MDR/PGP and LAX genes, with distinct expression patterns and subcellular

of IAA efflux proteins Efflux carrier activity and/or synthesis may be regulated by phos-phorylation and by regulatory molecules, such as flavonoids (as reviewed by Muday and DeLong 2001; Benjamins et al 2005) Finally, increasing evidence suggests that an in-terplay between hormonal signaling pathways may regulate gravity response, with inter-actions between auxin and ethylene signaling being the best developed

3.6.1 Mechanisms that May Control Localization of IAA Efflux Carriers

The polar localization of PIN proteins has been suggested to be mediated by dynamic cy-cling of these proteins between internal compartments and the plasma membrane (Geldner et al 2001; Geldner et al 2003; as reviewed in Murphy et al 2005) A recent report has used live imaging of PIN1::GFP in the Arabidopsis inflorescence meristem to identify rapid changes in protein localization that modulate auxin transport directionality and that are linked to floral meristem initiation (Heisler et al 2005) Pharmacological ap-proaches also support the idea that PIN protein localization is dynamic The drugs mon-ensin and brefeldin A (BFA), which are inhibitors of vesicle movements, were shown to reduce auxin transport though alterations in auxin efflux in the absence of protein syn-thesis (Wilkinson and Morris 1994; Morris and Robinson 1998; Delbarre et al 1998) The asymmetric plasma membrane localization of PIN1 was altered by treatment with BFA, which led to accumulation of PIN1 in internal compartments termed BFA bodies (Geldner et al 2001; Geldner et al 2003) These BFA bodies contain endosomal markers (Geldner et al 2003) and PIN1 protein accumulation in these structures is fully reversible upon removal of BFA (Geldner et al 2001), suggesting that dynamic cycling of PIN1 be-tween endosomes and the plasma membrane could control the localization of this and other auxin transport proteins, as shown in Figure 3.3 BFA treatment has now been shown to cause accumulation of PIN2, PIN3, and PIN4 in BFA bodies (Paciorek et al 2005), suggesting that multiple IAA efflux proteins use similar mechanisms to reach their appropriate localization on the plasma membrane

Animal cells possess BFA-sensitive ARF-GEFs (ADP ribosylation factor-guanine nu-cleotide exchange factors) that direct vesicle movements through several pathways (Donaldson and Jackson 2000) The Arabidopsis gnom mutant, which has a defect in a gene encoding an ARF-GEF, exhibits altered PIN1 localization in developing embryos (Steinmann et al 1999) GNOM was later shown to be the target for BFA in PIN cycling, as the GNOM transgenic plants with a mutated BFA binding site (GNOMM–L-myc)

showed resistance to BFA-regulated, auxin-mediated developmental processes such as root gravitropic bending and lateral root formation and to accumulation of PIN1-GFP in BFA bodies (Geldner et al 2003) Together, these results indicate that GNOM is a target in BFA inhibition of PIN1-dependent auxin transport, and suggest a mechanism for dif-ferential localization of IAA efflux carriers Accordingly, mutations in SCARFACE (SFC), a gene recently shown to encode an ARF-GAP (GTPase activating protein) that enhances cleavage of ARF bound GTP to GDP, thereby negatively regulating ARF activ-ity (Randazzo and Hirsch 2004), result in altered BFA-dependent PIN1::GFP cycling and defects in auxin transport and dependent physiological processes (Sieburth et al 2006)

that the PIN3 protein may function in this way (Friml et al 2002; see also Chapter 2) PIN3 is expressed in root columella cells and PIN3 localization changes in response to reorientation of roots relative to gravity, consistent with redistribution of IAA from the root tip (Friml et al 2002) This protein also cycles in a BFA-dependent manner (Friml et al 2002) Therefore, relocalization of PIN3 in the root cap may redirect auxin flow at the tip of roots reoriented relative to gravity Consistent with this role, pin3 mutant roots respond to gravitropic reorientation with a slower response than wild-type (Friml et al 2002)

Recent work has examined the physiological significance of the cycling of PIN pro-teins in controlling auxin transport polarity, with a specific focus on the role of this process in changing the auxin transport polarity in gravity-responding roots Paciorek et al (2005) tested the possibility that IAA controls the cycling of PIN proteins In their ex-perimental system, the active auxins IAA, NAA, and 2,4-D prevent the BFA-induced ac-cumulation of PIN1 into BFA bodies, as did the yucca mutant, which has elevated en-dogenous IAA levels (Paciorek et al 2005) In addition, it was shown that the auxins inhibit uptake of FM 4-64, a fluorescent dye taken up by cells during endocytosis (Paciorek et al 2005) To determine whether the cycling of PIN2 is affected by the local IAA concentrations, Paciorek et al (2005) used the endogenous gradients in IAA across a gravity-stimulated root and found that on the lower side of the root, where IAA levels are higher, there is less accumulation of PIN2 in BFA bodies and less endocytosis of FM 4-64 Treatment with NPA, which prevents formation of the IAA gradient, resulted in a similar PIN2 accumulation in BFA bodies and levels of endocytosis on the two sides of Figure 3.3. A model of interactions of BFA and auxin efflux inhibitors with PIN1 cycling and auxin efflux A magnification of vesicular cycling in the basal portion of a polarized plant cell is shown A PIN1 cycles between the endosome and plasma membrane in untreated, polarized, IAA-transporting cells B Interruption of PIN1 cycling in BFA-treated cells results in PIN1 accumulation in an endomembrane compartment, which reduces IAA transport BFA binds to GNOM, which co-localizes with endomembrane aggregations of PIN1 BFA washout restores PIN1 cycling and IAA efflux

the root (Paciorek et al 2005) Taken together, these results suggest a mechanism by which IAA may reinforce its own changing polarity during root gravitropism

The pharmacological approach using BFA, described above, provides strong evidence for the cycling of PIN proteins as a mechanism to change auxin transport polarity during gravity response, and has uncovered some intriguing potential regulatory mechanisms Yet several aspects of these studies need to be carefully considered First, the dose of BFA needed to cause PIN1 accumulation in BFA bodies (Geldner et al 2001) is much higher than that needed to reduce root growth, gravity response, and lateral root formation (Geldner et al 2001; Geldner et al 2004) and to alter vesicular movements (Niu et al 2005; Parton et al 2003) Second, these BFA bodies are not observed in untreated cells and, although they contain endosomal markers (Geldner et al 2003), the biological sig-nificance of these structures is not yet clear Aside from changes in endocytosis of FM 4-64, which correlate with changes in BFA sensitivity to BFA body formation (Paciorek et al 2005), there has been no evidence of regulated endosomal cycling of PIN proteins in the absence of BFA Finally, although BFA has been shown to alter IAA transport in in-florescence tissues (Geldner et al 2003) and cultured plant cells (Morris and Robinson 1998), the effect of BFA on IAA transport in the tissues in which BFA affects PIN cy-cling and growth and development have not been reported These points highlight the im-portance of additional studies to understand the intriguing idea of endosomal cycling as a regulatory mechanism to control IAA transport polarity

3.6.2 Regulation of IAA Efflux by Synthesis and Degradation of Efflux Carriers

Changes in abundance of IAA efflux proteins may also enhance the effects of PIN pro-tein cycling to amplify gradients in IAA Although PIN propro-tein cycling has been shown to occur in the absence of protein synthesis (Geldner et al 2001), other experiments have also shown that there are transcriptional controls of efflux carriers that accompany change in auxin transport (Peer et al 2004; Vieten et al 2005) In particular, the expres-sion of the PIN1-6 genes has been examined and shown to be controlled by changing auxin levels as a result of application of auxins and auxin efflux inhibitors (Peer et al 2004; Vieten et al 2005) These results are consistent with transcriptional controls of ef-flux carriers that may alter the capacity of plant tissues to transport auxin, and with an additional level of feedback of auxin on its own transport Induced synthesis of IAA transport proteins in cells on the lower side of roots in response to gravistimulation could then serve to enhance transport on the lower side of horizontal roots, whereas decreased expression and enhanced turnover of auxin transporters in cells of the upper side could reduce auxin transport on this upper side A schematic diagram of auxin transport changes at the root tip is shown in Figure 3.2E, F

Addition of auxin prevents the loss of EIR1/PIN2 protein and PIN2-GFP fusions (Abas et al 2006) Similarly, in gravity-stimulated roots, the loss of PIN2-GFP on the upper side of the root can be detected with kinetics that parallel the gravitropic response (Abas et al 2006) Together, these results suggest that efflux carriers are regulated at the level of syn-thesis, breakdown, and localization

3.6.3 Regulation of Auxin Transport by Reversible Protein Phosphorylation

The activity of many highly regulated proteins is controlled by reversible phosphorylation Therefore, it is not surprising that changes in localization and/or activity of auxin transport proteins due to protein phosphorylation may also regulate auxin transport (as reviewed in DeLong et al 2002; Muday et al 2003 ) Inhibitor studies have implicated protein kinases in regulating auxin transport and its sensitivity to auxin transport inhibitors (Bernasconi 1996; Delbarre et al 1998) Genetic evidence for phosphorylation control of auxin trans-port comes from studies of the pinoid (pid) and roots curl in NPA1 (rcn1) mutants, which have defects in genes encoding a protein kinase and a protein phosphatase regulatory sub-unit, respectively (as reviewed in DeLong et al 2002) The PID gene encodes a member of the AGC-family of serine/threonine kinases (Christensen et al 2000), and pid mutants ex-hibit altered auxin transport in inflorescences and a floral development defect resembling that of the pin1 mutant (Bennett et al 1995; Christensen et al 2000; Benjamins et al 2001). Overexpression of PID reduced elongation of roots and hypocotyls, DR5:GUS expression in the root tip, gravitropism, and lateral root initiation (Christensen et al 2000; Benjamins et al 2001) Additionally, the main root meristem was also found to collapse after a few days of germination, followed by the emergence of lateral roots (Benjamins et al 2001)

Treatments with the IAA efflux inhibitors, NPA and TIBA, increased root elongation and prevented the collapse of the primary root meristem, suggesting that auxin transport is increased in 35S::PID seedlings and auxin transport inhibitors help to reduce auxin transport to normal levels in these seedlings Finally, tissue-specific overexpression of

PID in the shoot led to increased lateral root initiation, which was blocked by the

appli-cation of NPA at the root shoot junction, consistent with PID regulating auxin flow from the shoot into the root (Benjamins et al 2001) Consistent with this finding, overexpres-sion of PID in root hair and tobacco cells enhanced auxin efflux (Lee and Cho 2006) In inflorescences, pinoid loss-of-function and PINOID overexpression have been suggested to have opposite effects on the polar targeting of the PIN1 auxin efflux facilitator protein (Friml et al 2004), consistent with the hypothesis that reversible protein phosphorylation by PID may act at the level of protein targeting (Muday and Murphy 2002)

Analysis of the rcn1 mutant has shown that protein phosphatase 2A (PP2A) activity regulates root auxin transport and gravitropic curvature The RCN1 gene encodes a reg-ulatory A subunit of PP2A, and the rcn1 mutant has reduced PP2A activity in vivo and in

vitro (Garbers et al 1996; Deruère et al 1999; Muday et al 2006) Roots of rcn1

seed-lings have elevated basipetal auxin transport and exhibit a significant delay in gravitro-pism (Rashotte et al 2001) Reduced PP2A activity causes the phenotypes observed in

rcn1 roots and hypocotyls because these effects can be mimicked by treating wild-type

Hypocotyls of rcn1 also exhibit altered gravity response and auxin transport (Muday et al 2006) As in the root tip, RCN1-controlled PP2A activity appears to act as a nega-tive regulator of basipetal auxin transport (Rashotte et al 2001) Paradoxically, loss of

RCN1 function impedes gravitropic response in roots but enhances curvature in

hypocotyls Consideration of the differences in gravity response mechanisms in these two tissues suggests a hypothesis to explain the apparent contradiction As discussed in Chapters and 2, roots sense gravity very locally in the columella cells in the root cap (Blancaflor et al 1998), and auxin is redistributed from the root tip to one side of the root after gravity stimulation rather than being laterally transported across the root tip (as re-viewed in Blancaflor and Masson 2003) In roots, uniformly increased basipetal transport may impede the redistribution of auxin at the root tip, which is required to form a lateral auxin gradient and to achieve maximal gravitropic bending (Rashotte et al 2001)

Consistent with this hypothesis, treatment of rcn1 roots with low doses of NPA reduces auxin transport and enhances gravity response to wild-type levels (Rashotte et al 2001) In contrast, gravity perception in stems occurs in the starch sheath parenchyma tissues that run the length of the hypocotyl (Fukaki et al 1998; see also Chapters and 2) Lateral auxin transport then is believed to occur in multiple tissues along the length of the hypocotyl (Blancaflor and Masson 2003) Increased basipetal auxin transport would provide more auxin to the lateral transport stream and would thereby increase gravitropic bending In contrast, the mdr1 mutant has reduced hypocotyl IAA transport (Noh et al. 2001), but has enhanced gravi- and phototropic responses These differences may be due to specific effects of the mdr1 mutation on transporter localization or function (Noh et al. 2003), rather than the rcn1 mutation which affects bulk polar auxin flow

The possibility that the rcn1 gravitropic phenotype was due to altered ethylene re-sponse was examined (Muday et al 2006), but this possibility is not consistent with sev-eral results The rcn1-2 allele was identified in a screen for increased ethylene response in etiolated seedlings and was originally designated eer1 (enhanced ethylene response) (Larsen and Chang 2001; Larsen and Cancel 2003) Enhanced ethylene response in rcn1 is a hypocotyl-specific phenotype and is accompanied by ethylene overproduction (Larsen and Chang 2001) The rcn1 hypocotyl gravitropic phenotype was found to be ethylene-independent as the rcn1-2 etr1-1 and rcn1-2 ein2-1 mutants showed gravity re-sponses that are identical to the rcn1 single mutant (Muday et al 2006) Additionally, al-though silver treatment of wild-type seedlings reduces the gravity response, silver treat-ment of rcn1 seedlings further enhanced the gravity response, consistent with the enhanced gravitropic phenotype of rcn1 being independent of ethylene signaling (Muday et al 2006) These results indicate that an intact ethylene signaling pathway is not re-quired for the enhancement of gravity response in rcn1 hypocotyls The etiolated growth phenotype of rcn1 is likely due to the elevated ethylene synthesis that is only found in dark-grown seedlings (Muday et al 2006)

which mutant phenotypes include altered gravity response, suggest other targets of phos-phorylation beyond the PIN proteins

3.6.4 Regulation of Auxin Transport by Flavonoids

Prime candidates for endogenous auxin transport inhibitors are flavonoids These pheno-lic compounds displace the binding of synthetic IAA efflux inhibitors, such as NPA, dur-ing in vitro assays (Jacobs and Rubery 1988) The flavonols quercetin and kaempferol had the greatest activity, suggesting that specific members of this chemical family func-tion as auxin transport inhibitors (Jacobs and Rubery 1988) The role of flavonoids as regulators of auxin transport have been examined by in vivo studies in tt4 mutants, which have a defect in the CHS gene encoding chalcone synthase, the first enzyme in flavonoid synthesis These tt4 mutants have elevated auxin transport in young seedlings, roots, or inflorescences, consistent with the absence of an endogenous negative auxin transport regulator (Murphy et al 2000; Brown et al 2001; Buer and Muday 2004; Peer et al 2004; Buer et al 2006) Roots of multiple alleles of tt4 mutants exhibit a lag in gravitropic cur-vature compared to wild-type roots (Buer and Muday 2004; Buer et al 2006) Chemical complementation of tt4(2YY6) by naringenin reinstated flavonoid production and re-stored a wild-type gravity response, consistent with a role for flavonoids in controlling the flow of auxin needed for root gravitropism (Buer and Muday 2004)

Mutations that alter flavonoid synthesis affect the abundance of the mRNA encoding members of the PIN gene family (Peer et al 2004; Lazar and Goodman 2006), suggest-ing that flavonoids may regulate synthesis of auxin transport proteins, not just the activ-ity of existing proteins Consistent with flavonoid abundance affecting transcription, re-cent evidence indicated that flavonoid biosynthetic enzymes and flavonoid products accumulate in the nucleus (Saslowsky et al 2005)

Changing environmental conditions modulate flavonoid synthesis (Winkel-Shirley 2002) and these changes in flavonoid accumulation may regulate plant growth and devel-opment, including gravity responses (as reviewed in Taylor and Grotewold 2005) Reorientation of plants relative to gravity leads to enhanced flavonoid accumulation in the epidermal tissues of Arabidopsis root tips (Buer and Muday 2004), which are the site of basipetal IAA transport Images of roots and the time course of this induction are shown in Figure 3.4 This induction is on both the upper and lower sides of gravistimu-lated roots, suggesting that its function may be to uniformly reduce the activity or abun-dance of a set of efflux carriers, and thereby accentuate the formation of a gradient of IAA across the root resulting from enhanced transport on the lower side Therefore, in-duction of flavonoid synthesis in response to environmental stimuli may alter auxin trans-port to facilitate plant gravity response

3.6.5 Regulation of Auxin Transport by Other Signaling Pathways

62

40 µm from the root tip of vertically-grown controls versus gravity-stimulated roots measured over time is reported Arrows indicate times of gravity-stimulated root bending in Col and tt4(2YY6) B–C Col root tips of 2.25 h vertical controls and (D–E) gravity-stimulated roots The optical slices in C and E are approximately 15 µm below the root-tip surface that is shown in B and D The scale bar = 40 µm

in Chapter 2, biochemical and cell biological approaches have identified several signal-ing molecules that are produced in response to a changsignal-ing gravity vector and may mod-ulate auxin transport It has long been suspected that calcium is a signaling molecule that changes in concentration in response to changes in the vector of gravity (as reviewed in Sinclair and Trewavas 1997) Attempts to demonstrate changes in cytoplasmic calcium concentration using calcium ratio imaging in Arabidopsis roots did not detect calcium concentration changes in response to gravitropic stimulation (Legue et al 1997) Evi-dence in support of calcium as a signal in gravitropic response comes from young seed-lings of Arabidopsis expressing aequorin in the cytoplasm (Plieth and Trewavas 2002)

When a population of seedlings is reoriented relative to gravity, there is an enhanced aequorin signal consistent with elevated cytoplasmic calcium concentration (Plieth and Trewavas 2002) Several experiments have suggested that gravity stimulation may be am-plified by cascades involving Ca2+/calmodulin (Sinclair et al 1996; Lu and Feldman 1997), and a number of older studies suggested a relationship between auxin transport and calcium concentration (dela Fuente 1984; Allan and Rubery 1991) Therefore, the possibility that calcium signals are integral to gravitropic response remains intriguing but requires additional experimental test

The role of several other signaling molecules has been more clearly demonstrated, as discussed in Chapter Changes in pH have been observed after gravitropic stimulation in both Arabidopsis roots (Scott and Allen 1999; Fasano et al 2001; Boonsirichai et al. 2003) and the maize pulvinus (Johannes et al 2001) Proton movements have been tied to auxin transport through examination of mutants and transgenics with altered expres-sion of the H+-pyrophosphatase, AVP1 (Li et al 2005) Altered AVP1 expression changes vacuolar pH and alters IAA transport and PIN1 localization (Li et al 2005)

Additionally, inositol lipids have been implicated in the gravity signal transduction pathway in maize and Arabidopsis A transient increase in the InsP3lipid signal have been observed in gravity-stimulated maize and oat pulvini (Perera et al 1999; Perera et al 2001) Gravity-stimulated pulvini undergo rapid initial changes in InsP3 levels on both sides, followed by a greater and more persistent elevation on the lower side (Perera et al 1999) This later InsP3elevation on the lower side is necessary for gravitropic bending of the pulvinus, as treatment with phospholipase C inhibitors prevent formation of the InsP3 gradient and reduced gravitropic bending (Perera et al 1999) In maize, free IAA has been measured and shown to develop an asymmetry across the gravity-stimulated pulvi-nus that follows the changes in InsP3levels (Long et al 2002)

More recently, additional support for the role of InsP3comes from transgenic studies in Arabidopsis with plants constitutively expressing the human type I inositol polyphos-phate 5-phosphatase (InsP 5-ptase), an enzyme that specifically hydrolyzes InsP3(Perera

et al 2006) In the transgenic plants, basal InsP3levels are reduced by greater than 90% compared to wild-type plants With gravistimulation, InsP3levels in inflorescence stems of transgenic plants show no detectable change, whereas in wild-type plant inflores-cences, InsP3levels increase approximately threefold within the first to 15 of

compared to the controls (Perera et al 2006) Together, these results suggest that InsP3 synthesis may be a signal that modulates auxin transport to allow differential growth in response to changes in the gravity vector

3.6.6 Regulation of Gravity Response by Ethylene

Increasing evidence suggests that ethylene may regulate gravitropism either directly or through modulation of auxin’s role in this process Exogenous application of ethylene gas or the ethylene precursor 1-aminocyclo-propanecarboxylic acid (ACC) has been used to test the role of ethylene in gravity response In some experiments ethylene or ACC treat-ment clearly reduced the early phase of gravitropic response of roots and shoots (Wheeler and Salisbury 1981; Wheeler et al 1986; Lee et al 1990; Kiss et al 1999; Madlung et al 1999; Buer et al 2006), whereas others showed no effect (Kaufman et al 1985; Harrison and Pickard 1986; Woltering 1991) A third set of experiments suggested that ethylene positively regulated shoot gravitropism (Chang et al 2004) The experiments that identi-fied a role for ethylene in gravity response examined the initial rate of gravitropic curva-ture (Wheeler and Salisbury 1981; Wheeler et al 1986; Lee et al 1990), suggesting that the absence of kinetic data at early times after gravitropic stimulation in several of the ex-periments may explain their negative results (Kaufman et al 1985; Harrison and Pickard 1986; Woltering 1991) It has been shown that the application of ethylene inhibitors such as AVG/AgNO3 also reduced the initial gravitropic curvature (Wheeler et al 1986; Lee

et al 1990; Muday et al 2006) both in roots and shoots The similar effect observed by the ethylene precursor and inhibitors suggests that ethylene may both positively and neg-atively regulate gravitropism

Mutants altered in ethylene signaling and/or synthesis have been used to examine the role of ethylene in gravitropic curvature The gravitropic responses of the etr1 roots (Buer et al 2006) and hypocotyls (Muday et al 2006) and ein2-1 roots are wild type (Roman et al 1995; Rahman et al 2001b; Buer et al 2006) The gravitropic response of shoots of the tomato mutants, Never-Ripe (Nr) and epi, which have reduced ethylene response and enhanced synthesis, respectively, were examined Both mutants exhibit delays in shoot gravitropic response but with only a small reduction in Nr (Madlung et al 1999), consis-tent with a role for ethylene in the early events of gravitropic response The study of Madlung et al (1999) revealed a concentration-dependent modulation of shoot gravitro-pism by ethylene, with Nr being insensitive to the effect of exogenous ethylene on hypocotyl gravitropism Similarly, etr1 and ein2 roots and hypocotyls are insensitive to the inhibition of gravitropism by ACC treatment (Buer et al 2006; Muday et al 2006) These results suggest that ethylene negatively regulates gravity response, and that for plants grown on agar the endogenous levels of ethylene are low enough that there are no detectable differences between wild-type and ethylene-insensitive mutants The one ex-ception to this conclusion are the agravitropic hypocotyls of ein2, although this pheno-type may be linked to EIN2 activities that are ethylene-independent (Muday et al 2006)

One mechanism by which auxin and ethylene may interact is at the level of hormone synthesis with auxin-inducing ethylene synthesis and/or ethylene-inducing auxin synthe-sis Auxin is a positive regulator of ethylene biosynthesis in many plants, including

rate-limiting step in ethylene synthesis is catalyzed by ACC synthase, which is encoded by the

ACS gene family with some family members being auxin-inducible in dark-grown

seedlings and plants (Abel et al 1995; Yamagami et al 2003) Recently, one ACC syn-thase gene was shown to be asymmetrically induced across a gravity-stimulated snap-dragon flower spike, suggesting that asymmetric synthesis of ethylene may be part of gravitropic response in some tissues (Woltering et al 2005) The regulation of auxin syn-thesis by ethylene has been uncovered by the mutants designated weak ethylene insensi-tive (wei2 and wei7) The WE12 and WE17 genes encode ethylene-regulated enzymes of Trp synthesis, whose activity positively regulates IAA synthesis (Stepanova et al 2005) Another explanation for the interaction between auxin and ethylene is that ethylene may inhibit IAA transport In some plant species, ethylene has been shown to inhibit the polar IAA transport in shoot tissues (Morgan and Gausman 1966; Suttle 1988), but a di-rect effect of ACC treatment on IAA transport was not detected in Arabidopsis hypocotyls (Muday et al 2006) or roots (Buer et al 2006) Lateral IAA transport has also been found to be inhibited in both shoots (Burg and Burg 1966) and gravity-stimulated corn roots (Lee et al 1990) These results suggest that ethylene-mediated inhibition of auxin transport may play an important role in regulating the gravity response A recent article from Buer et al (2006) asked whether ethylene might inhibit gravity response through induction of flavonoid synthesis resulting in reduced IAA transport Several al-leles of the flavonoid-deficient mutant tt4 exhibit a delayed gravity response in roots and are insensitive to the inhibition of gravitropism at early time points More interestingly, ACC has been shown to induce flavonoid accumulation in Arabidopsis roots through a mechanism that requires EIN2 and ETR proteins (Buer et al 2006) Taken together, these results suggest that the ethylene regulation of root gravity response may occur through al-tering flavonoid synthesis, assuming that enhanced flavonoid accumulation will reduce IAA transport However, Buer et al (2006) did not find any effect of ACC on root basipetal auxin transport, indicating that this interaction may be more complex in nature or too difficult to detect in the tips of the small roots of Arabidopsis.

