Erl

DWa

b

a

ARRAA

KHMOPRS

cdeGdhedriSTu

h0

Plant Science 225 (2014) 117–129

Contents lists available at ScienceDirect

Plant Science

j ourna l ho me pa ge: www.elsev ier .com/ locate /p lantsc i

ndogenous hydrogen sulfide enhances salt tolerance by coupling theeestablishment of redox homeostasis and preventing salt-induced K+

oss in seedlings of Medicago sativa

iwen Laia,1, Yu Maoa,1, Heng Zhoua, Feng Lib, Mingzhu Wub, Jing Zhanga, Ziyi Hea,eiti Cuia, Yanjie Xiea,∗

College of Life Sciences, Laboratory Center of Life Sciences, Nanjing Agricultural University, Nanjing 210095, ChinaZhengzhou Tobacco Research Institute of CNTC, Zhengzhou 450001, China

r t i c l e i n f o

rticle history:eceived 7 May 2014eceived in revised form 6 June 2014ccepted 7 June 2014vailable online 16 June 2014

eywords:ydrogen sulfideedicago sativaxidative damageotassium efflux

a b s t r a c t

Despite the external application of hydrogen sulfide (H2S) conferring plant tolerance against vari-ous environmental cues, the physiological significance of l-cysteine desulfhydrase (L-DES)-associatedendogenous H2S production involved in salt-stress signaling was poorly understood. To address this gap,the participation of in planta changes of H2S homeostasis involved in alfalfa salt tolerance was investi-gated. The increasing concentration of NaCl (from 50 to 300 mM) progressively caused the induction oftotal l-DES activity and the increase of endogenous H2S production. NaCl-triggered toxicity symptoms(175 mM), including seedling growth inhibition and lipid peroxidation, were alleviated by sodium hydro-sulfide (NaHS; 100 �M), a H2S donor, whereas aggravated by an inhibitor of l-DES or a H2S scavenger.A weaker or negative response was observed in lower or higher dose of NaHS. Further results showedthat endogenous l-DES-related H2S modulated several genes/activities of antioxidant defence enzymes,

edox homeostasisalt tolerance

and also regulated the contents of antioxidant compounds, thus counterbalancing the NaCl-induced lipidperoxidation. Moreover, H2S maintained K+/Na+ homeostasis by preventing the NaCl-triggered K+ efflux,which might be result form the impairment of SKOR expression. Together, our findings indicated thatendogenous H2S homeostasis enhance salt tolerance by coupling the reestablishment of redox balanceand restraining K+ efflux in alfalfa seedlings.

© 2014 Elsevier Ireland Ltd. All rights reserved.

Abbreviations: AsA, ascorbic acid; CAT, catalase; CHES, 2-(N-yclohexylamino)ethane-sulphonic acid; DHA, dehydroascorbate; DHAR,ehydroascorbate reductase; PAG, dl-propargylglycine; DTT, dithiothreitol; EDX,nergy-dispersive X-ray detector; GORK, guard cell outward rectifying K+ channel;SH, glutathione; GR, glutathione reductase; H2DCF-DA, 2′ ,7′-dichlorofluoresceiniacetate; H2S, hydrogen sulfide; hGSH, homoglutathione; hGSSGh, oxidizedomoglutathione; HT, hypotaurine; ICP-OES, inductively coupled plasma-opticalmission spectrometer; IRK, inward-rectifying K+ channel; l-DES, l-cysteineesulfhydrase; mBBr, monobromobimane; MDHAR, monodehydroascorbateeductase; NaHS, sodium hydrosulfide; NEM, N-ethylmaleimide; NMT, non-nvasive micro-test technology; NO, nitric oxide; ROS, reactive oxygen species;KOR, Shaker-like K+ outward-rectifying channel; SOD, superoxide dismutase;BARS, thiobarbituric acid reactive substances; TCA, trichloroacetic acid; UPLC,ltra performance liquid chromatography.∗ Corresponding author. Tel.: +86 25 84396542; fax: +86 25 84396542.

