DISSECTING THE HORMONAL CONTROL OF THE SALT STRESS RESPONSE IN ARABIDOPSIS ROOTS 2

Here, we show that ethylene promotes radial expansion of cortex cells through the canonical ethylene signaling pathway, while its precursor ACC may have an ethylene-independent function

Chapter 4 RESULTS AND DISCUSSION 2

4.1 Abstract

High salinity is an important agricultural contaminant that causes damage to the plant However, the distinctive roles of different cell types in the transition process from normal growth to stress acclimation are largely unknown Here, we show that ethylene promotes radial expansion of cortex cells through the canonical ethylene signaling pathway, while its precursor ACC may have an ethylene-independent function of inhibiting cell

elongation during salt stress By using mutants that have radial patterning defects, we show that salt-mediated induction of ethylene biosynthetic pathway members at the transcriptional level depends on the endodermis In order to find the components that work downstream of ethylene in regulating salt-mediated cell swelling, we performed microarray experiments with ethylene signaling mutants at early stages of salt stress Bioinformatic analysis revealed cell-type specificity in the expression pattern of the downstream targets of ethylene during salt stress, indicating the cell-type specific

function of ethylene in regulating the salt response Further, we demonstrated that local synthesis of auxin in the early elongation zone serves as a downstream component of ethylene signaling during salt stress Based on these observations, we that ethylene promotes salt-mediated cortical cell swelling through auxin signaling in an endodermis-dependent manner

4.2 Introduction

Plants, through intricate compositions of cell and tissue types, are good planners In favorable habitats, they plan their lives in a simple and effective way However, when they are facing stressful environments, they need to quickly coordinate their different tissue layers, conduct complex regulation to change their developmental and

physiological plane to adapt to such environments Salt stress is one of the most common environmental stresses It creates both osmotic and ionic stress to affect plant growth Decades of research into the effects of salinity on plant physiology and development have generated a wealth of information However, the distinctive roles of different cell types in the transition process from normal growth to stress adaptation are largely unknown In

this study, we used a particular salt-sensitive plant, Arabidopsis thaliana, focusing on one

environmental stress, high salinity, in order to understand how plants make this transition and how different cell types contribute to this process

High salinity has complex effects on root physiology These effects are mainly caused by both osmotic stress and ionic stress When the rhizosphere soil is contaminated with toxic soluble molecules, like NaCl, the water potential of the soil become lower, making it more difficult for plants to take up water from the environment When the cells are

suffering from dehydration, the turgor pressure of the cells against their cell walls is reduced, which can reduce the rigidity of plants and make them more vulnerable to wounding Along with water deprivation becoming more and more severe, many

biological processes, such as photosynthesis, are disrupted (Allen et al., 2001)

Furthermore, due to their similar chemical properties, the high amount of Na+ will break the K+/Na+ balance, leading to K+ deprivation by engrossing ion channels, which are normally used to transport K+ into the cell (Rubio et al., 1995) It has been shown that K+

is crucial for the activity of many enzymes; lack of K+ will largely affect plant

development and growth (Shabala et al., 2008)

When exposed to salt stress, the Arabidopsis root undergoes dramatic morphological changes, which include the inhibition of primary root elongation, reduction of meristem

size, and inhibition of lateral root formation in a dosage-dependent manner (Burssens et al., 2000; West et al., 2004; Wang et al., 2009) Besides generally affecting root growth,

high salinity also leas to cell type–specific morphological changes For example, the epidermis shows an immediate suppression of hair outgrowth, whereas the cortex

undergoes radial expansion, which may act to prevent additional uptake of salt into the

vasculature (Burssens et al., 2000; Dinneny et al., 2008; Dinneny, 2009).

It has been shown that high salinity increases ethylene production (Achard et al., 2006)

Interestingly, like high salinity, ethylene and its precursor ACC can also inhibit root cell elongation and promote cell radial expansion Previous studies have shown that ethylene regulates root growth, especially through the inhibition of cell elongation This occurs largely through the production and transportation of another important phytohormone,

auxin (Ruzicka et al., 2007; Swarup et al., 2007) However, there is a report showing that

the canonical ethylene signaling pathway may not be necessary for promoting radial cell

expansion This report suggested that ACS5, a member of ACS family, can regulate radial cell expansion by directly binding to two leucine-rich repeat receptor kinases, FEI1

and FEI2 (Xu et al., 2008).

