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

TABLE OF CONTENTS iii 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 funct

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

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

TABLE OF CONTENTS

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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 1 LITERATURE REVIEW 1

1.1 Introduction 2

1.2 Salt stress in plants 3

1.2.1 Early effects 3

1.2.2 Long term effects 6

1.3 Salt stress signaling in plants 6

1.3.1 CBL-CIPK signaling network 7

1.3.1.1 Salt Overly Sensitive (SOS) pathway 7

1.3.1.2 CBL1/CBL9-CIPK23-AKT1 network and K+ transportation 9

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

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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 2 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

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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 3 RESULTS AND DISCUSSION 1 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

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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 4 RESULTS AND DISCUSSION 2 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

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4.3.6 Auxin signaling serves as downstream signaling of ethylene in regulating

salt-mediated 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 5 CONCLUSION 137

REFERENCES 140

SUMMARY

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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 specific 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

cell-type-SUMMARY

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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

LIST OF FIGURES

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LIST OF FIGURES

Figure1 Diagram of Arabidopsis root structure……… …24

Figure 2 Time-lapse imaging system and semi-automated image analysis……….50 Figure 3 Salt treatment caused dynamic changes in growth………52 Figure 4 Generation of a spatiotemporal gene expression map of the response……….56 Figure 5 Expression pattern of genes in the spatiotemporal salt response data set with

known tissue-specific expression in roots……….57

Figure 6 Spatiotemporal expression patterns observed during the salt response……….61 Figure 7 Fluorol Yellow staining shows an enrichment of suberin deposition during the

salt response……… 63

Figure 8 Analysis of the hormone secondary signaling network regulating

salt-dependent transcriptional programs……… …………66

Figure 9 ABA biosynthesis and signaling are regulated at the spatiotemporal level

during salt stress……….……… 68

Figure 10 ABA biosynthesis and signaling are necessary to promote full growth

recovery during salt stress……….…….70

Figure 11 Cell-layer specific ABA signaling controls spatially growth……… 73 Figure 12 JA may be activated by cell damage at inner tissue layers resulting in

suppression of growth recovery……….76

Figure 13 GA signaling was dynamically regulated during salt stress………79

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Figure 14 BR signaling is dynamically suppressed during salt stress and is sufficient to

promote early growth recovery……… …82

Figure 15 Model for spatiotemporal dynamics in hormone signaling and growth control

during the salt-stress response……… 87

Figure 16 Arabidopsis roots undergo drastic cell shape changes during salt stress……95

Figure 17 Cell shape change is irreversible due to the rigid plant cell walls…….…… 96 Figure 18 Ethylene is a potent cell shape regulator that may be involved in salt-mediated

cortical cell swelling ………98

Figure 19 Mutations in the genes that are involved in ethylene canonical signaling

pathway impair salt-mediated cortical cell radial expansion but not inhibition of cell elongation……….100

Figure 20 The expression of ACS2, ACS6, ACS7 and ACS8 is regulated by salt in a

spatiotemporal fashion………103

Figure 21 Endodermis is important for salt-mediated cortical cell swelling….………106

Figure 22 Salt responsive ACSs are mis regulated under shr background………108

Figure 23.shr defect in salt-mediated cell swelling can be rescued by both exogenous

and endogenous applied ACC……… 109

Figure 24 Post transcriptional regulation may be involved in salt elevating ethylene

production………111

Figure 25 Ethylene signaling is dynamically activated during salt stress……… 114

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Figure 26 Large number of salt-responsive genes are mis-regulated in the ethylene

receptor mutant, etr1-1 background………118

Figure 27 GO analysis of ETR1-dependent salt responsive EIN3 direct target…… 119 Figure 28 Spatiotemporal expression of ETR1-dependent salt responsive genes…….120 Figure 29 Auxin signaling is involved in regulating salt-mediated cortical cell swelling

downstream of ethylene signaling pathway……….123

Figure 30 The re-location of PIN2 protein is correlated with cell radial expansion,

AUX1 may promote cell elongation during salt stress………124

Figure 31 Expression profile of TAA1 gene after salt stress; Cell width phenotype after

salt treatment in taa1 mutants ………128

Figure 32 The abundance of TAA1 protein starts to increase in the early stage and

becomes stable at later time points of salt stress in epidermis of transition zone…… 130

Figure 33 Model for NaCl promoting cortical cell swelling through ethylene and auxin

signaling pathways……… 136

LIST OF ABBREVIATIONS AND SYMBOLS

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LIST OF ABBREVIATIONS AND SYMBOLS

Chemicals and reagents

LIST OF ABBREVIATIONS AND SYMBOLS

RT-PCR Reverse transcription polymerase chain reaction

Chapter 1 LITERATURE REVIEW

1.1 Introduction

All organisms need to interact with their surroundings to survive Most of plants are sessile organisms Compared to non-sessile organisms, such as most animals, when facing adverse conditions, plants don’t have many choices but to adapt to them