The most direct evidence for interaction between ethylene signaling and auxin trans-port comes from the studies of Arabidopsis mutants having mutations in auxin transtrans-port proteins Both the auxin influx mutant, aux1, and the auxin efflux mutant, agr1/ eir1/pin

2/wav6, have ethylene-insensitive root elongation (Roman et al 1995; Pickett et al.

1990) The restoration of ethylene sensitivity in both aux1 and eir1 by exogenous appli-cation of NAA and IAA indicates that cytoplasmic auxin is needed at sufficient levels for ethylene response (Rahman et al 2001b)

3.7 Overview of the Mechanisms of Auxin-Induced Growth

This chapter has focused on the mechanisms by which asymmetries in auxin are estab-lished in response to changing orientation of plants relative to gravity Yet, to understand how these auxin gradients control growth, a brief discussion of auxin signal transduction is required, although this topic has been reviewed in greater detail elsewhere (Leyser 2002, 2006)

microarray experiments identifying hundreds of genes whose expression is rapidly mod-ified in response to auxin treatment (Pufky et al 2003) The most rapidly expressed genes are induced even in the absence of protein synthesis These primary auxin-responsive genes include SAURs (small auxin up-regulated), GH3s, and AUX/IAAs (Auxin/IAA in-ducible genes) gene families (as reviewed in Hagen and Guilfoyle 2002) Consensus pro-moter elements were found in these auxin-responsive genes and used to identify proteins, named Auxin Response Factors (ARFs), that bind to them (as reviewed in Hagen and Guilfoyle 2002) The in vivo function of these proteins has been demonstrated by isola-tion of Arabidopsis mutants altered in expression or funcisola-tion of ARF and AUX/IAA genes. These mutants have a diversity of auxin-dependent phenotypes, including agravitropic roots and/or hypocotyls (as reviewed in Liscum and Reed 2002) The nph4 (nonpho-totropic hypocotyl) mutant, which has a defect in the ARF7 gene, exhibits reduced hypocotyl phototropic and gravitropic responses (Stowe-Evans et al 1998; Harper et al 2000) Several mutants with defects in IAA genes also have agravitropic phenotypes, in-cluding iaa14/slr1-1, iaa17/axr3-1, and iaa19/msg2 (as reviewed in Liscum and Reed 2002) ARF proteins contain DNA binding domains and dimerization domains, and have been shown to act as transcriptional regulators (Tiwari et al 2003) AUX/IAA proteins have similar dimerization domains and form homo- and hetero-dimers with ARF pro-teins, thereby modulating the ability of ARFs to bind to the promoter of auxin-responsive genes (Kim et al 1997)

Until relatively recently, it has been unclear how the auxin signal influences the for-mation of transcription factor complexes that are needed to modulate the expression of auxin-responsive genes Recent experiments have demonstrated that auxin-dependent proteolytic destruction of AUX/IAA proteins is a critical factor (as reviewed in Leyser 2006) Specifically, mutations in the TIR1, AXR1, and AXR6 genes lead to auxin-resistant plants The proteins encoded by these genes have now been shown to be part of protein complexes that ubiquitinylate substrate proteins, thereby targeting them for destruction (as reviewed in Leyser 2006) The current model for this process is that TIR1 binding to auxin facilitates formation of complexes with AUX/IAA proteins, resulting in their ubiq-uitination Ubiquitinylated proteins are then targeted for destruction by the proteosome Consequently, ARF proteins are released from inhibitory complexes with AUX/IAA and bind to the promoter elements of auxin-responsive genes, regulating their expression (Dharmasiri et al 2005; Kepinski and Leyser 2005) Although this mechanism was rather unexpected, the data clearly show that destruction of regulatory transcription factor sub-units is an efficient system for rapid gene expression changes in response to changing auxin levels

strik-ing study, which ties auxin-induced gene expression to differential growth durstrik-ing pho-totropism and gravitropism, utilized Brassica oleracea (Esmon et al 2006) These seedlings were of sufficient size to isolate opposite flanks of hypocotyls after exposure to lateral light stimulation or after being placed horizontally mRNA was isolated from each flank and used to hybridize an Arabidopsis microarray A number of genes were shown to be differentially expressed on the two sides of the stimulated hypocotyls Additionally, the expression of these same genes was enhanced in the hypocotyls of auxin-treated etiolated

Arabidopsis seedlings in a NPH4/ARF7-dependent fashion (Esmon et al 2006) Several

of these genes were shown to encode proteins that are directly tied to growth, including two expansin genes, EXPA1 and EXPA8 (Esmon et al 2006)

Finally, the functional significance of differences in auxin-regulated gene expression across gravity-stimulated tissues needs to be evaluated in the context of differences in gravity response between roots and shoots In light-grown shoot tissues, auxin is gener-ally limiting for growth (Yang et al 1993; Gray et al 1998), so the elevated auxin con-centrations on the lower side of shoots reoriented relative to gravity could operate to in-duce transcription of genes encoding proteins that enhance growth, consistent with the report described above (Esmon et al 2006) In contrast, although roots also redirect auxin to the lower side, the opposite response is initiated, resulting in growth with the gravity vector Although root growth is negatively regulated by auxin under most conditions (Pickett et al 1990), it is not completely clear whether this growth response is due to the elevated auxin on the lower side or the reduced auxin levels on the upper side Detailed kinetic analysis of roots after gravitropic reorientation in many species indicates that most exhibit enhanced growth on the upper side, rather than the predicted growth inhibi-tion on the lower side (as reviewed in Wolverton et al 2002) These kinetic studies also revealed that shortly after gravitropic reorientation, there is enhanced growth on both sides of the root, which is sustained on the upper side of the root, followed by a reduced growth rate on the lower side of the root once curvature initiates (Buer and Muday 2004)

The complexity of this response has led to the suggestion that parts of the response may be auxin-independent (Wolverton et al 2002), but it is also possible that the signal-ing mechanisms (includsignal-ing transcription changes described above) could control this process Since auxin positively and negatively regulates the expression of responsive genes, depending on the complexes of ARF and AUX/IAA proteins involved (Hagen and Guilfoyle 2002), scenarios can be envisioned to support this more complex response of roots The lower auxin levels on the upper side of a reoriented root could relieve auxin’s repression of genes that encode proteins that positively regulate growth, just as the en-hanced auxin levels on the lower side could repress synthesis of growth-inducing pro-teins Additional experiments will be needed to determine the complex interplay of sig-nals that change in response to gravity stimulation and their mechanisms for controlling the complex process of root gravitropism

3.8 Conclusions

combination of genetic and biochemical approaches These findings offer tremendous in-sight into the novel mechanisms that control the synthesis, targeting, activity, as well as the proteolytic degradation of proteins that act in concert to drive auxin redistribution Finally, the understanding of how the auxin signal is transduced to control synthesis of proteins needed for asymmetric growth rounds out our understanding of how the asym-metries in auxin induce differential growth

3.9 Acknowledgments

Several grants facilitated this work, with GKM supported by the U.S Department of Agriculture (No 2006-35304-17311) and AR supported by the National Science Foundation (IBN 0316876)

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Jack L Mullen and John Z Kiss*

79

4.1 Phototropism: General Description and Distribution

That sunlight can affect the development of plants has been known for thousands of years, and one of the more striking effects is the growth of plants toward areas of more intense light This response, phototropism, is caused by a difference in growth rate across part of the plant with the direction of the growth differential determined by the gradient in light intensity Phototropism is widespread among plants and is found in algae, mosses, and ferns, as well as seed plants In flowering plants, stems are generally positively pho-totropic, curving toward the direction of highest light intensity However, there are some species, including climbing plants with tendrils, which have negatively phototropic shoots, allowing them to grow toward neighboring plants (Darwin 1875; Strong and Ray 1975) Some recent reviews of phototropism include Liscum and Stowe-Evans (2000), Kimura and Kagawa (2006), and Whippo and Hangarter (2006)

Leaves are also frequently phototropic, though the effect of light on leaf growth is more complex than for stems Leaves of many species engage in a diurnal phototropic re-sponse, following the movement of the sun Much of the curvature occurs in the leaf peti-ole, which is capable of sensing the light directly or, if shaded, responding to light sensed by the leaf blade (Haberlandt 1914) Some species have specialized turgor-driven motor tissues, called pulvini, which allow for greater reversibility in the response The leaf blade itself may also reorient to be normal to the incident light and can track the sun across the sky in this position (Lang and Begg 1979; Koller and Levitan 1989) Since the direction of the light is nearly perpendicular to the leaf surface throughout the day, this response requires impressive sensitivity to changes in photostimulation

Although its importance is not clear, phototropism has also been observed in root sys-tems, with roughly half of species examined showing some response (Hubert and Funke 1937) Root phototropism could be useful in positioning lateral roots near the soil sur-face, where light may penetrate, though the response is generally smaller than the grav-itropic response of roots (Kiss et al 2002) The majority of roots showing a response are negatively phototropic (Hubert and Funke 1937; Okada and Shimura 1992) However, roots of Arabidopsis can respond either positively or negatively to unidirectional light, depending on the light quality (Kiss et al 2003) Thus, it appears that multiple light-signaling pathways interact with other growth responses such as gravitropism, even be-lowground in the root systems

4.2 Light Perception

The light environment in which plants naturally live and grow can be a very heteroge-neous one, with both diffuse and directional light varying throughout the landscape Spatial differences in the light intensity impinging upon a plant manifest as a gradient in light intensity across the particular plant organ, due to scattering and absorption by the plant tissue (Vogelmann et al 1996) This leads to a gradient in photoreceptor activation in the plant organ, providing directional information regarding the light stimulus The presence of screening pigments, such as carotenoids, in the plant can increase the light gradient and have been found to increase the magnitude of phototropic responses (Piening and Poff 1988) In leaves of solar-tracking plants, however, gradients in light in-tensity across the organs not play an important role in the phototropic response In these plants, the leaf surface is perpendicular to the direction of the light beam, and pho-totropic leaf reorientation occurs when the direction of the light beam becomes oblique (Schwartz and Koller 1978) This vectorial phototropic response is sensed by cells above the veins of the leaves, and the ability to sense light direction is related to the angle be-tween the light beam and the directions of the major axes of the veins Sensing of the di-rection of light, in this case, has been postulated to be due to localization of photorecep-tors to the end walls of the cells along the leaf veins (Koller et al 1990)

Although the phototropins are the primary photoreceptors responsible for initiating phototropic responses, another family of blue-light-absorbing photoreceptors in plants, the cryptochromes, is also involved in regulating the response (Figure 4.1) The principal roles of cryptochromes are in photomorphogenesis and the entrainment of circadian rhythms (Cashmore 2003), but they have also been found, in mediating blue-light-induced stomatal opening, to act synergistically with phototropins (Mao et al 2005) Cryptochromes are not necessary for the induction of phototropic responses (Lasceve et al 1999; Sakai et al 2000) However, at low fluence rates, cryptochrome-deficient mu-tants have reduced phototropic responses (Ahmad et al 1998; Lasceve et al 1999; Whippo and Hangarter 2003) Cryptochromes are also important in regulating light-induced growth inhibition in hypocotyls, together with the phototropins (Parks et al 2001) Because phototropism is caused by complex changes in growth rate, alterations in growth inhibition by cryptochromes is likely to intersect phototropic signaling Indeed, Whippo and Hangarter (2003) have shown that at high light intensities, the cryptochromes, along with the phototropins, inhibit phototropism Thus, the cryptochromes may interact with phototropins to modulate the phototropic pathway (Figure 4.1 and Color Section) and also control the inhibition of growth of hypocotyls in a fluence-rate-dependent manner, which can limit the potential phototropic response

In most cases, red light does not induce phototropism in flowering plants Red and far-red light are sensed by the phytochromes (Figure 4.1)—photoreceptors which are involved in numerous aspects of the plant life cycle, including seed germination, flowering, and cir-cadian rhythms (Schepens et al 2004) The phytochrome gene family has five members (PHYA–E) in Arabidopsis Although unidirectional red light does not induce phototropism in Arabidopsis hypocotyls (Liscum and Briggs 1996), red-light pretreatments are known to greatly enhance blue-light phototropism, in a process mediated by phytochromes (Janoudi et al 1992; Liu and Iino 1996; Hangarter 1997) Phytochromes can also absorb blue light and they modulate blue-light phototropic responses, even in the absence of a red-light pre-treatment (Correll et al 2003) At low fluence rates of unidirectional blue light, phy-tochromes (particularly PHYA and PHYB) reduce the latent period and enhance the mag-nitude of the phototropic response (Janoudi et al 1997; Hangarter 1997) However, at higher fluence rates of blue light, phytochrome (primarily PHYA) causes an attenuation in the response (Whippo and Hangarter 2004) Phytochromes can interact with the cryp-tochromes (Ahmad et al 1998; Mas et al 2000); and like the crypcryp-tochromes, phycryp-tochromes also regulate light-induced growth inhibition in hypocotyls (Folta and Spalding 2001) Therefore, in addition to a possible role in directly modulating the phototropism signaling pathway, phytochromes also appear to act in coordination with the cryptochromes and pho-totropins (Figure 4.1) to regulate shoot growth rate more generally in response to light stim-uli, a process that also modulates the overall phototropic response observed

Some algae, mosses, and ferns engage in red-light phototropism, mediated by phy-tochrome (Wada and Sei 1994; Esch et al 1999), in addition to blue-light phototropism The green alga Mougeotia and a group of ferns have independently evolved a chimeric photoreceptor that is a hybrid between phytochrome and phototropin, termed neochrome (Figure 4.1), which controls the red-light phototropism in these plants (Kawai et al 2003; Suetsugu et al 2005) However, neochromes have not been found in mosses or flowering plants This chimeric photoreceptor broadens the response by allowing strong absorption of both red light and blue light The signaling of neochrome for the two wavelengths is synergistic, so that the photoreceptor has increased sensitivity to weak white light; this allows plants with these pigments (e.g., polypodiaceous ferns) to sense and respond to low-light signals in their naturally shaded light environment (Kanegae et al 2006)

There have been a few reports of phototropism mediated by normal phytochromes in flowering plants In roots of Arabidopsis, there is a positive red-light phototropic sponse controlled by phytochromes A and B, in addition to the negative blue-light re-sponse (Kiss et al 2003) In shoots, there are reports of phytochrome-regulated phototro-pism in mesocotyls of maize (Iino et al 1984) and negative far-red phototrophototro-pism in cucumber (Ballare et al 1992, 1995), as well as positive far-red phototropism in the par-asitic plant Cuscuta planiflora (Orr et al 1996)

4.3 Signal Transduction and Growth Response

transduction) following light perception Nevertheless, in recent years, numerous mutants in light-signaling intermediates, particularly in the phytochrome pathways, have been iso-lated and used to investigate light signaling in plants (Møller et al 2002)

In terms of signaling intermediates that are downstream from the phototropins, RPT2 (root phototropism) and NPH3 (non-phototropic hypocotyl) have been shown to bind to PHOT1 and to function very early in some blue-light-based signaling pathways (Sakai et al 2000; Inada et al 2004) For instance, RPT2 is involved in phototropism and stomatal opening but not in chloroplast movements, and although NPH3 is involved in phototro-pism, it is not needed for stomatal opening or chloroplast relocation RPT2 and NPH3 are considered to be in the same biochemical family, but the exact structure and precise phys-iological functions of these proteins are not known Although NPH3-deficient mutants of

Arabidopsis showed no phototropism in shoots or roots (Sakai et al 2000), the cpt1

mu-tant of rice, orthologous to NPH3, retained some root phototropism, suggesting that other members of the gene family may also play a role in this plant (Haga et al 2005)

The growth response of phototropism involves differential elongation on opposite sides of a plant organ, which eventually leads to phototropic curvature It is well-established that the plant hormone auxin plays an integral role in the differential growth that results in curvature In fact, the discovery of auxin and the classical Cholodny-Went model for auxin transport have been tied to research in tropisms, especially phototropism The NPH4 locus, which is important for both phototropism and gravitropism, encodes an auxin response factor involved in auxin-sensitive transcriptional regulation (Harper et al 2000) Much recent focus in auxin research, especially in conjunction with understand-ing the role of auxins in tropisms, has centered on the auxin transport proteins in the PIN (termed such because of the pin-shaped inflorescence stem in mutants) family (Blakeslee et al 2005; Paponov et al 2005; see also Chapter of this book) Understanding the link among auxin transport proteins, NPH3/RPT2, and the actin cytoskeleton will be impor-tant in determining the precise molecular role of these molecules in linking light percep-tion in phototropism to the differential growth effects mediated by auxin (Maisch and Nick 2007)

4.4 Interactions with Gravitropism

lev-els, as expression of PHOT1 (Sakamoto and Briggs 2002; Kong et al 2006) and several phytochromes (Sharrock and Clack 2002) is down-regulated by light In the case of grav-itropic adaptation, however, it is unclear where in the signaling pathway the modulation occurs Part of the reduction in curvature may also be due to autotropic straightening, a reversal of curvature which can occur following tropistic responses (Orbovic and Poff 1991; Stankovic et al 1998) There can also be differences in the spatial distribution of the responses, adding complexity to the overall growth pattern In roots, phototropic cur-vature develops farther from the root tip than gravitropic curcur-vature (Mullen et al 2002; Kiss et al 2003), whereas in shoots, there can be differences in the extent of the region of curvature between gravitropic and phototropic responses (Tarui and Iino 1999)

Nondirectional light, more generally, may modulate gravitropic responses apart from the integration of gravitropic and phototropic growth responses There have been observa-tions of red light sensitizing hypocotyls to gravitropic stimulation (Britz and Galston 1982; Woitzik and Mohr 1988) However, it appears more common for red light to inhibit grav-itropism In hypocotyls of Arabidopsis, red light acting via phytochrome appears to inhibit gravitropism, causing a randomization of shoot orientation (Figure 4.1; Liscum and Hangarter 1993; Poppe et al 1996; Robson and Smith 1996) This inhibition of a counter-ing gravitropic response may explain how red light enhances blue-light phototropism (Hangarter 1997; Parks et al 1996) Red-light attenuation of shoot gravitropism has also been observed in pea and tobacco (McArthur and Briggs 1979; Hangarter 1997) And in mosses, at least, red light does not act to inhibit gravitropism at the level of perception, as red-light treatments did not repress amyloplast sedimentation (Kern and Sack 1999) This signaling pathway may also be acting during blue-light phototropism, as phytochrome ab-sorption of blue light can also cause randomization of shoot orientation (Lariguet and Fankhauser 2004) Light inhibition of gravitropism has also been observed in leaves (Mano et al 2006), although whether it is also mediated by phytochrome is unclear

4.5 Importance to Plant Form and Function

At a whole-plant level, the positioning of branches and leaves through phototropic and gravitropic responses will play an important role in the overall functioning of the organ-ism Yet tests of the roles of specific phototropic responses at specific stages of the life cycle of a plant remain limited, although it appears that a key function of phototropism is to help the plant maximize photosynthesis Experiments with Arabidopsis phot mutants suggest that PHOT1 and PHOT2 may be important at different developmental stages, consistent with their different sensitivities to light intensity (Galen et al 2004; Galen et al 2007) In young seedlings, one of the important roles of phototropism may be orient-ing the root system away from the surface to aid in dealorient-ing with dry conditions (Galen et al 2007)

increases light interception by leaves, it also increases the heat load for these plants, which could be disadvantageous, particularly if water is limited In fact, some solar-tracking species can change their leaf orientation from being perpendicular to the direc-tion of light to parallel to the light, depending on water stress (Shackel and Hall 1979; Forseth and Ehleringer 1980) Also, in many arid environments the leaves of plants that not solar track become more vertical with increasing light intensity, apparently as a protective mechanism against photodamage from excess light (King 1997; Valladares and Pugnaire 1999; Falster and Westoby 2003)

In young seedlings, the primary shoot generally grows upward in a seemingly straight-forward integration of positive phototropism and negative gravitropism, and the primary root grows downward in a similarly straightforward combination of positive gravitropism and negative phototropism (Okada and Shimura 1992) However, the bulk of a mature plant is made up of lateral organs—branches and leaves in the shoot and lateral and ad-ventitious roots belowground—which grow at nonvertical orientations even in the ab-sence of a phototropic stimulus It is the growth of these lateral branches that gives plants their characteristic form, and they have large effects on plant productivity

Because these nonvertical orientations appear to be actively maintained via gravitropic responses, Digby and Firn (1995) have termed the growth orientation of these organs to be their gravitropic set-point angle, or GSA (see also Chapter of this book) The GSA of both shoots and roots can be altered by red light (Gaiser and Lomax 1993; Digby and Firn 2002; Mullen and Hangarter 2003) Thus, the fine-tuning of organ positioning in mature plants by directional light cues requires a more complex integration of growth re-sponses, involving not only phototropism, gravitropism, and their interactions, but also light-dependent changes in GSA Because the GSA is a developmentally regulated vari-able (Digby and Firn 1995), light regulation of GSA allows for differences in response depending on the age of the specific organ This may allow different parts of a mature plant to tailor their growth in an appropriate manner for the specific environmental con-ditions they encounter

4.6 Conclusions and Outlook

Tremendous progress in understanding the mechanisms of phototropism has been made in recent years Although phototropism has been intensively studied since the time of Darwin’s classic experiments, it is only within the past decade that we have identified the phototropins as the primary pigments involved in light perception in phototropism It has become increasingly obvious that the other two major groups of photoreceptors, the cryp-tochromes and phycryp-tochromes, interact with phototropins and play both direct and indirect roles in phototropic responses Much of the current research focuses on a better under-standing of the cellular and molecular events downstream from the primary photorecep-tors There is increased recognition that the interaction between and among the primary photoreceptors is important in phototropism and other important light-regulated develop-mental processes (Mas et al 2000; Folta and Spalding 2001; Whippo and Hangarter 2003; Lariguet and Fankhauser 2004; Kumar and Kiss 2007)

pho-totropism and gravitropism is to use the microgravity conditions during spaceflight (Correll et al 2005; Kiss et al 2007; see also Chapter of this book) In these experi-ments on the international space station, we plan to study the role of phytochromes in phototropism and to determine whether red light affects phototropism directly or indi-rectly by the attenuation of gravitropism However, through the increased use of the new tools of molecular and systems biology in the next decade, we should gain an even bet-ter understanding of the basic mechanisms of phototropism, its relationship to gravitro-pism, and its importance to plants

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Gabriele B Monshausen, Sarah J Swanson, and Simon Gilroy*

91

5.1 Introduction

The sessile nature of the plant lifestyle requires exquisite sensitivity to environmental sig-nals These stimuli provide the information that controls much of plant behavior, ranging from decisions about when to grow or reproduce to whether to mount a defense response against a pathogen The directional cue of gravity is clearly a central component of this wealth of information that regulates normal plant development However, other mechan-ical stimuli, ranging from the buffeting by wind and rain, impedance of the soil, and even the weight of an organ itself provide similarly important information that governs plant morphogenesis (Figure 5.1)

Indeed, plants are highly sensitive to mechanical cues in their environment This is per-haps most obvious when seeing the rapid mechanoresponse in organs specialized for touch sensing, such as the closing of the Venus fly trap (Dionaea muscipula) triggered by the mechanical stimulus of an insect alighting on the leaf, or the twining of a tendril in response to the touch signal from contacting a support However, plants exhibit many and varied responses to touch In general, plants grown with mechanical stimulation develop a shorter stature, more robust and stronger support tissues, and altered organ architecture and growth habit (Braam 2005 and references therein) Similarly, the directional cues of-fered by mechanical stimulation (be it touch or gravity) lead to highly controlled direc-tional growth responses manifested as thigmo- and gravitropism Considering the physi-cal nature of both the gravity and touch stimulus, it seems likely that they share common mechanotransduction elements It has even been proposed that gravity sensing is derived from an ancestral touch perception apparatus (Trewavas and Knight 1994)

