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Research Article

Hydrogen sulfide regulates abiotic stress tolerance and biotic stress resistance in Arabidopsis

Running title: Involvement of H2S in abiotic and biotic stresses

Haitao Shi1, Tiantian Ye1,2, Ning Han3, Hongwu Bian3, Xiaodong Liu4 and Zhulong Chan1*

1 Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden, Chinese

Academy of Sciences, Wuhan 430074, China 2 University of Chinese Academy of Sciences, Beijing 100039, China 3 Institute of Genetics, State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences,

Zhejiang University, Hangzhou 310058, China 4 College of Agronomy, Xinjiang Agricultural University, Urumqi 830052, China

* Correspondence: zhulongch@wbgcas.cn

Edited By: Ildoo Hwang, POSTECH Biotech Center, Korea

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: [10.1111/jipb.12302] This article is protected by copyright. All rights reserved. Received: August 26, 2014; Accepted: October 17, 2014

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Abstract

Hydrogen sulfide (H2S) is an important gaseous molecule in various plant developmental processes and plant stress

responses. In this study, the transgenic plants with modulation expressions of two cysteine desulfhydrases and

exogenous H2S donor (sodium hydrosulfide, NaHS) and H2S scavenger (hypotaurine, HT) pre-treated plants were

used to dissect the involvement of H2S in plant stress responses. The cysteine desulfhydrases overexpressing plants

and NaHS pre-treated plants exhibited higher endogenous H2S level and improved abiotic stress tolerance and

biotic stress resistance, while cysteine desulfhydrases knockdown plants and HT pre-treated plants displayed lower

endogenous H2S level and decreased stress resistance. Moreover, H2S up-regulated the transcripts of multiple

abiotic and biotic stress-related genes, and inhibited reactive oxygen species (ROS) accumulation. Interestingly,

MIR393-mediated auxin signaling including MIR393a/b and their target genes (TIR1, AFB1, AFB2, and AFB3)

was transcriptionally regulated by H2S, and was related with H2S-induced antibacterial resistance. Moreover, H2S

regulated 50 carbon metabolites including amino acids, organic acids, sugars, sugar alcohols and aromatic amines.

Taken together, these results indicated that cysteine desulfhydrase and H2S conferred abiotic stress tolerance and

biotic stress resistance, via affecting the stress-related gene expressions, ROS metabolism, metabolic homeostasis,

and MIR393-targeted auxin receptors.

Keywords: Hydrogen sulfide; cysteine desulfhydrase; abiotic stress; biotic stress; MIR393; auxin receptor.

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INTRODUCTION

Along with nitric oxide (NO) and carbon monoxide (CO), hydrogen sulfide (H2S) has emerged as the third

important gaseous molecule in various plant developmental processes and stress responses (Rausch and Wachter

2005; Wang 2010; Lisjak et al. 2013; Calderwood and Kopriva 2014; Hancock and Whiteman 2014). H2S promotes

root organogenesis (Zhang et al. 2009), seed germination (Zhang et al. 2010), lateral root formation (Fang et al.

2014), and enhances photosynthesis (Chen et al. 2011a). Moreover, H2S confers protective roles in responses to

heat, drought, salt, osmotic, and freezing stresses (Wang et al. 2010; Zhang et al. 2010b, 2011; Jin et al. 2011, 2013;

Shen et al. 2012, 2013; Xie et al. 2014).

Understanding the complex effects of H2S in plants requires further detailed analyses at the physiological,

biochemiscal and molecular levels. In recent years, most of the in vivo roles of H2S in plants were obtained from

pharmacology experiment through exogenous application of H2S donor (sodium hydrosulfide, NaHS), H2S

scavenger (hypotaurine, HT) and H2S inhibitors (potassium pyruvate, PP; and hydroxylamine, HA) (Li et al. 2013;

Shi et al. 2013, 2014a). However, the treatments with H2S modulating chemicals may have limitation in exactly

reflecting the in vivo roles of H2S in plants. So far besides exogenous application of H2S in pharmacological

experiments, it would be better to use plants with altered in planta H2S production as candidates for such an

approach.

To date, several proteins have been reported to be directly responsible for H2S generation in Arabidopsis,

including L-cysteine desulfhydrase (LCD), D-cysteine desulfhydrase 1 (DCD1), D-cysteine desulfhydrase 2

(DCD2), and DES1 (Bloem et al. 2004; Riemenschneider et al. 2005; Papenbrock et al. 2007; Álvarez et al. 2010,

2012a, 2012b; Shen et al. 2013). The knockout mutant of AtLCD (lcd) with decreased endogenous H2S level has

been used to investigate the role of H2S in plant response to drought stress (Jin et al. 2013), and expression of

AtLCD and AtDCD1 in Escherichia coli showed increased cadmium resistance (Shen et al. 2012). Álvarez et al.

(2012a, 2012b) found that cysteine-generated sulfide in the cytosol affected autophagy and transcriptional profile

of Arabidopsis, and knockout mutant of the DES1 shows high resistance to biotrophic and necrotrophic pathogens.

However, mechanisms of H2S mediated plant development and abiotic stress response remain obscure (Lisjak et al.

2013). Additionally, the connection between H2S and plant biotic stress resistance is largely unknown (Bloem et al.