E-mail address: yjxie@njau.edu.cn (Y. Xie).1 These authors contributed equally to this work.

ttp://dx.doi.org/10.1016/j.plantsci.2014.06.006168-9452/© 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

It is well known that salt stress is a major environmental fac-tor that leads to significant inhibition of plant growth and decreaseof productivity [1,2]. Normally, the excess salinity imposes numer-ous negative influences on plant cells, including osmotic stress, andthe overproduction of reactive oxygen species (ROS) as well as lipidperoxidation [3–6]. Salt stress also results in the over-accumulationof sodium ions (Na+) in plants cells, which competitively inhibitsthe uptake of potassium (K+), thus leading to a deficiency of K+ [4,7].Increasing evidence suggests that the prevention of Na+-triggeredK+ leakage, thereby restraining the decreased ratio of K+/Na+, iscritical for plant salt tolerance [8–10]. Potassium channels or trans-porters are responsible for the K+ transport into or out of root

cells [11]. Several reports showed that the outward-rectifying K+

channels, such as shaker-like K+ outward-rectifying channel (SKOR)and guard cell outward rectifying K+ channel (GORK), are involvedin K+ efflux. SKOR is expressed in the root stele and responsible

1 ience

flimN

tmsttm(ied[rprs(r

fiaeleaatrunhmcid(tshMrmau

(ssouswtHpwHpsrt

18 D. Lai et al. / Plant Sc

or K+ release to the xylem [12], while GORK is found to regu-ate K+ release in guard cell and stomatal closure [13]. Meanwhile,nward-rectifying K+ channels (IRK) have also been identified as

echanisms for long-term net K+-selective influx, and may reducea+ toxicity [14].

On the other hand, several lines of evidence have shownhat plant tolerance against salt stress is closely related to the

aintenance of cellular redox balance [1,4]. Redox-regulationystems could regulate the cascades of uncontrolled oxidation,hus protecting plant cells from oxidative damage and main-aining endogenous ROS homeostasis [15]. Plant redox-regulation

achinery was composed of several non-enzymatic antioxidantsglutathione, GSH; ascorbic acid, AsA, etc.) and the efficient antiox-dant defence systems (catalase, CAT; superoxide dismutase, SOD,tc.) [16,17]. Particularly, homoglutathione (hGSH) is more abun-ant than GSH in legumes, in which glycine is substituted by alanine18]. GSH/hGSH pool is regarded as an important parameter thateflects the redox state of cells, which is subsequently vital forlant tolerance to heavy metals-triggered oxidative stress [19]. Theecycling pathways of GSH and AsA include several key enzymes,uch as ascorbate peroxidase, monodehydroascorbate reductaseMDHAR), dehydroascorbate reductase (DHAR), and glutathioneeductase (GR) [15].

Like nitric oxide (NO) and carbon monoxide, hydrogen sul-de (H2S), a colorless gas with foul odor of rotten eggs, is knowns the gaseous transmitter in mammalian studies [20,21]. Forxample, H2S has been reported to participate in several physio-ogical processes, such as blood pressure regulation and neuronalxcitability [22,23]. Meanwhile, anti-inflammatory, vaso-relaxantnd neuron-protective functions of H2S in mammalian studies havelso been described [24–26]. Due to its association with adap-ive responses against multiple stress conditions, H2S has furthereceived increasing attention by plant researchers. It has been doc-mented that H2S is involved in the anti-oxidative response againstumerous environmental stimuli, including copper, aluminum,eat, drought, and osmotic stresses [27–31]. H2S was generatedainly by the desulfhydrases in higher plants. Hereinto, two spe-

ific desulfhydrases have been identified as the key enzymesnvolved in plant H2S biosynthesis, namely l-cysteine desulfhy-rase (l-DES, EC4.4.1.1; in particular) and d-cysteine desulfhydrased-DES, EC4.4.1.15) [32–35]. The transcript abundance of l-DES orotal enzyme activity of l-DES was induced/increased by droughttress, salicylic acid, abscisic acid, etc. [36,37]. On the otherand, the transcripts encoding l-DES have not been cloned inedicago sativa. Mutation of Arabidopsis l-DES (DES1) gene dis-

upts the cytosolic H2S generation and strongly affects plantetabolism [38]. However, the physiological functions of l-DES

nd its generated H2S involved in the plant salt tolerance are stillnknown.

