Functional characterization of giant killer in flower development and meristem regulation in arabidopsis thaliana 2

CHAPTER 1: Introduction 1.1 General Introduction In the course of human civilization, we are constantly exploring ways to harness and domesticate various plant species for our use as food sources, raw materials and bioenergy. In view of our current pressing issues on global warming and climate changes, extensive understanding of our sessile co-habitant of Mother Earth has become increasingly crucial in the development of a sustainable environment to live in. There are plenty of examples showing that plant research has significant impact on humankind. Research studies on our staple crops like oryza sativa (rice) have contributed in reducing poverty and hunger (beta.irri.org/news/). Since the imminent threat on fossil fuels depletion would have immediate consequences on global economic and political stability, scientific studies on Elaeis guineensis (oil palm) and Jatropha curcas (Barbados nut) for biodiesel development will certainly help in soothing the jitters triggered by the global oil crisis (Fairless, 2007; Kennedy, 2007). In general, scientific findings generated from the plant research will ultimately help in transforming human society and will provide numerous incentives for the future development of the mankind. Nevertheless, scientific research that uses economic or agricultural plant species often has a murky outlook. It is commonly suffering from various technical glitches, which may be caused by the species undefined genome sequences, their long generation time, and their intractability to molecular and genetic approaches. In light of this, extended and detailed analysis on a model plant like Arabidopsis thaliana would invariably provide useful insights and lay down a concrete foundation for the understanding of other plant species. For instance, study of floral organ identity genes in Arabidopsis thaliana has expedited the process of identifying their conserved counterparts in Dendrobium crumenatum (orchid), an ornamental plant that has significant economic implications (Xu et al., 2006). Arabidopsis thaliana is a small-size flowering plant (also known as mouse-ear cress) belonging to the mustard family. It is amenable to various molecular and genetic manipulations for scientific research (Meyerowitz and Pruitt, 1985). Its genome had been fully sequenced in year 2000 (Arabidopsis Genome Initiative, 2000) and documented to be at a relatively small size of 120 Mb with about 25, 000 genes sprawling across chromosomes. More than two decades ago, several phenotypically striking floral homeotic mutants had been identified by Meyerowitz and colleagues (Bowman et al., 1989; Yanofsky et al., 1990; Bowman et al., 1991). Detailed characterization and analysis of these mutant Arabidopsis flowers, together with the works done in Antirrhinum majus, lead to the formulation of the well-known ABC model in flower development (Coen and Meyerowitz, 1991). Arabidopsis thaliana has since eased its way through to become the most popular model in studying plant development, biochemistry, and various molecular pathways including stem cell regulation, some of which were later found to be conserved even in animal kingdom. With its relative short generation time of 7-8 weeks, it has out-competed other plants like Antirrhinum majus, Petunia hybrida, Nicotiana tabacum (tobacco), and oryza sativa in the understanding of plant-specific events, such as complex hormonal regulation, flowering, pathogenesis, etceteras. Transgenic Arabidopsis plants can be obtained by several transformation methods like Agrobacterium tumefaciens-mediated transformation by the transferred DNA of the tumor-inducing Ti plasmid (T-DNA) and gene gun bombardment (Clough and Bent, 1998; Taylor and Fauquet, 2002; Clough, 2005; Zhang et al., 2006; Ueki et al., 2009). A large collection of T-DNA mutagenesis lines is available from wellestablished Arabidopsis databases and stock centers to study the knock-out or knockdown effect of genes (www.arabidopsis.org; http://arabidopsis.info; Raina et al., 2002; Pan et al., 2003). Common gene silencing methods using double stranded RNA and microRNA work well in Arabidopsis and have contributed significantly in the understanding of numerous gene functions (Baulcombe, 2004; Schwab et al., 2005; Mansoor et al., 2006; Schwab et al., 2006; Ossowski et al., 2008; Bartel, 2009). In addition, it is easy to perform genetic analysis in Arabidopsis thaliana with its diploid genome and heterosexual reproduction mode. Furthermore, detailed analysis of many cellular and developmental processes is made possible in Arabidopsis by utilizing inducible systems like heat shock promoter, ethanol inducible system, and steroid hormone receptors inducible systems. In the glucocorticoid receptor (GR) post-translational activation system (Aoyama and Chua, 1997), hormone binding domain of rat glucocorticoid receptor is fused with a gene of interest, which normally encodes for a DNA binding factor. The translated protein fusion is retained in cytosol due to the binding of GR by heat shock protein. Upon receiving synthetic steroid dexamethasone (DEX), the fusion protein would then be translocated into the nucleus to provide the gene activity of interest. 