In this study, by focusing on one particular morphological change, cortical cell swelling during salt stress, we provided a detailed analysis on the distinctive functions of different cell types in this process We show that the endodermis is important for the promotion of ethylene production by salt The epidermis is crucial for the activation of auxin

biosynthesis by the canonical ethylene signaling pathway in order to promote cell

expansion during salt stress We also show that ethylene signaling target genes that are enriched in cortex that may be directly involved in regulating salt-mediated cell

expansion

of roots recovering growth rates in the homeostasis phase, the inhibition of cell

elongation was partially released (Figure 16A) Due to the rigidity of plant cell walls, all

of these morphological changes are irreversible after the cells enter into the maturation zone, allowing us to observe them after they occur (Figure 17A) Among these

morphological changes, the inhibition of cortical cell elongation and radial expansion were very consistent, easy to observe with microscopy, and quantifiable Figure 17 shows quantification of cortical cell length (Figure 17B) and cell width (Figure 17C) along the longitudinal axis from the transfer point to the root tip From the transfer point, the first four cells gradually become shorter, while the cell width of the first four cells largely remained unchanged (Figure 17B and C) This indicates that the inhibition of cell

elongation may happen earlier than radial expansion during salt stress When comparing

Figure 17B to Figure 3B, we found the primary root growth pattern and the profile of cell length shared a certain level of similarity This suggests that inhibition of cell elongation contributes to inhibition of growth during salt stress.

We next focused on cortical cell shape changes to understand the roles of different cell types in acclimation during salt stress To simplify the analysis, we used the ratio

between cell width and cell length (Figure 17D) of the 10 cells that showed the most dramatic cell shape changes (Figure 17B and C, marked in grey) to represent the cortical cell shape changes

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

A) Plasma membrane marker WAVE131: YFP showed morphological changes in

Arabidopsis roots at different time points during salt stress Yellow arrows point

at the first cell undergoing ectopic radial expansion Noted after 4 hours of salt treatment, the cells in the early elongation zone started to expand radially Scale bar= 200 µm

B) Cortical cell length from quiescent center to elongation zone

C) Cortical cell width from quiescent center to elongation zone Cell width increased

in the early elongation zone after 4 to 5 hours of salt treatment

Figure 17 Cell shape change is irreversible due to the rigid plant cell walls

A) Salt-mediated cortical cell swelling is easy to observe under confocal microscopy

FM 4-64 was used as counterstain to highlight the cell shape

B) Quantification of cortical cell length from the transfer point Grey area highlights the cells that are most strongly affected by salt

C) Quantification of cortical cell width from the transfer point Grey area highlights the cells that are most strongly affected by salt

D) Phenotype was presented as the ratio between cell width and cell length of those cells that show most dramatic changes Error=SEM

4.3.2 Ethylene is involved in the morphological changes of the roots during salt stress

Ethylene and its precursor ACC have been described to cause an increase in the width of the root (Smalle and Van Der Straeten, 1997) and a rapid decrease in cell elongation (Lee

et al., 2001) To test if the ethylene precursor ACC can cause morphological changes in

cortex cells similar to those caused by salt treatment, we treated Arabidopsis wild type

Columbia-0 (Col-0) seedlings with 140mM NaCl and different concentrations of ACC

As shown in Figure 18A, ACC can cause a concentration-dependent effect on cortical cell swelling: from 0.5 µM to 1.3 µM, ACC can cause very similar cell shape changes with 140mM NaCl As mentioned before, high salinity elevates ethylene production