Previous studies revealed that exogenous sodium hydrosulfideNaHS, a H2S or HS− donor) promotes alfalfa seed germination uponalt stress by reducing oxidative damage, which might have a pos-ible interaction with NO [39]. However, the biological significancef endogenous H2S and its related enzymatic source in the mod-lation of salt tolerance in alfalfa seedling remain obscure. In thistudy, by using the inhibitor of l-DES and a scavenger of H2S asell as checking the kinetics changes of endogenous H2S produc-

ion, our aim was to characterize the impact of NaCl on endogenous2S homeostasis and subsequent NaCl toxicity symptoms, witharticular emphasis on the temporal signature of this event. Mean-hile, the physiological and biochemical function of endogenous2S homeostasis that acts as a critical bio-modulator to improve

lant tolerance against salt stress was also examined. Our resultsupport the hypothesis that H2S could serve as an endogenousegulon to enhance salt tolerance by helping reestablishment ofhe redox balance including the improvement of hGSH/hGSSGh in

225 (2014) 117–129

particular, as well as preventing the K+ leakage in alfalfa seedlingroots.

2. Materials and methods

2.1. Plant materials, growth conditions and treatments

Alfalfa (M. sativa L. cv. Biaogan, 2n = 4x = 32, semi-dormant geno-type, fall dormancy rating of 6) seeds were surface-sterilized with5% NaClO for 10 min, rinsed extensively in distilled water and ger-minated for 1 day at 25 ◦C in the darkness. Identical seeds werethen selected and transferred to the plastic chambers and culturedin quarter-strength Hoagland’s solution (pH 6.0) for another 4 daysin the growth incubator (25 ± 1 ◦C, 14 h photoperiod with a lightintensity of 200 �mol m−2 s−1).

For physiological assays, 5-day-old alfalfa seedlings weregrown hydroponically in plastic chambers with quarter-strengthHoagland’s solution in the presence or absence of varying concen-trations of NaCl, NaHS (a H2S or HS− donor), Na2SO4 (100 �M),NaHSO3 (100 �M), NaHSO4 (100 �M), CH3COONa (100 �M),DL-propargylglycine (PAG; 2 mM; an inhibitor of l-DES), orhypotaurine (HT; 10 mM; a scavenger of H2S) alone for 6 h orthe indicated times as presented in figure legends, respectively,followed by the exposure to NaCl stress at the indicated concen-trations for the indicated times as presented in figure legends. Theconcentrations of these chemicals were determined in pilot experi-ments from which significant responses were obtained. The samplewithout chemicals was regarded as control (Con). The pH of nutri-ent medium or treatment solutions was adjusted to 6.0. At theindicated time points, the mature zone or intact alfalfa seedlingroots were chosen for used immediately or quick-frozen in liquidnitrogen, and stored at −80 ◦C for further analysis.

2.2. Determination of fresh weight, primary root elongation anddry weight

The primary root elongation was determined according to themethod described previously [6]. Fresh weight and dry weight werealso measured.

2.3. Determination of endogenous H2S content, total activities ofl-DES and d-DES

Endogenous H2S content was determined by the formationof methylene blue from dimethyl-p-phenylenediamine in H2SO4according to the method described previously [27]. Root samples(0.2 g) were extracted in 1 ml of phosphate buffer solution (pH 6.8,50 mM) containing 0.1 M EDTA and 0.2 M AsA. The supernatantwas mixed with 0.5 ml of 1 M HCl in a test tube to release H2S,and H2S was absorbed in 1% (w/v) zinc acetate (0.5 ml) trap whichis located in the bottom of the test tube. After 30 min reaction,0.3 ml 5 mM N,N-dimethyl-p-phenylenediamine dihydrochloridedissolved in 3.5 mM H2SO4 was injected into the trap. Then 0.3 ml of50 mM ferric ammonium sulphate in 100 mM H2SO4 was injectedinto the trap. The amount of H2S in zinc acetate traps was deter-mined spectrophometrically at 670 nm, after leaving the mixturefor 15 min at room temperature. Solutions with different concen-trations of Na2S were prepared, treated in the same way as theassay samples, and were used for the quantification of H2S.