Therefore, perception, response and adaptation are fundamental processes that occur in plants Most plants are multicellular organisms Different cell types need to coordinate to conduct those processes This makes environment stimuli powerful tools to dissect the interaction between different cell types in plants

Salt stress is one of the most severe environmental stresses More than 6% of the world’s total land area is contaminated with salt And this problem is becoming even worse due to

the irrigation or land cleaning (Munns et al., 2008) High salinity has a negative effects

on plant growth and development Understanding how plants respond to salt is not just an interesting biological question but also can help us to improve food security by breeding crops that are more resistant to salt

Decades of research into the effects of salinity on plant physiology and development and how plants respond to salt at organ or organism level have generated a wealth of

information However, our understanding of salt response at cell and tissue levels is still

rudimentary Due to the simplicity, Arabidopsis roots have been use as a model system to study cell-type specific responses to salt stress (Dinneny et al., 2008) Based on these studies, we can clearly see that, at least in Arabidopsis, most salt-stress regulation occurs

in a tissue or cell specific fashion ( Dinneny 2010)

In this review, we are going to summarize the research progress on salt stress and salt

response in plant, introduce Arabidopsis root structure and development, and discuss how the cell-type specific analyses in Arabidopsis roots has contributed to our knowledge of

the interactions between cell types

1.2 Salt stress in plants

The negative effects of high salinity in plant growth are believed to result from water

stress, ion toxicities, ion imbalance or a combination of these factors ( Kurth et al., 1986)

Here we are going to discuss about how salt affects plant growth and development in the order of when those events occur

1.2.1 Early effects

The growth of plant cell is determined by the plastic response of the wall to the

mechanical force exerted by the turgor pressure against the cell wall (Kroeger et al.,

2011) So plants need to constantly uptake water from the environment to maintain the turgor pressure of the cells High salinity decreases water potential of the soil, therefore reducing water availability to plants Here we use salt-induced osmotic stress to refer the situations where insufficient water availability that is caused by high salinity limits plant

growth and development (Zhu et al., 1997) It has been shown that most early effects of

salt in plant growth are caused by osmotic stress (Munns 2002)

Salt shock triggers dramatic changes in growth rates First, the growth is strongly

inhibited just after few minutes of salt treatment, then it recovers gradually in the next 30 mins or more before reaching the plateau (regaining the homeostasis) The time taken to recover and the growth rates in the new homeostasis stage are salt concentration

dependent This growth profile is solely driven by the change of water availability, since when applied with water directly, leaves can grow normally even under salt stress

(Munns et al., 2000)

The rapid and transient reductions in growth rates after salt shock are also observed in roots Compared to leaf growth, root growth recovers better after sudden exposure to high salinity: Maize roots grow slower than growing under normal condition at the early time points of 150 mM NaCl treatment, while during later time points the initial growth rates

was restored (Rodríguez et al., 1997) Again since other osmotica such as KCl or

mannitol can trigger the similar effects, it is believed that the effects are caused by induced osmotic stress It has been shown that the inhibition of cell elongation is due to plants adjusting water relations in cytoplasm at the expanse of the ability of cell walls to

salt-expand ( Iraki N et al., 1989)

However, other studies also show that supplemental Ca2+ can alleviate the deleterious

effects of salt stress in cotton root growth (Cramer et al 1987) Even at 150 mM NaCl,

elongation of sorghum roots was reduced by only 20% if supplemental Ca2+was present,

but by 80% if it was not (Colmer et al 1996) This was believed to result from Na+

inhibiting Ca2+ uptake from soil or displacing membrane-bound Ca2+ (Kurth et al., 1986)

However, recent studies show that the reason why supplemental Ca2+ can increase the salt tolerance is that Ca2+ can help to keep the balance between K+ and Na+ (Luan et al.,

eventually affects the final growth output The imbalance between these two ions

triggers the CBL-CIPK signaling by changing calcium levels in the cytosol, and it will further interact with and regulate the proteins that involved in the uptake and

translocation of K+ and Na+, maintaining the ‘‘balance’’ of these cations in plants under

stress conditions ( Luan et al., 2009)

High salinity not only inhibits the cell elongation, it can also promote the cell radial

expansion in cotton roots (Kurth E et al., 1986) It is also observed in Arabidopsis

(Burssens et al., 2000) However, the timing of this event was not well characterized in these studies We found cortical cells in Arabidopsis roots start to expand radially after 4

hours of 140 mM NaCl treatment (See Chapter 4 Figure 16) And we did not observe a similar phenomenon after 300 mM mannitol treatment (Data not show) These indicate cell radial expansion is a salt specific effect

In summary, although most of early effects are caused by osmotic stress, there are still some effects, such as ectopic cell radial expansion, can be the results of ionic stress or the combination of osmotic and ionic stress