(Achard et al., 2006) It is possible that the cortical cell swelling under high salinity is

due to elevated ethylene production

To test this hypothesis, we treated Col-0 with amino oxyacetic acid (AOA) in the

presence or absence of 140 mM NaCl using methods similar to the previous experiment AOA inhibits enzymes that require pyroxidal phosphate, including ACS, the key enzyme for ethylene biosynthesis The results are shown in Figure 18B Compared to seedlings under salt treatment alone, there was a significant reduction in cell swelling in the

seedlings that were treated with both salt and AOA Together with the results from the

previous experiment, this indicates that ethylene production has a function in

salt-mediated cortical cell swelling

Figure 18 Ethylene is a potent cell shape regulator that may be involved in salt-mediated

cortical cell swelling

A) ACC shows a concentration-dependent effect on cell shape Noted that the effect could be saturated since 1.3µM ACC caused less cell swelling than 1 µM ACC B) ACC synthase inhibitor AOA is sufficient to decrease salt-mediated cell swelling Asterisks represent significant differences based on Student’s t-test P value<0.05

FM 4-64 was used as counterstain to highlight the cell shape Grey arrows point to the cortical cell file Scale bar= 50µm

It has been reported that the canonical ethylene signaling pathway might not be necessary

for promoting cell radial expansion (Xu S et al., 2008) To test if ethylene signaling has a

function in promoting cortical cell radial expansion, we investigated the inhibition of cell

elongation and cell radial expansion separately in etr1-1, ein2-1, and ein3-1/eil1-1 As

shown in Figure 19, all three of these mutants showed significantly reduced radial

expansion compared to wild type after salt stress Surprisingly, we found that all three mutants had normal salt response in terms of the inhibition of cell elongation, indicating that mutations in these three ethylene signaling components cannot affect cell elongation during salt stress

One possible explanation is that the canonical ethylene signaling pathway is required for promoting radial cell expansion, but ethylene or ACC regulates cell elongation through

an alternative signaling pathway It is also possible that ethylene has a function in

promoting radial cell expansion, while ACC regulates cell elongation in an independent fashion

ethylene-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

A) etr1-1 shows less cell radial expansion, but similar rate of cell elongation

compared to wild type

B) ein2-1 has similar phenotype with etr1-1 mutant

C) The cell radial expansion phenotype in ein3-1 / eil1-1 double mutants is less severe than in etr1-1 mutants, but the difference between wild type and ein3-1 / eil1-1 is still significant

FM 4-64 was used as counterstain to highlight the cell shape Grey arrows point to the cortical cell file Scale bar= 50µm Error= SEM Asterisks represent significant

differences based on Student’s t-test P value<0.0001

4.3.3 Salt promotes cell swelling by elevating ethylene production

4.3.3.1 Salt enhances ACSs expression

We investigate how salt regulates ethylene production ACC synthase is the rate-limiting enzyme in the ethylene synthesis pathway The ACC synthases form a large gene family, most of whose members have been found to function in a spatial- and temporal-specific

manner in response to many environmental stimuli (Tsuchisaka A, et al., 2004) By using

the spatio-temporal transcriptional map of salt response, we found that out of eight

functional isoforms of ACS (Tsuchisaka A, et al., 2009), four isoforms were responsive

to salt treatment: ACS2, ACS6, ACS7 and ACS8 (Figure 20 A to D) As shown in Figure

20 A to D, their spatio-temporal expression patterns were all different from each other In

addition, different tissue layers showed different responses to salt For example, ACS2

showed a strong decrease in expression in the endodermis after 3 hours of salt treatment; however, in the stele the expression gradually increased until 8 hours of salt treatment In order to profile their expression patterns at the organ level at different time points, we validated the microarray data by Q-PCR with whole roots As shown in Figure 20 E to H,

we found that ACS2 expression is activated by salt in the early stage of salt stress; the

expression of all four genes was immediately increased after one hour of salt treatment

These results suggest that the increased expression of the ACS genes could contribute to

the elevated ethylene production under salt stress

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

spatiotemporal fashion

A-D) eFP representation of expression for salt responsive ACSs They all have higher

expression level in the inner tissue layers

A) ACS2, AT1G01480 Noted that this gene is highly enriched in endodermis

B) ACS6, AT4G11280

4.3.3.2 The endodermis is crucial for cortical cell swelling by mediating ethylene production at the transcriptional level during salt stress