In this chapter we will therefore describe some of the broad classes of mechanore-sponse seen in plants, discuss some of the models for how mechanosensing is likely op-erating at a cell and molecular level, and then ask how the plant integrates multiple stim-uli, in this case touch and gravity, to generate the appropriate tropic response

5.2 Plant Mechanoresponses

Plant mechanoresponses can be divided into two broad categories: those associated with highly specialized mechanosensory organs such as tendrils or the traps of carnivorous plants, and those reflecting a more ubiquitous mechanosensory system that seems to af-fect most parts of the plant The specialized touch sensory systems have clearly evolved to trigger a single specialized response, such as the twining of a tendril The more “gen-eral” touch sensitivity, however, may well relate to the systems required to monitor and control the mechanical stresses inherent in normal turgor-driven cell expansion

5.2.1 Specialized Touch Responses

hinge region between the lobes (Fagerberg and Allain 1991), rapid turgor loss in motor cells in this region (Hill and Findlay 1981), and an inherent tendency of the lobes to “snap” shut due to the elasticity and geometry of the trap itself (Forterre et al 2005) The sensor that triggers this closure is a series of modified hairs on the inner trap surface Activating the trap requires multiple stimulations on these hairs as the insect climbs across the leaf The requirement for several signals is likely a safeguard to prevent clo-sure of the trap by random mechanical stimuli, such as the impact of a raindrop, which tend to be solitary events The multiple stimulations then trigger action potentials that are transmitted to the hinge region to effect closure (Jacobsen 1965; Simons 1981) The con-tinued struggling of the insect, likely supplemented with some chemical sensory events, then closes the trap even tighter, forming a chamber in which the insect is digested (Fagerberg and Allain 1991)

Similar mechanosensory triggers are seen in other carnivorous plants, such as the suc-tion trap of the bladderworts (Utricularia) and the leafrolling sticky trap of the sundews (Drosera) (Darwin 1893; Lloyd 1942) In this latter case the mechanosensor is respon-sive to microgram stimuli, yet is able to ignore the presumably much larger mechanical signals from the impact of rain or wind (Darwin and Darwin 1880; Darwin 1893)

Although these plants have received much attention due to their dramatic carnivorous responses, we are largely ignorant of how their mechanosensors operate The rapidity of triggering the response seen in Dionaea and Utricularia (which responds in the millisec-ond range) strongly suggests the involvement of a mechanosensory ion channel Indeed, the inherent speed and signal amplification of channels makes them the top candidates for most rapid mechanosensory responses (Gillespie and Walker 2001, and see below) However, at present the molecular identity of plant mechanosensors remains unknown Identification of this initial signaling system will be an important step toward answering the many perplexing questions raised by the touch response in these carnivorous plants For example, how does Dionaea suppress its response until multiple touch signals have been received, and how can Drosera tell the difference between an insect and a raindrop impacting on the leaf?

the vasculature Again, we are still ignorant of the identity of the initial mechanosensor and associated signaling events that should be coupled to such a hydraulic signaling system

One general theme emerging from all these studies on thigmonastic responses is that the most rapid changes monitored upon mechanical stimulation are generally the electri-cal changes thought to transmit the touch information to the motor response These elec-trical changes reflect ion transport phenomena at the plasma membrane, highlighting the central role of ionic signaling in the early phase of a plant mechanoresponse

5.2.2 Thigmomorphogenesis and Thigmotropism

Even in plants with no obvious specialized mechanosensory structures, mechanical per-turbation leads to morphogenetic changes that are highly adaptive Thus, in shoots, me-chanical stimulation causes inhibition of elongation and radial swelling (Biddington 1986; Telewski and Jaffe 1986) Such changes can be local; for example, trees will strengthen regions of the trunk or branches undergoing compression or tension with the production of reaction wood (compression and tension wood; Hellgren et al 2004) These modified areas are rich in cells exhibiting strengthened, often highly lignified cell walls to reinforce mechanically stressed tissues

However, changes in growth habit upon localized mechanical perturbation can also be systemic (Biddington 1986; Coutand et al 2000), suggesting that a mobile signal is inte-grating the overall thigmomorphogenetic response Although mechanosensitive channels, Ca2+-dependent signaling cascades (see below), ethylene, and perhaps GA (Mitchell 1996) are strong candidates for components of the mechanosensory signal transduction system regulating and integrating these changes in growth, we still have remarkably lit-tle molecular data on how these thigmomorphogenetic responses occur

In addition to this general change in form upon mechanical stimulation, plants also show highly oriented changes in growth where the direction of response is determined by the direction of the stimulus (i.e., a thigmotropic response) Thigmotropism can be seen in many parts of the plant but is perhaps most familiar in the specialized touch-responsive organs known as tendrils Many plants use leaves or shoots modified into ten-drils to secure themselves to supports to allow increased height without the need for ex-tensive deposition of metabolically expensive strengthening agents such as lignin These tendrils are highly touch-sensitive, responding to stimuli of as little as 250 µg (Simons 1992) Upon sustained mechanical stimulation they rapidly (often within seconds) begin to exhibit differential growth across the organ, leading to coiling around the contacted ob-ject (Jaffe and Galston 1968) In Bryonia dioica, octadecanoids and auxin seem to regu-late this cell expansion (Stelmach et al 1999) The direction of coiling is often deter-mined by the direction of the mechanical stimulus, leading to a thigmotropic response of the organ However, thigmotropism is not limited to such highly specialized touch-sensitive organs For example, thigmotropism is also exhibited by roots growing into ob-stacles in the soil, a response we will discuss in more detail later in this chapter

expect from such a sensor Therefore, in the next section we will discuss the principles of mechanoperception that can be derived from the touch sensors seen across the kingdoms and ask how well the molecular insight gained from these animals and bacteria might translate to identification of a plant mechano- or gravisensory system

5.3 General Principles of Touch Perception

Any protein embedded in a membrane experiences mechanical force exerted by the lipid bilayer All bilayers have a characteristic lateral pressure profile, with outward (positive) directed pressure in the hydrophobic core of the membrane and large tension at the membrane–water interfaces, though the specific distribution and magnitude of pressures vary considerably with lipid composition (Poolman et al 2004) (Figure 5.2) In the rest-ing state, a transmembrane protein adopts a conformation that is at equilibrium with the surrounding mechanical forces of the lipid bilayer However, when the membrane is stretched or deformed by a mechanical stimulus, the distribution of forces is altered and the equilibrium perturbed In a mechanosensitive protein, this change in force is thought to trigger a conformational change, which results in activation (or deactivation) of the protein (Hamill and Martinac 2001; Janmey and Weitz 2004; Kung 2005) (Figure 5.2) Though in-plane membrane forces may conceivably activate any number of transmem-brane proteins, so far only the mechanosensitive ion channels of bacteria and the TRPC1 channel of Xenopus oocytes have been shown to be directly gated by membrane tension (Maroto et al 2005; Moe and Blount 2005) (see below) However, several other candi-date mechanosensitive ion channels from animals and yeast have recently been identified (TRPY1, TREK1 and 2) (Bang et al 2000; Chemin et al 2005; Zhou et al 2005)

How relevant is this model of mechanoperception for plant cells where the flexion of the plasma membrane must go hand-in-hand with the deformation of the cell wall? The cell wall is under hydrostatic pressure from the protoplast, so that cell wall deformation would require the application of an external force large enough to overcome turgor pres-sure Given that turgor is in the range of to 40 bars (Tomos and Leigh 1999; Franks 2003), it may at first glance seem that only extreme forms of mechanical stimulation would provide sufficient force to be detected via changes in membrane tension However, even subtle stresses such as a gentle breeze can make leaves or stems sway and such organ bending can only occur when at least a subset of cells change shape (i.e., when the cell wall and plasma membrane are deformed) This is possible because parts of the plant act as levers In this way, even very moderate mechanical forces can be sufficiently amplified and focused onto the responding cells to exceed turgor pressure, flex the plasma membrane, and thus directly gate transmembrane proteins via changes in lipid force distribution

above, the end result—the altered force distribution at the protein–lipid interface and en-suing activation of the mechanosensitive protein—is the same in both models (Kung 2005)

According to another model, tether-mediated activation of membrane proteins occurs when stress-deformed cytoskeletal/ECM components pull on the protein and thereby di-rectly change the protein conformation [e.g., by shifting an autoinhibitory domain which releases block and triggers activation (trapdoor model) (Hamill and Martinac 2001; Kung 2005)] (Figure 5.3) or by partially unfolding the attached protein and thus revealing pre-viously hidden catalytic/binding sites (Janmey and Weitz 2004; Vogel 2006) Several mechanosensitive ion channels of vertebrate and invertebrate organisms are proposed to Figure 5.2. Forces exerted through the membrane can gate ion channels A The force distribution through-out the depth of the membrane leads to a steep gradient in gating force on the interface between the channel protein and the lipid head groups Membrane tension can favor channel opening if the cross-sectional area of the open state of a channel is larger than the closed (B), or if membrane tension leads to thinning of the bi-layer and the conformational change leading to the open state of the channel reduces the profile of the mem-brane-spanning, hydrophobic residues on the channel surface (C)

be activated by mechanical force transmitted through their linkage to the cytoskeleton (e.g., TRPA1) (Lin and Corey 2005) and/or the ECM (e.g., MEC channel complex; see below), though the precise mechanism of gating is not yet fully understood

In plant cells, any force large enough to overcome turgor and flex load-bearing ele-ments of the cell wall and alter cellular shape will also deform the cytoskeleton However, some plant organs are known to be extraordinarily sensitive to mechanical stimuli, re-sponding to as little as a 10-µN weight (~1 mg) with altered growth (Jaffe and Galston 1968) Such weak forces are unlikely to change cell shape but may conceivably deform non-load-bearing elements embedded in the wall matrix, such as cell wall proteins If these elements are linked via transmembrane proteins to the cytoskeleton, force may be transmitted from the cell exterior not only to plasma membrane mechanosensors, but— because the cytoskeleton is ideally suited to long-distance transfer of stresses—to mechanosensitive proteins in endomembranes as well (Ingber 2003, 2006)

ing Thus, the cytoskeleton may not only act to convey forces to interior regions of the cell by tugging on an attached mechanosensor, but may itself translate stress information into biochemical reactions via altered binding to associated proteins (Figure 5.3d) For example, specific cytoplasmic proteins show differential binding to the cytoskeleton de-pendent on the state of cytoskeletal tension (Sawada and Sheetz 2002; Tamada et al 2004) Similarly, stretching of the cytoskeleton promotes activation of a tyrosine kinase (Scr) via interaction with an actin-binding protein (AFAP) (Han et al 2004) Strain-induced alterations in binding affinity of cytoskeletal proteins are thought to be mediated via conformational changes, especially the unfolding of protein domains and resulting exposure of cryptic binding (or catalytic) sites

Indeed, mechanically induced partial unravelling of proteins is a potentially wide-spread signalling mechanism By using force spectroscopy to investigate the mechanical properties of multimodular proteins, such as the giant muscle protein titin and the ECM adhesion protein fibronectin, it was shown that, when exposed to increasing levels of ten-sion, the modules unfolded one-by-one in a sequence determined by the mechanical sta-bility of each domain (Zhuang and Rief 2003; Vogel 2006) (see Figure 5.3) The stasta-bility should be dictated by features such as disulfide and hydrogen bonds that determine the secondary structure of the protein These features will in turn be modulated by cellular environment such as pH and ionic strength and local redox potential (Vogel 2006) Thus, the magnitude of stress experienced by the protein may well be encoded in the degree of protein unfolding and signalled to the cell by way of revealing different recognition (binding or catalytic) sites in each unravelled module (Vogel 2006) This is also an in-triguing possibility for plant cells If deformation of cell wall components results in ex-posure of new binding sites, interactions with plasma membrane receptors may be newly formed or broken to trigger signalling to the cell interior

In addition to such direct effects on protein conformation, two other themes that emerge from this overview of mechanosensory channel function are that gating through tension in the lipid bilayer and through tethering to either the ECM and/or the cytoskele-ton are the most prominent modes regulating the opening and closing of mechanosensory channels We will therefore describe in more detail two of the most intensively studied channel types, the MscL channels of Escherichia Coli and the MEC channels of

Caenor-habditis elegans, to explore the molecular mechanisms that underlie each of these modes

of channel regulation These channels may well provide clues to the structure of the elu-sive plant mechanosensory complex

5.3.1 Gating through Membrane Tension: The Mechanoreceptor for Hypo-osmotic Stress in Bacteria, MscL

The MscL of E coli is perhaps the best understood of these mechanosensors. Mechanically gated MscL activity was first observed in patch clamp experiments con-ducted on giant E coli spheroplasts, and subsequent purification of MscL and reconsti-tution in artificial lipid bilayers demonstrated that the channel retained its characteristic mechanosensitive conductance (~3 nS) and gating kinetics even in a cell-free environ-ment (Sukharev et al 1993; Sukharev et al 1994) These findings convincingly estab-lished that changes in membrane tension alone can be sufficient to activate a mechanosensitive ion channel

Sequence analysis revealed that the E coli (Eco-)MscL encodes a 136 amino acid membrane protein with cytoplasmic N- and C-terminal regions and two ␣-helical trans-membrane domains (designated TM1 and TM2) connected by a periplasmic loop (Blount et al 1996) Determination of the crystal structure of a homolog of Eco-MscL, the MscL of Myobacterium tuberculosis (Chang et al 1998), showed that five MscL subunits form a homopentamer, with TM1 domains forming the funnel-shaped permeation pathway and TM2 helices interacting with the lipid bilayer and surrounding the central barrel of TM1 domains (Figure 5.4a) On the cytoplasmic side of the channel, the five C-terminal he-lices assemble into a bundle not required for gating but presumed to act as a size-exclusion filter (Anishkin et al 2003) Though unresolved in the crystal structure, the five amphiphilic N-terminal (S1) domains were predicted to organize as ␣-helices, inter-acting to form another bundle situated between the channel pore and the C-terminal “hanging basket” (Sukharev et al 2001b; Sukharev et al 2001a)

How these different channel domains contribute to the mechanical gating of MscL? According to a current model based on computer simulations and experiments using cys-teine substitutions to stabilize specific domain interactions, the channel is activated by the sequential tension-induced opening of two gates (Sukharev et al 2001b) Upon stretch of the membrane, the channel pore increases in diameter from ~0.2 nm to about 3.5 nm (Doyle 2004) This dramatic change in channel conformation is thought to occur as the transmembrane domains TM1 and TM2 undergo significant tilting, thereby swing-ing away from the central channel axis This iris-like expansion is considered the initial gating event because it would open the hydrophobic constriction at the narrow end of the funnel-shaped TM1 pore acting as a barrier to ion permeation However, removal of this constriction is not sufficient to elicit full conductance of the channel In fact, the initial conformational change only seems to draw the channel into a low subconducting state (Anishkin et al 2005) (Figure 5.4a and Color Section) Further extension is required to activate the channel completely The transition to the fully open state is thought to depend on the disruption of the N-terminal bundle connected to the TM1 domains via linker re-gions Only when the TM barrel fully expands at close to lytic tensions is force transmit-ted via the linkers to this bundle, pulling it apart and thereby opening the second gate to release huge amounts of ions and small solutes (Sukharev et al 2001b)

5.3.2 Gating through Tethers: The Mechanoreceptor for Gentle Touch in

Caenorhabditis elegans

mov-ing forward when the tail is touched Laser ablation experiments have revealed that six touch receptor neurons are responsible for sensing these mechanical stimuli (Chalfie et al 1985) These six neurons have processes that lie embedded in the hypodermis near the cuticle of the worm and seem to be attached to the hypodermis via extensive connections to the ECM (Garcia-Anoveros and Corey 1997)

By patch-clamping these neurons in vivo while gently touching the nematode cuticle, Figure 5.4 (also see Color Section). Structures of mechanosensitive channels gated by membrane tension (MscL) and tethering to the cytoskeleton and extracellular matrix (Mec) In the bacterial MscL channel (a), five subunits form the channel, each contributing two transmembrane domains (green) In the closed state, the S1 domain (red) sits close to the inner face of the pore When the membrane experiences tension, the transmembrane helices tilt and the pore expands in an iris-like fashion S1 is drawn closer to the membrane, partially occluding the pore As the channel fully opens, the S1 domains are dispersed, leaving the pore un-obstructed In the Mec channel of C elegans (b), the Mec-4 and Mec-6 subunits form the conducting pore, which is gated through a complex of proteins that interact with the extracellular matrix and the cytoskeleton

O’Hagan et al (2005) were able to show that mechanical stimulation triggers a rapidly ac-tivating mechanoreceptor current that is carried mostly by Na+and depolarizes the plasma membrane The membrane depolarization is then thought to elicit an increase in cytosolic Ca2+by activating voltage-sensitive L-type Ca2+channels (Suzuki et al 2003), highlight-ing that a mechanosensory channel need not be directly Ca2+-permeable to elicit the

touch-induced Ca2+increases proposed to transduce the mechanical signal in plants (see below) Saturating mutagenesis analysis has identified 18 genes required for this C elegans mechanosensitivity (named mechanosensory abnormal, or MEC proteins; Ernstrom and Chalfie 2002), some of which encode transcription factors, cytoskeletal proteins, or pro-teins of the ECM (Tavernarakis and Driscoll 1997) At least four of the MEC propro-teins (MEC-2, -4, -6, and -10) form the channel complex producing the mechanoreceptor current (O’Hagan et al 2005) (Figure 5.4b and Color Section) MEC-4 and MEC-10 are pore-forming channel subunits and belong to the DEG/ENaC (degenerin/epithelial sodium chan-nel) family of amiloride-sensitive Na+-conducting channels found exclusively in animals (Lai et al 1996; Goodman et al 2002; Kellenberger and Schild 2002) MEC-6 is a single-pass transmembrane protein with a cytoplasmic N-terminus and large extracellular C-terminal domain (Chelur et al 2002), whereas MEC-2, a monotopic protein with a stomatin-like region, does not span the plasma membrane but is associated with the cytoplasmic side of the lipid bilayer (Goodman et al 2002) Both MEC-2 and MEC-6 interact with MEC-4 and MEC-10 to regulate channel conductance (Chelur et al 2002; Goodman et al 2002)

Interestingly, no mechanically induced activation of current was observed when the wild-type channel complex was heterologously expressed in Xenopus oocytes (Goodman et al 2002) This observation suggests that mechanical gating is not accomplished by changes in membrane tension alone, but requires additional force-transmitting elements such as proteins of the ECM and the microtubule cytoskeleton physically tethered to the channel complex It has been proposed that the touch-induced movement of the two pu-tative tethering sites (ECM-channel extracellularly and microtubule-channel intracellu-larly) relative to each other provides gating tension and activates the channel (reviewed by Tavernarakis and Driscoll 1997) Indeed, both the pore-forming channel subunits MEC-4 and MEC-10 as well as MEC-6 have large extracellular regions thought to be im-portant for interaction with the ECM proteins MEC-1, MEC-5, and MEC-9 (Chelur et al 2002; Emtage et al 2004)

The precise role of the prominent microtubule cytoskeleton in mechanosensation is less clear Mutations in the genes MEC-7 and MEC-12 cause loss of mechanoresponse. These genes encode ß- and ␣-tubulins, respectively, which are required to form micro-tubule protofilaments These micromicro-tubules are proposed to be tethered to the channel complex via MEC-2 (Tavernarakis and Driscoll 1997) (Figure 5.4b) However, mutants in MEC-7 still show (attenuated) touch-triggered mechanoreceptor currents, suggesting that direct linkage of the channel complex to microtubules is not an absolute requirement for gating (O’Hagan et al 2005)

5.3.3 Evidence for Mechanically Gated Ion Channels in Plants

mem-brane from a variety of cell types derived from different tissues in different plant species Applied pressures of to 10 kPa have been shown to trigger activation of channel cur-rents in Arabidopsis, Allium, Vicia, Valonia, Lilium, and others Apart from this common mechanism of gating, however, the observed channel activities are quite diverse with re-gard to selectivity, conductance, or voltage dependence Some channels are unselective (Spalding and Goldsmith 1993) and others have been demonstrated to show preference for anions (Falke et al 1988; Cosgrove and Hedrich 1991; Heidecker et al 1999; Qi et al 2004) or cations (Garrill et al 1994), whereas certain channels show high selectivity for K+or Ca2+(Cosgrove and Hedrich 1991; Ding and Pickard 1993; Liu and Luan 1998;

Dutta and Robinson 2004) Measured conductances range from to 15 pS for Ca2+ -selective channels (Cosgrove and Hedrich 1991; Ding and Pickard 1993; Dutta and Robinson 2004) to 13 to 97 pS for anion channels (Falke et al 1988; Cosgrove and Hedrich 1991) Although most channels are seemingly independent of membrane poten-tial (Falke et al 1988; Garrill et al 1994; Liu and Luan 1998; Heidecker et al 1999; Dutta and Robinson 2004; Qi et al 2004), others show significantly reduced open prob-abilities at either negative or positive voltages (Cosgrove and Hedrich 1991; Spalding and Goldsmith 1993), whereas some are active only at higher voltages, irrespective of the po-larity (Ding and Pickard 1993)

Unfortunately, most of these channels have not been analyzed beyond the initial char-acterization Thus, even though the molecular identity remains elusive, information on how the regulation of these mechanosensors are modulated by other factors such as pH, lipid environment, or the cytoskeleton could greatly enhance our understanding of how plant cells tune their mechanosensitivity during differentiation or in response to chang-ing environmental conditions

Despite all the differences shown by the plant mechanosensitive conductances de-scribed above, one interesting common feature of all these channels is that their acti-vation is feasible in excised membrane patches devoid of cell wall or intact cytoskele-ton This observation suggests that plant mechanosensory channels are sensitive to changes in lipid bilayer tension and their gating does not absolutely depend on tethering to force transmitting elements, though both cell wall and cytoskeleton may have a role in modulating membrane tension in vivo The pressures used to open mechnosensitive conductances in all these plant experiments are also comparable to those that gate the bacterial MscL and MscS channels, consistent with a role for membrane tension in their regulation

5.4 Signal Transduction in Touch and Gravity Perception

5.4.1 Ionic Signaling

From the above discussion, it is clear that a major theme emerging from our current un-derstanding of mechanosensors in animals and microbes is that channels and ionic fluxes play a central role in the transduction of external force to intracellular biochemical sig-nal There is also an increasing body of evidence that ionic signals are intimately associ-ated with the touch and gravity responses in plants Principal among these mechanically related signals are changes in Ca2+and pH.