2004, 2007; Lisjak et al. 2013).

In this study, the transgenic plants with modulated expressions of two cysteine desulfhydrases (AtLCD and

AtDCD1) together with exogenous pre-treatments with H2S donor (NaHS) and scavenger (HT), were used to

dissect the involvement of H2S in plant abiotic and biotic stress responses. Further physiological, molecular, and

metabolic studies were performed to investigate the underlying mechanisms of H2S-mediated abiotic and biotic

stress responses, and the involvement of MIR393-targeted auxin receptors were highlighted in H2S-mediated

disease resistance.

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RESULTS

Effect of abiotic and biotic stresses on cysteine desulfhydrases and the endogenous H2S content in

Arabidopsis

Through real-time quantitative PCR analysis, we found that the transcript levels of LCD and DCD1 increased upon

all stress treatments tested (cold, dehydration, salt, hydrogen peroxide (H2O2) and bacterial pathogen) except

abscisic acid (ABA) which had no effect on DCD1 transcript amount (Figure 1A). All treatments for 6 hours

increased transcript amounts of LCD and DCD1 than those of treatments for 3 hours except pathogen infection,

which induced highest transcript levels of LCD and DCD1 at 3 hours (Figure 1A). Consistently, the enzyme

activity of LCD significantly increased after all these stress treatments, and the enzyme activity of DCD was

significantly induced after dehydration, salt, H2O2 and bacterial pathogen treatments (Figure 1B). In addition, the

endogenous H2S content significantly increased with 1.2-3 fold changes after stress treatments (Figure 1C).

Modulation of cysteine desulfhydrase expression affects endogenous H2S level

To further investigate the in vivo role of LCD and DCD1, we isolated the T-DNA mutant of LCD (lcd mutant,

SAIL_739_C08/CS835466), and constructed LCD and DCD1 overexpressing plants and DCD1 RNAi knockdown

plants. Consistently, the expression of LCD and DCD1 and the activities of LCD and DCD showed higher levels in

the overexpressing plants, but exhibited lower levels in the knockdown plants (Figure S1A-D). LCD and DCD1

overexpressing plants exhibited significantly higher concentration of H2S, while LCD and DCD1 knockdown

plants displayed significantly lower concentration of H2S (Figure 2). Moreover, 100 μM NaHS and 100 μM HT

treatments significantly changed endogenous H2S level as expected (Figures 2, S2). Additionally, the accumulation

of cysteine was negatively regulated by LCD and DCD1 expressions, but not affected by NaHS and HT treatments

(Figure S3A). However, modulation of H2S content had no significant effect on reduced glutathione (GSH) content

(Figure S3B). Modulation of cysteine desulfhydrase expression and endogenous H2S level affect abiotic stress

tolerance and reactive oxygen species (ROS) metabolism

Under stress conditions, although the endogenous H2S levels were up-regulated in all lines, LCD and DCD1

overexpressing plants and NaHS-treated plants showed higher concentrations of H2S compared with WT plants,

while LCD and DCD1 knockdown plants and HT-treated plants displayed lower levels of them than WT plants

(Figure S4). When subjected to drought, salt, and freezing, LCD and DCD1 overexpressing plants and

NaHS-treated plants showed better growth and higher survival rate than those of wild type (WT) plants, while LCD

and DCD1 knockdown plants and HT-treated plants exhibited worse growth and lower survival rate in comparison

to WT plants (Figure 3A-B). Moreover, the increased abiotic stress tolerance of LCD and DCD1 overexpressing

plants were restored by HT treatment, and the descreased abiotic stress tolerance of LCD and DCD1 knockdown

plants were restored by NaHS treatment (Figure S5), indicating that H2S might be largely related with the affection

of cysteine desulfhydrase on abiotic stress tolerance.

Additionally, the transcripts of several abiotic stress-related genes (including CBF1, CBF3, CBF4, DREB2A,

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DREB2B, RAB18, RD22, RD29A, and RD29B) exhibited higher levels in the LCD and DCD1 overexpressing

plants and NaHS-treated plants (Figure 4 and Table S1), while lower in the LCD and DCD1 knockdown plants and

HT-treated plants (Figure 4 and Table S1). This result indicated the significant effect of H2S on transcript levels of

stress-related genes.

Under control condition, no significant differences of ROS level and several antioxidants were observed among

wild type and various lines with different endogenous H2S levels (Figure 5A-I). When abiotic stresses were applied,

LCD and DCD1 overexpressing plants and NaHS-treated plants displayed lower levels of H2O2, O2•-, and oxidized

glutathione (GSSG), but higher activities of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and

glutathione reductase (GR) and higher levels of GSH and GSH redox state than those of WT plants (Figure 5A-I).

On the contrary, LCD and DCD1 knockdown plants and HT-treated plants exhibited the opposite effects on the

ROS accumulation (Figure 5A-I).