Although the expression patterns of the four salt-responsive ACS genes show distinctive

spatio-temporal profiles, ACS isoforms function redundantly (Tsuchisaka A, et al., 2009)

It is possible that even a multiple-mutant of all four salt-responsive ACS genes would not show any phenotype during salt stress We wanted to test if altered root tissue patterning

could change the expression pattern of the ACSs which could lead to altered ethylene

production If elevated ethylene production during salt stress is responsible for cortical cell swelling, we may see different levels of cortical cell swelling under salt stress when modulating root tissue patterning

Here, we utilized two mutants that have defects in root radial patterning, shortroot (shr) and scarecrow (scr) Figure 21A shows a diagram of the root structures of wild type and

these two mutants Instead of two layers of ground tissue, these two mutants only have

one, which is called the mutant layer In scr, the mutant layer has a mixture of cortical and endodermal identities; in shr, the mutant layer has cortical identity, and as a

consequence, this mutant lacks an endodermis(Di Laurenzio et al., 1996) We first

checked the cell swelling phenotype in these two mutants, as shown in Figure 21B

Although the roots of these two mutants have very similar radial structures, they have

very different responses to salt in cortical cell swelling Compared to wild type, the shr

mutant showed a significant reduction of cortical cell swelling

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

A) Diagram of Col-0, scr, shr radial section.

B) shr-salk shows less cell swelling phenotype, while scr-3 mutant has a very

different response to salt, indicating the lack of an endodermis but not the presence of only one layer of ground tissue caused less cell swelling

FM 4-64 was used as counterstain to highlight the cell shape Grey arrows point to the cortical cell file Scale bar= 50µm Asterisks represent significant differences based

on Student’s t-test P value<0.0001 Error= SEM

As we expected, we found ACS2, ACS6, ACS7 and ACS8 were mis-regulated in the shr background (Figure 22) ACS2 and ACS6 have impaired responses to salt in the mutants; while ACS7 and ACS8 were expressed higher under standard conditions, and repressed instead of activated by salt in the shr background

Since SHR is a transcription factor, it is possible that SHR might directly regulate these

salt-responsive ACSs However, none of these genes are direct or indirect targets of SHR (Levesque et al., 2006) These results suggest that the spatial information, but not the SHR protein itself, is crucial for ACS2, ACS6, ACS7 and ACS8 responding to salt stress.

The abundance of ACS transcripts is lower in shr during salt stress, which could lead to

lower production of ethylene In order to test whether the reduction of cell swelling is due

to the reduced ethylene production in the shr mutant, we treated the mutant with 140mM

NaCl in the presence or absence of 0.5 µM ACC The result clearly shows that exogenous

ACC can rescue shr defects in salt response (Figure 23A, B) We also introduced an eto1 (ethylene overproducer 1) mutation into the shr mutant to increase ethylene synthesis in the shr mutants The result from this experiment shows that the eto1 mutation can also rescue shr cell swelling phenotype under salt stress

Since shr mutants lack an endodermis, it is possible that the ethylene that synthesized in

the endodermis during salt stress is important for cortical cell swelling

Figure 22 Salt responsive ACSs are mis regulated under shr background

A) ACS2, AT1G01480 Noted that this gene does not respond to salt in shr mutant

B) ACS6, AT4G11280 The response of this gene to salt is largely impaired in shr

C) ACS7, AT4G26200 The expression of this gene is higher in shr mutant under

standard condition, and is repressed by salt

D) ACS8, AT4G37770 The mutation in SHR only has a minor effect on this gene

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

and endogenous applied ACC

A) 0.5 µM ACC is sufficient to rescue shr cortical cell swelling phenotype

B) shr/eto1 shows normal salt-response regarding cortical cell swelling

FM 4-64 was used as counterstain to highlight the cell shape Grey arrows point to the cortical cell file Scale bar= 50µm Asterisks represent significant differences based on Student’s t-test P value<0.0001 Error= SEM

4.3.3.3 Post-transcriptional regulation of ACSs may be involved in salt-mediated cortical cell swelling

Our data suggest that salt stress can regulate ACSs at the transcription level; however, as

