DISSECTING THE HORMONAL CONTROL OF THE SALT STRESS RESPONSE IN ARABIDOPSIS ROOTS 1

          DISSECTING THE HORMONAL CONTROL OF THE SALT STRESS RESPONSE IN ARABIDOPSIS ROOTS YU GENG A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2013               ACKNOWLEDGEMENT   ACKNOWLEDGEMENT First I would like to thank my supervisor José R Dinneny He is the one who gave me the opportunity to start this amazing journey He is also the one giving me lots of help and support in the past four years He makes me became a better scientist I appreciate Department of Biological Science in National University of Singapore for allowing me to be a part of the top university in Asia I appreciate Temasek Life Sciences Laboratory and Carnegie Institution for Science for their generous financial support There is no way I can finish my Ph D training without them I would like to thank all the members in Dinneny’s lab for the discussions and help I am very lucky to work with these people I would like to thank people from Yu Hao’s lab in TLL, people from Wang Zhiyong’s lab in Carnegie, David Ehrhardt and Heather Cartwright for their valuable advice and help Finally, I would like to thank my family and friends for them to understand me, stand by me no matter what decision I made Aug 2013 Geng Yu i   TABLE OF CONTENTS   TABLE OF CONTENTS ACKNOWLEGEMENT i TABLE OF CONTENTS ii SUMMARY vii LIST OF TABLES ix LIST OF FIGURES x LIST OF ABBREVIATIONS AND SYMBOLS xiii Chapter LITERATURE REVIEW 1.1 Introduction 1.2 Salt stress in plants 1.2.1 Early effects 1.2.2 Long term effects 1.3 Salt stress signaling in plants 1.3.1 CBL-CIPK signaling network .7 1.3.1.1 Salt Overly Sensitive (SOS) pathway 1.3.1.2 CBL1/CBL9-CIPK23-AKT1 network and K+ transportation 1.3.2 Osmotic stress signaling 10 1.3.3 The transcriptional programs and phyto-hormones in salt stress response .11 1.3.3.1 ABA biosynthesis and signaling pathways, and its function in salt stress…………………………………………………………………………… 11 ii   TABLE OF CONTENTS   1.3.3.2 Jasmonates ( JAs) biosynthesis and signaling pathway and its function in salt stress 16 1.3.3.3 Ethylene (ET) biosynthesis and signaling pathway and its function in salt stress .18 1.3.3.4 Gibberellic acid (GA) and Brassinosteriod (BR), two positive growth regulators and their functions in salt stress 20 1.4 Arabidopsis root development 23 1.5 Cell type specific studies in Arabidopsis roots 25 1.6 Objective and significance of this study 28 Chapter MATERIAL AND METHODS .31 2.1 Plant materials .32 2.2 Plant growth conditions 32 2.3 Live-imaging and data analysis 33 2.4 Sample preparation, RNA isolation, microarray hybridization and data analysis: Agilent array 33 2.4.1 Protoplasting of roots and isolation of GFP-enriched cell populations by FACS 35 2.4.2 RNA isolation, microarray hybridization and data analysis: Affymetrix ATH1 arrays 35 2.5 Development of an eFP browser-style visualization tool for the spatiotemporal map 37 iii   TABLE OF CONTENTS   2.6 Secondary signaling network construction 37 2.7 Quantitation of ABA content .38 2.8 High-Throughput qRT-PCR 39 2.9 Fluorescence microscopy and image analysis 41 Chapter RESULTS AND DISCUSSION 43 3.1 ABSTRACT 44 3.2 INTRODUCTION 44 3.3 RESULTS 48 3.3.1 Growth regulation by salt stress is a multi-phasic process 48 3.3.2 Generation of a spatiotemporal global transcriptional map of the salt-stress response ……………………………………………………………………………53 3.3.3 Predominant expression patterns highlight both autonomy and coordination in the stress response of each cell layer 58 3.3.4 A cluster-comparison method identifies secondary hormone signaling events during salt stress 64 3.3.5 ABA biosynthesis and signaling are dynamically activated during the early stage of salt stress 67 3.3.6 ABA biosynthesis and signaling promote growth recovery during the late phases of the salt response……………………………………….