5.4.2 Ca2+Signaling in the Touch and Gravity Response

Changes in the levels of cytosolic Ca2+are recognized as a ubiquitous regulatory system and are also among the most widely reported initial changes in response to mechanostim-ulation (Gillespie and Walker 2001) For plants there is a wide body of literature support-ing the idea that mechanical stimuli, rangsupport-ing from wind and rain to localized mechano-stimulation of a single cell, elicit complex patterns of Ca2+ change Most of these measurements have been made noninvasively using transgenic plants expressing the lu-minescent Ca2+-sensitive protein aequorin Such analyses confirm touch-related Ca2+ in-creases in plants ranging from Arabidopsis and tobacco (Knight et al 1991; Knight et al. 1992; Knight et al 1993; Plieth and Trewavas 2002) to the moss Physcomitrella patens (Haley et al 1995; Russell et al 1996) and even the characean algae (Blancaflor and Gilroy, unpublished), suggesting the presence of an ancient mechanosensory system Ca2+chelation attenuates the stunting of growth associated with thigmomorphogenesis

(Jones and Mitchell 1989), tentatively suggesting a functional role for such Ca2+changes The site of these Ca2+fluxes (trans-plasma membrane versus release from

intracellu-lar stores) remains to be unequivocally determined Thus, Ca2+ chelators and channel blockers that should inhibit influx at the plasma membrane have been reported to either block or fail to affect touch-induced Ca2+increases (Haley et al 1995; Legue et al 1997) Further, evidence for release from intracellular stores comes largely from the ability of ruthenium red to inhibit Ca2+changes (Knight et al 1992; Legue et al 1997) Ruthenium red is thought to block channel-mediated Ca2+ release from mitochondria and the ER

(Denton et al 1980; Campbell 1983) but its action on Ca2+channels in plants is not well characterized Ruthenium red is, however, known to affect other plant processes; for ex-ample, it binds strongly to unesterified pectins (Moffatt et al 2002) In addition, in some reports (Haley et al 1995) no effect of ruthenium red was observed on the mechanically induced Ca2+transients

Despite this uncertainty as to its precise source, the mechanically induced Ca2+increase

has been confirmed in plants using sensors other than aequorin Thus, plants expressing the Ca2+-sensitive, green fluorescent, protein-based sensor cameleon (Allen et al 1999) show

mechanically induced Ca2+transients (Figure 5.5 and Color Section), as plants loaded with the Ca2+-sensitive dye Indo-1 (Legue et al 1997) In this latter study, the apical cells

type The high sensitivity of the surface cells of the root cap fits well with their position, being the first cells to encounter obstacles as the root forces itself through the soil

It is important to note that many other stimuli have been shown to elicit increases in intracellular Ca2+, ranging from cold shock to symbiont elicitors (reviewed in Hetherington and Brownlee 2004) These Ca2+changes often occur in exactly the same

cell as touch-induced Ca2+transients (Figure 5.5) One possible explanation for this re-sponse to multiple stimuli is that Ca2+is simply acting as an “on switch” informing the

cell that something has happened, with the specificity of the response being encoded by other signaling systems (Scrase-Field and Knight 2003; Plieth 2005) Alternatively, the spatial and/or temporal footprint of the Ca2+ change (the so-called Ca2+signature) may encode information about the stimulus evoking the response

There has long been literature supporting the informational content of Ca2+changes in animal cells (Dolmetsch et al 1997, 1998) and there is some evidence supporting a sim-ilar phenomenon in plants For example, in stomatal guard cells, Ca2+spiking is seen in response to stimuli that induce closure of the stomatal aperture (McAinsh et al 1995; Allen et al 1999) The frequency of spiking appears to be critical, with Ca2+transients that occur either too frequently or too slowly being less effective in triggering the re-sponse (Allen et al 2001) These observations suggest that, at least in the guard cell, the temporal character of a Ca2+ increase is important for the response that is elicited Similarly, in the touch response the magnitude of Ca2+increase has been reported to cor-relate with the magnitude of mechanical stimulation (Haley et al 1995), consistent with the idea that the Ca2+change could be carrying information about the kind of mechani-cal stimulation that the cell is experiencing However, we clearly need more detailed analysis to distinguish a role for Ca2+in the “signature” versus “on switch” modes of ac-tion in plants in general and the mechanoresponse in particular

A role for Ca2+in signaling touch response appears to extend to the specialized touch-sensitive systems such as the tendril described at the beginning of this chapter For exam-ple, a Gd3+-sensitive, voltage-dependent Ca2+release channel (BCC1) has been electro-physiologically identified in ER isolated from the tendrils of Bryonia dioica (Klusener et Figure 5.5. Touch- and cold-induced calcium signatures in root cap cells a An outline of the cells in an

Arabidopsis root tip; box indicates area of the root cap observed in (b) and (c) b Changes in cytoplasmic

al 1995) Touch-induced tendril coiling can be inhibited in this system by Gd3+and ery-throsine B, a putative inhibitor of the Ca2+-ATPases that pump Ca2+into the ER Thus, there is some tentative evidence placing a Ca2+release channel involved in mechanore-sponse on an intracellular membrane in this system Interestingly, BCC1 may also be reg-ulated by cytosolic pH and levels of reactive oxygen species (ROS) (Klusener et al 1997) Considering the proposed roles for these two agents in response to mechanical perturbation and gravistimulation (see below), BCC1 may be hinting at one mechanism whereby gravity and touch signaling may modulate each other

Although the link between touch and Ca2+is supported by a wealth of experimental data, an equivalent connection between Ca2+and gravity signaling is less clear There is much circumstantial evidence linking Ca2+ to the gravity response For example, appli-cation of Ca2+chelators such as EGTA and BAPTA abolishes the growth component of the graviresponse (Lee et al 1983a, 1983b; Björkman and Cleland 1991) and the auxin fluxes that accompany tropic curvature (Young and Evans 1994; see also Chapter 3) Similarly, a range of pharmacological agents thought to disrupt diverse aspects of Ca2+ signaling have been reported to alter gravitropism (Fasano et al 2002; Massa et al 2003) There is also extensive evidence implicating the Ca2+-dependent regulatory protein calmodulin (CaM) in the graviresponse For example, CaM levels are enriched in the root tip (the site of gravity perception) (Allan and Trewavas 1985; Stinemetz et al 1987), are enhanced upon gravistimulation (Sinclair et al 1996), and the CaM levels in the root tip correlate with the responsiveness of the organ to gravity (Stinemetz et al 1987) Calmodulin transcripts have also been shown to be recruited into polysomes on the lower side of the gravistimualted pulvinus (Heilmann et al 2001), suggesting that gravistimu-lation should change the abundance of CaM across the stimulated organ Calmodulin an-tagonists inhibit the asymmetric Ca2+ and proton fluxes associated with graviresponse (Lee et al 1983b, 1984; Björkman and Leopold 1987) and impair gravisensing and tropic response at levels that not inhibit growth (Stinemetz et al 1992; Sinclair et al 1996) Although it is always important to view such pharmacological data with caution due to unknown targets and side effects of the antagonists used, this body of data, taken with the other evidence for a role for CaM described above, does point toward an important role for this Ca2+-dependent protein in gravisignaling and response, and therefore, by impli-cation, a role for Ca2+

The identification of a possible role for inositol-1,4,5-trisphosphate (InsP3) in grav-isignaling/response is also consistent with Ca2+playing an important role in this process Classically, the activation of the phospholipids-cleaving enzyme phospholipase C is thought to produce the second messengers diacylglycerol and InsP3 The precise

signal-ing role for diacylglycerol in plants is still unclear but InsP3seems to play a similar role to its function in animal cells in triggering signaling-related Ca2+release from intracel-lular stores (Wang 2004) In the graviresponsive pulvinus from maize and oat, InsP3 be-comes elevated within minutes of gravistimulation, although a clear asymmetry in levels between upper and lower side of the organ takes several minutes to appear (Perera et al 1997, 1999, 2001) Phosphatidyl-inositol-phosphate (PIP) kinase activity similarly in-creased, suggesting that levels of the substrate for phospholipase C (phosphatidylinositol-4,5-bisphosphate) might be fuelling the elevated InsP3levels Perera et al (2006) showed

gravsitmula-tion in Arabidopsis inflorescences They then constructed Arabidopsis plants expressing a human inositol polyphosphate 5-phosphatase that should attenuate these InsP3levels These plants showed 90% reduction in basal InsP3content and a disruption of gravitropic response kinetics, further implicating InsP3, and so perhaps Ca2+release, in gravitropic signaling/response

Despite this strong circumstantial evidence for a role of Ca2+in gravisignaling, direct measurements for this change remain ambiguous Although Gehring et al (1990) re-ported gravistimulation-induced Ca2+increases in maize coleoptiles, it has been difficult to unequivocally relate these changes to Ca2+ increases associated with the gravity

re-sponse (Firn and Digby 1990) Legue et al (1997), using plants loaded with the Ca2+ -sensing fluorescent dye Indo-1, and Sedbrook et al (1996), using plants expressing ae-quorin, were unable to detect Ca2+ increases upon gravistimulation, yet Legue et al (1997) saw clear touch-induced changes under identical conditions In contrast, Plieth and Trewavas (2002) have reported Ca2+ increases induced by gravistimulation These measurements were made in Arabidopsis seedlings transformed with the Ca2+sensor

ae-quorin and gravistimulated by rotation through 135 degrees The kinetics of these changes were different from plants rapidly rotated through 360 degrees, which was used to provide a control for the mechanical stimulation inherent in the rotation However, con-sidering the exquisite sensitivity of plants to mechanical stimulation, it still remains pos-sible that the Ca2+increases associated with the gravistimulation were also reflecting the mechanical stimulation of the rotation to 135 degrees These kinds of caveats about ex-perimental results highlight the difficulties of separating touch from gravity signaling and response This problem is not only limited to experimental design but also to the bi-ological responses to these stimuli, a theme we will discuss in more detail in the section describing transcriptional responses to touch and gravity below

If the circumstantial evidence so strongly points to a role for Ca2+ signaling in the gravity response, why has it been so hard to clearly demonstrate the change? One possi-bility is suggested by the experiments of Plieth and Trewavas (2002) In order to detect Ca2+ changes upon reorientation of their aequorin-expressing plants, these researchers had to resort to making simultaneous measurements on 500 to 1,000 seedlings and reconstituting the aequorin with the most sensitive version of its cofactor known (Cp-coelentrazine) The need for such high sensitivity and numbers suggests the Ca2+signal is either localized to a very few cells and/or localized within those sensory cells The el-evated levels of CaM and CaM-like proteins in the root cap gravisensory cells will likely sensitize them to very small changes in Ca2+that may be at the limits of current Ca2+

de-tection systems

Similarly, in animal cells it is well characterized that highly localized Ca2+ fluxes, which are extremely difficult to detect, can elicit dramatic effects on cellular response For example, in neurons Ca2+ flux specifically through L-type Ca2+ channels at the plasma

such localized fluxes remain the reason no Ca2+signal has been localized to the gravisens-ing cells of the plant Alternatively, the circumstantial evidence for Ca2+signaling may be misleading and gravisignaling may reside in some other transduction pathway

Also as a note of caution, the coelentrazine cofactor for aequorin required to reconsti-tute the active Ca2+ sensor is itself exquisitely sensitive to ROS In response to ROS it generates a signal similar to that expected of an increase in Ca2+(Lucas and Solano 1992; Plieth 2005; Molecular Probes 2006) Therefore, experiments using this approach require careful controls for possibly confounding effects of ROS production Such controls are especially important as there are reports that transient changes in ROS production are as-sociated with the auxin fluxes generated during the graviresponse (Joo et al 2001; Joo et al 2005)

Similarly, sustained mechanical stress induces ROS production in suspension-cultured soybean and parsley cells within to 10 (Yahraus et al 1995; Gus-Mayer et al 1998), suggesting that ROS may also complicate aequorin-based measurements involving me-chanical stimulation ROS are now thought to participate in many plant response systems (Mori and Schroeder 2004), and the finding that they in turn affect ROS-gated Ca2+ chan-nels (Pei et al 2000; Demidchik et al 2003; Foreman et al 2003) makes them strong can-didates for regulators in mechano-/graviperception To make the story even more com-plex, ROS may well be involved both at the level of intracellular signaling elements (Mori and Schroeder 2004) and also as factors modulating cell wall properties associated with growth (Campbell and Sederoff 1996; Brady and Fry 1997; Coelho et al 2002; Kerr and Fry 2004)

5.5 Insights from Transcriptional Profiling

The initial identification of touch-responsive (TCH) genes in Arabidopsis was achieved through differential cDNA screening of plants stimulated by touch or wind (Braam and Davis 1990) The identity of many of these genes as encoding Ca2+-binding proteins re-inforced the theme of Ca2+-dependent signaling in the mechanical response of the plant Thus, TCH1 encodes CaM2 (one of the Arabidopsis CaMs) and TCH2 and TCH3 encode CML24 and CML12, both CaM-like proteins (Braam and Davis 1990; Sistrunk et al 1994; Khan et al 1997; McCormack and Braam 2003) Similar analysis has now identi-fied a range of genes showing mechanosensitive expression, including other CaMs (Ling et al 1991; Perera and Zielinski 1992; Gawienowski et al 1993; Botella and Arteca 1994; Ito et al 1995; Botella et al 1996; Oh et al 1996)

sim-ilarity in the growth responses these stimuli elicit and/or the multiple pathways within which each protein operates Indeed, many of the transcripts identified as touch- and/or gravity-responsive are also known to be regulated by other stimuli such as cold, light, and pathogens However, a small subset were found to be either touch- or gravity-specific For example, these researchers found 65 genes that changed rapidly and selectively in re-sponse to gravistimulation, with changes in five being evident within to minutes (Kimbrough et al 2004) Similarly, 26 genes showed a mechanostimulation-specific pro-file These gravity- or touch-specific genes covered a wide range of functional categories, from transcription factors to transporters and wall-modifying proteins Correspondingly similar studies profiling mechanoresponsive genes in seedlings (Moseyko et al 2002) or aerial tissues (Lee et al 2005) led to similar conclusions Thus, Moseyko et al (2002) found that of the 8,300 genes they probed, 183 were significantly altered after 30 minutes of gentle mechanical stimulation, with a significant overlap to those regulated by gravi-stimulation When comparing transcriptional profiles in aerial tissues after 30 minutes of touch or darkness, Lee et al (2005) found that although 2.5% of the total genes were touch-inducible (589 genes had touch-inducible expression; 171 had reduced expression), 53% were also altered upon transfer of seedlings to darkness Indeed, all but of the 68 genes most strongly up-regulated by darkness were also touch-inducible Again, the range of gene functions in these groups was diverse, including putative signaling elements, wall modification, and defense responses These widespread changes in transcription suggest an exquisitely sensitive mechanical response system that feeds into much of the physiol-ogy and developmental pathways that are shared by many other signal/response systems

It seems likely that changes in mRNA stability as well as transcriptional regulation are playing some role in governing message abundance, especially over the very short (1- to 2-min) time frames where Kimbrough et al (2004) reported alterations in transcript level Indeed, Gutierrez et al (2002) found that although only approximately 1% of Arabidopsis genes have unstable transcripts, touch-induced genes were among the most highly repre-sented group in their analysis Unstable transcripts are thought to be the hallmark of genes requiring rapid changes in steady-state transcript abundance, consistent with the rapid and widespread transcriptional changes seen in response to touch

The alterations in the five most rapidly changing gravity-responsive transcripts were abolished in plants where InsP3 signaling is likely curtailed through ectopic expression of a human inositol 5-phosphatase (Salinas-Mondragon et al 2005) However, the other widespread changes in gene expression induced by gravistimulation were unaffected in these plants Thus, there may well be a functional link between rapid InsP3-dependent

signaling (Perera et al 1997, 1999, 2001) and very rapid mRNA abundance changes seen upon gravistimulation There must also be an alternative pathway acting to modulate the expression of the large number of other responsive genes Equivalent analysis with re-spect to touch-related gene expression has yet to be reported

sensitiv-ity to expression (Iliev et al 2002), as does a similar sequence for CBF2 (a transcriptional activator with a central role in cold response; Zarka et al 2003) The microarray data de-scribed above now present the possibility of assessing how widespread these touch- and gravity-related transcriptional regulatory motifs might be Such analysis might provide one avenue to start to dissect the transcriptional machinery responsible for these genome-wide changes in expression in response to either gravity or mechanical perturbation

The general conclusion from all these transcriptional profiling analyses is that both mechanical- and gravistimulation induce very rapid and extensive changes in mRNA pro-files In addition, these stimuli share a huge overlap in the transcriptional changes they cause, likely reflecting the similarity in developmental responses they elicit This is in contrast to recent work on plant hormonal regulation of growth and development where Nemhauser et al (2006) concluded that these regulators controlled similar developmen-tal outcomes through largely non-overlapping transcriptional responses

The other striking feature is the differences in the precise suites of genes reported as being under mechanical or gravitational regulation in each study This may in part reflect differences in timing and tissues under analysis However, for the case of mechanical stimulation it is important to note that touch is a complex stimulus to quantify and apply, and so the variation is almost certainly also reflecting the different ways mechanical stim-ulation was conducted in each study It seems likely that the transcriptional response is highly tailored to the kind of mechanical stimulation, hinting at a signaling system capa-ble of encoding information such as magnitude, duration, and location of the touch stim-ulation coupled to an extremely adaptable response circuit

An additional theme from such studies is that genes for putative Ca2+-binding proteins are disproportionately up-regulated upon touch stimulation (Lee et al 2005), suggesting an alteration in the Ca2+response system, perhaps as part of an adaptation mechanism to the initial Ca2+signals associated with touch sensing In general, as the levels of Ca2+ sig-naling components are elevated, two outcomes are likely: (1) the sensitivity of the system to future Ca2+ increases should be increased, and (2) the emphasis of Ca2+-dependent cellular responses will be shifted toward those involving the now-elevated Ca2+response

elements Thus, the touch history of the plant may well shift both its sensitivity and pre-cise response to future touch stimulation However, as a note of caution, although CML24 mRNA has been shown to be highly induced by touch (nine-fold increase at 30 minutes after stimulation) and to be expressed in regions of the plant likely experiencing mechan-ical strain (such as branch points and organs undergoing rapid elongation), recent analy-sis indicates no detectable change in protein abundance upon touch stimulation (Delk et al 2005)

the gravity signal is rapidly transduced to a broad range of response elements Interest-ingly, a similar proteomic analysis of the mechanoresponse does not show an extensive overlap in the proteins regulated at the post-transcriptional level in the mechanical and gravity response systems (see Chapter 2) Thus, one possibility is that post-transcriptional modification represents one theme of how specificity and selectivity may be imposed on the touch versus gravity response and possibly how these response systems may interact

5.6 Interaction of Touch and Gravity Signaling/Response

progresses Only a few of the peripheral cells of the root cap are in contact with the bar-rier and these must be transmitting touch information to the systems that control root growth to suppress gravitropic response (Massa and Gilroy, 2003a; Figure 5.6) Upon reaching the end of the obstruction and the mechanical stimulation, gravitropism domi-nates and the root resumes normal downward growth

The root tip-to-barrier angle is intermediate between vertical and flat (44 degrees in wild-type Arabidopsis; Massa and Gilroy 2003b), suggesting a compromise between rap-idly moving around the object by growing across its surface and the gravitropic response sending the tip of the root vertically downward A complete down-regulation of the gravity-sensing system during contact with the barrier would result in growth flat against its surface Therefore, it is likely that the intermediate angle forms as an adaptive com-promise between the graviresponse and the touch stimulation to successfully allow navi-gation around an obstacle (Fasano et al 2002) Indeed, a brief touch to the root cap de-sensitizes roots to gravity, as assayed by inhibition of subsequent gravitropic growth on a clinostat (Massa and Gilroy 2003a) Likewise, Mullen et al (2000) showed that mechan-ical stress during gravistimulation can delay the development of gravitropic curvature

Further data in support of the idea that gravisignaling is modulated by mechanosignal-ing come from experiments observmechanosignal-ing root growth along a barrier where some or all of the cells in the root cap are killed via laser ablation, thereby removing the gravisensing cells Ablating the whole cap or only the graviperceptive columella cells causes a loss of the DEZ curvature phase upon encountering the barrier, while the initial CEZ curvature is unaffected (Massa and Gilroy 2003a) Such observations suggest that the differential growth in the DEZ comes about because of a modified gravitropic response, as this is de-pendent on the presence of an intact columella However, an intact root cap is not needed for the initial CEZ curvature, suggesting that the differential growth in this region occurs as a result of cells sensing strain caused by the compressive forces that occur after initial contact (Evans 2003; Massa and Gilroy 2003a) That is, the cells in the CEZ that respond by producing tropic curvature may also be the cells experiencing the mechanical stimu-lation, rather than secondarily responding to a signal produced in the cap, where the di-rect touch stimulation is occurring Although the precise mechanism for the interaction between touch and gravity signaling during these responses remains to be determined, there are clues to potential components responsible for such signal processing/integration in the physical machinery of the gravity sensing system

The first step in the graviresponse is the perception of the gravity vector by the plant There are two schools of thought about how this initial event is achieved: the statolith the-ory and the gravitational pressure thethe-ory (see Chapters and for a complete descrip-tion) The statolith theory states that intracellular sedimenting particles are responsible for sensing gravity In higher plants, statoliths are dense, starch-filled amyloplasts inside specialized cells (Sack 1997; Kiss 2000) In contrast, the gravitational pressure theory states that the entire protoplast acts as the gravity sensor and the tension and compression by the protoplast against the extracellular matrix initiates the graviresponse (Wayne and Staves 1996; Staves et al 1997a, 1997b)

mechani-cally stimulated, the motility of the sedimenting statoliths is reduced (Massa and Gilroy 2003a) This reduced motility would provide a system whereby touch stimulation could reduce gravitropic response by simply reducing the sedimentation rate to the gravity re-sponsive component of the sensory cell However, it appears that such a model of sens-ing based on large-scale amyloplast sedimentation may be too simplistic a view of these initial sensory events Thus, perception and presentation times (measures of the time to generate and export the initial gravitropic signal) fall in the seconds to < minute range, times much less than required for amyloplasts in the sensory cells to completely sediment to the new, lower face (Hejnowicz et al 1998; Perbal et al 2002; Perbal and Driss-Ecole 2003) Such rapid generation of signal implies that the amyloplasts are in contact with some network that rapidly converts the force of their sedimentation to a biochemical sig-nal The cytoskeleton remains a prime candidate for such a network in most gravisensing models (Blancaflor 2002; see also Chapters and 2)

Filamentous actin in particular has been proposed to play a critical role in gravisensi-tivity, based on its abundant presence in columella cells (Yoder et al 2001; Blancaflor 2002) The “restrained gravisensing” model (Baluˇska and Hasenstein 1997; Hejnowicz et al 1998; Driss-Ecole et al 2000; Perbal et al 2002) suggests that amyloplasts are physically connected to the actin network, which then in turn connects to downstream components of the signaling cascade Alternatively, in unrestrained gravity sensing, any connections between the cytoskeleton and the statoliths are nonexistent, too weak, or too transient to provide direct mechanotransduction In this model, the dense actin web in a columella cell is deformed locally by sedimenting amyloplasts, resulting in distant effects in the cell such as activation or inactivation of mechanoreceptors on the plasma membrane

The reduced amyloplast motility induced by mechanical stimulation would therefore deliver less force to the actin network and so reduce gravitropic signal generation Recent data suggest that this view of the role of actin may also be too simple For example, dis-rupting the actin network (Blancaflor and Hasenstein 1997; Staves et al 1997a) does not block the gravitropic response but, rather, enhances organ bending (Blancaflor and Hasenstein 1997; Yamamoto and Kiss 2002; Blancaflor and Masson 2003; Hou et al 2003; Hou et al 2004), suggesting that actin may operate to down-regulate gravitropic signaling In this case mechanostimulation may actually be enhancing the interactions of actin with statoliths to inhibit gravitropic response

The graviresponse is also accompanied by an immediate cytoplasmic pH increase after reorientation that is required for maximal gravitropic bending (Scott and Allen 1999; Fasano et al 2001) This pH increase is extended by treatments which disrupt actin fila-ments (Hou et al 2004) It is possible that actin regulates transport processes at the plasma membrane, leading to the down-regulation of the pH signal, facilitating a reset-ting of the gravitropic signaling system (Hou et al 2004) The pH change itself should have far-reaching effects in the cell, as a change in pH will alter the activity of most pro-teins in the cytoplasm Thus, pH changes may represent a way to effect a large-scale change of cell activities upon gravistimulation, and touch may be acting through actin to modulate this system

al 2002; Muday and Murphy 2002; Friml 2003) Auxin plays an important role in tropic response (see Chapter 3) and actin may affect polar auxin transport by changes in cyto-plasmic pH Actin may also operate through altering the distribution, targeting, and turnover of auxin efflux and uptake transporters

How might touch stimulation affect all these features of the gravity signaling system, such as actin dynamics or pH fluxes? The touch-induced Ca2+increase described above has the potential to directly affect many cellular activities, including actin structure (Blancaflor 2002), proton pumping (Kinoshita et al 1995), and auxin transport/signal transduction (Benjamins et al 2003) Thus, the ionic signaling associated with both touch and gravity signaling may well form a nexus at which information from both systems is incorporated to control pH and auxin flux and so generate an integrated tropic response

5.7 Conclusion and Perspectives

It is clear that plants integrate a tremendous amount of environmental information to dic-tate the appropriate growth response Under laboratory settings, the gravitropic response can represent an extremely powerful and often dominating influence on growth habit However, the integrative nature of plant signaling means that many other factors will likely influence growth in the field Thus, thigmotropic (this chapter) and hydrotropic (Chapter 6) signals are known to modulate gravitropic response through reduction in gravitropic sensitivity For roots, where touch stimulation is likely almost constant, these other stimuli may dominate, with gravitropism perhaps providing a default directional cue to orient growth in the absence of other stimuli Indeed, a putative gravitropic sensor reported in the elongation zone of the maize root (Wolverton et al 2002; also see Chapter 1) could well reflect a mechanical strain sensor eliciting a thigmotropic response to the stress from the mass of the unsupported root in these experiments

Although much work has been directed to analysis of the interactions of gravitropism with these other stimuli in primary roots, it will be very informative to understand how signals such as touch interact with the mechanisms that define gravitational set-point angles of lateral organs where growth is not simply directed straight up or down (Chapter 2)