Cysteine desulfhydrase expression and H2S regulate defense resistance against bacterial pathogen

As expected, NaHS and HT treatments significantly increased and decreased endogenous H2S level, respectively

(Figure S6). Through quantification of bacteria number in the pathogen-infected Arabidopsis leaves, we found that

the LCD and DCD1 overexpressing plants showed significantly less bacterial than WT at 3 day post infection (dpi),

while LCD and DCD1 knockdown plants exhibited more bacterial than WT (Figure 6A). Consistently, H2S donor

(NaHS)-treated plants exhibited improved defense resistance, while H2S scavenger (HT)-treated plants showed

decreased defense resistance (Figure 6A). Additionally, the transcript levels of ENHANCED DISEASE

SUSCEPTIBILITY 1 (EDS1), PHYTOALEXIN DEFICIENT 4 (PAD4), PR1, PR2, PR3, PR4, and PR5 were higher

in the LCD and DCD1 overexpressing plants and NaHS-treated plants, but were lower in the LCD and DCD1

knockdown plants and HT-treated plants (Figure 6B and Table S1). Therefore, these results indicated that cysteine

desulfhydrase expression and H2S regulated defense resistance against Pseudomonas, partially via modulating SA

signaling-related genes.

The involvement of MIR393-mediated auxin signaling in H2S-mediated antibacterial resistance

The transcript levels of MIR393a and MIR393b were up-regulated by H2S effect, while the transcript levels of

TRANSPORT INHIBITOR RESPONSE 1 (TIR1), AUXIN SIGNALING F BOX PROTEIN 1 (AFB1), AFB2, and

AFB3 were negatively regulated by H2S effect (Fig. 7A and Table S1). In accordance with the real-time assay, the

GUS activities of pMIR393a:GUS and pMIR393b:GUS transgenic plants were significantly activated by

exogenous NaHS treatment, but were significantly decreased by exogenous HT treatment (Figure 7B). Under

control condition, 35S:MIR393a-6, 35S:MIR393b-1, and tir1afb1afb2afb3 plants exhibited enhanced antibacterial

resistance with less bacterial numbers in plant leaves at 3 dpi, while 35S:TIR1-5 and 35S:mTIR1-9 transgenic plant

leaves showed more bacterial numbers at 3 dpi in comparison to WT plants (Figure 7C). To further investigate the

involvement of MIR393-targeted auxin receptors in H2S-mediated antibacterial resistance, we determined the

antibacterial resistance of 35S:MIR393a, 35S:MIR393b, tir1afb1afb2afb3, 35S:TIR1, and 35S:mTIR1 plants in

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comparison to WT after the treatments of NaHS and HT. Unlike the WT plants, the improved antibacterial

resistance of 35S:MIR393a-6, 35S:MIR393b-1, and tir1afb1afb2afb3 plants and the decreased antibacterial

resistance of 35S:TIR1-5 and 35S:mTIR1-9 transgenic plants were not significantly affected by either NaHS or HT

treatment (Figure 7C). However, the endogenous H2S level in these transgenic plants exhibited no significant

difference compared with that in WT plants under both control and NaHS/HT-treated conditions (Figure 7D).

These results suggested that H2S induced resistance might be mainly mediated by MIR393-dependent auxin

signaling.

Modulation of metabolic homeostasis by H2S effect

To gain more insight into the metabolic homeostasis which might be affected by H2S, multiple carbon metabolites

were assayed through gas chromatography time-of-flight mass spectrometry (GC-TOF-MS) analysis. Totally, 54

metabolites including 16 amino acids, 13 organic acids, 18 sugars, 5 sugar alcohols and 2 aromatic amines were

reproducibly examined in WT (without treatment, and with NaHS and HT treatments), LCD and DCD1

overexpressing plants and knockdown mutants (Figure 8 and Table S2). Generally, 50 of 54 metabolites (except

aspartic acid, lysine, hexadecanoic acid, and melibose) were significantly affected by NaHS/HT treatment and in

the plants with altered LCD or DCD activity. Interestingly, most of the metabolite concentrations were higher in

NaHS-treated and LCD-OX/DCD1-OX plants and lower in HT-treated and LCD/DCD1 knockdown plants (Figure

8 and Table S2), indicating the modulation of H2S in several carbon metabolites including amino acids, organic

acids, sugars, sugar alcohols and aromatic amines.

DISCUSSION

Previous studies dissecting the in vivo roles of H2S in plants were mainly obtained from pharmacology experiment

using H2S donor, scavenger and inhibitors (Li et al. 2013; Shi et al. 2013, 2014a). Therefore, it is difficult to

distinguish the effects of H2S from its upstream (cysteine) and other downstream metabolites of cysteine (such as

glutathione) (Wu et al. 2010; Cao et al. 2013, 2014; Calderwood and Kopriva 2014). In this study, modulation of

cysteine desulfhydrase expression affected endogenous H2S and cysteine levels, but had no significant effect on the

accumulation of GSH under control condition (Figures 2, S1, S2). However, exogenous treatments of H2S donor

(NaHS) and scavenger (HT) specifically affected the endogenous H2S, and did not affect the accumulations of

cysteine levels and GSH under control condition (Figures 2, S3). Since the endogenous sulfate metabolism is very

complex, the upstream and downstream metabolites of H2S might form part of a feedback mechanism to reset its

homeostasis. In this study, using the transgenic plants with modulated endogenous H2S level together with the

exogenous treatments of H2S donor (NaHS) and scavenger (HT), we assigned the protective role of H2S in both

abiotic stress tolerance and biotic stress resistance (Figures 3, 6). This result is in accordance with the enhanced

abiotic stress tolerance by H2S donor (NaHS) in various plant species, and this is the first report to show the

protective role of H2S in antibacterial resistance.