1.2.2 Long term effects

When salt sensitive plants suffer from high salinity for days, the damages become visible

in older leaves, as observed in white lupin when exposing to 100 mM or higher

concentration of NaCl (Munns et al., 1988) Salt injury is due to salt sensitive plants

lacking the ability of transporting Na+, Cl- , or both into vacuoles or vacuoles ceasing to

take up incoming salt (Munns et al., 1993) When Na+ or Cl- accumulate in the cytoplasm, they will inhibit enzyme activity When they build up in cell walls, the cells will be

dehydrated and eventually killed (Munns & Passioura 1984) As NaCl accumulates

overtime, the older leaves will die There may not be enough photosynthetic surfaces for plants to survive

1.3 Salt stress signaling in plants

Salt stress can trigger the osmotic and ionic stress signaling Here we will describe the ionic stress signaling, also known as the CBL-CIPK signaling network, and then we will present the osmotic stress signaling

1.3.1 CBL-CIPK signaling network

As mentioned before, the increase of Na+ concentration in cytoplasm triggers CBL-CIPK signaling network by changing free cytosolic Ca2+ concentration

1.3.1.1 Salt Overly Sensitive (SOS) pathway

SOS pathway is a part of the CBL-CIPK network It mainly contains SOS3, SOS2 and SOS1 They are all identified by forward genetic approach

SOS3 (CBL4) encodes a myristoylated EF hand calcium-binding protein that works as a primary calcium sensor to perceive the increase in cytosolic Ca2+in plants under salt stress The EF hand structure can bind Ca2+ The myristoylation at the N-terminus of this protein is important for its ability to associate with the plasma membrane It has been shown that the mutation that abolishes the calcium binding ability leads to increased salt

sensitivity And the inhibition of myristoylation in wild type plant can mimic sos3 mutant

phenotype, indicating that this lipid modification is important for SOS3 to fulfill its

function during salt stress (Ishitani et al., 2000).

Upon binding to Ca2+, SOS3 interacts and activates its downstream component, SOS2 (CIPK24) However, SOS2 is not the only target It has been shown that SOS3 can interact with other proteins, such as SOS2-like proteins, to mediate osmotic stress

induction of abscisic acid (ABA) biosynthesis (Zhu 2002)

CBL10 also function through SOS2 cbl4/cbl10 double mutants show much stronger phenotype than both of the single mutants and overexpression of CBL10 can partially rescue the sos3 mutant, indicating that they work redundantly

Based on GUS reporter expression, CBL4 is mainly expressed in roots And CBL10 is

mainly expressed in the vasculatures of leaves and shoot tissues When exposed to 75

mM NaCl, both cbl10 and cbl4 mutants show strong phenotype in both root and rosette leave growth However, cbl10 mutants have longer roots than cbl4; cbl4 mutants have bigger rosette leaves than cbl10 These results suggest that CBL10 mainly works in shoot tissues and leaves, whereas CBL4 mainly works in roots (Quan et al., 2007).

CBL10 may have additional functions related to Na+ sequestration into vacuoles (Kim et

al., 2007)

SOS2 encodes a serine/threonine protein kinase It has two domains: a kinase domain and

a regulatory domain The kinase domain is essential for SOS2 to activate downstream

components The regulatory domain contains a NAF motif (Albrecht et al., 2001) and a

protein phosphatase interaction (PPI) motif Based on in vitro analysis, the regulatory domain has an auto-inhibitory function in regulating N-terminal kinase domain SOS3 and CBL10 activate SOS2 kinase activity by binding to the NAF motif in the regulatory domain, disabling the inhibitory effect Phosphorylation is also very important for SOS2 enzyme activity ABI2 is a PP2C type protein phosphatase It is a negative regulator of ABA signaling It has been reported that ABI2 can bind the PPI motif, dephosphorylate

SOS2 to deactivate it This indicates a crosstalk between ABA signaling and SOS2

pathway (Gong et al., 2004)

SOS1 is one of SOS2’s substrates It is the first locus that identified in SOS pathway It

is a Na+/H+ antiporter that located in the plasma membrane It controls ion homeostasis

by transporting Na+ out of cells and loading Na+ into xylem vessels (Shi et al., 2002)

Based on the protein structure, it has been predicted that SOS1 may also work as a sensor

to sense Na+ concentration and form a regulatory loop by regulating and being regulated

by SOS2 (Zhu 2002)

1.3.1.2 CBL1/CBL9-CIPK23-AKT1 network and K+ transportation

The majority of the salt-induce cell damage is due to ion imbalance between Na+ and K+

Therefore, enhancing the K+ transportation into the cell is as important as pumping Na+ out of the cytosol Here we will summarize the research progress on CBL1/CBL9-