……………… 69 3.3.7 Cell-type specific manipulation of ABA signaling highlights the importance of specific tissues in mediating growth responses to salt stress .72 iv   TABLE OF CONTENTS   3.3.8 Jasmonate activates defense pathways during salt stress and inhibits growth recovery………………………………………………………………………………74 3.3.9 Dynamic repression of GA and BR signaling during salt stress controls growth quiescence 77 3.4 DISCUSSION 84 Chapter RESULTS AND DISCUSSION 88 4.1 ABSTRACT 89 4.2 INTRODUCTION 90 4.3 RESULTS 93 4.3.1 During salt stress Arabidopsis roots undergo dramatic morphological changes 93 4.3.2 Ethylene is involved in the morphological changes of the roots during salt stress 97 4.3.3 Salt promotes cell swelling by elevating ethylene production 102 4.3.3.1 Salt enhances ACSs expression .102 4.3.3.2 The endodermis is crucial for cortical cell swelling by mediating ethylene production at transcriptional level during salt stress 105 4.3.3.3 Post-transcriptional regulation of ACSs is involved in salt-mediated cortical cell swelling 110 4.3.4 Salt activates ethylene signaling in the early elongation zone dynamically during salt stress 113 4.3.5 Salt-responsive ethylene signaling downstream components show cell-type specificity in their expression 116 v   TABLE OF CONTENTS   4.3.6 Auxin signaling serves as downstream signaling of ethylene in regulating saltmediated cell swelling .122 4.3.6.1 Possible functions of auxin transportation in salt-mediated cell swelling……………………………………………………………………… 123 4.3.6.2 Auxin local synthesis in epidermis of the early elongation zone is important for salt-mediated cortical cell swelling .127 4.4 DISCUSSION .131 Chapter CONCLUSION 137 REFERENCES 140 vi   SUMMARY   SUMMARY Plants have to face a constantly changing environment as they grow in the soil In order to survive, growth and signaling need to be dynamically and precisely regulated during the transition from non-stress conditions to stress conditions However, little is known about how the progress through this transition is controlled In this study, by using live-imaging, we identified that growth regulation in Arabidopsis roots during salt stress is a multiphasic process: a brief period of quiescence was induced, followed by recovery and homeostasis In order to dissect the transcriptional regulation behind this phenomenon, we built a high-resolution spatiotemporal transcriptional map for salt stress using fluorescence-activated cell sorting and microarray-based transcriptome profiling By using this map and genetic analysis, we were able to characterize the key hormonal signaling pathways that are involved in transcriptional and growth regulation during salt stress and identified the time window and spatial domain of where they act By using confocal microscopy, we found that primary roots undergo drastic cell-typespecific morphological changes during salt-induced growth quiescence We focused on one of these morphological changes, cortical cell swelling, to understand the distinct functions of different cell types during salt stress By using genetic and bioinformatic analyses, we found that ethylene plays an important role in regulating salt-mediated cortical cell swelling Salt elevates ethylene production in an endodermis-dependent manner, and ethylene signaling activates auxin signaling in the epidermis and possibly triggers cell wall modifications in cortical cells to promote radial expansion vii   SUMMARY   Together, our data reveal a sophisticated assortment of regulatory programs acting together to coordinate spatially patterned biological changes involved in the immediate and long-term response to a stressful shift in environment viii     A END/PER B C D Figure 11 Cell-layer specific ABA signaling controls spatially growth A) Expression of the UAS::erGFP reporter from the four different GAL4-VP16 enhancer-trap lines used in this study J0951 drives expression in the epidermis (EPI) and lateral root cap (LRC), J0571 expresses in the cortex (COR) and endodermis (END), Q2500 expresses in the END and pericycle (PER) cell layers and Q0990 expresses in the stele (STE) B) Relative root length of various transactivation lines after transfer to salt stress conditions Relative root length calculated as the ratio between the length under salt-stress conditions and that under standard conditions after growth for days P value< 0.0001 C) Live imaging analysis of the Q2500>>abi1-1 transactivation line and associated control under salt-stress conditions Control genotype standard 72       (n=16), Control genotype 140 mM NaCl (n=24), Q2500>>abi1-1 standard (n=16), Q2500>>abi1-1 140 mM NaCl (n=24) D) Live imaging analysis of the J0951>>abi1-1 transactivation line and associated control under salt-stress conditions Control genotype standard (n=16), Control genotype 140 mM NaCl (n=24), J0951>>abi1-1 standard (n=16), J0951>>abi1-1 140 mM NaCl (n=24) Growth data are presented as a percent difference in growth rate relative to standard