H2S and abiotic stress tolerance in Arabidopsis

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When subjected to abiotic stress conditions, plant ROS level (mainly including H2O2 and O2•-) was largely induced,

along with induced unstabilization of plasma membrane and decreased cell turgor (Mittler et al. 2004, 2011; Miller

et al. 2010). Consistently, plants have evolved complex ROS defence system, including several enzymatic

antioxidant enzymes (SOD, CAT, POD, GR, etc) and non-enzymatic antioxidant molecules (such as glutathione) to

regulate endogenous ROS level. In this study, increased endogenous H2S and exogenous application of NaHS

significantly activated ROS detoxification system, including enzymatic antioxidant enzymes and non-enzymatic

glutathione to make cellular ROS at the relatively low level under abiotic stress conditions, thus conferring

improved abiotic stress tolerance (Figure 5). When the endogenous H2S level decreased, significantly increased

level of ROS and decreased levels of antioxidants resulted in decreased abiotic stress tolerance (Figure 5). Thus,

the modulation of endogenous and exogenous H2S in ROS detoxification system might be directly connected with

the effect of H2S on abiotic stress tolerance.

In Arabidopsis, CBF1, CBF2, CBF3 (also known as DREB1b, DREB1c, DREB1a, respectively) and CBF4

have important roles in cold, salt and drought stress responses via binding to the C-repeat

(CRT)/dehydration-responsive element (DRE) cis-acting element of several stress-responsive genes (Seki et al.

2001; Haake et al. 2002; Achard et al. 2008; Novillo et al. 2012). When constitutively overexpressing

AtCBF1/2/3/4, the expression of several downstream genes such as COR (cold related), LTI (low-temperature

induced), KIN (cold inducible), RD (responsive to dehydration) and ERD (early responsive to dehydration) were

largely induced, and the transgenic plants conferred improved abiotic stress tolerance (Seki et al. 2001; Haake et al.

2002; Achard et al. 2008; Novillo et al. 2012). Moreover, ABA2 and NCED3 are responsible for ABA generation

in Arabidopsis (Chan 2012). Abiotic stress responses in plants are initiated via both ABA-dependent and

ABA-independent signal transduction pathways (Jin et al. 2011; Novillo et al. 2012). CBF4 is ABA-independent,

and RAB18, RD22, RD29A, and RD29B are ABA dependent stress-responsive genes (Jin et al. 2011; Novillo et al.

2012). In this study, the modulation of H2S in the transcripts of multiple abiotic stress-related genes (CBF1, CBF3,

CBF4, DREB2A, DREB2B, RAB18, RD22, RD29A, and RD29B) might be directly related with the abiotic stress

tolerance.

H2S and biotic stress resistance in Arabidopsis

The transcripts of EDS1 and PAD4, which are responsible for SA biosynthesis in Arabidopsis, were constitutively

activated in the plants with higher H2S levels, but were significantly reduced in the plants with lower H2S levels

(Figure 6B). SA accumulation is necessary for the activation of SA downstream gene expression such as PRs

(Wang et al. 2007; Kazan and Manners 2009; Fu and Dong 2013). Consistently, the plants with higher H2S levels

showed increased expression of SA-dependent PR genes, resulting in improved immunity resistance, while the

plants with lower H2S levels exhibited decreased immunity resistance (Figure 6). Álvarez et al. (2012a) found that

knockout mutant of the DES1 showed improved resistance to biotrophic and necrotrophic pathogens, SA

accumulation and PR1 induction. The controversy might be attributed to at least three aspects as described

following. Firstly, the effect of LCD1 and DES1 on sulfate metabolism is different. Although they have the same

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effects on cysteine content, they modulate endogenous GSH and H2S levels differently (Figures 2, S3; Álvarez et al.

2012a). Modulation of LCD1 expression affected H2S levels (Figures 2, S4) but had no significant effects on

endogenous GSH level (Figure S3) and DES1 transcript (Figure S7), while DES1 knockout mutant displayed

higher concentration of GSH, but no report about its effect on H2S level (Álvarez et al. 2012a). Secondly,

28-day-old plants were used for disease resistance assay in this study, whereas 6-7-week-old plants were used by

Álvarez et al. (2012a), and the different growth stage might have differences in sulfate metabolism including

cysteine, GSH, and H2S levels. Additionally, Álvarez et al. (2012a) found that DES1 was localized in cytoplasm.

LCD is predicted to be localized in cytoplasm, but DCD1 is predicted to be localized in chloroplast, cytosol, and

mitochondrion. The differential subcellular localization might also contribute to the contradictory results.

Wang et al. (2007) showed that SA and auxin act individually or through antagonistic crosstalk, and the finely

tuned balance between them is critical for plant-pathogen interaction. Shen et al. (2013) showed that the transcripts

of multiple miRNAs including MIR167, MIR393, MIR396, and MIR398 were significantly up-regulated by H2S.