CIPK23-AKT1 network and its function in increasing K+ uptake during salt stress

CBL1 is involving in regulating salt, cold, osmotic, and even wounding responses It has

been shown that mutation in CBL1 leads to low tolerance to salt, drought and cold Interestingly overexpression of CBL1 can enhance the survival under salt and drought stress, but lower the tolerance to cold (Cheong et al., 2003) CBL9 works as a negative regulator of ABA biosynthesis Mutation in CBL9 leads to hypersensitivity to salt,

drought and ABA, duo to the elevated ABA contents in seedlings (Pandey et al., 2004)

Despite the specific functions of these two CBLs they share one common target called CIPK23 It has been shown in several studies that CBL1 and CBL9 both target CIPK23 that functions in the regulation of K+ uptake and stomatal movements Interestingly both

cbl1 and cbl9 mutants do not have K+ uptake defects but cbl1/cbl9 double mutants show

strong K+ deprivation phenotype, which indicates that CBL1 and CBL9 work redundantly

in regulating CIPK23 (Luan et al 2009) CIPK23 was first identified from a mutant screen Mutations in CIPK23 lead to drought tolerance due to the defect in stomatal

opening Phenotypic and biochemical analyses also show that CIPK23 positively controls

K+ uptake Further genetic analyses indicate that CBL1and CBL9 work upstream of

CIPK23 (Cheong et al., 2007) One potential target of the CBL1/9-CIPK23 pathway is

Arabidopsis K+ transporter 1 (AKT1) Patch-clamping studies using root hair cells

demonstrate that AKT1 activity in the cipk23 and cbl1/cbl9 double mutant is significantly reduced (Li et al., 2006), suggesting that AKT1 is a downstream component of this

pathway

In summary, increasing Na+ concentration in the cytosol triggers free Ca2+ flow in the cell, activating CBL-CIPK signaling network In addition, CBL-CIPK signaling network reduces Na+ contents by pumping Na+ out through Na+/H+ antiporter 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 cytoplasm to overcome osmotic stress Glycine betaine (GB),

proline and mannitol are major osmolytes Overexpressing GB biosynthesis gene leads

plants to tolerate stresses, including salt stress (Rhodes et al., 1993) It was also observed

that GB-treatment can significantly increase the tolerance to high salinity The amino acid proline is another well-known osmolyte that accumulates in cytosol under stress and

is correlated with osmotic adjustment to improve plant salinity tolerance P5CS1

(Pyrroline-5-Carboxylate Synthase 1) is important for proline synthesis It has been shown that mutations in this gene impair the tolerance to high salinity and exogenous

proline can rescue the defects (Székely et al., 2008) P5CDH (pyrroline-5-carboxylate dehydrogenase) controls proline degradation Mutations in P5CDH gene lead to proline

hypersensitivity During salt stress reactive oxygen species (ROS) accumulate in plants This triggers a series of aatural antisense short interfering RNA (nat-siRNA) processing

steps that lead to the degradation of P5CDH transcripts This process, at some level, increases the proline concentration in plants (Borsani et al., 2005) Overexpression of celery mannonse-6-phosphate reductase in Arabidopsis protects plants only from salt

stress, but not from drought stress in growth and photosynthesis, suggesting mannitol can

protect chloroplasts against salt (Munns et al., 2008)

1.3.3 The transcriptional programs and phyto-hormones in salt stress response 1.3.3.1 ABA biosynthesis and signaling pathways and its function in salt stress

ABA is a well-known stress induced hormone It was discovered in the early 1960s It was thought that ABA is involved in the abscission of fruit and dormancy of woody plants However, the role of ABA in these processes is still not very clear ABA

biosynthesis increases by water deprivation The increasing ABA contents help plants to

acclimate the stress by mediating stomatal closure and activating some transcriptional programs In seeds, ABA is important for the dormancy In this section first we will introduce ABA biosynthesis and degradation pathway Then we will talk about ABA perception and signaling transduction

ABA is derived from C40 carotenoids, although it only has 15 carbon atoms It is not a surprise that many mutants that have defects in carotenoid synthesis also have defects in

seed dormancy like most of the viviparous mutants in maize The core ABA synthesis

pathway starts from zeaxanthin Zeaxanthin epoxidase (ZEP) catalyzes the conversion of

zeaxanthin to violaxanthin Mutations in ZEP lead to a severe reduction in ABA contents, often resulting in stomatal closure and seed dormancy defects In Arabidopsis, ZEP is encoded by ABA1 gene It was first identified from a reporter based mutant screen

Mutations in ABA1 gene impair the expression of ABA reporter genes, such as RD29A,

KIN1, RD22 and ADH under salt stress aba1 mutants are smaller and have shorter life

span compared to wild type plants They are also prone to water loss and this defect can

be rescued by applying exogenous ABA (Xiong et al., 2002) How violaxanthin converts

to 9’-cis-Neoxanthin and 9’-cis-violaxanthin is still not clear Nine-cis-epoxycarotenoid dioxygenase (NCED) enzymes cleave the cis-isomers of violaxanthin and neoxanthin to a