ACS proteins have a very short half-life, it is possible that salt also regulates ACSs at the post-transcriptional level It has already been shown that calcium-dependent protein kinases (CDPKs) can increase ethylene production by stabilizing ACS proteins

(Sebastian, C.H., et al., 2004) However, previous studies failed to identify which CDPKs

are responsible for this process LeCDPK2, the homolog of Arabidopsis CDPK1 in tomato has been shown to stabilize type I ACSs, which including ACS1, ACS2 and

ACS6 (Kamiyoshihara et al., 2010) Interestingly, the CDPK1 gene is co-expressed with

the ACS2 gene during salt stress in our spatio-temporal transcriptional map (Figure 24A) Furthermore, CDPK1 is involved in stress responses in Arabidopsis, and cell expansion

and cell wall synthesis in Medicago truncatula roots (Ivashuta et al., 2005), which

indicates that it might be involved in the stabilization of ACSs, especially ACS2, during

salt stress The analysis of cdpk1mutants showed that knockout of the gene caused

impaired inhibition of cell elongation and less radial expansion in cortical cells during salt stress compared to wild type (Figure 24B), suggesting that CDPK1 is involved in regulating salt-mediated cell swelling

These results indicate that the stabilization of ACS proteins might be important for

cortical cell swelling As a type I ACS, the C-terminal tail of ACS2 is crucial for its degradation Here, we fused GFP to the C-terminus of ACS2, resulting in slow turnover

of the fusion protein Endogenous expression of ACS2 is very weak under standard condition Since ACS2 expression is enriched in the endodermis, we used the

endodermis-specific promoter SCR to drive ACS2 expression As shown in Figure 24C,

expressing the ACS2-GFP fusion protein in the endodermis using the SCR promoter is sufficient to cause cortical cell swelling under normal conditions

These data support the hypothesis that the stabilization of ACS proteins is important for salt-mediated cortical cell swelling

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

A) eFP representation of expression for CDPK1

B) Mutation in CDPK1gene causes less salt-mediated cell swelling

C) Introducing the low turnover version of ACS2 protein in endodermis is sufficient

to mimic the effect of salt on cell shape changes

FM 4-64 was used as counterstain to highlight the cell shape Grey arrows point to the cortical cell file Scale bar= 50µm Asterisks represent significant differences based on Student’s t-test P value<0.0001 Error= SEM

4.3.4 Salt activates ethylene signaling in the early elongation zone dynamically during salt stress

Since the genetic analysis of ein3-1/eil1-1 double mutant shows that EIN3 transcription

factor is crucial for salt-mediated radial expansion When ethylene signaling becomes

active, EIN3 protein is accumulated in the nucleus Here we used the Pro35S::EIN3:GFP

to investigate the spatio-temporal changes of ethylene signaling during salt stress

First, we focused on the temporal profile of the activation of ethylene signaling Figure

25 shows the region of the meristem and early elongation zone From these data we can see that ethylene signaling started to be activated as early as 30 minutes after salt

treatment After one hour treatment the signal became most active After two hour

treatment, the nuclear-localized GFP signal started to disappear (Figure 25A) Figure 25C shows the quantification of average fluorescent intensity which we can clearly see a transient accumulation of GFP signal We also examined the temporal activation profile

of EIL1 protein during salt stress We observed very similar activation profile with EIN3 protein (data not show) With 10µM ACC treatment, we can see the GFP signaling is

very stable in Pro35S::EIN3:GFP in the first 2 hours( Figure 25B) Therefore, the

transient accumulation of EIN3-GFP is not due to the stability feature of the protein but the transient activation of ethylene signaling by salt Together, these results indicate that ethylene signaling is activated transiently at early stage during salt stress

Next, we switched our focus to the spatial profile of ethylene signaling Again we used

Pro35S::EIN3:GFP as an indicator of ethylene signaling As shown in Figure 25B,

ethylene signaling was activated ubiquitously over all the tissue layers in the early elongation zone, which is the region that radial expansion first occurred after 1 hour salt treatment We did not observe any GFP signal in the meristem This spatial pattern is not due to the ability of EIN3 protein responding to ethylene From Figure 25B, we can see that under 10µM ACC treatment, EIN3 protein was ubiquitously accumulated after 1 hour