Among these miRNAs, MIR393 has been shown to be involved in plant-pathogen interaction (Navarro et al. 2006;

Robert-Seilaniantz et al. 2011). Overexpression of MIR393, a microRNA that targets auxin receptors (TIR1, AFB1,

AFB2, and AFB3), renders plants more resistant to bacterial pathogens, while over-expression of AFB1 results in

plants more susceptible to bacterial pathogens (Navarro et al. 2006; Chen et al. 2011b; Robert-Seilaniantz et al.

2011; Bian et al. 2012; Liu et al. 2013). Besides SA-related genes, the transcript levels of MIR393a/b and their

target genes (TIR1, AFB1, AFB2, and AFB3) were up-regulated and down-regulated by H2S, respectively (Figure

7A, B). Since the transcript of MIR393 is induced by IAA (Chen et al. 2011b) and H2S (Shen et al. 2013 and this

study), the connection between auxin and H2S is further investigated in this study. DR5:GUS is a widely used

auxin-related marker line under the control of the auxin-responsive DR5 promoter (Ulmasov et al. 1997), and

NaHS and HT treatments had no significant effects on the GUS activities of DR5:GUS line (Figure S8). These

studies suggested that H2S affected only the transcripts of MIR393 and its targeted auxin receptors, but not auxin

responses. As shown in Figure 7C, MIR393a/b and their target auxin receptors mediated antibacterial resistance

was not affected by NaHS and HT treatments, suggesting that MIR393-repressing auxin signaling might be largely

contributed to H2S-mediated antibacterial resistance. Thus, H2S conferred plant immunity resistance via

modulating both the transcript levels of SA-related genes and MIR393-mediated auxin signaling.

H2S and metabolic homeostasis in Arabidopsis

Not only stress-related genes, but also several metabolites were significantly affected by H2S effect (Figure 8 and

Table S2). Notably, higher endogenous H2S level largely activated the accumulation of compatible solutes such as

proline, arabinose, sucrose, lactulose, allose, fructose, lactose, tagatofuranose, talose, mannose, galactinol, dulcitol

(Figure 8 and Table S2). Proline and some carbohydrates are important compatible solutes to respond to abiotic

stress for osmotic adaptation (Krasensky and Jonak 2012). Thus, higher levels of proline and the carbohydrates

might provide beneficial effect in response to stress conditions. On the contrary, lower endogenous H2S level

exhibited lower concentrations of these compatible solutes (Figure 8 and Table S2), which might be related with

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the decreased stress resistance. Among these amino acids, the mutant and HT-treated plants displayed significantly

higher concentration of alanine, but lower concentrations of other amino acids, indicating the complex role of H2S

in regulating amino acid pools. Asparagine accumulation shows that nitrogen re-distribution and mobilization are

important features of the salt stress response (Maaroufi-Dguimi et al. 2011). In addition, asparagine was an amino

group donor for the synthesis of the photorespiratory intermediate glycine, and Nagy et al. (2013) found this was

also a good indicator of drought stress in drought tolerant and sensitive wheat cultivars. In the meanwhile, the

modulation of other amino acids and organic acids by H2S treatment indicated common changes of H2S in carbon

metabolism, and might contribute to the affected stress resistance.

Based on the above results, a model for H2S-mediated stress responses in Arabidopsis was proposed (Figure 9).

In response to various abiotic and biotic stresses, the H2S synthetic pathway was largely activated and H2S level

was significantly increased. The increased endogenous H2S and exogenous application of H2S donor, on one hand

constitutively activated the expression of several abiotic stress-related genes and SA signaling genes, and

accumulate of compatible solutes such as proline and soluble sugars; on the other hand, changes of antioxidant

enzyme activities and glutathione redox state under stress conditions, conferred improved both abiotic stress

tolerance and biotic stress resistance (Figure 9). When the endogenous H2S was down-regulated, the opposite

results were observed. Moreover, MIR393-targeted auxin receptors were regulated by H2S, and were largely

contributed to H2S-mediated antibacterial resistance (Figure 9).

Taken together, this study assigned the protective role of H2S in Arabidopsis responses to abiotic and biotic

stresses, via regulating multiple stress-related gene transcripts, ROS metabolism, metabolic homeostasis and

MIR393-targeted auxin signaling pathways.

MATERIALS AND METHODS

Plant materials and growth conditions

Arabidopsis thaliana (ecotype Columbia-0) seeds were sterilized with 70% (v/v) ethyl alcohol, and 10% (w/v)

NaClO and then washed with deionized water. The seeds were sown on Murashige and Skoog (MS) medium plate

containing 1% (w/v) sucrose or in soil after stratification at 4 °C for 3 days in darkness. The plants were then kept

in the growth chamber, which was controlled at 23 °C and at an irradiance of about 150 µmol quanta m-2 s-1, with

65% relative humidity under 16 hour light and 8 hour dark cycles. Nutrient solution of modified Hoagland solution

(Tocquin et al. 2003) was watered twice in the bottom of the pots with plants every week. The lcd mutant

(SAIL_739_C08/CS835466) was obtained from the Arabidopsis Biological Resource Center, and the transgenic

lines of pMIR393a:GUS, pMIR393b:GUS, 35S:MIR393a-6, 35S:MIR393b-1, 35S:TIR1-5 and 35S:mTIR1-9 have

been described in Chen et al. (2011b), and the tir1afb1afb2afb3 mutant through crossing tir1 (CS3798), afb1

(SALK_070172), afb2 (SALK_137151), and afb3 (SALK_068787) has been described in Liu et al. (2013).