5.8 Acknowledgments

The authors gratefully acknowledge support by grants from NSF (MCB 02-12099, IBN 03-36738, and DBI 03-01460) and NASA (NAG2-1594) We also thank Greg Richter for critical reading of the manuscript

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Gladys I Cassab*

123

6.1 Introduction

Plants have evolved an elaborate and sophisticated set of growth responses to the envi-ronment that allow them to survive adverse conditions The degree to which plants de-pend on environmental cues to orchestrate growth and development is unmatched in the animal kingdom Of all of the environmental signals, gravity—which is a constant factor on Earth—profoundly impacts the form, structure, and function of plants (Hangarter 1997) However, gravitropic response of plant organs can be affected by the abundance of incoming signals from the environment, and therefore plants modify their growth ac-cordingly, taking into account all these variables Responses of plants to environmental stimuli such as moisture (hydrotropism), temperature (thermotropism), oxygen gradients (oxytropism), electric fields (electrotropism), touch (thigmotropism; see Chapter 5), and wounding (traumatropism) are short-term tropic responses that are commonly accom-plished within a few hours by differential growth These tropisms have been mostly ob-served and analyzed in roots, although there are a few studies on pollen tubes, coleop-tiles, and shoots Most of these tropisms were documented more than 100 years ago (Hart 1990); nevertheless, research in this field has so far received little attention despite the biological significance of these growth behaviors in plant survival

6.2 Hydrotropism

Even though the lack of sufficient water is the single most crucial factor influencing world agriculture, interest in hydrotropism has fluctuated over the years Studies on hy-drotropism have been scarce since Knight and von Sachs (in 1811 and 1872, respectively) showed that roots move toward water (Takahashi 1997) In particular, von Sachs (1877) demonstrated that in seedlings grown in a freely hanging sieve basket, the emergent roots became diverted from the vertical and grew along the bottom of the basket (wet substrate) (von Sachs 1887) (Figure 6.1)

Around that time, Darwin, Pfeffer, and Weisner (who introduced the term hydrotro-pism) were all convinced that moisture gradients affected root orientation (Hart 1990) Interestingly, the idea that plant roots penetrate the soil in search of water to maintain their growth was first presented as the explanation for the downward orientation of roots (Dodart, around 1700, reviewed in Hart 1990) However, in comparison to studies on the roles of other directional signals (such as gravity and light) on the general orientation of plant organs, studies on hydrotropism have been surprisingly sparse In fact, genetic analysis of hydrotropism lagged 19 years behind the first reports of Arabidopsis

ropic mutants (Eapen et al 2003; Olsen et al 1984) Here, we discuss the potential of a genetic approach for understanding the molecular mechanisms governing root hydrotro-pism and their interaction with gravitrohydrotro-pism

6.2.1 Early Studies of Hydrotoprism

Hydrotropism studies have always been hard to interpret because both thigmotropism and gravitropism interact with hydrotropism Mechanical stimuli can generally be avoided (see Chapter 5), but gravity is ubiquitous on Earth Consequently, several tools, such as those involving agravitropic mutants, clinorotation, or microgravity in space have been utilized to differentiate the hydrotropic from the gravitropic response (Takahashi 1997; see also Chapter 9) Significantly, experiments with the pea mutant ageotropum, whose roots were agravitropic but responded positively to hydrotropism, indicated that there are independent sensing and response pathways for these two tropisms (Jaffe et al 1985) Hence, ageotropum was a model system for the study of hydrotropism for many years In particular, roots of ageotropum responded to a gradient in water potential as small as 0.5 MPa (Takano et al 1995)

hydrotropically but their growth was not affected (Jaffe et al 1985; Takahashi and Scott 1993) Cytoplasmic Ca2+ has been postulated to be a transducer for the gravity signal (Plieth and Trewavas 2002) However, changes in this ion after amyloplast sedimentation have not been properly documented (Blancaflor and Masson 2003; see also Chapters and 5) Even so, hydrotropic response was completely blocked in ageotropum roots with a Ca2+chelator and lanthanum, a Ca2+channel blocker (Takano et al 1997) These ob-servations suggest that Ca2+may function in the hydrotropic response, independently of

amyloplast sedimentation It has been proposed that mechanotransductive Ca2+channels (Chapter 5) might be triggered by temperature, gravity, touch, and water stress (Pickard and Ding 1993), which might regulate tropic modification of growth (Pickard and Ding 1992)

Pollen tube guidance on the stigma has been also considered the most frequently oc-curring hydrotropic response in higher plants (Lush et al 1998) Guidance toward the stigma by a water gradient may be the first step in a multistage process of guidance to the ovules

6.2.2 Genetic Analysis of Hydrotropism

Up to now, various screening procedures have been implemented to isolate mutants af-fected in response to gravity, light, and obstacle touching However, hydrotropism has not been common in genetic studies because of the complexity of establishing a large-scale screening system that offers an appropriate stimulus–response interaction (Eapen et al 2005) For this reason, the design of a screening method for the isolation of Arabidopsis mutants with abnormal responses to a water potential gradient is noteworthy (Eapen et al 2003) The screening method consists of a vertically oriented square Petri dish with a nor-mal nutrient medium (NM) in the upper part, in which Arabidopsis seeds are plated, and a water stress medium (WSM) in the lower part A gradient in water potential develops over time, and wild-type Arabidopsis roots stopped their downward growth and devel-oped a hydrotropic curvature when the water potential was 0.53 MPa By developing this hydrotropic response, Arabidopsis roots avoided the substrate with lower water potential; that is, they never reached the area containing the WSM and consequently arrested their gravitropic growth (Fig 6.2A) Mutants were selected on two conditions: by their contin-uous root gravitropic response into the medium with lower water potential (lack of hy-drotropic response), and by their inability to sustain continuous growth into the severe water-deficit conditions of the WSM (Fig 6.2A) With this selection, hydrotropic mu-tants were distinguished from mumu-tants resistant to severe water deficit conditions The initial screening resulted in the isolation of two negative hydrotropic mutants, which were named no hydrotropic response (nhr) Importantly, in a different system with an air hu-midity gradient, nhr1 roots responded negatively to this stimulus, developing a curvature in response to gravity instead, confirming that their directional growth toward water is impaired (Eapen et al 2003)

water availability Therefore, we assumed it feasible to screen for super-hydrotropic re-sponse mutants in Arabidopsis For this approach, we used the model of the screening system for nhr mutants, but the WSM is placed in the upper part and the NM in the lower section of the dish Roots from one mutant line isolated in this system named super

hy-drotropic response (suh1) continuously grow under water deficit for 10 days in order to

reach the moderate water potential conditions present in the lower section of the dish (Saucedo and Cassab, unpublished results; Fig 6.2B and Color Section) Thus, both the screen for impaired and enhanced hydrotropic response appear to be fruitful avenues of research toward dissecting the complex signaling phenomena behind the hydrotropic response

6.2.3 Perception of Moisture Gradients and Gravity Stimuli by the Root Cap and the Curvature Response

ized as yet In addition, the mechanisms that the root cap utilizes to intermingle different stimuli to resolve in which direction root growth should occur are unknown

Nonetheless, the recent isolation of hydrotropic mutants, in combination with analy-sis of a number of gravitropic mutants, have provided some hints of the signal transduc-tion mechanisms of these tropisms For example, the gravitropic and waving response of the nhr1 mutant is increased, suggesting that the lack of a hydrotropic response results in an enhancement of these other root growth responses (Eapen et al 2003) Moreover, roots from the starchless mutant pgm1-1, which has a reduced gravitropic response (Kiss et al. 1989), showed an enhanced responsiveness to moisture gradients (Takahashi et al 2003) Gravistimulated nhr1 seedlings also attained a root curvature of 80 degrees hours be-fore wild-type plants (Eapen et al 2003) In contrast, pgm1-1 roots reached a 20-degree hydrotropic curvature hour before the wild-type (Takahashi et al 2003)

These observations suggest that once the sensing system of the root cap is affected for one stimulus, the integration and assessment mechanism for other signals is enhanced, and thus these other root growth responses can occur faster However, the differences in the rate of gravitropic bending of nhr1 versus hydrotropic bending of pgm1-1 roots seem also to reflect variations in the timing of perception between both stimuli It has been shown that the perception time for a gravity stimulus can be as short as second (Hejnowicz et al 1998) In contrast, the perception time for osmotic stimulation is up to minutes (Stinemetz et al 1996) The variability observed in the perception time of both tropisms might be the consequence of their distinctive mechanisms of perception It is widely accepted that perception of gravity occurs in specialized cells of the root cap (sta-tocytes or columella), which contain motile amyloplasts that can sediment in response to gravity and can therefore elicit gravisensing (Sack 1997; see also Chapter 1)

The capacity of the root cap to perceive and respond to moisture gradients apparently produces a dominant signal that abates the gravity response Recently, it has been found that this signal triggers the degradation of amyloplasts in columella cells of both

Arabi-dopsis and radish, and hence roots exhibit hydrotropism with fewer impediments from

gravitropism (Takahashi et al 2003) Transient touch stimulation of Arabidopsis root tips likewise restrains gravitropic growth but, in this case, by limiting amyloplast sedimenta-tion in columella cells (Massa and Gilroy 2003; see also Chapter 5) Therefore, columella cells can integrate the signaling triggered by moisture gradients, touch receptors, and possibly even other stimuli in order to generate the appropriate tropic response

Interestingly, and unlike wild-type plants, hydrostimulated nhr1 roots maintained their amyloplasts in the columella (Ponce and Cassab, unpublished observations) This might sug-gest that the negative hydrotropic response of nhr1 is related to the absence of the signaling cascade that triggers amyloplast degradation during hydrotropic stimulation In the screen-ing system for the isolation of super-hydrotropic response mutants, wild-type seedlscreen-ings ceased growing after six days in the WSM and lacked amyloplasts in their columella cells, supporting the observation made by Takahashi et al (2003) that water-stressed roots also de-grade amyloplasts In contrast, suh1 roots exhibited amyloplasts in the columella, indicating that the root cap seems to combine both a hydrotropic and a gravitropic response in order to reach the medium with higher water potential in the bottom part of the plate

Root hydrotropic responsiveness has also been studied by using an agar KCl system in some Arabidopsis agravitropic, auxin-insensitive, and ABA-related mutants (Takahashi et al. 2002) In this study, Arabidopsis wild-type roots began hydrotropic curvature against the gravity vector after 30 minutes of stimulation and reached 80 to 100 degrees within 24 hours In contrast, roots of axr1-3 and axr2-1 mutants showed a greater hydrotropic response com-pared with those of wild type Both mutants are insensitive to auxin and show altered root gravitropism, with axr2 roots being particularly agravitropic (Lincoln et al 1990; Nagpal et al 2000) axr2 roots developed a curvature even in the absence of a moisture gradient, which might suggest that this response is a consequence of their random root growth direction

Additionally, mutants affected in basipetal polar auxin transport from the root cap to the root elongation zone, such as wav6 and aux1 (Blancaflor and Masson 2003; Swarup et al 2005), showed hydrotropic curvature However, it was previously shown that an auxin transport inhibitor blocked the hydrotropic curvature of ageotropum roots (Takahashi 1994) Therefore, this analysis suggests that auxin may regulate gravitropism and hydrotropism differently, although both tropisms might depend on the formation of an asymmetric auxin gradient for differential growth Furthermore, the relatively random root growth direction of these mutants makes the interpretation of their hydrotropic re-sponse complex, and the results not provide strong evidence for or against a role of auxin and auxin transport in the hydrotropic response

6.2.4 ABA and the Hydrotropic Response

gravity or waviness response (Lu and Federoff 2000; Kang et al 2002) On the other hand, it has been postulated that ABA maintains a higher growth rate on the side with lower water potential in hydrotropically responsive roots This suggestion arose from the observation that the hydrotropic response of roots in an agar KCl system of two

Arabidopsis ABA mutants, aba1-1 and abi2-1, was slightly reduced compared with those

of wild type (Takahashi et al 2002) In contrast, both aba1-1 and abi2-1 mutant roots re-sponded positively to hydrotropism in the screening system with a water potential gradi-ent (Eapen et al 2003) However, there are some differences between these two hy-drotropic systems, namely, light conditions, seedling age, and the substrate of the water potential gradient, which may account for this discrepancy

Light has been shown to influence the gravity response in roots, shoots, and other or-gans (Hangarter 1997) So far, there are no studies that have examined an interaction of ABA with the development of an asymmetric auxin gradient for differential growth, which may be an important factor during hydrofacilitation Yet, it was recently reported that cells in the columella and quiescent center of Arabidopsis showed low levels of ABA, which were not increased by water stress, suggesting a non-stress-related role for ABA in these cell types (Christmann et al 2005) Further, there are some reports of ABA-activated gene expression in the root cap (Hong et al 1988; Nylander et al 2001), and so the root cap might be required for proper regulation of the ABA-dependent processes of cell division (Dewitte and Murray 2003) Therefore, an important role of ABA in devel-opmental programs such as tropisms is anticipated A detailed analysis of more ABA mu-tants and the cloning of the NHR1 gene might thus provide evidence as to whether ABA functions on gravitropic and/or hydrotropic response in roots

6.2.5 Future Experiments

Our understanding of hydrotropism has lagged many years behind that of gravitropism and phototropism However, the future characterization of the genes in the hydrotropic mutants isolated so far might help to unravel the players in this particular growth re-sponse Many questions remain open, particularly those related with the sensing system for moisture gradient and the mechanism that merges and assesses the diverse stimuli im-pacting on the root in order to generate the proper tropic response

Thus far, the NHR1 gene seems to block root gravitropic growth and allow roots to di-rect their growth toward water, since in nhr1 mutant roots gravity response is enhanced (Fig 6.3) Further, it remains to be determined whether signals such as Ca2+, calmodulin, pH increases, reactive oxygen species, inositol 1,4,5-trisphosphate, auxin, ethylene, flavonoids, cytokinins, and brassinosteroids participate in hydrotropism as they seem to in gravitropism (reviewed in Chapter 2) Thus, there is considerable potential for further research to uncover the mystery of how roots are able to amplify a signal, such as water

6.3 Electrotropism

(McGillavray and Gow 1986) and algae (Brower and Giddings 1980), as well as in pollen tubes (Marsh and Beams 1945), roots (Fondren and Moore 1987; Schrank 1959), and shoots (Schrank 1959; Lee et al 1983) of higher plants Upon gravistimulation, the cur-rent flow along the upper flank of the distal elongation zone (DEZ) reversed to efflux from the root (Iwabuchi et al 1989; Collings et al 1992), and changes in intracellular po-tentials in this zone occurred within a minute (Ishikawa and Evans 1990a) These changes arose before the development of the gravitropic curvature Gravistimulation also modi-fied the pattern of electric current surrounding the root tip (Behrens et al 1982; Björkman and Leopold 1987; Iwabuchi et al 1989), and within the root cap triggered rapid depolarization of statocytes (Behrens et al 1985), suggesting that electrical/ionic signals may be an important component of the gravity sensing/response system

Electrotropism was enhanced by treatments that interfere with gravitropism, like de-capping the roots or pretreating them with Ca2+chelator Likewise, roots of ageotropum were more responsive to electrotropic stimulation than were roots of normal peas (Ishikawa and Evans 1990b), suggesting that the early steps of gravitropism and elec-trotropism occur by independent mechanisms Nonetheless, the motor mechanisms of the two responses may have features in common since auxin and auxin transport inhibitors reduced both gravitropism and electrotropism (Moore et al 1987)

The kinetics of electrotropic curvature in Vigna mungo L roots revealed that curvature Figure 6.3. Root hydrotropism opposes gravitropism in Arabidopsis A Perception of gravity (a weak sig-nal) appears to occur in columella cells of the root cap, triggered by amyloplasts that can sediment because of gravitational force After the sensory system identifies this stimulus, it connects with the hormone system (perhaps by transient Ca2+fluxes and/or alkalinization of columella cytoplasm) and an asymmetrical signal

occurred in the same root toward both the anode and cathode, but the two responses took place in two different regions of the root—the central elongation zone (CEZ) and the DEZ, respectively (Wolverton et al 2000) Furthermore, both responses are related to the electric field rather than one being a secondary response to induced gravitropic stimula-tion, since these oppositely directed responses could be reproduced individually by a lo-calized electric field application to the region of response The electrotropic responses of plant organs described thus far are quite clear, but very little is known about the actual mechanisms of stimulus reception and directional guidance

6.4 Chemotropism

In green plants, the ultimate energy source for growth is light rather than a chemical input The directional cues from light, supplemented by the orientation afforded by gravi-tropic growth, help place the aerial part of the plant in the optimal position to intercept sunlight Gravity and light are thus two of the main directional signals engaged in the reg-ulation of vegetative growth However, chemicals still act as significant vehicles of com-munication between individuals in their reproductive behavior or, in the case of insectiv-orous plants, as signals for obtaining nutrients

Chemotropism is a directional growth response that is driven by a chemical stimulus; the term chemotaxis is used to depict the locomotory responses of motile organisms or gametes Both forms of response can be positive (toward a beneficial or attractive sub-stance) or negative (away from a harmful or unattractive subsub-stance) (Hart 1990) Responses to chemical substances have been well documented in lower plants, particu-larly in unicellular organisms and gametes In multicellular organisms there is usually a greater homeostasis of the cellular environment, and chemoresponses to external chemi-cals seem limited to rather specialized situations A very wide range of chemichemi-cals is im-plicated in these types of responses, indicative of the variety of adaptive advantages that can be provided in these situations

In higher plants, considerable chemical interaction does indeed occur Many bacteria and fungi release hormones and hormone-like substances into the soil, and these can have significant effects on root growth (Bilderback 1985) In some older physiological texts, it was suggested that roots can develop chemotropic responses to soil nutrients However, these suggestions were based upon studies in which chemicals were unilaterally applied to individual roots of several species (Newcombe and Rhodes 1904), and these methods not constitute a robust chemotropic directional assay (i.e., directional growth in re-sponse to the repositioning of the stimulus) However, there is a recent report in which the root cap seems to sense extracellular glutamate to trigger a reduction in the rate of cell production and/or cell expansion (Filleur et al 2005), suggesting a specific response which is likely to involve the action of a specific receptor, but these authors did not re-port on a directional response to an actual gradient

tighten-ing of the trap (Ziegler 1962) However, nothtighten-ing is known about any of the chemorecep-tors involved

Since the early work by Molisch and DuBary in the 1890s (Hart 1990), the directional growth of the pollen tube down the style and into the ovary has usually been regarded as the classic example of chemotropic growth in higher plants Two molecules that have neurotransmitter properties in animals were recently found to be involved in pollen tube growth, GABA (␥-amino butyric acid) and nitric oxide (NO) Through genetic analysis in Arabidopsis, a gradient of GABA was shown to be involved in the final stages of pollen tube guidance to the ovule (Palanivelu et al 2003) GABA is a four-carbon ␻-amino acid, best known for its role in animal neuronal synapses, but was actually first discovered in plants (Steward et al 1949) In the pistil, GABA concentrations increase along the pollen tube path, reaching maximal concentrations in the inner integument cells directly surrounding the micropyle, the target of the pollen tube for delivery of sperm cells (Palanivelu et al 2003) This gradient is interrupted in pollen-pistil

interac-tion (pop2) mutants, resulting in aberrant pollen tube growth and guidance POP2

en-codes a transaminase, which converts GABA to succinate pop2 pistils have more than 100-fold greater GABA levels than wild-type tissues, thus reducing the magnitude of the gradient This elevation apparently results in improper targeting (Palanivelu et al 2003) (Fig 6.4A and Color Section) Hence, intracellular degradation of GABA in wild-type pollen tubes by POP2 presumably increases the GABA gradient, allowing tubes to distinguish the micropyle from the rest of the ovule The fact that a GABA gradient ex-ists along the path of pollen tube supports a chemotropic growth of pollen tubes In an-imals, GABA binds to G-protein-coupled GABA receptors; homologs of these proteins have been identified in plants The heterotrimeric G-protein alpha subunit, GPA1, is ex-pressed in the pollen tube, although it is not known whether it participates in pollen tube guidance (Ma 2003)

A role of nitric oxide (NO) was recently demonstrated in the regulation of pollen tube growth in Lilium longiflorum, especially in the reorientation response (Prado et al 2004) (Fig 6.4B and Color Section) A NO gradient may play a role in finding a suitable path for the pollen tube, suggesting that this may be another directional response Nonetheless, so far nothing is known about the nature of the NO chemoreceptor

A female gamethophyte protein from maize has also been shown to be required for pollen tube attraction (Márton et al 2005), indicating that a wide range of different sub-stances can elicit a chemoresponse in pollen tubes Furthermore, directional guidance of pollen tubes into the style does not appear to be influenced by gravity, indicating that their chemotropic response might only interact with their hydrotropic, electrotropic, and oxytropic ones (see following section)

6.5 Thermotropism and Oxytropism

species using seedlings planted in moist sawdust in a metal box heated on one side by a gas burner and cooled on the other side by water

Others followed Wortmann’s work, but a consensus on the existence of thermotropism was not reached until recently Fortin and Poff (1990) showed that primary roots of maize reoriented from their original vertical direction when exposed to a 4.2oC cm–1 thermal

gradient applied perpendicular to the gravity vector, indicating that a thermal gradient can be sensed by roots This positive root thermotropic curvature might represent the inte-grated sum of thermotropism and gravitropism (Fortin and Poff 1991) However, when roots were placed horizontally under g with a vertical thermal gradient, the thermal stim-Figure 6.4 (also see Color Section). Chemotropism in pollen tubes A concentration gradient of GABA af-fects the final stages of directional guidance of pollen tubes to the ovule A In the wild-type ovule, the inner integument possess a much higher level of GABA than other tissues of the ovule or the septum, and the pollen tube grows toward the micropyle In the pop2 mutant ovule, GABA concentrations in the entire ovule, including the funiculus and outer integument, are very high, and that in the inner integument is even higher The pollen tube of pop2 mutants fails to direct its growth toward the micropyle, and grows randomly in the ovary (after Ma 2003) B Lily pollen tube showing three consecutive reorientation responses, which were in-duced by moving the NO source to the locations marked with arrows The growth axis of the pollen tube al-ways developed right angles after each challenge by the NO source facing the pollen tube-tip

(From Feijó JA, Costa SS, Prado AM, Becker JD, Certal AC 2004 Signaling by tips Current Opinion in

ulus at 15 oC was stronger than the gravity stimulus and the root curved toward the top of the dish (Fortin and Poff 1991), providing additional evidence that thermal gradient sens-ing is accountable for the thermotropic response The thermoreceptors in plants remain to be discovered but, in animals, temperature-sensitive channels can relay thermal informa-tion Thermotropism could be of adaptive value for root optimal development since hori-zontal temperature gradients are common in agroecosystems (Fortin and Poff 1990)

Molisch (1884) was the first to demonstrate an aerotropic response of plant roots, with Pfeffer (1906) reviewing research on this topic and proposing the term oxytropism As with many of the tropisms discussed in this chapter, research in oxytropism was ignored for 60 years until several reports of altered root system growth resulting from limiting O2

availability appeared (listed in Porterfield and Musgrave, 1998) Recently, oxytropism has been reexamined in a microrhizotron capable of producing and maintaining an O2

gradient of 0.8 mmol mol–1 mm–1 using two cultivars of pea: Weibul’s Apolo and

ageotropum (Porterfield and Musgrave 1998) Oxytropic curvature was seen all along the

O2gradient in both cultivars of pea, with growth toward the higher O2concentrations re-gardless of the starting position within the O2gradient Roots of ageotropum showed a

curvature of 90 degrees into the O2gradient, in contrast to the gravity-sensing cultivar, which only curved 45 degrees The curvature of the Weibul’s Apolo cultivar indicates that roots normally integrate both the gravity and oxygen signal, resulting in the diageotropic and plagiotropic growth seen in response to soil flooding (Huck 1970)

Oxytropism may allow roots to evade O2-deprived soil strata and may also be the basis of an auto-avoidance mechanism, diminishing the competition between roots for water and nutrients as well as oxygen (Porterfield and Musgrave 1998) In addition, it has been shown that pollen tube guidance is influenced not only by chemical or water gradients, but also by oxygen gradients Pollen tubes of several species showed a clear tropic re-sponse to oxygen gradients in an in vitro system (Blasiak et al 2001) The biological sig-nificance of this phenomenon in vivo has not been analyzed yet, but may be critical for orienting the pollen tubes toward the stigma, or in maintaining basipetal growth in the style