1.2 Regulation of shoot apical meristem, inflorescence meristem and floral meristem in Arabidopsis thaliana In plants, there are two distinct apical stem cell pools governing the growth and differentiation at the shoot tip and root tip. The shoot apical meristem (SAM) and root apical meristem (RAM) can give rise to various organs and tissues to sustain postembryonic growth and differentiation in plants (Veit, 2004). During the vegetative growth phase, the cells in shoot apical meristem can divide and differentiate into leaf primordia. Upon receiving flowering transition signal (for transition to reproductive phase), the SAM turns into an inflorescence meristem (Figure 1), which divides and gives rise to flower primordia or secondary inflorescence meristems subtended by bracts. Each of these floral primordia is developed from a floral meristem (FM) and can be further developed into four specific floral organs, which are perianths (sepals and petals) and floral reproductive organs (stamens and carpels). The FM is terminated after the pre-determined organ primordia are generated. A normal Arabidopsis flower consists of four sepals (calyx), four petals (corolla), six stamens, and a pistil formed by two fused carpels (Zik and Irish, 2003). In contrast to floral meristems, shoot apical meristems and inflorescence meristems are indeterminate, which means it can continue to grow and differentiate into different organs depending on environmental cues it receives. The stem cells in shoot apical meristems reside in the L1 and L2 layers of the dome-shaped apical region termed central zone (CZ) (Carles and Fletcher, 2003; Sharma et al., 2003). The cells flanking the lateral sides of the central zone form the peripheral zone (PZ). These cells at the peripheral zone differentiate and give rise to new organ primordia. The rib zone (RZ) is located beneath the central zone and consists of cells that sustain the growth of stem. The regulation of stem cell niche in shoot apical meristem is a well-coordinated process in Arabidopsis (Sablowski, 2007). WUSCHEL (WUS) and SHOOT MERISTEMLESS (STM) are two key regulators for shoot apical meristem development (Clark et al., 1996; Gallois et al., 2002; Lenhard et al., 2002). WUS is expressed in a small population of cells at L3 layer beneath the L1 and L2 layers where the stem cells reside. It encodes for a homeodomain-containing transcription factor that plays a pivotal role in maintaining the stem cell pools. This population of WUS-expressing cells is commonly regarded as the organizing centre of shoot meristem. In wus mutants, the stem cell pools in shoot apical meristem is depleted precociously, which leads to premature termination of meristem activity (Mayer et al., 1998). STM is also a homeodomain-containing protein which is expressed in the meristem. It is suggested to play a less prominent role in meristem maintenance in comparison to WUS. WUS expression in the organizing centre is restricted by CLAVATA3 (CLV3). CLV3 is a secreted polypeptide that is expressed in L1 and L2 layers of the central zone (Rojo et al., 2002). The domain of cells that are expressing CLV3 is commonly regarded as stem cell pools in the meristem. CLV3 moves between cells and serves as a ligand for a receptor complex consists of CLV1 and CLV2 (Clark et al., 1993; Fletcher et al., 1999). Ectopic CLV3 activity causes instant suppression of WUS expression and a decrease of meristem activity (Reddy and Meyerowitz, 2005). In clavata mutants, the WUS expression domain is expanded and the meristem is enlarged. Interestingly, WUS has an instrumental role in the activation of CLV3 expression. This WUS-CLV regulatory feedback loop is instrumental in keeping the meristem size in check (Brand et al., 2000; Schoof et al., 2000). The growth of the indeterminate shoot apical meristem is eventually replaced by the growth of inflorescence meristem upon transition to reproductive development. The process of flowering transition (from vegetative meristem to inflorescence meristem) is accomplished by both internal and external signals, which converge on primary regulators for floral identity. Recently, the protein product of FLOWERING LOCUS T (FT) gene has been strongly suggested to be the much sought-after flowering signal (Huang et al., 2005; Corbesier et al., 2007). FT is expressed in leaves and its