To determine the effects of different stresses on the expression and activities of AtLCD and AtDCD1,

28-day-old Arabidopsis Col-0 plants were treated by cold stress (4 °C), dehydration (put the plants in a

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un-covering the plate in the growth chamber), or watered by 50 µM ABA, 200 mM NaCl or 20 mM H2O2 in the

pots with plants for 3 and 6 hours, or the plant leaves were infected by Pst DC3000 as Shi et al. (2012) described at

OD600 = 0.001 for 1, 3 and 6 hours. The plant leaves were then harvested at indicated timepoints for further

analysis.

Transgenic plasmid construction and plant transformation

For the AtLCD and AtDCD1 overexpressing constructs, the coding regions of AtLCD and AtDCD1 were amplified

and cloned into pBARN vector under the control of CaMV 35S promoter with basta resistance (LeClere and Bartel

2001). For the amiR-AtDCD1 knockdown transgenic construct, amiR-AtDCD1 fragment were amplified from the

plasmid pRS300 by PCR using specific primers from WMD3 (http://wmd3.weigelworld.org/cgi-bin/webapp.cgi)

(Schwab et al. 2006), and the amiR-AtDCD1 fragment was then cloned into pBARN vector. The primers for vector

constructs were listed in Table S3. The above recombinant constructs were then transformed into Agrobacterium

tumefaciens strain GV3101 and introduced into Columbia-0 plants using the floral dip method (Clough and Bent

1998). Homozygous transgenic plants were selected on MS medium using basta resistance and were confirmed by

PCR analysis.

RNA isolation and real-time quantitative PCR

Total RNA was extracted from plant leaves using TRIzol reagent (Invitrogen, California, USA) and RQ1

RNase-free DNase (Promega, Wisconsin, USA) was added to avoid possible genomic DNA contamination.

First-strand cDNA was then synthesized using reverse transcriptase (TOYOBO, Osaka city, Japan) from 2 µg of

total RNA. Real-time quantitative PCR was carried out using CFX96TM Real Time System (BIO-RAD, California,

USA) with iQTM SYBR® Green Super mix (BIO-RAD, California, USA) as Shi et al. (2012) described. Ubiquitin

10 (UBQ10) was chosen as the reference gene in the treatments according to geNorm software (Czechowski et al.

2005), and housekeeping genes of ACTIN2 and EIF4A were used as the control. The gene transcript levels were

standardized with ubiquitin 10 (UBQ10) using comparative ΔΔCT method, and all experiments were repeated at

least three times. The specific primers for real-time quantitative PCR were listed in Table S4.

Determination of H2S concentration

H2S concentration was assayed as Chen et al. (2011a) described that based on formation of methylene blue. Briefly,

1 g of plant leaves were ground in 10 mL of extraction buffer (50 mM phosphate buffer, pH 6.8, 0.2 M ascorbic

acid, and 0.1 M EDTA). The homogenate was mixed with 1 ml of 1 M HCl to release H2S, and H2S was absorbed

in a 1 mL of 1% (w/v) zinc acetate trap. After 30 min of reaction, 0.5 mL of 5 mM dimethyl-p-phenylenediamine

dissolved in 5 mM H2SO4 was added to the trap, and then 0.5 ml of 50 mM ferric ammonium sulphate in 100 mM

H2SO4 was added into the trap. The concentration of H2S in zinc acetate traps was examined at 667 nm of

absorbance.

Determination of abiotic and biotic stress resistances

Plant abiotic stress tolerance was assayed as Shi et al. (2012) described, and survival rate was determined at 4 d

after recovery from the abiotic stress treatment. For drought stress treatment, 14-day-old plants were withheld

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water for 21 days and then re-watered for 4 days. For salt stress treatment, 14-day-old plants were watered with

NaCl solution, and the NaCl concentration was increased stepwise by 50 mM every two days to 150 mM for

another 21 days. For freezing stress treatment, 2-week-old seedlings were cold acclimated at 4 °C for 14 days, then

the plants were placed at -8 °C for 8 hours, and then transferred to a growth chamber at 22 oC for another 4 days.

For disease resistance assay, 28-d-old plant leaves were infected with the pathogen strain of Pst DC3000 at OD600

= 0.001 as described by Shi et al. (2012) described, and the bacterial growth from infected Arabidopsis leaves was

monitored at 0 and 3 days post infection (dpi).

Determination of ROS level and antioxidant activities

0.5 g of plant leave samples were harvest from ten independent plants in each experiment and used for the ROS

assay. H2O2 and O2•- contents were quantified using the titanium sulfate method and the Plant O2•

- ELISA Kit

(10-40-488, Dingguo, Beijing, China), respectively, as Shi et al. (2013) described.