conditions Error is SEM The data in Figure 11A,B were generated by Lina Duan 3.3.8 Jasmonate activates defense pathways during salt stress and inhibits growth recovery Based on our bioinformatic analysis, JA signaling was predicted to promote the expression of genes in different salt-regulated clusters (Figure 8B) Clusters 12, 20 and 23 were stably up-regulated after hours of salt response Cluster 22 was the exception and was repressed by salt, however this cluster was also strongly associated with an IAA-responsive sub-module that was predicted to repress the expression of this cluster and this may modify the effects of JA signaling on gene expression All three clusters tended to be induced by salt stress in the inner tissue layers of the root indicating some spatial control of this signaling pathway Indeed, based on the spatiotemporal map, JAI3, which functions as a repressor of the JA pathway, showed EPI enriched expression throughout the salt response (Figure 12A) Clusters 20 and 23 were both enriched for genes associated with biotic defense (Cluster 20, callose deposition in cell wall during defense response, P-value < 1E-9, glucosinolate biosynthetic process P-value < 1E-7, Cluster 23, response to biotic stimulus, P-value < 73       1E-6) By using PI staining we observed that large proportion of cells in early elongation zone was damaged during salt stress (Figure 12B) The damage in cell wall may be the trigger of those biological processes They are not obviously related to the regulation of growth; however, previous studies have shown that JA can also negatively regulate both meristem size and cell elongation (Chen et al., 2011) Thus, we asked whether loss of JA signaling affected the growth of the root during salt stress using the JA resistant mutant allele jai3-1, which encodes a negative regulator of the pathway that is resistant to JAmediated protein degradation (Chini et al., 2007) Under salt stress, jai3-1 mutant roots entered the recovery phase hour earlier than wild type and showed an increased growth rate during the first hours of the homeostasis phase (Figure 12C) Both results are consistent with a role for JA in growth repression during these later phases of the salt response 74       A C B Std NaCl Figure 12 JA may be activated by cell damage at inner tissue layers resulting in suppression of growth recovery A) JAI3, which encodes a repressor of JA signaling showed enriched expression in the EPI layer This correlated with an overall low level of induction of JAregulated genes during the salt stress response B) Cells in meristem and elongation zone were damaged after salt stress Propidium iodide was used as an indicator of apoptosis C) Live-imaging analysis of salt response in the primary root in Col-0 and jai3-1 mutants Col-0 standard (n=8), Col-0 140 mM NaCl (n=14), jai3-1 standard (n=8), jai3-1 140 mM NaCl (n=16) 75       3.3.9 Dynamic repression of GA and BR signaling during salt stress controls growth quiescence We next turned our attention to hormone pathways known to promote growth in the root GA sub-modules were associated with salt-regulated clusters (cluster 19 and 21), which showed early transcriptional repression and late recovery during the stress response (Figure 8C) Both clusters showed peak expression in the COR and/or EPI tissue layers suggesting that GA levels may decline in these regions of the root While we were not able to reproducibly detect the various forms of GA in our study, other studies have shown that GA levels fall upon salt treatment and the RGA protein, which is a repressor of GA-mediated growth, is stabilized (Achard et al., 2006) The ProRGA:GFP:RGA reporter can be used to track GA signaling, as GA perception leads to the degradation of the GFP:RGA protein Indeed, we observed that GFP:RGA fluorescence intensity increased between five to eight hours after salt treatment and diminished by 48 hours (Figure 13A and 13B), which correlated well with the predicted dynamics of GA signaling The reduction in GFP:RGA fluorescence late in the salt response suggested that GA signaling may partially recover at these times and play a positive role in the reactivation of growth We tested this hypothesis by performing live imaging of salt-treated roots supplemented with paclobutrazol (PAC), a GA-biosynthesis inhibitor (Rademacher, 2000) In contrast to the control salt treatment where roots recovered growth