6.6 Traumatropism

Wounding is a mechanical process that harms cells in a localized region, but which also typically results in alterations in the activities of cells in other regions (Imaseki 1985) A less well-known effect of wounding is the induction of differential growth in the wounded organ within the first hour or so after damage Pfeffer introduced the term traumatropism in 1893 to describe such wound-induced, directional growth responses Along with his experiments on thigmotropism, Darwin (1881) also analyzed the responses of roots to wounding The root response to injury was found to be similar to touch, that is, if the in-jury was close to the root tip, the root curved away from the side that was wounded, oc-casionally even to the extent of forming a 90-degree curvature; but if the wound was just beyond the tip, the root bent toward the wounded side

positive curvatures of 30 to 40 degrees (Bünning 1959) The negative and positive trau-matropic response of both roots and shoots indicates the participation of message trans-mission from the wound site to the cells showing differential growth Such a wound mes-sage may be transmitted chemically (through the action of hormones or proteins), electrically (through the generation and propagation of action potential), and/or mechan-ically (through activated mechanosensory Ca2+ channels) It remains to be determined whether only one or the combined action of these three wounding messages regulates traumatropism The significance of traumatropism to the plant may be to offer initial pro-tection to injury, reinforced later on by the systemic long-distance signaling induced in the wound response (Schilmiller and Howe 2005)

6.7 Overview

Sensory systems in plants may consist more of a network of interconnected response chains rather than a series of separate stimulus reception-transduction pathways Dissection of this complex network will require the development of innovative method-ologies in conjunction with present technmethod-ologies to resolve the mechanism of perception and assessment that controls organ-bending responses to these diverse stimuli

6.8 Acknowledgments

This work is dedicated to Professor Barbara G Pickard, whose comprehensive know-ledge of the field has been the source of many invaluable suggestions from which my studies on hydrotropism have profited greatly We warmly thank Yoloxóchitl Sánchez for drawing the figures and Manuel Saucedo for his enormous contribution in the isolation of the super-hydrotropic mutants We are grateful to all past and present members of the laboratory for their contributions and discussions on hydrotropism We also gratefully ac-knowledge financial support by the Mexican Council for Science and Technology (CONACYT grant No 462022Q), the Universidad Nacional Autónoma de México (Dirección General de Asuntos del Personal Académico Grant No IN224103), and the University of California Institute for Mexico and the United States (UC Mexus)

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Markus Braun* and Ruth Hemmersbach

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7.1 Introduction

Over the last few decades, single-cell systems have increasingly attracted attention as model organisms for research on gravity-related biological processes Gravity is the only constant environmental stimulus and provides the most reliable cue that free-swimming and sessile organisms learned to use for orientation This capacity is found in organisms ranging from single cells up to multicellular animals and plants In all these systems grav-itropism and gravitaxis involve the pure physical step of susception followed by physio-logical steps comprising gravity perception, signal transduction, and signal transmission, eventually resulting in a gravity vector-related response in the form of a reorientation of growth or movement direction

In this review, free-swimming protozoa like flagellates (e.g., Euglena) and ciliates (e.g., Paramecium and Loxodes) are referred to as single-cell systems, as are tip-growing cell types such as protonemata of mosses and ferns and the rhizoids and protonemata of algae Although such cells are part of a multicellular organism, they extend away from it They have to cope directly with the environment and they have to adapt to it in a most beneficial way in order to survive Communication of single-cell systems with other cells of the organ is limited and they are not integrated into complex signal transduction net-works and response pathways required for multicellular response Thus, for single-celled systems, the stimulus response is dependent only on the cell’s own orientation All single-cell systems share a number of advantageous features for study The unobstructed access to the cell body permits a great variety of experimental approaches and allows easy iso-lation for biochemical and molecular analyses The signaling pathways are relatively short and all phases occur in a single cell

This chapter accentuates the substantial contribution single-cell systems have added to our understanding of the intracellular mechanisms underlying gravity sensing and the molecular basis of the gravity-dependent signaling pathways

7.2 Definitions of Responses to Environmental Stimuli that Optimize the Ecological Fitness of Single-Cell Organisms

Microorganisms (bacteria and protists), animals, and plants respond to environmental stim-uli in a multitude of ways The capacity of free-living organisms to orient the direction of their movement with respect to the source of an external stimulus is called taxis: positive taxis if the direction of movement is toward the source of the stimulus, negative taxis if it is away from it, and diataxis or transverse taxis if it is at an angle to the stimulus direction

Correspondingly, sessile organisms show growth responses called tropisms defining a steady-state bending of an organ with respect to the direction of the stimulus source The kind of environmental stimulus to which the organisms respond is indicated as a prefix to the appropriate term: for example, phototaxis or phototropism (directional response to light; see also Chapter 4); chemotaxis and chemotropism (directional response to a chem-ical, e.g., nutrients; Chapter 6); thermotaxis and thermotropism (response to a thermal gradient; Chapter 6)

In this chapter we concentrate on directional responses with respect to the gravita-tional field, which are called gravitaxis and gravitropism In addition, a special gravity-dependent kinetic response has been described for some protists (ciliates) These systems have the capacity to regulate their swimming velocity depending on their swimming di-rection They manage to speed up during upward swimming and to decelerate during downward swimming Consequently, they compensate for at least part of their passive sinking (sedimentation) rate (Machemer et al 1991; Ooya et al 1992; Hemmersbach-Krause et al 1993a) Gravikinesis is calculated by comparing the upward and downward swimming velocities of a cell (population) with its sedimentation velocity (Machemer and Bräucker 1992):

7.3 Occurrence and Significance of Gravitaxis in Single-Cell Systems

Gravitactic behavior has been reported for several protozoan species These unicellular organisms are heavier than water and most of the species studied so far show negative gravitaxis, which guides them to the surface Well-studied examples are the negative gravitaxis of the heterotrophic ciliates Paramecium and Tetrahymena (for review, see Bean 1984; Häder et al 2005; Hemmersbach and Häder 1999; Hemmersbach et al 1999), the oxygen-dependent gravitaxis of the microaerophilic ciliate Loxodes (Fenchel and Finlay 1986), and the light-dependent gravitaxis of the autotrophic green algae

Euglena and Chlamydomonas (Bean 1984; Häder 1987) These examples clearly show

that the direction of gravitaxis increases the ecological fitness of the organisms

Besides ciliates and flagellates (Figure 7.1), gravity effects have also been studied in other unicellular organisms: amoeba, acellular slime molds, swimming reproductive stages such as zoospores and sperm cells, and bacteria These systems have been exposed to different acceleration levels in order to analyze the impact of gravitational forces on different physiological processes (behaviour, proliferation, etc.) Due to the fact that in these cases a clear hypothesis as to the mechanism of graviperception is still missing, we will only briefly mention these in this chapter (for more details, see Häder et al 2005) before describing the more thoroughly understood single-cell systems such as Euglena,

Loxodes, and Chara

The migrating plasmodium of the single-celled slime mold Physarum polycephalum (Myxomycetes, acellular slime mold) shows gravitaxis and has been used as model sys-tem to study the impact of gravity on actomyosin-driven movements (Block et al 1986)

Gravikinesis=upward swimming rate − downwaard swimming rate

Though distinct behavioral responses to altered gravitational stimulation have been re-ported, the gravireceptor candidates in Physarum remain speculative: nuclei and mito-chondria have been proposed, both occurring in high numbers in this giant single cell

The accumulation of zoospores near the surface of the medium raises the still-unsolved question of whether this behaviour is guided by gravity or by oxygen (Cameron and Carlile 1977) The gravity-induced, altered physiology of sperm cells is of high in-terest due to possible impacts on reproduction and thus, for example, food supply during long-term space flights (Engelmann et al 1992; Tash and Bracho 1999) In the case of bacteria, effects of microgravity on, e.g., growth rate, sporulation and phage productivity have been reported (for reviews, see Mennigmann and Lange 1986; Cogoli and Gmünder 1991); however, reasons and mechanisms remain speculative According to the model de-rived by Klaus and coworkers (1997), it seems likely that a “cumulative effect of gravity may have a significant impact on suspended cells via their fluid environment, where an immediate, direct influence of gravity may otherwise be deemed negligible.”

7.4 Significance of Gravitropism in Single-Cell Systems

Among the few single-cell systems that respond gravitropically are the rhizoids of characean green algae, which share similar functions with roots of higher plants They anchor the organism in the substrate by penetrating mud and soil, thereby enabling stabi-lized upward growth of the shoots Other gravitropically responding cell types, including moss caulonemata and protonemata and characean protonemata, seek to grow upward in darkness in order to find optimal ecological conditions where the plants harvest light, re-Figure 7.1. Unicellular systems used to study gravity sensing A Paramecium biaurelia (170 µm), seen in phase contrast B Loxodes striatus (150 µm), bright field; arrows indicate Müller organelles (MO) C.

generate a multicellular body, and proliferate These cells elongate by polarized growth or tip growth enabling them to extend rapidly and so penetrate easily all kinds of sub-strates When accidentally covered with sediment or sand, protonemata of Chara arise from cells at the nodal complexes of the green thallus by asymmetric cell division These cells use negative gravitropic tip growth to grow back to the light Light induces termi-nation of tip growth and a depolarization of the cells The cells undergo a complex pat-tern of divisions that reconstitute the green thallus This process ensures survival of the plant in a difficult environment characterized by unpredictably changing conditions

7.5 What Makes a Cell a Biological Gravity Sensor?

Gravity is a weak but ubiquitous force that acts on masses Therefore, work has to be done in the form of moving a mass in the gravitational field in order to create sufficient energy to activate a biological sensor Theoretically, many cells possess organelles with suffi-cient masses whose gravitationally induced movements could create suffisuffi-cient energy However, most cell types of the various tissues usually actively prevent organelles from sedimenting by keeping them in place via cytoskeletal anchorage This genetically de-fined high degree of cytoplasmic organization is quite stable, even against moderately in-creased acceleration forces Thus, organelles not move in the gravitational field or their movements are obviously not transduced into a physiological response Therefore, in addition to the presence of organelles with sufficient mass and size, a gravity-sensing apparatus must exist in a gravisensitive cell type that facilitates sedimentation of specific masses and mediates the activation of gravity-specific receptors (i.e., the transduction of the physical stimulus into a physiological gravity perception signal)

As discussed in Chapters and 2, little is known about the mechanism of gravity sens-ing in higher plant statocytes, and identification of components of a gravity-sensor appa-ratus is limited to starch-filled amyloplasts These amyloplasts act as statoliths whose gravity-directed sedimentation precedes graviperception and gravitropic curvature Several studies suggest the involvement of actin microfilaments that might act as trans-ducers of tensional forces generated by the gravity-induced sedimentation of statoliths (Sievers et al 1991a; Yoder et al 2001; Blancaflor 2002; Perbal and Driss-Ecole 2003) to mechano-sensitive receptors in cortical ER membranes or in the plasma membrane of higher plant statocytes (Ding and Pickard 1993; Kiss 2000) However, experimental evi-dence for the role of the actin cytoskeleton in susception and perception of gravity re-mains controversial For example, treating statocytes with actdisrupting drugs in-creased the sedimentation rate of statoliths (Sievers et al 1989) and actually enhanced the gravitropic response (Hou et al 2003, 2004; Yamamoto and Kiss 2002)

7.6 Gravity Susception—The Initial Physical Step of Gravity Sensing

A gravisensitive cell must provide the molecular and cellular conditions that facilitate the activation of a receptor by gravity The pure physical action that is induced by a change of the direction or amount of acceleration resulting in the activation of a gravireceptor has been termed susception Sedimentation of masses is a prerequisite for any interaction of gravity with cellular components Two hypotheses have been put forward to explain how gravity is sensed in a cell (Salisbury 1993; Sack 1997; Kiss 2000) Candidates for sedi-mentable masses are either intracellular statoliths or the entire protoplast In higher plants, the statolith-based hypothesis (the starch-statolith theory) is favored since the dis-covery of starch-filled amyloplasts (statoliths) in specialized columella and endodermal cells of higher plants (reviewed in Sack 1997; see also Chapter 1) However, the absence of obvious statoliths in several graviresponsive cells, including the internodal cells of

Chara, and the observation that starchless mutants of Arabidopsis can sense gravity,

were decisive for the formulation of the alternative, protoplast-based hypothesis (the protoplast-pressure theory, also discussed in Chapter 1; Pickard and Thimann 1966; Wayne et al 1990; Wayne and Staves 1996)

In the protoplast-pressure theory, the hydrostatic pressure of the entire protoplast is suggested to trigger conformational changes of gravireceptor molecules at the plasma membrane The cell then perceives the direction of gravity by sensing the differential ten-sion and compresten-sion between the plasma membrane and the extracellular matrix at the top and at the bottom of the cell, respectively In the following, we will provide evidence that both models of gravity sensing are realized in single-cell systems

7.7 Susception in the Statolith-based Systems of Chara

Characean rhizoids and protonemata are among the best-studied gravisensory cell types in which the cytoskeleton-based susception apparatus is well-understood In downward-growing Chara rhizoids, BaSO4-crystal-filled vesicles rather than starch-filled

amylo-plasts serve as statoliths The high density of BaSO4and the vesicle size of to µm make these statoliths ideal for indicating the direction of gravity to the cell Removal of statoliths from the tip abolishes gravitropic responsiveness (Sievers et al 1991b), which clearly indicates that gravity signaling is triggered by these intracellular sedimentable particles Thus, the sensory system for gravitropism is not obscured by alternative and re-dundant mechanisms as appears to be the case for higher plants

Statoliths in Chara rhizoids not passively fall into the tip In fact, they are actively kept in an area 10 to 35 µm above this region (Figure 7.2) (Hejnowicz and Sievers 1981; Braun 2002) Myosin-like proteins, which were found attached to the surface of statoliths (Braun 1996a), interact with predominately axially arranged actin microfilaments to pre-vent statoliths from settling into the tip by exerting net-basipetal forces (Braun and Wasteneys 1998) In tip-upward-growing protonemata of Chara, actomyosin forces pre-vent statoliths from sedimenting toward the cell base by acting net-acropetally (Hodick et al 1998; Braun et al 2002)

highly dynamic actin cytoskeleton that is regulated by the concerted action of numerous associated proteins (Braun et al 2004) Inhibitor studies have shown that disrupting the actin cytoskeleton in rhizoids and in protonemata terminated the actomyosin-driven po-larized growth and caused statoliths to drop into the tip or toward the nucleus, respec-tively (Hejnowicz and Sievers 1981; Bartnik and Sievers 1988; Sievers et al 1996) After removing the drug, statoliths were quickly repositioned and gravity-oriented tip growth was restored as soon as the actin cytoskeleton was rearranged and fully functional (Braun 2001)

Experiments conducted under microgravity conditions provided by parabolic flights of sounding rockets (TEXUS, MAXUS) and during Space Shuttle missions (IML-2, S/MM05), as well as experiments in simulated weightlessness provided by two-dimensional (fast-rotating) and three-two-dimensional clinostats, have unravelled the specific contributions of gravity and actomyosin to the interplay of forces that underlie the statoliths-based gravity-sensing (susception) apparatus of Chara rhizoids and protone-mata (Buchen et al 1993, 1997; Cai et al 1997; Hoson et al 1997; Braun et al 2002) When the influence of gravity was abolished during the microgravity phases of sounding rocket flights (Volkmann et al 1991; Buchen et al 1993) and randomized during rotation on clinostats (Hoson et al 1997; Braun et al 2002), actomyosin forces generated a dis-placement of statoliths against the former direction of gravity Thus, in vertically-oriented rhizoids and protonemata at ⫻ g, the statoliths are kept in a dynamically stable equilib-rium position by actomyosin forces which exactly compensate the effect of gravity on the statoliths (Figure 7.3)

which were displaced to different cell regions by optical laser tweezers or by centrifuga-tion revealed the surprising complexity of forces by which actomyosin controls statolith positioning (Braun et al 2002) Individual acropetal and basipetal movements of sta-toliths can be observed in both cell types, indicating that stasta-toliths interact with the mainly axially oriented actin microfilaments with opposite polarities When statoliths were centrifuged into the subapical region, statolith transport back to the original posi-tion was observed which is not notably influenced by gravity (Sievers et al 1991b; Braun and Sievers 1993) Active transport is generated along actin microfilaments and statoliths not sediment onto the lower cell flank until they have reached the statolith region near the tip, where sedimentation is not constrained by microtubules (Braun and Sievers 1994) Microtubules are excluded from the apical region of rhizoids and protonemata

The actomyosin component of movement is always the strongest pointing toward the statolith region This ensures that statoliths are always kept in, or are retransported to, their original position In the statolith region itself, however, gravity plays the decisive Figure 7.3. Gravitropic phases in characean rhizoids and protonemata In rhizoids, the position of the sta-toliths (St) is balanced by net-basipetally acting actomyosin forces (Factin) and gravity (Fgravity) Upon

grav-istimulation, actomyosin forces guide the sedimenting statoliths toward the gravireceptors located in a belt-like area of the plasma membrane 10 to 35 µm above the tip The Spitzenkörper (SpK) remains arrested at the tip and the calcium gradient (indicated by darker and lighter grey dotted areas) is always highest at the tip When statoliths contact the gravireceptors (GR), graviperception takes place and is followed by a local reduction of cytosolic Ca2+that results in differential extension of the opposite cell flanks (double-headed

role as an additional passive transport component that contributes to the resting position-ing of statoliths as long as the rhizoid tip points downward Any changes in the orienta-tion of the cells with respect to the direcorienta-tion or the amount of the acceleraorienta-tion results in a disturbance of this balance and a displacement, namely, the lateral sedimentation of sta-toliths (Figure 7.3)

In lateral directions, statolith position is only weakly controlled by the actomyosin sys-tem in both cell types (Leitz et al 1995; Buchen et al 1997) Recently, the forces required to move statoliths in the lateral direction were determined during the 13-minute micro-gravity phases of two MAXUS rocket flights In rhizoids growing vertically downward, lateral acceleration forces in a range of 0.1 ⫻ g were sufficient to displace statoliths to-ward the membrane-bound gravireceptors Based on the known size of statoliths and the density differences between statoliths and the surrounding cytoplasm, it was calculated that molecular forces in a range of ⫻ 10–14N must be exerted on a single statolith in

the lateral direction to overcome the cytoskeletal bonds and induce sedimentation, thus eliciting graviperception (Limbach et al 2005)

The need for the complex actomyosin-based control of statoliths position for gravity perception became fully comprehensible only after it was discovered that only the sub-apical plasma membrane area of the statolith region, 10 to 35µm from the cell tip, is able to trigger the gravitropic response upon statolith sedimentation (Braun 2002) Forcing statoliths to sediment outside these areas by using optical laser tweezers or centrifugation did not result in a gravitropic response (Braun 2002) After reorienting rhizoids by 90 de-grees, the statoliths sediment mainly along the gravity vector and settle onto the lower cell flank of the statolith region where graviperception takes place and the graviresponse is initiated However, when cells were stimulated at angles different from 90 degrees, sta-toliths did not simply follow the gravity vector (Figure 7.3) (Hodick et al 1998) Instead, even in almost fully inverted cells, statoliths were actively redirected against gravity and were guided to the small gravisensitive area of plasma membrane in this “statolith re-gion” of the tip

Gravistimulation of tip-upward-growing protonemata causes an actin-mediated acropetal displacement of statoliths into the apical dome, where they sediment very close to the tip (Figure 7.3) (Hodick et al 1998) In contrast to rhizoids, the gravisensitive plasma membrane area in protonemata is limited to an area to 10 µm basal to the tip (Braun 2002) During the upward bending of protonemata, the statoliths periodically sed-iment along the gravity vector and leave the gravisensitive site, which deactivates the gravireceptor The periods when the statoliths exit the gravisensitive region are reflected by phases of straight growth Actomyosin-mediated transport of statoliths back to the gravisensitive membrane area reinitiates the gravitropic response from time to time until the vertical orientation is resumed (Figure 7.3) On one hand, these actin-mediated sus-ception mechanisms guarantee a highly efficient readjustment of the growth direction and, on the other hand, avoid inexpedient responses to transient stimuli

sur-rounding medium triggers graviperception), graviorientation should be abolished in neutral-buoyancy experiments, whereas an intracellular gravisensor, such as a starch statolith-based system, should still be functional under these conditions Recent experi-ments have shown that gravitropism in this moss takes place in media that are denser than the cytoplasm of the apical cell (Sack et al 2001) This result provides evidence that grav-ity sensing in Ceratodon strictly relies on sedimentation of intracellular masses rather than on the mass of the entire protoplast Like in Chara rhizoids and protonemata, the statolith-based sensory system is not complemented by alternative mechanism

7.8 Susception in the Statolith-based System of Loxodes

The ciliate Loxodes (Figure 7.1) maintained its positive gravitaxis when the density of the external medium was in the same range as or even higher than the density of its cyto-plasm (1.03 g/cm3) As gravikinesis of Loxodes was slightly reduced under isodensity conditions, Neugebauer et al (1998) proposed that in Loxodes an intracellular gravisens-ing mechanism is complemented by a protoplast pressure-based mechanism (Figure 7.4) By analogy to the situation in Chara, Loxodes uses BaSO4 as the statolith material

Loxodes possesses to 25 so-called Müller organelles (Müller vesicles), which are 7- to

10-µm-wide vacuoles containing a body of BaSO4 (3–3.5 µm in diameter) fixed to a

mod-Figure 7.4. Models of graviperception in different protists Ca2+- and K+-mechanoreceptor channels are

lo-cated in specific areas of the plasma membrane (ant = anterior cell pole) Receptor channels are activated by the mechanical gravity-induced load of the protoplast (direction of gravity indicated by arrows) In Loxodes, specialized gravireceptor organelles (the Müller organelles) complement gravity sensing by using a statolith-based perception mechanism Activation of the receptor channels affects the membrane potential and regu-lates the activity of motion organelles: cilia in Paramecium and Loxodes (not shown) and a single flagellum in Euglena

ified ciliary stick It has been suggested that gravity is perceived by bending the ciliary complex, which induces changes in membrane potential, regulating the activity of the body cilia (Fenchel and Finlay 1984, 1986)

7.9 Susception in the Protoplast-based Systems of Euglena and Paramecium

Starting in the nineteenth century, different hypotheses have been proposed to explain the mechanism of gravitaxis in unicellular organisms either involving a pure physical mech-anism, a physiological process, or a combination of both (for reviews, see Bean 1984; Machemer and Bräucker 1992; Häder et al 2005) Although the physical mechanisms as-sume a passive alignment of the cell in the water column caused by, for example, the cell being tail-heavy, physiological mechanisms predict the existence of an active gravirecep-tor To summarize the results from decades of experimentation—some of which will be mentioned below—a physiological gravity signal transduction pathway exists in unicel-lular systems and, thus, the existence of a gravity-sensing mechanism can be predicted in free-swimming organisms For example, after immobilizing Paramecium cells (Figure 7.1), Kuznicki (1968) observed not only sedimentation of the cells but also their variable orientation, in contradiction to a purely physically determined mechanism (buoyancy principle) of gravitaxis (Fukui and Asai 1985)

In contrast to the results obtained from Ceratodon and Loxodes, increasing the density of the medium impaired graviorientation of Euglena (Figure 7.1) (density 1.046 g/ml_1) and Paramecium (density 1.054 g/ml_1) The capacity for orientation was completely

dis-turbed under isodensity conditions In addition to indications for a physiologically guided mechanism of graviperception, these results support the hypothesis of protoplast-based graviperception in Paramecium and Euglena It was speculated that membrane-located gravisensors are early inventions of evolution, whereas the Müller bodies of Loxodes are later acquisitions due to adaptation to special living conditions (e.g., living in the sedi-ment of lakes)

7.10 Graviperception in the Statolith-based Systems of Chara

weight of sedimented statoliths by lateral centrifugation did not enhance the gravitropic response However, graviperception was terminated within seconds when the contact of statoliths with the plasma membrane was interrupted by inverting gravistimulated cells This result provides evidence that graviperception in characean rhizoids relies on di-rect contact allowing as-yet unknown components on the statoliths’ surface to interact with membrane-bound receptors rather than on pressure or tension which is caused by the weight of the statoliths (Limbach et al 2005) A mechanoreceptor was postulated to rep-resent the receptor because the gravitropic responses of many plant organs seem to obey the sine law of gravitropism (Galland 2002) The pressure that statoliths exert on the re-ceptor at different gravistimulation angles would explain the sinusoidal dependency, but the observation that the number of statoliths which settle on the receptor area in characean rhizoids decreases with the steepness of the angle can equally well account for this dependency