Total l-DES activity was determined according to previousmethod with some modifications [37]. Soluble proteins wereextracted by adding 1 ml of 20 mM Tris–HCl (pH 8.0) to 0.2 g of

samples. Centrifuged at 12,000 × g for 15 min, the protein contentof the supernatant was adjusted to 100 �g ml−1 to obtain equalamounts of protein in each assay sample. Total l-DES activity wasdetermined by the release of H2S from l-cysteine in the presence

ience 2

o1atwHdwdstwcbm

2(

dftibtt0pwc

2

ipAtZ

2

uswbatpbLDo

2

pptwwa

D. Lai et al. / Plant Sc

f dithiothreitol (DTT). The assay contained in a total volume of ml: 0.8 mM l-cysteine, 2.5 mM DTT, 100 mM Tris–HCl (pH 9.0),nd 10 �g protein solution. The reaction was initiated by the addi-ion of l-cysteine. After incubated for 15 min at 37 ◦C, the reactionas terminated by adding 100 �l of 30 mM FeCl3 dissolved in 1.2 NCl and 100 �l of 20 mM N,N-dimethyl-p-phenylenediamine dihy-rochloride dissolved in 7.2 N HCl. The formation of methylene blueas determined at 670 nm by a spectrophotometer. Solutions withifferent concentrations of Na2S were also prepared, treated in theame way as the assay samples, and were used for the quantifica-ion of enzymatically formed H2S. By contrast, total d-DES activityas determined in the same way with following modifications: d-

ysteine instead of l-cysteine was used, and the pH of the Tris–HCluffer was adjusted to 8.0 [34]. Protein was determined by theethod of Bradford [40].

.4. Determination of thiobarbituric acid reactive substancesTBARS) contents

Lipid peroxidation was determined by measuring TBARS pro-uction as previously described [41,42]. Approximately 500 mgresh tissue was ground in 0.25% 2-thiobarbituric acid in 10%richloroacetic acid (TCA) using a mortar and pestle. After heat-ng at 95 ◦C for 30 min, the mixture was quickly cooled in an iceath and centrifuged at 10,000 × g for 10 min. The absorbance ofhe supernatant was read at 532 nm, and corrected for unspecificurbidity by subtracting the absorbance at 600 nm. The blank was.25% 2-thiobarbituric acid in 10% TCA. The concentration of lipideroxides together with oxidatively-modified proteins of plantsas thus quantified in terms of TBARS level using an extinction

oefficient of 155 mM−1 cm.

.5. Histochemical analyses

As described previously, histochemical detection of lipid perox-dation and loss of plasma membrane integrity in root apexes wereerformed with Schiff’s reagent and Evans blue, respectively [42].fter staining, all the roots stained were washed three times, and

hen observed under a light microscope (model Stemi 2000-C; Carleiss, Germany).

.6. Confocal determination of ROS production

Endogenous ROS level was assayed by confocal microscopysing 2′,7′-dichlorofluorescein diacetate (H2DCF-DA). Alfalfaeedlings were collected at the indicated time points and loadedith 50 �M H2DCF-DA for 20 min before washing in 20 mM HEPES

uffer (pH 7.8) three times for 5 min each, and then analyzed using TCS-SP2 laser scanning confocal microscope (LSCM; Leica, excita-ion at 488 nm, emission at 500–530 nm). All manipulations wereerformed at 25 ± 1 ◦C. ROS production in root tips was quantifiedased on 20 overlapping confocal planes of 2 �m each using theeica software package. Each treatment had n = 6 per experiment.ata are presented as relative units of pixel intensities via regionf interest analysis, provided by the Leica software.

.7. Antioxidant enzyme assays

Seedling roots (100 mg) were homogenized in 2 ml of 50 mMotassium phosphate buffer (pH 7.0) containing 1 mM EDTA and 1%olyvinylpyrrolidone for the CAT, SOD and GR assays, with the addi-

ion of 1 mM AsA for DHAR and MDHAR assays. The homogenateas centrifuged at 12,000 × g for 20 min at 4 ◦C and the supernatantas used as the crude enzyme extract. SOD and CAT activities were

nalyzed by the methods described in previous reports [43]. GR,

25 (2014) 117–129 119

DHAR, and MDHAR activities were analyzed as described previ-ously [44]. Protein was determined by the method of Bradford [40].

2.8. Real-time RT-PCR analysis

Total RNA of fresh-weight alfalfa roots (100 mg) was isolatedby grinding with mortar and pestle in liquid nitrogen until a finepowder appeared and using Trizol reagent (Invitrogen) accord-ing to the manufacturer’s instructions. The RNA samples weretreated with RNAase-free DNase (TaKaRa Bio Inc., Dalian, China) toeliminate traces of DNA, followed by the quantification using theNanoDrop 2000 (Thermo Fisher Scientific, Wilmington, DE, USA).Afterwards, total RNA (2 �g) was reverse-transcribed using an oligod(T) primer and M-MLV reverse transcriptase (BioTeke, Beijing,China). Real-time quantitative RT-PCR reactions were performedusing a Mastercycler® ep realplex real-time PCR system (Eppen-dorf, Hamburg, Germany) with SYBR® Premix Ex TaqTM (TaKaRa BioInc., China). Using specific primers (Suppl. Table S1), the expressionlevels of the genes are presented as values relative to the cor-responding control samples under the indicated conditions, withnormalization of data to the geometric average of internal controlgene Actin2.