translated product is found to be transported from leaves to shoot apex though phloem to trigger flowering upon long-day exposure. In addition to FT, phytohormones like gibberellins (GAs) also play an important role in the induction of flowering transition (Blazquez et al., 1998). These flowering signals then activate a number of major regulators that are important for inflorescence meristem identity which include LEAFY (LFY), APETALA1 (AP1) and CAULIFLOWER (CAL). LFY is a transcription factor that has an important role in promoting floral determinacy, as well as inflorescence meristem determinacy (Schultz and Haughn, 1991; Weigel et al., 1992; Weigel and Nilsson, 1995). In lfy mutant, the floral meristems are transformed into inflorescence shoots. Conversely, overexpression of LFY transforms the inflorescence meristem into a terminal flower. AP1 belongs to a MADS (MCM-1, AGAMOUS, DEFICIENS, SRF)-domain protein family. It is a key determinant for both inflorescence meristem identity and floral organ identity (Irish and Sussex, 1990; Mandel et al., 1992; Mandel and Yanofsky, 1995). CAL is another MADS-domain protein closely related to AP1 (Kempin et al., 1995) . In ap1 cal double mutant, the floral meristem becomes indeterminate and produces ectopic meristems to form a cauliflower-like structure at the inflorescence apex. In contrary, AP1 overexpression transforms the inflorescence meristem into a terminal flower. Figure 1: Inflorescence meristem and floral meristem in Arabidopsis thaliana Upper panel: schematic diagram of inflorescence meristem and floral primordium in Arabidopsis thaliana. L1, L2, and L3 layer of the meristem is shown. CLV3 expression domain is highlighted in pink, and WUS expression domain is highlighted in green. Lower panel: a section of the meristem obtained from Ler wild-type plant. 1.3 Regulation of flower development in Arabidopsis thaliana During the processes of floral organ identity specification and floral organ differentiation, there are well-coordinated interplays of transcription factors. The classical ABC model was proposed nearly two decades ago to explain the organ identity determination in flower development (Coen and Meyerowitz, 1991). The ABC model predicts that the combinatorial action from ABC floral homeotic genes is largely responsible for the specification of the floral organ identity. The ABC genes, A class for APETALA1 (AP1) and APETALA2 (AP2), B class for APETALA3 (AP3) and PISTILLATA (PI), and C class for AGAMOUS (AG), have been extensively studied and have been shown to encode transcription factors (Bowman et al., 1989; Yanofsky et al., 1990; Bowman et al., 1991; Jack et al., 1992; Goto and Meyerowitz, 1994; Gustafson-Brown et al., 1994; Jofuku et al., 1994). Apart from its role in inflorescence meristem identity, AP1 also serves as an A-class gene for organ identity determination. AP1, together with another A-class gene AP2, is responsible for the specification of the first whorl of floral primordia into sepals (Irish and Sussex, 1990; Jofuku et al., 1994). AP2 encodes for a member of AP2/EREBP class of transcription factors. In combination with B-class genes activity, AP1 and AP2 promote development of the second whorl of floral primordia into petals. In ap1 mutants, the first whorl organs undergo homeotic conversion, which turns sepals into bracts subtended by floral meristems. In the meantime, the second whorl organs in ap1 mutant are lost. In ap2 mutants, sepals are converted into carpels or missing, whereas the petals in the second whorl are lost. B-class genes such as AP3 and PI act synergistically with both A-class and C-class genes to specify the development of second whorl and third whorl into petals and stamens, respectively (Jack et al., 1992; Goto and Meyerowitz, 1994) . In the absence of B-class genes, the second whorl organs are converted into sepals (in place of petals) and the third whorl organs are transformed into carpels (in place of stamens). Both AP3 and PI encode for members of MADS domain transcription factors. The C-class gene AG also encodes a member of the MADS domain transcription factors, and AG is necessary for the specification of stamens and carpels, the floral reproductive organs (Bowman et al., 1989; Yanofsky et al., 1990). In ag-1 mutants, the flower undergoes homeotic conversion to adopt a sepal-petal-petal reiteration instead of the normal sepal-petal-stamen-carpel structure. The complete lack of reproductive organs in ag-1 flowers places AG at the top of hierarchy of genes controlling reproductive development. This conclusion is supported by microarray expression profiling of wild-type and ag mutant flowers showing that more than 1, 000 genes are expressed downstream of AG (Wellmer et al., 2004). 