between to 10 hours after treatment, PAC treated roots were strongly inhibited in their recovery (Figure 13C) To test the effects of increased GA signaling on the salt-stress response, 76       we performed live-imaging analysis of the della quadruple mutant (Tyler et al., 2004) The effects of this mutant were subtle, but did reveal increased growth at later time points (Figure 13D) Together these data showed that GA biosynthesis and signaling are likely necessary during the late phases of the salt response to promote recovery and suggested that dynamic changes in the biosynthesis of this hormone are critical for determining the temporal pattern of growth regulation during salt stress 77       A B C D Figure 13 GA signaling was dynamically regulated during salt stress A) Confocal images showing the maximum intensity projection of fluorescence from a root tip expressing the ProRGA1:GFP:RGA1 reporter Fluorescence is shown in roots after transfer to standard or salt-stress media for various lengths of time Fluorescence signal shown using the Rainbow LUT setting in ImageJ to more clearly show differences in intensity (blue, low; red, high) B) Quantitation of GFP intensity at different time points after salt treatment in ProRGA1:GFP:RGA1-expressing roots 78       C) Effect of PAC treatment on growth under standard or salt-stress conditions PAC treatment strongly inhibited the ability of the root to recover growth rates after salt treatment Col-0 standard (n=8), Col-0 140 mM NaCl (n=15), Col-0 10µM PAC (n=8), Col-0 140 mM NaCl with 10µM PAC (n=15) D) Effect of the della quadruple mutant on the salt response Significant differences in growth rates were only observed during the recovery phase La(er) standard (n=8), La(er) 140 mM NaCl (n=16), della quadruple standard (n=8), della quadruple 140 mM NaCl (n=16) Growth data are presented as a percent difference in growth rate relative to standard conditions Error is SEM The data in Figure13A and B were generated by Rui Wu BR signaling is predicted to promote the expression of genes in the same salt responsive clusters as GA (Figure 8C) Thus, similar to GA, we predicted that BR signaling was dynamically suppressed during salt stress and contributed to the temporary reduction in growth rate observed during these time points BR perception leads to dephosphorylation of the BRASSINOZOLE RESISTANT (BZR1) transcription factor, nuclear localization of the protein and regulation of downstream transcriptional targets such as DWARF4 (DWF4) (Wang et al., 2002; He et al., 2005) We utilized the ProBZR1:BZR1:YFP reporter line to profile changes in BR signaling over time during salt stress Fluorescence intensity in nuclei of cells in the elongation zone of the root showed a significant decrease after hour of treatment (Figure 14A and 14B) After 24 hours, the average fluorescence intensity recovered Quantification of the YFP signal in nuclei was difficult to interpret under salt stress as the size of the nucleus was affected by the treatment Thus, total fluorescence was also quantified per nucleus and clearly showed that the amount of BZR1:YFP in the nucleus was not significantly different at 24 hours of growth on standard or salt stress media (Figure 14C) These results suggested 79       that BR signaling was temporarily suppressed during the early phase of the salt response and recovered later These data are also consistent with the dynamic expression pattern of DWF4, which is directly repressed by BZR1 (He et al., 2005); DWF4 expression increased in the STE and peaked at hours of salt treatment, but then fell back to normal levels between 20 to 32 hours (see Supplemental Figure 10B online) Consistent with the hypothesis that genes in clusters 19 and 21 are targets of BR signaling during salt stress, we found significant enrichment of BZR1 targets in both gene sets (P-value < 6E-8 and 0.03, respectively) (Oh et al., 2012) To test whether recovery of BR signaling was important for promoting growth during salt stress, we examined the response of the bzr1-D mutant, which causes constitutive activation of many BR-dependent processes (Wang et al., 2002) Interestingly, brz1-D mutant roots showed a substantially reduced quiescent phase and recovered growth hours earlier than wild type (Figure 14D) In addition, growth rates during the homeostasis phase were also higher To test whether BR signaling was also necessary for growth