C15 product xanthoxin and a C25 metabolite It is the rate-limiting step in ABA

biosynthesis In Arabidopsis NCED is encoded by NCED gene family and five of them (AtNCED2,3,5,6 and 9) have already been cloned and studied (Tan et al., 2003)

AtNCED2 and AtNCED3 mainly work in the roots based on their expression pattern The

expression of AtNCED3 increases dramatically after salt or drought treatment

Interestingly, mutations in AtNCED3 lead to higher tolerance to NaCl and KCl but lower

tolerance to sorbitol, LiCl and drought treatment Exogenous ABA can rescue the

hypersensitivity of nced3 mutants to LiCl and drought, and ethylene treatment in wild type plants can mimic the LiCl hypersensitive phenotype of nced3 mutants, suggesting a crosstalk between ABA and ethylene in stress response AtNCED3 is responsible to

inhibit ethylene production by elevating ABA contents during stress condition (Ruggiero

et al., 2004) These results also indicate ethylene can regulate salt tolerance in plants

positively, but drought tolerance negatively Our results show that AtNCED3 expression

is dynamically activated only at the early stage of salt stress in endodermis specifically

(Geng et al., 2013), suggesting the spatio-temporal regulation of ABA synthesis during salt stress AtNCED2 also responds to drought, but our data suggest it is not strongly responsive to salt treatment compared to AtNCED3 ABA2 catalyzes the conversion of xanthoxin to abscisic aldehyde in Arabidopsis Since ABA2 protein is encoded by a single gene ABA2, loss-of-function of this gene largely impairs ABA synthesis under both non-stress and drought condition aba2 mutant plants are much smaller than wild

type, indicating that ABA is also important to plant growth under non-stress condition

(Nambara et al., 1998) Abscisic aldehyde oxidase (AAO) catalyzes the final step in

ABA synthesis, which is the oxidation of the abscisic aldehyde to abscisic acid In

Arabidopsis, the knockout of AAO3 gene, one of the four AAOs, causes severe defect in

ABA production in both dry seeds and rosette leaves under non-stress, drought and salt stress conditions These results demonstrate that compared to other AAOs, AAO3 plays a

major role in ABA synthesis (González-Guzmán et al., 2004)

CYP707A is important for ABA degradation in Arabidopsis It encodes ABA

8’-hydroxylases It has been shown that recombinant CYP707A protein coverts ABA to PA

in vitro (Nambara et al., 2005)

ABA receptors, Pyrabactin Resistance 1/ PYR-Like proteins/ Regulatory Components of ABA Receptor (PYR1/PYLs/RCARs), were recently identified by four independent

studies with chemical genetic and biochemical approaches (Ma et al., 2009; Park et al., 2009; Melcher et al., 2009; Santiago et al., 2009) These proteins belong to a family of

START domain proteins They contain a central hydrophobic pocket that is flanked by two mobile loops ABA binding to the hydrophobic pocket triggers the conformational changes of these two mobile loops It creates a new interaction surface for binding to the type 2C protein phosphatases (PP2Cs)

Many PP2Cs, such as ABA INSENSITIVE 1 (ABI1), ABI2, HOMOLOG OF ABI1 (HAB1), are well-studied negative regulators of ABA signaling It has been reported that

expressing PP2C ectopically inhibits the sensitivity to ABA (Kuhn et al., 2006) The

binding prevents PP2C from accessing their substrates: SNF1 (Sucrose-Non fermenting Kinase1)-related protein kinases OST1/SnRK2.6/SnRK2E, SnRK2.2/SnRK2D and

SnRK2.3/SnRK2I (Agepati et al., 2010) The interaction between PP2C and these

kinases leads to the inactivation of SnRK2s via dephosphorylation of multiple positions

in their activation domains These kinases are responsible for initiating the transcriptional programs that lead to ABA or drought responses via activating some transcription factors

(Fujita et al., 2009; Mizoguchi et al., 2010) These transcription factors include

ABFs/AREBs (ABA-responsive Element Binding Factor/Protein), basic region/leucine zipper (bZIP)-type transcriptional regulators like ABI5 Besides activating transcription factors directly, SnRK2s also have other functions in regulating ABA signaling OST1, also called SnRK2.6 can phosphorylate the anion channel SLAC1 and the cation channel

KAT1 to activate the former and deactivate the later (Raghavendra et al., 2010) SLAC1

and KAT1 are crucial for stomata movement Interestingly, these two ion channels are also regulated by CIPK23, indicating the integration of CBL-CIPK signaling network and core ABA signaling pathway There are other transcription factors that are key

components of core ABA signaling but not directly regulated by SnRKs ABI3 is one of them It belongs to B3 transcriptional regulator, which can bind to ABI5 and enhance its

action Genetic analyses reveal that together with FUS3 and LEC1, ABI3 is crucial for

chlorophyll and anthocyains accumulation, activation of the members of 12S storage

protein gene family (Parcy et al.,1997) ABI3 protein is unstable The ABI3-interacting protein (AIP2), which is E3 ligase, can mediate the degradation of ABI3 (Zhang et al.,