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Meyerowitz (2007). The homeotic protein AGAMOUS controls late stamen development by regulating a jasmonate biosynthetic gene in Arabidopsis. The Plant Cell 19(11): 3516-3529. 152 [...]... of AHL 22 effectively delays the flowering process in Arabidopsis In spite of these reports, the function of AT-hook 22 DNA binding protein has yet to be demonstrated in flower development and plant meristem regulation 1.6 Objective of the study The aim of this study is to augment the understanding of flower development and meristem regulation in Arabidopsis thaliana through functional study of a novel... changes of the chromatin In addition, my study has also provided new insight for the understanding of meristem integrity in Arabidopsis thaliana I have shown that ectopic GIK activity effectively disrupts stem cell 23 regulation, and causes aberrant expression of WUS in inflorescence and floral meristems 24 CHAPTER 2: Materials and Methods 2. 1 Materials 2. 1.1 Plant materials All Arabidopsis thaliana. .. proteins (two molecules for each protein) in the specification of carpels 11 Figure 2: The ABCE model of flower development Schematic diagram showing the ABCE model of flower development Combinatorial action of A, B, and E-class genes specify the development of perianth organ Whereas, the B, C, and E-class genes determine the identity of reproductive organs 12 1.4 Patterning and differentiation of lateral... al., 20 07) In addition, AT-hook proteins are also likely to participate in the regulation of gene expression in response to jasmonic acids (JA) in suspension culture of Catharanthus roseus cells (Vom Endt et al., 20 07) Recently, AHL 22 (AT-HOOK MOTIF NUCLEAR LOCALIZED PROTEIN 22) has been reported to be involved in the regulation of flowering and hypocotyl elongation (Xiao et al., 20 09) Overexpression of. .. proteins forming protein complexes to regulate and specify the development of floral organs In each whorl, there will be a distinct combination of the four homeotic proteins, henceforth gives rise to different organ identities For instance, in whorl 2, there might be a combination of AP1, PI, AP3 and SEP proteins to specify petals Whereas in whorl 4, the protein complex might be a combination of AG and. .. also involved in the development of sepals, petals, stamens, and carpels (Ditta et al., 20 04) In sep1 sep2 sep3 sep4 quadruple mutans, all flower organs are converted into leaf-like organs Following the unraveling of SEP proteins function, the ‘quartet model’ of flower organ identity has been proposed to explain the combinatorial effects of ABCE genes (Theissen, 20 01; Theissen and Saedler, 20 01) In this... MARs and AT-hook DNA binding proteins are suggested to be the key determinants in anchoring specific DNA sequences to nuclear matrix, a process that helps to generate chromatin loop domain and also to introduce structural changes in the chromatin (Reeves, 20 01) In animals, the MAR binding protein SATB1, which contains an AT-hook DNA binding motif, has been implicated in tissue- or cell type-specific regulation. .. gene involved in the biosynthesis of a lipid-derived phytohormone, jasmonic acid AG binds to the 5’ coding region of the DAD1 gene, and activates its expression at later stages of floral development (days 7-8 after the onset of AG expression) 1.5 Functions of AT-hook DNA binding proteins during development AT-hook DNA binding proteins may contribute to a functional nuclear architecture by binding to... incubated at 65 °C for 5 min and later placed on ice for 1 min After the cold incubation, the mixture was added with 1 µL of 10× RT buffer, 2 µL of 25 mM MgCl2, 1 µL of 0.1 mM DTT, 0.5 µL of RNase OUT enzyme, and 0.5 µL of Superscript III enzyme in a 10 µL incubation system The solution was incubated at 50 °C for 70 min and then at 85 °C for 5 min Next, the solution was added with 1 µL of RNase H and. .. purified using a gel purification column (Qiagen) Around 2 µg of the purified plasmid DNA was mixed with 2 µL of 10× transcription buffer, 2 µL of 10× NTP-DIG mix, 2 µL of 100 mM DTT, 1 µL of RNase inhibitor, and 40 U of SP6 RNA polymerase in a 20 µL incubation system The reaction mix was incubated at 37 °C for 3 h Next, the in vitro transcribed RNA mix was added with 2 µL of RNase-free DNaseI and incubated . meristem and floral meristem in Arabidopsis thaliana Upper panel: schematic diagram of inflorescence meristem and floral primordium in Arabidopsis thaliana. L1, L2, and L3 layer of the meristem is. instrumental in keeping the meristem size in check (Brand et al., 20 00; Schoof et al., 20 00). The growth of the indeterminate shoot apical meristem is eventually replaced by the growth of. protein would then be translocated into the nucleus to provide the gene activity of interest. 1 .2 Regulation of shoot apical meristem, inflorescence meristem and floral meristem in Arabidopsis