Determination of enzyme activities

LCD and DCD activities were determined by measuring the production rate of H2S from L-cysteine and D-cysteine

as Jin et al. (2013) described. The activities of antioxidant enzymes [SOD (EC 1.15.1.1), CAT (EC 1.11.1.6), POD

(EC 1.11.1.7), and GR (EC 1.6.4.2)] were assayed using Total SOD Assay Kit (S0102, Beyotime, Haimen city,

China), CAT Assay Kit (S0051, Beyotime, Haimen city, China), Plant POD Assay Kit (A084-3, Nanjing Jiancheng,

Nanjing city, China) and GR Assay Kit (S0055, Beyotime, Haimen city, China), respectively, according to

previously described protocols (Shi et al. 2013). The GSH content, GSSG content, and GSH redox state

[GSH/(GSH+GSSG)] were quantified using the GSH and GSSG Assay Kit (S0053, Haimen Beyotime, China) as

previously described (Shi et al. 2013).

Quantification of GUS activity

Quantification of GUS activity was performed as Jefferson et al. (1987) described by detecting the amount of

4-methylumbelliferone (MU) produced from the substrate 4-methylumbelliferyl-β-glucuronide (MUG).

Quantification of metabolites

The metabolite extraction from 0.2 g of plant leaves and sample derivatization were carried out as Lisec et al.

(2006) described. The metabolites were determined using GC-TOF-MS (Agilent 7890A/5975C, California, USA)

with a DB-5MS capillary (30 m × 0.25 mm × 0.25µm, Agilent J&W GC column, California, USA) according to

Shi et al. (2014b) described protocol. Various metabolites were identified by comparing retention time index

specific masses with reference spectra in mass spectral libraries (NIST 2005, Wiley 7.0).

Cluster analysis

The hierarchical cluster analysis of differentially expressed physiological parameters was performed using

CLUSTER program (http://bonsai.ims.u-tokyo.ac.jp/~mdehoon/software/cluster/) and Java Treeview

(http://jtreeview.sourceforge.net/) as Shi et al. (2014a, b) described.

Statistical analysis

All experiments in this study were repeated at least three times, and one sample was harvest from ten independent

12

plants in each experiment. Means ± SEs of three independent experiments are shown in the results. In all results,

Duncan’s range test was used to determine the significant difference among WT and other lines, and asterisk (*)

indicates the significant difference of p<0.05 compared to WT.

Acknowledgments

We thank Prof. Jianmin Zhou for providing the pathogen strain of Pst DC3000, Prof. Bonnie Bartel for providing

the pBARN plasmid, Prof. Detlef Weigel for providing the pRS300 plasmid, and Prof. Pingfang Yang for the help

in real-time quantitative PCR. This research was supported by the National Natural Science Foundation of China

(31370302), “the Hundred Talents Program” (54Y154761O01076 and 29Y329631O0263) to Zhulong Chan, and

by the National Natural Science Foundation of China (31200194), Youth Innovation Promotion Association of

Chinese Academy of Sciences (29Y429371O0437), the Knowledge Innovative Key Program of Chinese Academy

of Sciences (55Y455446O0544) to Haitao Shi.

13

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Figure legends

Figure 1. Endogenous H2S synthesis in responses to abiotic and biotic stress in Arabidopsis. (A)-(C) Effects of

abiotic stress on the expression of LCD and DCD1 (A), on the activities of LCD and DCD (B), and on the

endogenous H2S production (C). The transcript levels and activities of LCD and DCD at 0 h of treatment were

normalized as 1.0. The results shown are the means ± SEs (n=3), and * indicate the significant difference of p<0.05

in comparison to 0 hour of treatment.

Figure 2. Modulation of cysteine desulfhydrase expression and exogenous application of H2S donor and

scavenger affect endogenous H2S content.

For NaHS and HT treatments, 7-day-old soil-grown WT plants were watered with water solutions containing 100

μM NaHS and 100 μM HT for 7 days, respectively. The results shown are the means ± SEs (n=3), and * indicate

the significant difference of p<0.05 in comparison to WT.

Figure 3. Modulation of cysteine desulfhydrase expression and endogenous H2S affect abiotic stress

tolerance.

(A) The photograph showing 39-day-old Arabidopsis plants with control, drought, and 150 mM NaCl, treatments

and 32-day-old Arabidopsis plants that were recovery from freezing stress. For NaHS and HT treatments,

7-day-old WT plants were watered with water solutions containing 100 μM NaHS and 100 μM HT for 7 days, then

the 14-day-old plants were used for abiotic stress treatment for another 25 days (control and drought), 21 days (150

mM NaCl) or 14 days (freezing). (B) The survival rate of plants after recovery for 4 days after 25 days (control and

drought), 21 days (150 mM NaCl) or 14 days (freezing) of treatments. At least 117 plants in 9 pots were counted

for the survival rate assay in three independent experiments. The results shown are the means ± SEs (n=3). Asterisk

(*) indicates the significant difference of p<0.05 compared to WT.

Figure 4. Modulation of cysteine desulfhydrase expression and endogenous H2S level affect the expression of

stress-related genes.

For NaHS and HT treatments, 7-day-old WT plants were watered with water solutions containing 100 μM NaHS

and 100 μM HT for another 7 days, respectively. And then the 14-day-old Arabidopsis plants were harvest for

RNA isolation and real-time PCR analysis. For cluster analysis, all these gene transcript levels were quantified as

fold change in relative to the WT plants under control condition which was set as 1.0. The transcript levels of these

genes were shown in Table S1. Asterisk (*) indicates the significant difference of p<0.05 compared to WT.