2005) The ChIP-chip analysis identified 177 promoters bound by ABI3 Since the

primary function of ABI3 is to mediate seed development, not surprisingly most of ABI3

target genes are expressed in the seeds (Mönke et al., 2012)

The cis-acting element ABRE (ABA-responsive element, PyACGTGGC), in ABA

responsive genes’ promoter regions is crucial for their abilities to respond to ABA and osmotic stress It is believed that those genes respond to osmotic stress through ABA signaling However, a single copy of ABRE is not sufficient for ABA-responsive

transcription It needs to have either some coupling elements, such as CE1 and CE3 in

wheat HVA1 and HVA22 genes, or multiple copies, like in Arabidopsis RD29B

(Yamaguchi-Shinozaki et al., 2006)

ABA is crucial for salt tolerance Many ABA mutants show altered tolerance to salt stress

An interesting case we would like to highlight here is aba2 mutants which has a severe

defect in ABA synthesis The loss-of-function of this gene leads to lower sensitivity to salt on germination, but higher sensitivity at later stages of development and overall

survival rate (González-Guzmán et al., 2002) These results suggest that ABA plays an

important role in arresting the germination, promoting growth and increasing the survival rate under high salinity environment

1.3.3.2 Jasmonates (JAs) biosynthesis and signaling pathway and its function in salt stress

Jasmonate (JA) and its derivatives are lipid-based hormones They are involved in wide spectrum of biological processes that are essential for plant development, reproduction and defense response JA was named for methyl jasmonate (MJ), an organic volatile

compound derived from Jasminum grandiflorum long used in the perfume industry MJ is

only one of many jasmonate isomers

JA is the best-known and best-characterized member among all the JAs The biosynthesis

of JA starts from the release of α-linolenic acid (α-LeA 18:3) from chloroplast

membrane into the interior in response of environmental stimuli, such as wounding It

was oxidized by 13-lipoxygenase (LOX), forming

13-hydroperoxy-9,11,15-octadecatrienoic acid (13-HPOT) 13-HPOT is converted into 12-oxophytodienoic acid (OPDA) OPDA is the direct precursor of JA It is converted to JA in peroxisomes Furthermore, JA is conjugated to L-isoleucine, forms jasmonoyl-isoleucine (JA-Ile) which is a major biologically active jasmonate among a growing number of jasmonate

derivatives in the cytosolic compartment (Gfeller et al., 2010)

CORONATINE INSENSITIVE 1(COI1) is crucial for JA signaling transduction It

encodes an F-box component of an SCF (SKIP–CULLIN–F-box) complex These

complexes have E3 ubiquitin ligase activity The SCFCOI1 complex is responsible for the JA-mediated protein ubiquitination With the presence of JA-Ile, SCFCOI1 binds to

JASMONATE-ZIM-DOMAIN (JAZ) containing proteins and mediates their degradation through 26S proteasome JAZs regulate JA-activated transcriptional programs negatively

by inhibiting the bHLH transcriptional activator MYC2/JIN1 MYC2 binds the G-box (CACGTG) or the T/Gbox (AACGTG) in the promoters of JA-regulated genes There are other transcription factors that are also involved in JA-responsive gene regulation, such

as ERF1, AtERF2, AtERF4, ORA47 and ORA59 But it is not clear if they are regulated

by JAZs (Fonseca et al., 2009)

Like ABA, JA is believed to be a growth repressor, as it can repress primary root growth

and reduce meristem activity under non- stress condition (Chen et al., 2011) However, it

has been shown that exogenous JA can promote growth under salt tolerance through

elevating ABA and GA contents in chard plants (Kim et al., 2009) Since systemin

signaling can activate JA signaling, it is not a surprise that it can also increase salt

tolerance in tomato (Orsini et al., 2010)

1.3.3.3 Ethylene (ET) biosynthesis and signaling pathway and its function in salt stress

Ethylene, a simple hydrocarbon gas, is a plant hormone, acts as a pivotal mediator to respond to and coordinate internal and external cues in modulating plant growth

dynamics and developmental programs (Yoo et al., 2009) Ethylene is believed to play

roles in many core processes in root growth and development Impaired ethylene

perception interrupts root hair patterning and formation (Dolan and Roberts, 1995) Ethylene regulates gravitropic response by inducing the accumulation of auxin transport

regulator, flavonoid (Buer et al., 2006) The interplay between ethylene and auxin is also important for regulating root growth, especially cell elongation (Ruzicka et al., 2007; Swarup et al., 2007)