7.11 Graviperception in the Statolith-based System of Loxodes

In order to show that Müller vesicles function as gravisensory organelles in Loxodes, these structures were destroyed by laser ablation within individual cells Observation of the manipulated cells revealed that they were no longer able to orient to the gravity vec-tor, though their vitality and swimming velocity were unaffected (Hemmersbach et al 1998) As discussed earlier in this chapter, it has been proposed that Loxodes perceives gravity through bending of the ciliary complex (Figure 7.4), thus inducing changes in the membrane potential Such changes modify the activity of the body cilia, affecting the swimming behaviour (Fenchel and Finlay 1984, 1986) It is still unknown whether the gravity-induced “pull” of the BaSO4-granulum is directly transduced via ion channels in the plasma membrane, or whether it requires second messengers Besides a cellu-lar gravisensor, it is postulated from isodensity experiments that Loxodes also has a protoplast-based perception mechanism (Figure 7.4) In order to determine the minimum acceleration that is necessary to induce a graviresponse, Loxodes, Paramecium, and

Euglena were exposed to increasing acceleration steps from microgravity to 1.5 ⫻ g, or

vice versa, on a centrifuge microscope in space With this experimental approach, thresh-old values for gravitaxis have been determined: Loxodesⱕ 0.15 ⫻ g, Paramecium: 0.3 ⫻ g (Hemmersbach et al 1996a), Euglena: ⱕ 0.16 ⫻ g and 0.12 ⫻ g (Häder et al 1996, 1997) The existence of such thresholds implies that gravitaxis is the result of a physio-logical signal transduction chain

7.12 Graviperception in the Protoplast-based Systems of Paramecium and Euglena

prerequi-site for graviperception In Paramecium and other ciliates, mechano-sensitive ion chan-nels are distributed in a characteristic, bipolar manner in the plasma membrane: mechano-sensitive K+-channels mainly posteriorly and mechano-sensitive Ca2+-channels

mainly anteriorly (Figure 7.4; for a full discussion of mechano-sensitive channels, see Chapter 5)

In upward-swimming cells, the mechanical load of the protoplast is supposed to induce an outward-directed force onto the lower part of the cell membrane, thus opening mechano-sensitive K+-channels, which hyperpolarizes the cell In turn, membrane hyper-polarization promotes an increase of swimming velocity, which can be measured in upward-swimming cells In contrast, in a downward-swimming cell, the swimming ve-locity is reduced due to depolarization of the membrane potential by mechanical stimu-lation of mechano-sensitive Ca2+channels Such a distinct activation of two kinds of re-ceptor populations delivers a reasonable explanation for gravitaxis and gravikinesis (Baba et al 1991; Machemer and Bräucker 1992)

Correspondingly, in Euglena, stretch-activated gravireceptor channels are proposed to be located asymmetrically in the anterior cell membrane (Figure 7.4) In an upward-swimming (negative gravitactic) Euglena, these channels are closed If the longitudinal axis of the cell deviates from the vertical or if the cell swims downward, these channels open (Lebert and Häder 1996; Lebert et al 1997; Hăọder et al 2005), thereby inducing a depolarization of the plasma membrane, followed by activation of an intracellular signal transduction cascade that results in a reorientation of the cell

If graviperception in these systems occurs via gravisensory channels, it should be possi-ble to measure a “gravireceptor potential.” This was done by intracellular electrophysiolog-ical recording in Paramecium and in the ciliate Stylonychia Depending on the cell’s orien-tation with respect to the gravity vector, a hyperpolarization or a depolarization was registered after turning a cell upside-down (Gebauer et al 1999; Krause 2003) In the case of Euglena, a direct measurement of electrical potentials has not been successful However, an involvement of the membrane potential in graviperception in these cells was supported by experiments in which the lipophilic cation TPMP+(triphenyl-methylphosphonium) and the ionophore calcimycin were used to alter the cellular membrane potential Both com-pounds resulted in a loss of gravitaxis In another experimental approach, Richter et al (2001a) used voltage-sensitive dyes such as Oxonol to observe changes in membrane po-tential depending on the Euglena cell’s orientation within the gravity field

These experiments led to the discovery of a close relationship between precision of orientation and membrane potential Interestingly, the channel blocker gadolinium blocked gravitaxis in Euglena, suggesting that stretch-activated channels function as gravisensory ion channels in this species (Lebert et al 1997) Corresponding experiments with Paramecium showed no specific effect on gravitaxis, indicating that gadolinium is not specific for mechano-sensitive channels in this cell (Nagel and Machemer 2000).

Regardless of the nature of the mass and the gravireceptor, graviperception can only occur when sufficient energy is provided to the system to overcome thermal noise in the receptor Graviresponses are found in protists of different size and cell volume, ranging from Euglena gracilis (2.6 ⫻ 103 µm3) to Paramecium caudatum (327 ⫻ 103 µm3) or

ther-mal noise level (2 ⫻ 10–21 J) However, for smaller species such as Euglena or even

smaller systems such as human cells (lymphocytes), the signal-to-noise ratio may be lim-iting This ratio is even more critical if one considers threshold values in the range of 0.1 ⫻ g to 0.3 ⫻ g for graviperception Therefore, in these systems the involvement of sup-porting amplifying structures, such as the microfilament system of Loxodes, may facili-tate graviperception (Häder et al 2005)

7.13 Signal Transduction Pathways and Graviresponse Mechanisms in the Statolith-based Systems of Chara

Although several studies clearly indicate that gravireceptor molecules are located in the subapical plasma membrane area of the statolith region (Braun 2002), the nature of the membrane-bound gravireceptor and the immediate downstream physiological steps in-volved in graviperception in rhizoids and protonemata remain to be clarified Results ob-tained from the local application of ions and channel blockers by means of microcapil-laries suggest that membrane-potential changes might be among the earliest steps following gravireceptor activation (Braun, unpublished results) However, more data have been published in the last decade that illuminate the cellular and molecular processes that underlie the gravitropic response

The smooth, downward curvature response of a rhizoid is best described as “bending by bowing,” whereas the response of a protonema was described as “bending by bulging” (Braun 1996b), referring to the bulge that initially appears on the upper cell flank and in-dicates the drastic upward shift of the growing tip The Spitzenkörper (a tip-growth or-ganizing complex consisting of endoplasmic reticulum, actin filaments, and a dense net-work of vesicles) (Braun 1997) and, consequently, also the center of maximal growth are displaced upon gravistimulation of protonemata by intruding statoliths (Figure 7.3) Although rhizoids can be forced to respond to some extent like protonemata, this can only be done by pushing statoliths aymmetrically into the apical dome with optical tweezers or by centrifugal forces greater than 50 ⫻ g (Braun 2002) There is evidence from cen-trifugation experiments (Braun 1996b; Hodick and Sievers 1998) and from attaching par-ticles to the surface of gravitropically responding rhizoids (Sievers et al 1979) that the position of the growth center at the cell tip is relatively stable and that the Spitzenkörper is more tightly anchored by cytoskeletal forces in rhizoids than in protonemata

The specific properties of the actin cytoskeleton, which have been shown to be respon-sible for Spitzenkörper anchorage, are controlled by calcium Interestingly, calcium im-aging demonstrated a drastic shift of the steep, tip-high calcium gradient toward the upper flank during initiation of the graviresponse in protonemata (Figure 7.3), but not in rhi-zoids (Braun and Richter 1999) In accordance with this observation, dihydropyridine fluorescence (indicating the tip-focused distribution of putative calcium channels) was also displaced toward the upper flank in graviresponding protonemata, but not in rhizoids (Braun and Richter 1999)

sta-toliths sedimented onto the lower subapical cell flank It is speculated that this reduction is due to a local inhibition of calcium channels that is initiated by statolith-induced gravireceptor activation The subsequent reduction of the rate of exocytosis of secretory vesicles causes differential growth of the opposite cell flanks and results in the positively gravitropic curvature growth (Figure 7.3)

The data obtained from characean protonemata suggest that the early asymmetric dis-tribution of the calcium gradient (which precedes the negative graviresponse in protone-mata) results either from statolith-induced repositioning of calcium channels or, more likely, by differential activation and/or inhibition of apical calcium channels (Braun and Richter 1999) Such processes would lead to an asymmetric influx of calcium, thus alter-ing the pattern of exocytosis and causalter-ing an asymmetric incorporation of calcium chan-nels The asymmetric influx of calcium could mediate a repositioning of the Spitzen-körper and the growth center by differentially regulating actin anchorage or the activity of actin-associated proteins along the shifting calcium gradient (Braun and Richter 1999) The resulting polarity change would lead to the new growth direction (Figure 7.3)

Additional support for the proposed gravitropic response mechanism in protonemata comes from immunofluorescence labeling of spectrin-like proteins in the actin-rich area that contains the ER aggregate in the center of the Spitzenkörper The signal, which lo-calizes to the median cell axis during vertical growth, is drastically displaced toward the upper flank—the site of future outgrowth—during initiation of the graviresponse in pro-tonemata, clearly before curvature is recognizable (Braun 2001) In contrast, the same la-beling in rhizoids gives a signal that remains symmetrically positioned in the apical dome throughout the graviresponse These findings confirm that a repositioning of the Spitzenkörper is involved in the negative graviresponse of protonemata, but probably does not play a role in the positive graviresponse of rhizoids (Figure 7.3; Braun 2001) The tendency of protonemata to reorient toward the former growth axis after only short gravistimulation phases indicates that the new growth axis induced by the upward shift of the Ca2+gradient is rather labile and may require actin anchorage to stabilize the new growth direction (Braun and Richter 1999; Braun 2001)

7.14 Signal Transduction Pathways and Graviresponse Mechanisms in Euglena and Paramecium

The important role of gravity for cellular orientation and confirmation of the correctness of the term “gravi” for dedicated tactic and kinetic responses of motile microorganisms were shown by experiments in microgravity and in simulated weightlessness Exposing a culture of ciliates with a preferred upward orientation to the conditions of microgravity resulted in a random distribution after 80 seconds in a rocket experiment and after 120 seconds in a clinostat experiment (Hemmersbach-Krause et al 1993b) Similar behav-ioural responses were observed for Euglena (Vogel et al 1993) Long-term cultivation of

Euglena (Häder et al 1996), Paramecium, and Loxodes (Hemmersbach et al 1996a) for

be-come more pronounced (up to µg in the case of Paramecium) (Hemmersbach et al. 1996b; Bräucker et al 2001)

When considering likely elements of the gravitactic signal-transduction pathway, it was assumed that second messengers such as Ca2+, cAMP, cGMP, and calmodulin might play a role in this process, as they are clearly involved in the regulation of ciliary and fla-gellary activity; in the case of cAMP, a coupling to hyperpolarizing cellular events has been shown (Bonini et al 1986; Schultz and Schönborn 1994) Application of phospho-diesterase inhibitors (IBMX, caffeine, 8-bromo-cyclo-AMP) to Euglena cultures, with the aim of increasing intracellular cAMP levels, resulted in an increase in the precision of negative gravitaxis, whereas decreasing cAMP levels inhibited the capacity of the cells for gravitactic orientation Determination of the cAMP levels of Paramecium and

Euglena under different acceleration conditions revealed significant

acceleration-dependent changes (Tahedl et al 1998; Häder et al 2005) Indeed, experiments in hyper-gravity and microhyper-gravity showed changes in cAMP in Paramecium according to the pred-ication of the “statocyst” (protoplast-pressure) hypothesis: a decrease in cAMP was found in microgravity where no stimulation of the lower membrane should occur and an increase was found under hypergravity conditions due to an increased mechanical load on the lower membrane compared to ⫻ g conditions (Häder et al 2005)

Direct visualization of second messenger changes in living cells are promising ap-proaches to clarify the exact underlying mechanism and the time course of events Using the chlorophyll-free flagellate Astasia longa loaded with calcium Crimson dextran and excited by laser light, researchers have investigated a possible correlation between the cy-tosolic calcium concentration and the orientation of the cell In this context, a 180-degree turn of a negative-gravitactic Astasia culture induced an increase in the calcium signal, with a maximum after 30 seconds correlated with the reorientation of the cells When performed in space, this experiment revealed that microgravity-adapted Astasia cells showed an increase in calcium-dependent fluorescence signal when accelerated above their threshold for gravitaxis An image analysis system established a correlation between the calcium-dependent fluorescence signal and the swimming direction: cells moving in parallel to the acceleration vector showed a low signal and cells moving perpendicular to the vector displayed a high signal (Richter et al 2001b) Acceleration-dependent changes in intracellular calcium levels in Euglena were also observed in a recent parabolic flight experiment onboard the Airbus A300 Zero-G (Richter et al 2002) Together, these data support the existence of a calcium influx during reorientation of misaligned cells (Richter et al 2001b, 2002)

7.15 Conclusions

obviously very different, the presented experimental data reveal that especially the early phases of gravity sensing share common features The gravisensory processes can be re-duced to two principles: perception via intracellular statoliths and via the whole proto-plast Gravisensory ion channels and cascades of ubiquitous second messengers are pre-dicted to operate in most gravity-dependent signaling pathways, and have been identified in some cases Finally, the cytoskeleton has been shown to play a master role in the com-plex process of gravity sensing and graviorientation One fascinating question, further discussed in Chapters and 9, will hopefully be answered in the near future: What will be the impact of long-term exposure to microgravity conditions in multigeneration exper-iments on the physiology of specialized gravisensory cells?

7.16 Acknowledgments

The authors thank the several space shuttle crews, the teams of EADS ST, Kayser-Threde, Deutsches Zentrum für Luft- und Raumfahrt (DLR), NASA, Swedisch Space Corporation (SSC), Novespace, and the European Space Agency (ESA) for their dedi-cated work and for stimulating discussions This work was financially supported by Deutsches Zentrum für Luft- und Raumfahrt (DLR) on behalf of the Bundesministerium für Bildung und Forschung (50WB9998 and 50WB0515)

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Melanie J Correll and John Z Kiss*

161

8.1 Introduction—The Variety of Plant Movements

Although plants are generally considered stationary organisms, they move in response to a variety of environmental stimuli Plants can move their organs and direct their growth to avoid harmful situations or to find important resources for survival The movements of plants can be classified into three main categories: nastic responses, circumnutations, and tropisms

Nastic movements, sometimes called turgor movements, include motion of plants to external environments that not depend on the direction of the stimulus source Examples include motion due to changes in temperature (thermonasty), light (pho-tonasty), touch (hap(pho-tonasty), humidity (hydronasty), chemicals (chemonasty), as well as other stimuli Nastic movements may allow the plant to protect itself from harmful ele-ments or improve growth, development, and reproductive opportunities These move-ments can be seen in the opening and closing of some flowers due to the light/dark cy-cles during the day, or the movement of leaves of the “sensitive plant” (Mimosa) when touched (see Chapter 5) The mechanisms and level of interactions of nastic movements with other plant movements are unclear

In contrast, circumnutations are the endogenous oscillatory movements of plants around a central axis Circumnutation may be a simple consequence of growth and may serve as a mechanism for plant movements to adjust plant growth from movements that overshoot the plumb line of gravity (Barlow et al 1994; Antonsen et al 1995; Kiss 2006) The purpose of circumnutation is unknown and may simply be a consequence of general growth processes In Arabidopsis thaliana, circumnutation of organs has been shown to be modulated by the circadian clock, with greatest movement occurring during dawn (Niinuma et al 2005) The role of gravity in circumnutation is still unclear, although re-cent evidence suggests that gravity and circumnutation are inherently linked (Kitazawa et al 2005; Kiss 2006; Yoshihara and Iino 2006)

Tropisms are the directed growth of a plant organ in response to external stimuli Unlike nastic movements, tropisms depend on the direction of the stimulus Curvature of a plant organ toward the stimulus is termed positive tropism, and curvature away is termed negative tropism The best-characterized tropisms are directed growth in response to gravity (gravitropism; see also Chapters and 2), light (phototropism; Chapter 4), water (hydrotropism; Chapter 6), touch (thigmotropism; Chapter 5) and oxygen (oxytro-pism; Chapter 6), although many other tropisms have been reported

Since several types of plant movements can occur simultaneously, the ability to study one movement without the interactions from other movements is extremely difficult For example, on Earth, gravity is ubiquitous Thus, movements induced by gravity inevitably

interact with movements induced by other stimuli (Barlow 1995) The relatively strong gravitropic responses of plants can mask comparatively weaker tropisms, such as hy-drotropism or oxytropism Therefore, in order to better characterize tropisms, scientists have used several methods to reduce the effects of gravity on plant motion (Figure 8.1)

These methods include the use of the following:

• Plant mutants that have reduced responses to gravity;

• Experimental systems that prevent plants from receiving unidirectional gravity accel-eration (i.e., clinostats or random positioning machines);

• Microgravity/free-fall environments

The extent to which these techniques reduce the effects of gravity on plant movement has been a topic of much debate, with each method having unique advantages and disad-vantages (Sievers and Hejnowicz 1992; Kordyum 1997; Correll and Kiss 2002)

In this chapter, experimental results from microgravity experiments are compared with other ground-based studies on plant movements for experiments performed from ~1990 to the present Although we will concentrate on discussing ground-based and micrograv-ity research on tropisms, other movements such as circumnutation will also be briefly considered

8.2 The Microgravity Environment

The gravitational acceleration on Earth is approximately 9.8 m/s2, defined as 1g The term microgravity has been used to refer to levels that are less than 1g, typically 10–3g to

10–6g In this chapter, we consider microgravity to range from approximately 1% of

Earth’s gravitational acceleration (0.01g) to approximately millionth of the Earth’s gravitational acceleration (10–6g) Other terms that have been used to describe different

levels of gravitational accelerations include hypogravity (accelerations less than 1g but greater than 10–3g), weightlessness (net sum of forces acting on a body equaling zero),

zero-g (an object that does not experience any gravitational pull), and hypergravity (ac-celerations greater than 1g) (Schaefer et al 1993; Klaus 2001).

Since gravity can influence other nongravity-related movements of plants, scientists have attempted to reduce gravitational effects below biologically detectable levels to study certain types of plant movements One way to mitigate the effects of gravity is to travel away from the major source of gravitational interference, the Earth Unfortunately, to reduce the effects of gravity on plants to millionth of Earth’s gravity, you would have to travel approximately 6.4 million km away from Earth (Rogers et al 1997) To put this in context, the moon orbits the Earth at approximately 380,000 km Thus, studies on plant movement at this distance are unlikely in the near future, so other options are necessary to mitigate the effects of gravity on plants

object of interest (i.e., the plant) All of the studies with plants in microgravity discussed in this chapter are actually with plants in free fall

Some mechanisms to create free fall or a microgravity environment include the use of drop towers, parabolic flights of airplanes, rockets, and orbiting satellites such as the Space Shuttle and space stations (Figure 8.1 and Color Section) The quality and duration of microgravity created in these environments are variable For example, the longest drop tower (located in a mine shaft in Japan) is 460 m in length and only provides approxi-mately 10 sec of free fall (Tokyo 1993) Parabolic flights are when a plane or rocket climbs rapidly at a ~45-degree angle then descends at a ~45-degree angle (Figure 8.1C) The parabolic flight between these two maneuvers can reduce gravitational effects to ~10–2g for about 20 sec for airplanes (Pletser 1995) and ~10–5g for up to several minutes

for rockets (European Space Agency 2006) However, since many tropistic responses of plants can take up to several minutes before detection, the short duration of microgravity provided by drop towers and parabolic flights limits the types of studies that can be per-formed with these methods

Studies with drop towers and parabolic flights have been successfully used with pro-tists to study the gravitaxis (orientation with respect to the gravity vector) (Häder and Hemmersbach 1997; see Chapter 7) or to study elements in the gravity-induced-signal cascade, such as movement of statoliths in plant roots (Volkmann et al 1991) Parabolic flights are useful, relatively inexpensive laboratories to study the very early phases of gravity responses in plants

However, the best methods for studying tropisms and other plant responses in micro-gravity are through the use of biosatellites and orbiting spacecraft Biosatellites (un-manned missions into space) can provide long-term microgravity, but typically missions are of about two weeks’ duration Orbiting space stations such as Skylab, Mir, or the International Space Station (ISS) have offered the potential to study plant tropisms in a microgravity environment indefinitely

All of the methods used to create a reduced-gravity environment have limitations in their effectiveness as a microgravity laboratory For example, the quality of acceleration on orbiting craft depends on the orbital motion of the spacecraft, the position of the item on the spacecraft, and the aerodynamic drag on the craft Luckily, these accelerations are only about 10–6g in magnitude, which is relatively small and possibly below the sensory

threshold for plant responses to gravity (Shen-Miller et al 1968; Sobick and Sievers 1979; Merkys and Laurinavicius 1990; and see below) However, other accelerations can be created by vibrations in orbiting craft from fans, pumps, centrifuges, and crew activ-ity For the Space Shuttle, these accelerations were found to be about 10–4g, which may

affect some experimental results since the minimum threshold values for root and shoot curvature may range in the values of 10–4g and 10–3g, respectively (Shen-Miller et al.

1968; Sobick and Sievers 1979; Merkys et al 1986; Merkys and Laurinavicius 1990) Therefore, scientists using orbiting spacecraft for microgravity studies should consider these additional accelerations in their analyses

out-ages, and lack of biological replicates Several experiments have noted poor gas compo-sition or exchange rates which resulted in unusual growth responses of plants in micro-gravity (Porterfield et al 1997; Kiss et al 1998, 1999; Perbal et al 1996) In addition, many experiments performed in space have not been replicated, which limits the scien-tific interpretation of results (Cogoli 1996) The major impedances to studying plant tro-pisms in space are the limited opportunities and the associated costs Recent delays in Space Shuttle flights to the ISS have further limited the amount of science that can be performed in the future The future outlook for opportunities to study plant movements in space is also discussed in Chapter of this book

8.3 Ground-based Studies: Mitigating the Effects of Gravity

The problems associated with studying biological responses to gravity on space missions have encouraged scientists to develop alternative ground-based methods to simulate mi-crogravity Devices such as clinostats and random positioning machines (also known as three-dimensional clinostats) have been used to reduce gravity effects on plants for tro-pism studies (Figure 8.1) A clinostat generally rotates the specimen around one axis whereas a random positioning machine offers three-dimensional rotation (Salisbury 1993; Hoson et al 1997) The extent to which clinostats simulate microgravity has been debated, since results from clinostats not always correspond to those from true micro-gravity experiments (Brown et al 1976; Sievers and Hejnowicz 1992; Heathcote et al 1995a; Brown et al 1996; Kordyum 1997; Klaus 2001) A comparison of experimental results of plant and other organisms in microgravity with results from clinostats has been reviewed (Brown et al 1976; Albrecht-Buehler 1992; Kordyum 1997) and is discussed in more detail in the subsequent sections of this chapter

Another ground-based attempt to reduce gravity effects on plant movements is through the use of mutants in gravity perception For example, mutants of Arabidopsis that have reduced activity in the phosphoglucomutase gene (PGM) have reduced responses to grav-ity (Kiss et al 1989) The pgm plants have reduced amounts of starch-filled amyloplasts in the cells involved in the perception of gravity An otherwise cryptic positive, red light-induced phototropism in roots was discovered with studies using these plants (Ruppel et al 2001) Interestingly, hypergravity experiments performed with pgm mutants have shown that gravitropic orientation of organs of the pgm plants could be restored with ac-celerations of 5g for roots and 10g for hypocotyls (Fitzelle and Kiss 2001) Other starch-deficient mutants (e.g., agravitropic pea, Pisum sativum) have been used to study other tropisms such as hydrotropism or oxytropism that may be masked by gravitropic response (Jaffe et al 1985; Porterfield and Musgrave 1998)

8.4 Gravitropism

On Earth, the direction of the gravitational acceleration vector can indicate the location of water/nutrients (i.e., underground) and sunlight (i.e., aboveground) Not surprisingly, gravity is a relatively strong stimulus in terms of directing plant growth Typically, pri-mary roots grow toward the direction of gravitational acceleration (positive gravitro-pism), and shoots grow away from the direction of gravitational acceleration (negative gravitropism) Other organs in plants, such as inflorescence stems, leaves, and lateral roots and branches also display gravitropic responses, but their orientation may be an in-termediate angle relative to the gravity vector (Hangarter 1997; Kiss et al 2002; Mano et al 2006) After reorientation, a plant organ will curve to reach the appropriate orienta-tion, called the gravitational set point angle (Mullen and Hangarter 2003) The gravita-tional set point angle in plants can be observed in the patterns of branching in trees and orientation of lateral and primary roots