2.9. Determination of Na+ and K+ contents by inductively coupledplasma-optical emission spectrometer (ICP-OES)

Fresh root tissues were washed twice with EDTA-Na2 solutionand rinsed briefly in de-ionized water. Afterwards, samples wereoven-dried at 60 ◦C, then digested with HNO3 using a MicrowaveDigestion System (Milestone Ethos T, Italy). The Na+ and K+ contentswere determined using ICP-OES (Perkin Elmer Optima 2100DV)[44].

2.10. Electron probe X-ray microanalysis

Element ratio measurement was examined with a scanningelectron microscope (model S-3000N; Hitachi High-TechnologiesCorporation, Tokyo, Japan) equipped with an energy-dispersiveX-ray detector (EDX; Horiba Inc., Kyoto, Japan) as described pre-viously [44,45]. After being placed vertically in the hole of analuminum stub and immersed quickly into liquid nitrogen forfreeze-drying, the tender root tips were transferred quickly into avacuum evaporator and dried under the vacuum (5 × 10−4 Torr) forat least 12 h. The root tips were coated with a fine layer of pure evap-orated carbon, and then observed. Acceleration voltage of 10 kV wasused. Beam current was adjusted to a fixed (0.06 mA), and the work-ing distance from the EDX detector was 13.5 mm. For an analysisof relative elemental level within cortical and stelar cells of roottips accurately, a point analysis of each sample was conducted forat least four times. The results were calculated by expressing theatomic number for a particular element in a given point or region asa percentage of the total atomic number for all the elements mea-sured (K, Na, calcium, chlorine, phosphorus, oxygen) in the roottips.

2.11. Measurements of K+ fluxes with non-invasive micro-testtechnology (NMT)

Transient K+ fluxes were measured through non-invasive micro-test technology (NMT, Younger USA Sci. and Tech. Corp., Amherst,MA, USA, http://www.youngerusa.com; Xuyue-Sci. & Tech. Co. Bei-

jing, China, http://www.xuyue.net) [46]. 5-Day-old seedlings wasfloated in measuring solution (0.1 mM KCl, 0.1 mM CaCl2, 0.1 mMMgCl2, 0.5 mM NaCl, 0.2 mM Na2SO4, 0.3 mM MES, pH 6.0) forat least 15 min before measurement. Concentration gradients of

1 ience

teppflifiiacX

2(

aotcuscAf(

a5t

FsmTiA

20 D. Lai et al. / Plant Sc

arget ions were measured by moving the ion-selective micro-lectrode between two positions close to the plant material in areset excursion with a distance of 30 �m. Each cycle was com-leted in approximately 6 s. For transient measurements, steadyuxes of K+ in the mature zone were recorded for about 10 min

n control conditions, followed by salt shock treatment (with anal NaCl concentration in the buffer of 175 mM). The transient

on fluxes were monitored for 15–20 min to insure that no fluctu-tion was present and 6 seedlings per treatment. Ionic fluxes werealculated with the MageFlux acquisition program, developed byuyue-Sci. & Tech. Co.

.12. Thiol analysis by ultra performance liquid chromatographyUPLC)

Contents of GSH, reduced and oxidized homoglutathione (hGSHnd hGSSGh), and l-cysteine were measured according to the meth-ds reported previously, with minor modification [47,48]. Rootissues were extracted by 1 ml of 0.2 M HCl. The homogenate wasentrifuged at 13, 000 g for 15 min at 4 ◦C. The supernatant wassed for analysis. After neutralized by 0.2 M NaOH, the extractupernatant (200 �l) was mixed with 100 �l of 500 mM 2-(N-yclohexylamino)ethane-sulphonic acid (CHES) buffer (pH 8.5).fter that, 20 �l of 10 mM DTT was added and incubated for 30 min,

ollowed by the addition of 20 �l of 30 mM monobromobimanemBBr) to tab thiols.