Ethylene is originally synthesized from methoinine and there are two major intermediates among this process: S-adenosylmethionine (S-AdoMet) and 1-aminocyclopropane-1-

carboxylic acid (ACC) (Kevin L.-C W et al., 2002) In general, ACC synthase (ACS)

activity is very low in tissues that do not produce significant quantities of ethylene Upon stimulation, ACS activity is rapidly induced In contrast, ACO activity is constitutively present in most vegetative tissues Therefore, ACS is the rate-limiting enzyme and is the

major regulatory step in ethylene induction (Kim et al., 2003) ACS is encoded by a big

gene family Most of them were found to function in a spatial and temporal-specific

manner and respond to many environmental stimuli (Tsuchisaka et al., 2004)

Ethylene signaling transduction is well characterized Five membrane-bound receptors of ethylene are known as ETR1 (ETHYLENE INSENSITIVE 1), ETR2, ERS1

(ETHYLENE RESPONSE SENSOR 1), ERS2, and EIN4 (ETHYLENE INSENSETIVE 4) They form a complex with a serine-threonine kinase, CTR1 (CONSTITUTIVE

TRIPLE RESPONSE 1) The receptor complex serves as a negative regulator in ethylene signaling transduction It represses the downstream component EIN2 by phosphorylating

it with the absence of ethylene With the presence of ethylene, CTR1 is released from the receptor/CTR1 complex and becomes inactive As a result, EIN2 becomes active

Furthermore, EIN2 is cleaved into two parts The C-terminal of EIN2 enters into the nucleus and stabilizes EIN3/EIL1/EIL2 transcription factors, which will activate or

repress downstream genes to trigger ethylene responses (Stepanova et al., 2005, Qiao et

al., 2012)

Ethylene is long recognized as a stress hormone During salt stress, genes that involved

in ethylene biosynthesis and signaling transduction are up-regulated (Ma et al., 2006) Genetics studies show mutations in ethylene signaling components, such as ETR1, CTR1,

EIN2, EIN3, EIL1, can cause alterative salt sensitivity, which suggests that ethylene plays

important roles in salt response (Lei et al., 2011; Achard et al., 2006; Cao et al., 2007)

It has been shown recently that ETHYLENE RESPONSE FACTOR1 (ERF1) regulates salt tolerance positively The expression of ERF1 is massively increased under high salt

condition (150 mM NaCl) Although the overexpression of this gene only has a very mild effect on increasing the root growth under salt stress, the seed germination and survival

rates are dramatically increased And interfering the expression of this gene by RNAi

leads to hypersensitivity to salt (Cheng et al., 2013)

1.3.3.4 Gibberellic acid (GA) and Brassinosteriod (BR), two positive growth

regulators and their functions in salt stress

Biologically active gibberellins (bioactive GAs) plays important roles in various

processes in plant growth and development, including seed germination, stem elongation, leaf expansion, and flower and seed development Among over a hundred of identified GAs from plants, only GA1, GA3, GA4, and GA7, are believed to be major active forms

They are biosynthesized by very complicated pathway GAs are biosynthesized from geranylgeranyl diphosphate (GGDP) Three different classes of enzymes are required for the biosynthesis of bioactive GAs from GGDP in plants: terpene synthases (TPSs),

cytochrome P450 monooxygenases (P450s), and 2-oxoglutarate–dependent dioxygenases (2ODDs)

GA1 encodes one of the two TPSs It catalyzes the conversion of geranylgeranyl

pyrophosphate (GGPP) to copalyl pyrophosphate (CPP) of gibberellin biosynthesis The

ga1-3 mutant allele displays severe developmental defects, such as dwarfism, lack of

trichome, male sterile and difficulty in germination Another TPS, kaurene synthase, is encoded by GA2 It catalyzes the second step in the cyclization of GGPP to ent-kaurene

in the gibberellins biosynthetic pathway The ga2-1 mutant allele also shows dwarf phenotype (Yamaguchi 2008; Ogawa et al., 2003)

The first GA receptor was found in rice It was believed to be a membrane protein based

on biochemical studies (Lovegrove et al., 1998) Surprisingly, it is a soluble nuclear protein that is encoded by GIBBERELLIN INSENSITIVE DWARF1 (GID1) gene

(Ueguchi-Tanaka et al.,2005) There are three GID1 homolog in Arabidopsis (GID1A,

GID1B and GIDC) that work redundantly (Nakajima et al., 2006) With the presence of

GA, GID1 binds the central repressors of GA signaling, DELLA proteins and mediates

the degradation of them by the 26S proteasome There are five DELLAs in Arabidopsis:

GA-INSENSITIVE, GAI; REPRESSOR OF GA1-3, RGA; RGA-LIKE1, RGL1; RGL2 and RGL3 They have their unique roles but in some cases they also work redundantly For example, RGL2 has a function in inhibiting seeds germination but it also work with RGL1 and RGL2 to modulate floral development Besides that, RGA and GAI regulate vegetative growth negatively; RGL3 has a function in abiotic stress response It has been shown recently that GA works in a tissue-specific manner; endodermal is very important

for GA signaling to fulfill its functions in root growth (Davière et al., 2013)