recovery, we examined the brassinosteroid insensitive 1-5 (bri1-5) mutant, which mildly impairs BR signaling (Noguchi et al., 1999) As expected, we detected a subtle yet significant decrease in recovery phase growth, consistent with the proposed function of BR in promoting growth recovery during salt stress (Figure 14E) 80       Figure 14 BR signaling is dynamically suppressed during salt stress and is sufficient to promote early growth recovery (A) Maximum projection of fluorescence from confocal images of roots expressing the ProBZR1:BZR1:YFP reporter Fluorescence signal shown using the Rainbow LUT setting in ImageJ to more clearly show differences in intensity (blue, low; red, high) (B) Quantification of average fluorescence of nuclei in the epidermal tissue layer from the elongation zone Note that a significant decrease in average fluorescence was observed at hour of treatment Fluorescence recovered under 81       salt-stress conditions but dropped under standard conditions These differences may be due to changes in the size of the nucleus (C) Total fluorescence quantified in nuclei of the epidermal tissue layer in the elongation zone Note that measuring total fluorescence corrected for changes in nuclear volume and showed that significant differences in BZR1:YFP accumulation are only observed at hour after treatment (D) The bzr1-D mutation had a significant effect on the growth profile of the root under salt stress conditions, where the time to recovery was hastened by approximately hours Growth rates were also higher during the recovery phase Col-0 standard (n=8), Col-0 140 mM NaCl (n=16), bzr1-D standard (n=8), bzr1D 140 mM NaCl (n=16) (E) The bri1-5 mutant is weakly insensitive to BR treatment and showed a modest reduction in growth recovery compared to the Ws ecotype control Ws standard (n=8), Ws 140 mM NaCl (n=16), bri1-5 standard (n=8), bri1-5 140 mM NaCl (n=16) Growth data are presented as a percent difference in growth rate relative to standard conditions Error is SEM 82       3.4 Discussion We describe here the first cell-type resolution analysis of the full temporal landscape of an environmental response in plants from the initial moments of perception to the longterm adjustments leading to transcriptional and growth-rate homeostasis By analyzing both growth and transcriptional regulation at high-temporal resolution and defining transitions in biological functions using our multi-dimensional microarray data set, we were able to parse out several critical regulatory pathways important for the salt response (Figure 15) We have defined the hormonal signals predicted to have the largest role in transcriptional control, identified their target pathways and cell types, determined their time of action and placed this regulation in the developmental context of dynamic root growth control We have found that dynamic changes in root growth are not simply a consequence of the inability of the cells to expand, but instead are regulated processes orchestrated by hormonal growth repressors and activators that act during discrete phases to hasten or delay the time at which root growth resumes We speculate that the transition from quiescence to growth recovery may be under the control of many separate physiological pathways acting through different hormones to precisely advance the root through the stress response time course at the appropriate pace Hormonal control of root growth during salt stress: a model In work presented here as well as another study we have recently published (Duan et al., 2013), we show that ABA signaling acts through internal tissue layers to regulate growth In the lateral root, ABA signaling acts to suppress growth for an extended length of time, with growth recovery occurring after days of salt treatment At 100 mM NaCl, the 83       primary root does not strongly activate ABA signaling and we observed that disruption of ABA signaling largely has lateral root-specific effects on growth Here, we showed that at 140 mM NaCl, ABA signaling was briefly activated in the primary root with peak ABA levels correlating with growth quiescence Thus, both the primary and lateral roots can activate ABA signaling, but at different threshold concentrations of salt and for very different lengths of time Interestingly, while ABA signaling in the lateral root has clear functions in suppressing growth, in the primary root, our data suggests a