Combined with the previous results, we concluded that ethylene signaling is activated transiently in the early elongation zone at the early stage of salt stress

Figure 25 Ethylene signaling is dynamically activated during salt stress

A) Confocal analysis of GFP signal distribution in the epidermis of early elongation

zone and meristem of Pro35S::EIN3:GFP at different time points under salt stress

B) Confocal images of medial longitudinal section at the elongation zone and

meristem of Pro35S::EIN3:GFP after salt or ACC treatment

C) Average GFP intensity analysis of Pro35S::EIN3:GFP at different time points

under salt stress

FM 4-64 was used as counterstain to highlight the cell shape Error= SEM

4.3.5 Salt-responsive ethylene signaling downstream components show cell-type specificity in their expression

Recent study indicates that early ethylene response may show heterogeneity in different

tissue layers (Chang et al., 2013) The next question we investigated is what the cell-type

specific function of ethylene signaling in regulating salt-mediated cell swelling is

However, as shown previously, EIN3 is activated ubiquitously in the early elongation zone after salt stress So we ask if we could find the cell-type specific EIN3 downstream components that are involved in salt response, especially in salt-mediated cell swelling

First, we performed microarray experiments with Col-0 and etr1-1 mutants after 1 hour

salt treatment to screen out the genes that are responsive to salt at early stage in an

ethylene-dependent fashion The reason we chose 1 hour salt treatment is that EIN3 stabilization peaks at this time

As shown in Figure 26, by using LIMMA, we found over 35% salt-activated and 30% of

salt-repressed genes were mis-regulated in etr1-1 mutants, indicating that canonical

ethylene signaling pathway plays an important role in early salt response Next, by referencing the AGI numbers of the genes that were responsive to salt in an ethylene

cross-dependent fashion with our spatio-temporal transcriptional map, we were able to find the spatio-temporal expression profiles under salt treatment of 310 genes

There are 1313 direct targets of EIN3 (Chang et al., 2013) We found that in these

ethylene dependent salt responsive genes, near 20% are EIN3 direct targets ( n=54) GO analysis shows that 57% (n=28) of these loci (n=49) encode nuclear proteins (Figure 27) Except 8 genes encoding with unknown functions and 1 gene encoding a protein

regulating protein location, the rests all encode proteins that are involved in

transcriptional regulation or signaling cascade, which suggests that majority of salt

responsive EIN3 targets may serve as upstream components in salt response regulation

We divided salt responsive ethylene -dependent genes into 10 clusters by their expression patterns using K-means algorithm (Figure 28A) Most of the clusters show cell-type

specific response to salt Interestingly, we found most of the clusters show higher

expression at 1 hour in cortex where the most dramatic morphological change occurs The distribution of salt responsive EIN3 direct targets in those clusters shows a certain level of bias Cluster 10 is enriched genes that are activated by salt at inner tissue layers

In cortex, the activation of genes in this cluster is transient; at later stage most of those genes are repressed by salt However, in stele most of those gene expressions were

peaked at 3 hours and gradually decreased at later time points during salt stress Near 30% genes in this cluster are EIN3 direct targets These data suggest that although ethylene signaling is activated over all the tissue layers, the diverse temporal expression pattern of

downstream components may result different tissue layer having different functions in regulating salt response

Cluster 3 is the second biggest cluster It is enriched in the genes that are strongly

activated by salt immediately after salt treatment throughout all tissue layers but has highest expression in cortex As we described previously, cortex has the highest rate of radial expansion among those layers that show significant cell shape changes So it is possible that genes in cluster 3 are more directly involved in cell radial expansion than the genes in the other clusters We found 4 direct EIN3 targets in cluster 3 Interestingly, none of these four genes is involved in transcriptional regulation or signaling transduction

Only one gene in these four is properly annotated: ARABIDOPSIS THALIANA

EXPANSIN 10 ( AT1G26770) Its spatio-temporal expression profiles are shown in Figure