For determination of hGSSGh, N-ethylmaleimide (NEM) waspplied after neutralization. After removal of NEM, 100 �l of00 mM CHES (500 mM) was added. DTT was then added in ordero reduce disulphides to thiols, which could be labeled by mBBr.

ig. 1. Changes of root elongation (A), endogenous H2S production (B and D), total l-DES (Ceedlings were treated with different concentrations of NaCl (0, 50, 100, 175, 200, and 3easured 5 days later (A). Endogenous H2S content, total l-DES activity were determine

ime course determination of total d-DES activity (F) was also performed. The sample windependent experiments with at least three replicates for each. Bars with different lettesterisks indicate significantly different between treatments at the same time points at P

225 (2014) 117–129

After incubated with mBBr for 15 min under dim light, theprocess of conjugation was completed, and 660 �l of 10% (v/v)acetic acid was added to stabilize the mBBr derivatives. After cen-trifugation at 10,000 × g for 10 min, the supernatant was filteredthrough a 0.22 �m organic filter, and 4 �l of the mixture wassubjected to UPLC analysis (Agilent Technologies, 1290 series).Thiol derivatives were separated on a ZORBAX Eclipse Plus C18column (2.1 mm × 100 mm, 1.8-Micron; Agilent) at a flow rate of0.2 ml min−1. The linear gradient was from 0% solution B to 10%(v/v) solution B (90% methanol, 0.25% acetic acid, pH 4.3) within10 min. This composition was maintained for 0.8 min; thereafterthe column was washed with 100% solution B for 4 min and re-equilibrated with 100% solution A (10% methanol, 0.25% acetic acid,pH 4.3) for 5 min. Thiol derivatives were quantified by fluorescencedetection (excitation at 380 nm, emission at 480 nm). A standardsolution of 0.2 mM l-cysteine, GSH and hGSH were used for quan-tification, with retention times of 4.3, 6.3 and 9.3 min, respectively.

2.13. Determination of AsA and dehydroascorbate (DHA)

Contents of AsA and DHA were measured according to the pre-vious method [49]. Fresh root tissues were homogenized in cold6% TCA immediately, and centrifuged at 12,000 × g for 20 min at4 ◦C, and the supernatant was collected. For the determinationof AsA, distilled water (200 �l) was added into the supernatant(200 �l); while for DHA determination, 10 mM DTT (100 �l) and

0.5% (w/v) N-ethylmaleimide (100 �l) were added. Afterwards, fol-lowing reagents were then added to each sample: 10% TCA (400 �l),44% orthophosphoric acid (400 �l), 4% 2,2-dipyridyl in 70% ethanol(400 �l), and 0.3% (w/v) FeCl3 (200 �l). After vortex mixing, the

and E), and d-DES (F) activities in alfalfa seedling roots upon NaCl stress. 5-Day-old00 mM for A–C; 175 mM for D–F). Afterwards, corresponding root elongation wasd 6 h after various treatments (B and C), or at the indicated time points (D and E).thout chemicals was regarded as the control (Con). Values are means ± SE of threers are significantly different at P < 0.05 according to Duncan’s multiple range test.

< 0.05 according to t-test.

ience 225 (2014) 117–129 121

ma

2

psa

3

3b

seFm1teludawiroDsNi

twHputwbrr

3g

uwiw2tNNbwaiws(

Fig. 2. Effects of sodium hydrosulfide (NaHS) pretreatment on root elongation (A)and TBARS accumulation (B) in the roots of alfalfa upon NaCl stress. 5-Day-oldseedlings were pretreated with the indicated concentrations of NaHS for 6 h andthen exposed to 0 or 175 mM NaCl for another 120 h (A) or 12 h (B). Afterwards,root elongation (A) and TBARS accumulation (B) were measured. The sample with-out chemicals was regarded as the control (Con). Values are means ± SE of threeindependent experiments with at least three replicates for each. Bars with different

D. Lai et al. / Plant Sc

ixture was incubated at 37 ◦C for 60 min and the change inbsorbance at 525 nm was recorded.

.14. Statistical analysis

For statistical analysis, either t-test (P < 0.05) or Duncan’s multi-le range test (P < 0.05) was selected where appropriate. Values arehown as the means ± SE of three independent experiments witht least three replicates for each.