role in promoting growth ABA, like many other hormones, has the property that low levels of the hormone promote growth while high levels inhibit growth (Finkelstein and Rock, 2002) In this regard it is intriguing that the phase of the salt response where ABA biosynthesis is critical for promoting growth is during the Recovery and Homeostasis phases where we have measured much smaller differences in ABA accumulation compared to control conditions Our proposed role for ABA in promoting long-term growth recovery during salt stress is similar to that proposed in maize where it has been shown that under water stress, low levels of ABA (~1 micromolar) promote growth of the primary root through the suppression of ethylene signaling (Spollen et al., 2000) Our work also identifies important new roles for other plant hormones in the salt stress response and places these pathways in a temporal context Hyperactivation of BR transcriptional regulation using the bzr-1D mutant indicates that the timing of growth recovery during salt stress can be dramatically hastened by as much as five hours These 84       data, together with the observations we made using the BZR1-YFP reporter, indicates that a critical outcome of salt-stress signaling is the temporary suppression of the BR pathway Importantly, growth is still strongly repressed for several hours even in bzr1-D mutants, which indicates that some component of growth regulation may occur independent of BR hormone regulation The immediate suppression of growth that occurs with 140 mM NaCl treatment may be a consequence of the physical impediment to cell elongation that is expected to occur after a dramatic change in the osmotic environment of the root Our work shows that dynamic hormonal signaling is necessary to properly time the recovery of growth These events likely require tight coordination with the regulation of other biological processes important for long-term acclimation to a saline environment It will be critical for future studies to understand the mechanisms that control the rate of hormone biosynthesis and catabolism and how these pathways ultimately control the growth of the root through cell-type specific signaling Furthermore, while our secondary signaling network has identified little transcriptional evidence of cross-talk between the hormonal pathways, post-transcriptional cross-talk mechanisms and non-hormonal signaling pathways such as Calcium ions and Reactive Oxygen Species are likely to be important mechanisms for signal integration 85       Figure 15 Model for spatiotemporal dynamics in hormone signaling and growth control during the salt-stress response Diagram shows the inferred timing and spatial pattern of activity for ABA, JA, GA and BR signaling during the salt-stress response time course ABA levels peak during the ST and QU phases of the salt response, but lower levels present during the RE and HO phases promote growth recovery Based on experiments using tissue-specific suppression of ABA signaling, the inferred site of ABA in growth regulation is the END/PER tissue layers, however regulation of gene transcription appears to occur through signaling in all tissue layers JA levels are predicted to rise during the RE and HO phases, which are when the jai3-1 mutant has an effect on growth rates and when JAassociated transcriptional programs are induced during the salt response GA and BR signaling is co-regulated with peak levels occurring under non-stress conditions Based on the expression pattern of GA and BR-associated transcriptional targets and dynamic changes in fluorescent-based reporters of pathway activity, we infer that GA and BR signaling recovers during the RE and HO phases of the salt-stress response Reestablishment of pathway activity is necessary to promote growth recovery 86     ... 1. 3 .1. 2 CBL1/CBL9-CIPK23-AKT1 network and K+ transportation 1. 3.2 Osmotic stress signaling 10 1. 3.3 The transcriptional programs and phyto-hormones in salt stress response .11 1. 3.3 .1. .. indicating the profound roles of BR in cell elongation (Zhao et al., 2 012 ) The key components of BR signaling include the cell-surface receptor kinase, BRASSINOSTEROID INSENSITIVE1(BRI1), the co-receptor... SOS1 and enhances K+ uptake by activating AKT1 1. 3.2 Osmotic stress signaling During salt stress, plants increase the synthesis of osmoprotectant osmolytes to adjust the water potential in the