28B This gene is involved in cell wall loosening and multi-dimensional cell growth The

homolog of this gene in Brassica juncea is believed to be involved in stem swelling Based on the expression profile we obtained from eFP browser (Winter et al., 2007), the

expression of this gene is elevated in elongation zone shortly after salt treatment As mentioned before, our spatio-temporal transcriptional map shows that the expression of this gene in cortex peaked at 1 hour after salt treatment, indicating that it may be directly involved cortical cell radial expansion Further genetic analysis would shed light on the cellular mechanism about salt-mediated cell radial expansion

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

receptor mutant, etr1-1 background

Over 35% salt-activated and 30% of salt-repressed genes were mis-regulated in etr1-1

mutants, indicating the importance of ethylene canonical signaling pathway in salt response

Figure 27 GO analysis of ETR1-dependent salt responsive EIN3 direct target

The graph was a redrawn from a graph generated by TAIR GO annotation tool Majority

of these genes is localized in nucleus Noted that there are 8% of genes that are involved

in direct cell wall modification

Figure 28 Spatiotemporal expression of ETR1-dependent salt responsive genes

A) Centroid profiles for each of the 10 gene clusters identified in the spatiotemporal

data set The numbers on the left represent the cluster number; the numbers on the right represent the percentage of EIN3 direct target in that cluster

B) AT1G26770, ATEXP10, an EIN3 direct target, is dynamically activated in cortex

in the early stage of salt treatment

4.3.6 Auxin signaling serves as downstream signaling of ethylene in regulating mediated cell swelling

salt-In order to find out which downstream processes are involved in salt-mediated cell

swelling, we performed a Gene ontology (GO) category enrichment analysis on ethylene dependent salt responsive genes As shown in Figure 29A, auxin responsive genes are enriched in this group of genes, which suggests that auxin may serve as a downstream signaling component of ethylene in regulating salt response

Previous studies show that ethylene regulates root growth by inhibiting cell elongation largely through inducing auxin production and transportation in an ethylene canonical

signaling pathway-dependent fashion (Růzicka K et al., 2007; Swarup R, 2007) Here we

wanted to ask if auxin is also involved in the regulatory function of ethylene in

salt-mediated cell swelling DII-VENUS is a negative indicator of endogenous auxin signal (Brunoud et al., 2012) Since cells started to swell in the early elongation zone during salt stress, we measure the DII-VENUS intensity in this region at 4 hours and 8 hours after

salt treatment We can clearly see the decrease of VENUS signal after 8 hours of salt treatment This indicates the auxin signal in this region became more active over time during salt stress, while it remained unchanged in standard condition (Figure 29B, 29C)

4.3.6.1 Possible functions of auxin transportation in salt-mediated cell swelling

It has been shown previously that PIN2 is also required in ethylene regulating cell shapes However, we didn’t see any changes in PIN2 protein abundance at the early stage of salt stress We did observe PIN2 protein re-localization from the elongation region that is horizontal to the agar media to the elongation region toward the air after 4 hours of salt treatment ( Figure 30A,B,C) It was correlated salt-mediated cell swelling, indicating that auxin efflux carriers may have a function in regulating this process

Next, we wanted to know if auxin influx carriers are also involved in salt-mediated cell

swelling We examined aux1-7, an allele of AUX1 mutants Surprisingly, we found this

mutant has more swelling than wild type When we separated the cell width and cell length, we found that the cell width was normal in this mutant, but the cells were

significantly shorter than the wild type after salt stress (Figure 30C to E) These results

suggest that AUX1 may serve as a positive regulator in cell elongation during salt stress

Figure 29 Auxin signaling is involved in regulating salt-mediated cortical cell swelling downstream of ethylene signaling pathway

A) A redraw of a branch of a network generated by AGRI-GO analysis tool, showing

that auxin responsive genes are enriched in ETR1-dependent salt responsive genes

B) Confocal images showing the maximum intensity projection of fluorescence from

a root tip to elongation zone expressing the DII:VENUS 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) Yellow frames highlight the sampling region Scale bar=200µm

C) Quantitation of GFP intensity at different time points after salt treatment in

DII:VENUS -expressing roots Asterisk represents significant differences based

on Student’s t-test P value<0.05 Error= SEM