. Results

.1. NaCl-induced alfalfa seedling growth inhibition and H2Siosynthesis

To assess the toxicity symptom of alfalfa seedling upon NaCltress, root elongation was determined after seedlings werexposed to different NaCl concentrations for 5 days. As shown inig. 1A, seedling root elongation was inhibited in a dose-dependentanner by increasing NaCl concentrations (50–300 mM), with

75 mM NaCl exhibited approximately 42% of inhibition. To inves-igate whether H2S is associated with this process, changes ofndogenous H2S content and total activity of its synthetic enzyme-DES were further measured in alfalfa seedling roots. As expected,pon the exposure of 50–300 mM NaCl (6 h), both the H2S pro-uction and total l-DES activity were increased approximately in

dose-dependent fashion (Fig. 1B and C). For example, comparedith the untreated control samples, a treatment of 175 mM NaCl

ncreased H2S production or total l-DES activity by 52% or 46%,espectively. Compared with the moderate induction, higher dosef NaCl treatment (300 mM) resulted in the overproduction of l-ES-associated H2S production, which apparently preceded the

evere inhibition of root elongation. In the following experiments,aCl (175 mM) was applied to investigate the protective role of H2S

n NaCl toxicity.Subsequently, the time-course analysis of endogenous H2S con-

ent, total activities of l-DES and d-DES were performed. Comparedith the control samples, a moderate and substantial increase of2S production was observed 3 h after NaCl exposure, followed byeaking at 6 h and remaining higher levels than the control samplesntil 24 h after the beginning of treatment (Fig. 1D). The changes inotal activities of l-DES exhibited the similar tendencies (Fig. 1E),hereas total activities of d-DES were not significantly altered

y NaCl (Fig. 1F). Thus, these results indicated a possible inter-elationship between l-DES-related H2S homeostasis and salinityesponses in alfalfa seedlings.

.2. Exogenous NaHS alleviated NaCl-inhibited alfalfa seedlingrowth and lipid peroxidation

In the following experiment, the protective role of H2S was eval-ated by examining the effects of sodium hydrosulfide (NaHS), aell-known H2S or HS− donor used both in animal and plant stud-

es [27–29,50], on root elongation of alfalfa seedlings. As expected,e observed that the pretreatment with NaHS (varying from 10 to

00 �M; for 6 h) progressively alleviated the NaCl-caused inhibi-ion of root elongation in alfalfa seedlings, with 100 and 200 �MaHS in particular (Fig. 2A). Meanwhile, a high concentration ofaHS (1000 �M) treatment exhibited a negative response. A slightut no significant increase in alfalfa root elongation was observedhen NaHS was applied alone (ranging from 10 to 200 �M). We

lso observed that NaHS treatment alone at 1000 �M significantly

nhibited root elongation of alfalfa seedlings, which was similar

ith the previous results showing the overproduction of H2S andevere root growth inhibition upon a high dose of NaCl treatment300 mM; Fig. 1A–C).

letters are significantly different at P < 0.05 according to Duncan’s multiple rangetest.

TBARS is a reliable indicator for salinity-induced lipid peroxida-tion [36,39]. It was found that, compared with salt-stressed alonesample, pretreatment with the increasing doses of NaHS (rangingfrom 10 to 200 �M) caused a considerable decrease in the NaCl-induced TBARS levels at 12 h, with a maximal response at 100 �MNaHS (Fig. 2B). Meanwhile, NaHS at high concentration (1000 �M)treatment produced a negative response. Therefore, 100 �M NaHSwas applied to investigate the cytoprotective role of H2S in thefollowing investigation.

3.3. H2S/HS−, rather than other derivatives, contributes to thealleviation of NaCl-induced inhibition of root elongation

It should be noticed that the NaHS solution still containssodium-and other sulfur-containing components [39,51,52]. Inorder to discriminate the specific role of H2S/HS− from that ofother compounds derived from NaHS, a range of sodium- andsulfur-containing compounds were used as the negative controls ofNaHS, including Na2SO4, NaHSO3, NaHSO4 and CH3COONa. Resultsshowed that above chemicals failed to rescue NaCl-inhibited rootelongation (Fig. 3A). Meanwhile, compared with stress alone sam-ple, a similar or decreased endogenous H2S content was observed(Fig. 3B). We also noticed that the administration of NaHS (100 �M)strengthened the NaCl-triggered induction of endogenous H2S lev-els, within the similar physiological range. Therefore, these results

suggested that H2S or HS−, rather than other compounds derivedfrom NaHS, plays an important role in regulating the alfalfa rootelongation under NaCl stress.