Figure 5. Effects of cysteine desulfhydrase expression and endogenous H2S on ROS metabolism.

19

(A)-(I) Quantification of H2O2 content (A), O2•- content (B), SOD activity (C), CAT activity (D), POD activity (E),

GR activity (F), GSH content (G), GSSG content (H), and GSH redox state (I) in different Arabidopsis lines under

control and abiotic stress conditions. For NaHS and HT treatments, 7-day-old WT plants were watered with water

solutions containing 100 μM NaHS and 100 μM HT for 7 days before abiotic stress treatments, respectively. For

the assays of physiological parameters, 14-day-old Arabidopsis plants were treated by control, withholding water,

150 mM NaCl and 4 °C for 12 days. Asterisk (*) indicates the significant difference of p<0.05 compared to WT.

Figure 6. Modulation of cysteine desulfhydrase expression and endogenous H2S affect disease resistance

against bacterial infection.

(A) Growth of Pst DC3000 on Arabidopsis plants at 0 and 3 dpi. The results shown are the means ± SEs (n≥3).

Asterisk (*) indicates the significant difference of p<0.05 compared to WT. (B) Pathogen-related gene expressions

assayed by quantitative real time PCR. For NaHS and HT treatments, 7-day-old WT plants were watered with

water solutions containing 100 μM NaHS and 100 μM HT for 21 days before biotic stress treatments, respectively.

The 28-day-old Arabidopsis plants were used for disease resistance assay and real-time PCR analysis. For cluster

analysis, all these gene transcript levels were quantified as fold change in relative to the WT plants under control

condition which was set as 1.0. The transcript levels of these genes were shown in Table S1.

Figure 7. The involvement of MIR393-mediated auxin signaling in H2S-induced antibacterial resistance.

(A) The expression of MIR393a/b and their target genes assayed by quantitative real time PCR. For cluster analysis,

gene transcript levels were quantified as fold change in relative to the WT plants under control condition which

was set as 1.0. The transcript levels of these genes were shown in Table S1.

(B) Responses of pMIR393a:GUS and pMIR393b:GUS to exogenous NaHS and HT treatments. For GUS activity

assay, 7-day-old homozygous transgenic seedlings grown on MS-agar plates were transferred to new MS medium

with 100 μM NaHS or 100 μM HT for 0, 6, 12, and 24 hours. The relative GUS activities of pMIR393a:GUS and

pMIR393b:GUS transgenic plants at 0 hour of treatment were normalized as 1.0. Asterisk (*) indicates the

significant difference of p<0.05 compared to the GUS activity at 0 hour of treatment.

(C) Growth of Pst DC3000 on Arabidopsis plants at 0 and 3 dpi. For NaHS and HT treatments, 7-day-old WT

plants were watered with water solutions containing 100 μM NaHS and 100 μM HT for 21 days before abiotic

stress treatments, respectively. The 28-day-old Arabidopsis plants were used for disease resistance assay and

real-time PCR analysis.

(D) Quantification of endogenous H2S level in plants with altered MIR393-mediated auxin signaling. For NaHS

and HT treatments, 7-day-old WT plants were watered with water solutions containing 100 μM NaHS and 100 μM

HT for 7 days before abiotic stress treatments, respectively. The 14-day-old Arabidopsis plants were used for

quantification of endogenous H2S content. The results shown are the means ± SEs (n≥3). Bars with different letters

above the columns of figures indicate significant differences at p<0.05 (Duncan’s range test).

20

Figure 8. Effects of cysteine desulfhydrase expression and endogenous H2S level on metabolites.

For NaHS and HT treatments, 7-day-old WT plants were watered with water solutions containing 100 μM NaHS

and 100 μM HT for another 7 days, respectively. And then the 14-day-old Arabidopsis plants were used for

metabolic analysis. The concentrations of these metabolites were shown in Table S2. Asterisk (*) indicates the

significant difference of p<0.05 compared to WT.

Figure 9. A proposed model summarizing H2S-mediated abiotic and biotic stress responses in Arabidopsis.

21

Supporting Information

Table S1. The relative mRNA levels of genes in different lines that were used in Figures 4, 6B, 7A

Table S2. Concentrations of 54 metabolites affected by the modulation of LCD and DCD1 expressions and

endogenous H2S level

Table S3. Primers used for vector construction

Table S4. Primers used for real-time quantitative PCR

Figure S1. Characterization of LCD and DCD1 overexpressing and knockdown plants

Figure S2. Effects of NaHS and HT pre-treatments on endogenous H2S in WT plants

Figure S3. The effects of cysteine desulfhydrase expression and endogenous H2S on the accumulation of

cysteine (A) and GSH (B)

Figure S4. Modulation of cysteine desulfhydrase expression and exogenous application of H2S donor and

scavenger affect endogenous H2S content under control and stress conditions

Figure S5. The effects of NaHS and HT pre-treatments on cysteine desulfhydrase-mediated abiotic stress

tolerance

Figure S6. Effects of NaHS and HT treatments on endogenous H2S in WT plants

Figure S7. Modulation of cysteine desulfhydrase expression and endogenous H2S level had no significant

effect on DES1 transcript

Figure S8. Responses of DR5:GUS to exogenous NaHS and HT treatments

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Figure 4

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Figure 8

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Figure 9