It has been shown that the inhibition of GA signaling during salt stress results the

inhibition of primary root growth However, the inhibition of GA signaling is crucial for

plants to survive under salt stress condition, since GA biosynthesis mutants ga1-3 show higher resistance whereas quadruple-DELLA mutants are less resistant to salt (Achard et

al., 2006)

BRs are a group of polyhydroxylated steroidal hormones that are believed to be the positive regulator of plant growth Brassinolide (BL) is the final product of BR

biosynthetic pathway and the most active BR

DET2 and DWF4 are essential for BR signaling Biochemical analyses indicate that

DET2 is involved in converting (24R)-ergost-4-en-3-one (4-en-3-one) to (24R)- ergost-3-one (3-one), which is the second step in the BR specific biosynthesis pathway DWF4 was found to catalyze steps like CR to 22-OHCR, 4-en-3-one to 22-OH-4-en-3-one, and 3-one to 22-OH-3-one Recessive mutations of these two encoding genes result

5α-in the same dwarf phenotype Further analysis us5α-ing microscopy demonstrated that the phenotype is due to the smaller cell size but not less cell number, indicating the profound

roles of BR in cell elongation (Zhao et al., 2012)

The key components of BR signaling include the cell-surface receptor kinase,

BRASSINOSTEROID INSENSITIVE1(BRI1), the co-receptor kinase

BRI1-ASSOCIATED RECEPTOR KINASE1(BAK1), the GSK3-like kinase

BRASSINOSTEROID INSENSITIVE2 (BIN2) and two transcription factors

BRASSINAZOLE RESISTANT1 (BZR1) and BZR2 With the presence of BR, BRI1 and BAK1 inactivate BIN2 through other components, therefore abolishing the inhibitory effect of BIN2 on BZR1 and BZR2 Then these two transcription factors enter into the

nucleus from cytoplasm and activate downstream gene expressions (Zhu et al., 2013)

BR and GA are both positive growth regulators It has been shown recently that GA can regulate BZR1 activity through DELLA proteins in a BR perception and BIN2 de-

activation independent fashion (Zhu et al., 2013)

BRs can restore the level of chlorophyll and increase the activity of nitrate reductase enzyme during salt stress Nitrate reductase is very important for nitrogen supplement, growth and productivity of plants BRs have no effect on cell ultrastructure under normal conditions, but significantly reduce the damage induced by salt stress on nuclei and

chloroplast (Fariduddin et al., 2013).

1.4 Arabidopsis root development

As plants grow in soil and as soil is a very complex and heterogeneous system, plants need to acclimatize to fluctuating environmental conditions, Root is the organ that directly contacts soil, absorbs water and nutrients, senses the environmental cues and responds to them correspondently These features make roots become perfect model on studying the interaction between plants and environment

Arabidopsis roots are very simple They are rotationally symmetric Especially in young

roots, each tissue type only has one cell layer (Figure 1) The root can be artificially divided into 3 different regions: Meristem, elongation zone and maturation zone

In maturation and elongation zone, the Arabidopsis root can be mainly divided into four

cell types: Epidermis (EPI), Cortex(Cor), Endodermis (End) and Stele (Ste) Stele can be simply further divided into pericycle, procambian and vascular tissue In meristem, there

are three cell types: quiescent center (QC), Lateral root cap (LRC) and collumella (COL) Besides these mature cells, there are some stem cells surrounding the quiescent center in the meristem to generating new daughter cells

Each cell type is generated from distinct stem cells For example: Epidermal cells are generated by EPI/LRC initial cells; endodermis and cortex cells are generated by End/Cor initials; cells in stele are generated by Stele initials and collumella cells are generated by collumella initials These stem cells are all surrounding QC, which defined by auxin

maxima In wei8-1/tar2-1 mutants, auxin synthesis is disrupted; the mutants lose the

meristem in 7 days after germination These results indicate QC identity and auxin

maxima are required for meristem or stem cell maintenance As mentioned before,

Arabidopsis roots are rotationally symmetric and each tissue type consists of one cell

layer The radial patterning is also precisely controlled For example, the identities of cortical and endodermal cells are dependent on two GRAS transcription factors:

SCARECROW and SHORT-ROOT In both shortroot (shr) and scarecrow (scr) mutants,

there are only one instead of two ground tissue layers; we call that existing ground tissue

layer “mutant layer” Although they are all called mutant layer, the mutant layer in scr

mutants has mixed cortical and endodermal identity, the mutant layer in shr mutants only has cortical identity (Di Laurenzio et al., 1996) The overexpression of SHR leads to extra

ground tissue formation (Helariutta et al., 2000) These results suggest that both SHR and

SCR are important to promote cell periclinal cell division, but SHR is crucial for cell

specification Further studies also indicate that SHR is important for stele patterning since shr

mutants also show abnormal structure in stele