The temporal steps of gravitropism can be separated into three main stages: percep-tion, signal transducpercep-tion, and response Details of these stages are found in Chapters 1, 2, and and are described in Kiss (2000) Microgravity and simulated-microgravity envi-ronments have been used to study each stage of gravitropism as well as gravity-induced morphogenesis of plants and the straightening of a plant organ after a gravitropic re-sponse (autotropism) (Stankovic et al 1998) In the following sections, experiments that use microgravity as a tool to study the elements of gravity perception, signal transduc-tion, and response in gravitropism are described Gravimorphogenesis of plants has been reviewed in a paper by Takahashi (1997)

8.4.1 Gravitropism: Gravity Perception

Several models for gravity perception in plants have been proposed, although two mod-els currently dominate (Perrin et al 2005; Chapter 1) One model, the starch-statolith hy-pothesis, suggests that perception of gravity is mediated by the settling of dense or-ganelles (statoliths) after a plant is reoriented (Figure 8.2) (Kiss 2000) In flowering plants, the amyloplasts in specialized cells function as statoliths; in roots, the amyloplasts are in the columella cells; and in shoots they are located in the endodermal cells (re-viewed in Masson et al 2002) The other model for gravity perception in plants suggests that the entire mass of the protoplast is involved in perceiving gravity (Staves et al 1997) To date, neither of these models has been excluded and both may be acting simultane-ously Alternatively, another as-yet undiscovered mechanism of gravity detection may be involved in gravity perception in plants (reviewed in Wolverton et al 2002; Chapter 1)

For other plants, such as white clover (Trifolium repens) and lentil (Lens culinaris) roots, amyloplasts were grouped near the center of the cell in microgravity whereas amy-loplasts remained close to the distal half in 1g controls (Perbal and Driss-Ecole 1989; Lorenzi and Perbal 1990; Smith et al 1997) The grouping of amyloplasts was also found in plants grown on clinostats, but to a lesser extent (Smith et al 1997) Nonrandom grouping of amyloplasts in apical cells from moss (Ceratodon purpureus) was also found in clinostat- and microgravity-treated plants (Kern et al 2001) Although the mechanisms of the grouping of amyloplasts are unknown, it appears that the statocytes not move as a group in microgravity (Driss-Ecole et al 2000) It also appears that amyloplasts re-locate within minutes of microgravity treatments (Volkmann et al 1991; Driss-Ecole et al 2000) Future experiments in microgravity may help reveal the kinetics and mecha-nisms of amyloplasts’ movement in response to changes in acceleration and provide a bet-ter understanding of the grouping effect of amyloplasts in microgravity

Other studies on gravity perception mechanisms have been performed using plants that have reduced starch content and therefore smaller amyloplasts Plants grown in micrograv-ity that were then treated to 1g accelerations for 60 minutes showed greater curvature in roots from wild-type plants compared to roots from starch-deficient mutants (Kiss et al 1998) These results further suggest that the amyloplasts are involved in the mechanisms of gravity perception However, restoration of the gravitropic response in these mutants could be accomplished by treating plants with hypergravity, indicating that the starch-filled amyloplasts are not required for a gravitropic response (Fitzelle and Kiss 2001)

plants which develop in microgravity are still responsive to gravitational acceleration, suggesting that components of gravity perception not need gravity to be expressed Future studies in microgravity may identify new mechanisms of gravity perception in plants and identify downstream elements in the gravity signal transduction cascade

8.4.2 Gravitropism: Signal Transduction

Once the gravity signal is perceived in plants, downstream elements in the signaling cas-cade begin to react while the response (curvature) phase begins Elements downstream of sensing include changes in calcium, pH, flavanoids, ethylene, relocalization of auxin car-riers (PIN proteins), and auxin redistribution which results in differential elongation of a plant organ (Masson et al 2002; Chapter 2) Since all tropisms result in the differential growth of an organ, many of the downstream elements involved in gravitropism overlap with other plant tropisms Few studies have been performed in microgravity on the down-stream elements in plant tropisms because these elements are difficult to measure on Earth, even without the additional constraints imposed in the space environment

Some of the studies that have been performed using microgravity to identify down-stream elements in signal transduction are briefly described here An experiment with lentil roots was performed to identify the role of actin cytoskeleton in amyloplast move-ment in microgravity (Driss-Ecole et al 2000) Amyloplasts from roots treated with a drug that blocks actin polymerization (cytochalasin D) did move in response to micro-gravity, but the rate of movement was slowed compared to nontreated roots (Driss-Ecole et al 2000) These authors proposed that the microfilament bundles were not completely depolymerized with the cytocalasin D treatment, which allowed for limited movement of amyloplasts The limited rate of movement of the amyloplasts in the cytochalasin D sam-ples suggests that the cytoskeleton is involved, at some level, in gravity-induced re-sponses Other ground-based studies with drugs that disrupt the actin-cytoskeleton have also demonstrated that the cytoskeleton is involved in gravity responses, although results from these studies are often conflicting and depend on the organ, plant species, drug dosage, and experimental conditions (Friedman et al 2003; Palmieri and Kiss 2005)

Auxin transport is another downstream element in tropisms that has been studied using microgravity Polar auxin transport was inhibited in the internode segments of pea but was promoted in maize (Zea mays) coleoptiles (Ueda et al 1999) The mechanisms of auxin transport during tropism in microgravity have not been studied but, since seedlings can curve in response to stimuli in microgravity, it appears that auxin transport during tro-pisms does not need gravity to function Other studies with maize noted similar amounts of auxin in plants grown in microgravity and 1g controls (Schulze et al 1992)

Studies with downstream elements in the signal-transduction cascade of tropisms in microgravity may provide significant contributions to our understanding of gravity ef-fects on plant growth and movement Currently, plants have been developed with green fluorescent proteins that are expressed along with proteins in the downstream compo-nents of gravity signaling (e.g., PIN2-GFP plants) (Abas et al 2006) Studies monitoring the expression and localization of these proteins in microgravity may prove useful to identify how downstream elements in gravity-induced signaling respond to different gravitational accelerations

8.4.3 Gravitropism: The Curving Response

As the gravity signal-transduction process has reached a significant level of propagation downstream, the response or curvature of the organ can be detected Without gravity as a cue to direct plant growth, plants appear to grow randomly For example, roots of cress (Lepidium sativum) plants and hypocotyls of Arabidopsis show a vertical orientation in 1g but appear to grow randomly in microgravity (Figure 8.3) (Johnsson et al 1996a; Johnsson et al 1996b; Kiss et al 1998; Kiss et al 1999; Kiss 2000) Roots and hypo-cotyls also grew randomly when grown in simulated microgravity, such as on clinostats or random positioning machines (Kraft et al 2000) After microgravity-grown plants are exposed to acceleration, their organs will curve in response to the direction of the accel-eration vector (Kiss et al 1998; Kiss et al 1999) The following questions have been pro-posed to explore the sensitivity of plants to gravity:

1 What is the minimum time required for a plant to sense and respond to gravity (thresh-old duration or presentation time)?

Figure 8.3. Images of roots of lentil seedlings grown in a 1g control (A) and in microgravity (B) during spaceflight Roots of the 1g control are straight and oriented relative to gravity (toward the bottom of the fig-ure) In contrast, roots of the microgravity-grown seedlings appear to have a more random orientation

2 What is the minimum gravitational acceleration that can be detected in plants to in-duce a response (g-threshold)?

3 How the presentation time and acceleration dose interact to induce a response (threshold dose; gravitational acceleration ⫻ time)?

Various doses of acceleration have been provided to plants using clinostats, cen-trifuges, and microgravity to study the sensitivity of plants to gravitational acceleration (Brown et al 1995; Volkmann and Tewinkel 1996; Hejnowicz et al 1998; Perbal et al 2004) Originally, it was hypothesized that the reciprocity rule for tropism governed gravity-induced curvature in plants The reciprocity rule suggests that if g (gravitation ac-celeration) and t (time of stimulation) are varied reciprocally to maintain a constant prod-uct, then the curvature or response should be the same (Johnsson et al 1995) For oat (Avena sativa) coleoptiles, reciprocity appeared to hold true for lower accelerations (0.2 to 1g) and stimulation times of to 65 minutes for plants grown in microgravity and on clinostats (Johnsson 1965; Shen-Miller 1970; Johnsson et al 1995) However, the sensi-tivity of oat to gravity also depended on the stage of the plant (i.e, height; Shen-Miller 1970; Johnsson 1965; Johnsson et al 1995) For example, the threshold dose for taller plants showing detectable curvature was about 55 g·s, whereas for shorter plants it was approximately 120 g·s Therefore, the developmental age or size of the plant may have an influence on the threshold dose that can be sensed

For other plant species, the reciprocity rule did not represent the response For exam-ple, the curvature of cress roots in microgravity after a dose of 60 g·s for treatments of 0.1g for 600 seconds and 1g for 60 seconds should result in similar magnitudes if reci-procity were valid However, results indicated that the 0.1g⫻ 600 seconds treatment had about half (14 degrees) of the curvature compared to roots from the 1g ⫻ 60 seconds treatment (32 degrees; Volkmann and Tewinkel 1996, 1998)

Other factors may affect the sensitivity of a plant to gravity, such as the growth his-tory of the plant For example, roots from cress grown in microgravity were more sen-sitive to the stimulation time than were roots from plants grown in a 1g centrifuge, with threshold doses of 30 g·s and 60 g·s, respectively (Volkmann and Tewinkel 1996). Enhanced curvature was also found for lentil roots previously grown in microgravity compared to plants that were grown on a 1g centrifuge (Perbal et al 2004) Surprisingly, roots from transgenic and wild-type rapeseed (Brassica napus) during microgravity had no detectable curvature after hour of centrifugation at 1g, whereas roots from ground controls curved significantly after reorientation (Iversen et al 1996) These results were not attributed to altered growth rates between microgravity-grown plants and ground controls or the additional accelerations imposed by uncontrolled vibrations in the space craft, but may have been due to other technical difficulties imposed by the spaceflight environment

The threshold duration (i.e., the shortest duration of time capable of eliciting a de-tectable gravitropic response) in microgravity also appears to be variable For oat, the re-sponse was less than minute at 1g (Brown et al 1995) These results are consistent with

Arabidopsis roots, where less than minute at 1g was also found to cause curvature (Kiss

elicit a curvature response (Perbal and Driss-Ecole 2002) Future studies in microgravity can clarify the nature of the duration of stimulus needed to elicit a response in plants

The lowest acceleration that a plant can detect has been estimated to be approximately 10–4g for roots and 10–3g for shoots (Shen-Miller et al 1968; Merkys and Laurinavicius

1990) These results were obtained from studies with oat and lettuce (Lactuca sativa) plants grown on clinostats or in microgravity Since experimental limitations could not provide the lower levels of gravitational acceleration, these authors used mathematical methods to predict the minimum acceleration that could be detected Microgravity pro-vided by spaceflight environments may not be low enough to study the minimum accel-erations that plants can detect, mainly due to other acceleration on the craft from crew ac-tivity and centrifuges Therefore, directly identifying the lowest magnitude of acceleration that plants can detect may be a challenge with today’s technology

Results from ground-based studies to determine the sensitivity of plants to gravity using clinostats have been conflicting For example, the curvature of white clover roots due to gravity stimulation after previous growth in microgravity, 1g, or on a 2-D clinos-tat showed that roots only curved ~35 degrees from clinosclinos-tat-grown plants, ~75 degrees from 1g-grown plants, and ~60 degrees from microgravity-grown plants after 10 hours (Smith et al 1999) The authors suggest that the reduced curving response of roots grown on the clinostat treatment compared to both plants grown in microgravity and on 1g con-trols was due to root cap deterioration Other researchers have found that roots from microgravity-grown plants are more sensitive to gravity compared to clinostat-grown plants (Lorenzi and Perbal 1990; Perbal and Driss-Ecole 1994; Volkmann and Tewinkel 1996; Perbal et al 1997) Therefore, the application of clinostats to study gravity re-sponses in plants is limited

8.5 Phototropism

Light is another important stimulus that is involved in determining the direction of plant growth Therefore, plants have evolved a variety of photoreceptors to sense the quality, quan-tity, direction, and intensity of light In flowering plants, the photoreceptors can be grouped into the blue/UV-A photoreceptors (cryptochromes and phototropins) and the red/far-red photoreceptors (phytochromes) Phototropic responses are largely controlled by the pho-totropins with the other photoreceptors modulating the response (Briggs and Christie 2002) Hypocotyls and stems typically curve toward blue or white light (positive phototropism), whereas roots typically curve away from the light source (negative phototropism)

The sensitivity of plants to light is difficult to study on Earth due to the interacting ef-fects from gravity Therefore, microgravity has been explored to determine the sensitiv-ity of plants to different wavelengths and intenssensitiv-ity of light The first intensive study on plant phototropic responses in microgravity was performed on wheat (Triticum aestivum) seedlings (Heathcote et al 1995a) Wheat coleoptiles showed a significantly enhanced curvature response to or seconds of photostimulation in microgravity compared to ground controls, although no significant differences in curvature were found after 3, 501, or 1,998 seconds of photostimulation (Heathcote et al 1995a)

It appears that in microgravity the phototropic curvature may be enhanced relative to plants grown in 1g, but this enhancement may also depend on the total fluence of the il-lumination provided An enhanced curvature response to ilil-lumination was found from clinostat-grown plants relative to both space-grown and ground controls regardless of du-ration of stimulation (Heathcote et al 1995a) Since enhanced curving responses were not found for all durations of light stimulation in microgravity, it appears that in this case, again, the clinostat does not effectively represent the microgravity environment provided in space Other studies with maize also showed an enhanced curvature in response to di-rectional illumination from coleoptiles grown on clinostats relative to 1g controls (Nick and Schäfer 1988)

The phototropism of moss also was studied in microgravity during spaceflight (Kern and Sack 2001) Apical cells (protonemata) of moss can display both positive and nega-tive phototropism to red light The kinetics of alignment in the red light path (~1.5 µmol m–2s–1) was similar between protonemata from moss grown in 1g and in microgravity. However, for dark-grown cultures that were exposed to low irradiance of red light (~50 nmol m–2s–1), more of the protonemata (70%) had aligned in the light path (±45 degrees of the path) in microgravity compared to 1g controls These results suggest that, on Earth, gravitropism and phototropism both interact at low fluence of red light to orient moss protonemata Interestingly, dark-grown protonemata grew in spirals in microgravity and an experiment on Space Shuttle mission STS-107 was performed to study this response However, due to the Space Shuttle Columbia accident, there were relatively few results from this study (Kern et al 2005)

Other experiments have been performed to study phototropism in plants in the micrograv-ity environment, although results have been limited A future experiment is planned for the ISS using hardware developed by NASA and the European Space Agency (ESA) This exper-iment, called TROPI for tropisms, will monitor phototropic responses of roots and hypocotyls of Arabidopsis to various gravitational accelerations (Figure 8.4 and Color Section) in both red and blue light (Correll et al 2005) In addition, gene profile analyses on seedlings will be performed to study the interacting effects of light and gravity on gene expression patterns

8.6 Hydrotropism, Autotropism, and Oxytropism

were grown on a clinostat or in plants that were mutated in their response to gravity (Jaffe et al 1985; Takahashi and Suge 1991; Takahashi et al 1996) Hydrotropism was also ev-ident in lateral roots from cucumber (Cucurbita ovifera) seedlings grown on a clinostat or in microgravity (Takahashi et al 1999) The lateral roots from clinorotated and micrograv-ity seedlings grew at approximately 40- to 50-degree angles from the primary root axis, and this curvature was toward the water source, whereas roots from ground controls grew ~90 degrees from the primary root axis Therefore, although roots respond to moisture gradients, it appears that this response is largely masked by gravity-induced responses

The seemingly random orientation of plant roots out of rooting matrices for plants grown in microgravity may in fact be a result of oxytropism (Porterfield and Musgrave 1998) On two space shuttle experiments (STS-54 and STS-68) (Porterfield and Musgrave 1998), roots of Arabidopsis plants grown in microgravity grew out of the rooting matrices in the direction of the oxygen gradient The enzymatic activity, localization, and expression of mRNA of a protein associated with oxygen stress, alcohol dehydrogenase (ADH), were en-hanced in roots grown in microgravity compared to ground controls (Porterfield et al 1997) This observation suggested that roots may either be oxygen-deprived in micrograv-ity or expressing ADH in response to another stress induced by spaceflight

To test whether oxygen availability was limited to plants during microgravity, a novel sensor was designed that measured oxygen availability (not concentration) in micrograv-ity (Liao et al 2004) Results using this sensor during parabolic flights showed that oxy-gen availability changes during periods of microgravity and, therefore, roots from plants grown in microgravity are likely experiencing oxygen deprivation Experiments on the ground showed that indeed roots from both normal and agravitropic mutants of pea curved in response to oxygen gradients (Porterfield and Musgrave 1998) For example, roots from normal plants curved about half the amount of roots from agravitropic mu-tants after 48 hours of treatment These results suggest that roots respond to gradients of oxygen, but this is largely masked by gravitropic responses

Autotropism is the straightening of an organ after the g vector is randomized on a cli-nostat or is reduced, as is found in microgravity (Stankovic et al 1998) Cress roots that curved in response to various gravitational accelerations all underwent straightening once the acceleration was removed (Stankovic et al 2001) Roots from clinorotated plants also showed autotropism, suggesting that the straightening process is a process that does not depend on the prestimulus orientation

Microgravity offers a unique ability to identify and study tropic responses of plants Studies in microgravity can reduce the interacting effects of gravitropism with other tro-pisms, allowing the characterization of the tropism of interest without the ever-present gravity responses It also seems likely that plants curve in response to a variety of as-yet unidentified stimuli which may only be found when grown in low g conditions, or with studies that use plants that lack the ability to perceive gravity

8.7 Studies of Other Plant Movements in Microgravity

studied (sometimes simultaneously) with tropisms in spaceflight For example, both gravitropism and circumnutations of lentil roots were measured in an experiment on the space shuttle (Antonsen et al 1995; Perbal and Driss-Ecole 1994) These authors found that roots in microgravity displayed oscillations in growth along with gravitropic re-sponses that varied as the function of the stimulation time Ground-based studies with

Arabidopsis have shown that roots grown on a random position machine make large

loops in the right-handed direction, whereas in 1g, roots display a wavy, right-handed slant (Piconese et al 2003) Similarly, nastic movements in coleoptiles of wheat seedlings were observed in plants in microgravity (Heathcote et al 1995b) The extent of the interactions between oscillatory movements and gravitropism is unclear Some ev-idence with plants mutated in genes involved in gravity responses has suggested that the oscillatory movements are coupled to gravitropism with auxin distribution being an overlapping element (Piconese et al 2003; Kitazawa et al 2005; reviewed in Kiss 2006) In contrast, evidence from spaceflight experiments has indicated that a variety of plants have circumnutations in microgravity, suggesting that gravity is not necessary for cir-cumnutation (Brown et al 1990; Heathcote et al 1995b; Antonsen et al 1995) Future studies using microgravity may help to define the roles of gravity in these types of plant movements

8.8 Space Flight Hardware Used to Study Tropisms

The types of hardware that have been used to study plant tropisms in microgravity are variable since the spaceflight conditions can influence the hardware design For example, experiments that did not have crew available, such as experiments performed on sound-ing rockets, biosatellites, or some space shuttle/station experiments, needed to have fully automated hardware Other experiments had crew interaction, which increased the com-plexity associated with these types of studies Some examples of the different types of plant facilities used in microgravity to study plant tropisms are described in the next sec-tion In addition, hardware for plant growth studies in general has been reviewed (Lork 1988; Porterfield et al 2003)

The use of sounding rockets or biosatellites is a relatively cost-effective way to per-form simple experiments in microgravity Cress plants grown in sounding rockets (TEXUS program) were housed in containers that had temperature recording devices and an automatic fixation system (Tewinkel et al 1991) The fixation system automatically flooded the containers holding the plants with chemical fixatives Such in-flight fixation allowed further analysis on the ground to identify plastid location during the excursion into microgravity

Crew involvement in experiments allows for more complex experimental design and analyses For example, two tropism experiments, one with wheat, called Phototropic Transients (FORTRAN), and another with oat, called Gravitational Threshold (GTH-RES), were performed on the space shuttle implementing the International Microgravity Laboratory payload (IML-1; Heathcote et al 1995a; Brown et al 1995) These experiments were performed on the Spacelab module of the space shuttle mis-sion STS-42 in 1992 Plants in these experiments were transported to the shuttle in specially designed containers called Plant Carry-On Containers (PCOCs) Once on-board, the containers were stowed in either the Mid-deck Ambient Stowage Insert (MASI) or housed in the Gravitational Plant Physiology Facility (GPPF) In the GPPF, plants were placed in containers called Plant Cubes, which had a window that allowed infrared radiation but no visible light to penetrate, and a second window that allowed blue light to penetrate during photostimulation periods Photostimulation occurred when plants were placed on the Recording and Stimulus Chamber (REST) A culture rotor, which consisted of two centrifuges, provided a force of 1g, and a test rotor, which provided to 1g, were both available for video recording of curvature responses to different gravitational accelerations Ground controls for these experiments included a clinostat in a GPPF facility These procedures required crew time to transport con-tainers and perform the experiments, but also allowed for controls to be grown in space

Biorack is another facility that has been used to study tropisms in the space environ-ment It was developed by the ESA as a multi-user facility to study a variety of biologi-cal materials (Manieri et al 1996) The Biorack facility had a cooler/freezer, a glovebox, and two incubators Two types of containers have been available for studying biological specimens in the Biorack facility Type I containers hold a volume of 65 ml and Type II containers hold a volume of 385 ml Both Arabidopsis and lentil seedlings have been ger-minated and grown in Type I containers with Type II containers used as fixation devices (Perbal et al 1987; Kiss et al 1999) This hardware allowed for video monitoring of plant growth and curvature responses and, with the aid of the crew, the plants could be chemi-cally fixed to study the movement of amyloplasts in gravity-perceiving cells once the samples had been returned to Earth

8.9 Future Outlook and Prospects

With the advancement of molecular techniques and cellular imaging technologies, many of the key players involved in tropisms have been identified and localized within cells However, how all these elements integrate and orchestrate tropisms during plant develop-ment is far from understood Microgravity experidevelop-ments have only begun to clarify the sen-sitivity of plants to gravity and light It is likely that different species may be more sensi-tive to particular environmental stimuli, resulting in an enhanced tropistic response Future long-term experiments that will use space stations as microgravity laboratories to study tropisms are likely to reveal new information about how plants have evolved in a 1g envi-ronment on Earth and to identify new tropistic responses that are masked by gravity In ad-dition to increasing basic knowledge, this information, in the long term, will aid in the de-velopment of plants for use in bioregenerative life support systems during space missions

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Christopher S Brown*, Heike Winter Sederoff, Eric Davies,

Robert J Ferl, and Bratislav Stankovic

183

9.1 Introduction

Mankind will explore the solar system Some of that exploration will be done robotically, allowing vicarious human experience and study of extraterrestrial locations However, the U.S space program’s plans are replete with strategies to enable the first-hand human ex-ploration of space With the human system such an essential part of the long-term plan, technologies to keep humans alive and performing at full capacity in extraterrestrial en-vironments must be developed

To accomplish the long-term goal of a stable human presence on other planetary bod-ies in the solar system, the development and integration of a dependable life support sys-tem is critical Continued shipment and re-supply of the essentials for human survival— breathable air, clean water, and food—would be risky as well as prohibitively expensive They are risky in that the shipments would depend on an absolutely fail-proof launch, transit, and landing system They are expensive in that, with current technologies, the cost to get kg into low Earth orbit is about $10,000 That’s a $3,000 hamburger!

Therefore, success of the human expansion into the cosmos must coincide with the de-velopment of a life support system that is capable of regenerating all the essentials for survival Such systems already exist—they are called plants Using the primary processes of photosynthesis (air revitalization and biomass production) and transpiration (water pu-rification), plants have provided human beings with all the essentials for survival as well as many of the nonessentials that make life interesting

To date, developing the engineering and infrastructure to get us into space has resulted in important advances in transport and propulsion systems But this is not all that is needed for the successful human exploration of space As stated by astrophysicist Freeman Dyson:

“The chief problem for a manned mission [to space] is not getting there but learning how to survive after arrival Surviving and making a home away from Earth are problems of bi-ology rather than engineering Any affordable program of manned exploration must be cen-tered in biology, and its time frame tied to the time frame of biotechnology; a hundred years[ ] is probably reasonable To make human space travel cheap, we will need advanced biotechnology in addition to advanced propulsion systems.” (Dyson 1999)

The development of a plant-based, biologically regenerative life support system is therefore critical to providing the fundamental needs of a human crew However, to be fully supportive in a potentially changing environment, such a system must also be ble of responding to the changing needs of the personnel onboard Plants must be capa-ble of quickly reprogramming their metabolism, physiology, and growth to keep the crew