122 D. Lai et al. / Plant Science 225 (2014) 117–129

Fig. 3. H2S or HS− , but not other compounds derived from NaHS, are contributedto the alleviation of NaCl-inhibited root elongation (A) and increased H2S produc-tion (B). 5-Day-old seedlings were pretreated for 6 h with or without 100 �M NaHS,100 �M Na2SO4, 100 �M NaHSO3, 100 �M NaHSO4, or 100 �M CH3COONa. After-wards, seedlings were then exposed to 175 mM NaCl for another 120 h (A) or 6 h(B), then root elongation (A) and H2S content in roots (B) were measured, respec-tively. The sample without chemical was regarded as the control (Con). Values aremBm

3a

sa2es4bw

iec(ifsi

oio

Fig. 4. Effects of pretreatment with NaHS, PAG or HT on the morphology (A), rootfresh weight (B) and elongation (C) of alfalfa seedlings upon NaCl exposure. 5-Day-old seedlings were pretreated with NaHS (100 �M), PAG (2 mM) or HT (10 mM)for 6 h, respectively, and then exposed to 0 or 175 mM NaCl for another 120 h.Afterwards, photographs (A) were taken. Bar = 1 cm (A). The arrow indicated thelength of seedling roots before NaCl treatment. Meanwhile, corresponding parame-ters (B, C) were measured. The sample without chemicals was regarded as the control

eans ± SE of three independent experiments with at least three replicates for each.ars with different letters are significantly different at P < 0.05 according to Duncan’sultiple range test.

.4. NaCl-triggered toxic symptoms were aggravated by theddition of a l-DES inhibitor or a H2S scavenger

To further clarify the participation of endogenous H2S homeo-tasis in the regulation of NaCl-induced responses, we adopted

pharmacological approach by using DL-propargylglycine (PAG; mM, a l-DES inhibitor) or hypotaurine (HT; 10 mM, a H2S scav-nger) [52–54]. As expected, compared to the NaCl-treated aloneample, the pretreatment of PAG blocked the total l-DES activity by2.5% (Supp. Fig. S1), while HT reduced the endogenous H2S levely 37.0% (Supp. Fig. S2), further confirming that PAG or HT appliedith 2 mM or 10 mM was effective in our experimental conditions.

Compared with the alleviation of NaCl-triggered growthnhibition induced by NaHS pretreatment (Fig. 2), subsequentxperiments revealed that the pretreatment of PAG or HT, an effi-ient inhibitor of l-DES (Supp. Fig. S1) or endogenous H2S scavengerSupp. Fig. S2), could further aggravate the NaCl-induced growthnhibition, in terms of the morphological observation (Fig. 4A), rootresh weight (Fig. 4B), and root elongation (Fig. 4C). These resultsuggested the beneficial behaviors of endogenous H2S homeostasisn alfalfa seedling roots upon NaCl stress.

The protective role of H2S on the alleviation of NaCl-inducedxidative damage was also examined by the histochemical staining,n which the assessment of the loss of plasma membrane integrityr lipid peroxidation was performed by using Evans blue or Schiff’s

(Con). Values are means ± SE of three independent experiments with at least threereplicates for each. Bars with different letters are significantly different at P < 0.05according to Duncan’s multiple range test.

reagent, respectively [42]. As shown in Fig. 5A and B, the alfalfaseedling roots treated with NaCl alone were stained extensivelywith either Evans blue or Schiff’s regent, while those pretreatedwith NaHS had only slight staining. It was found that pretreatmentof PAG or HT exacerbated the NaCl-triggered staining pattern, withmore pronounced observation of blue color precipitates in bothroot tip and elongation zone (Evans blue staining). The changes ofTBARS levels (Fig. 5C) were approximately consistent with aboveobservations. Taken together, abovementioned results suggestedthat endogenous H2S homeostasis conferred the protection againstNaCl-induced oxidative damage in alfalfa seedling roots.

3.5. Reestablishment of redox homeostasis

NaCl-triggered oxidative stress was related to the overproduc-tion of ROS [4,5,15]. In this study, we found that NaCl-induced

enhancement of ROS-related DCF fluorescence intensity was fur-ther quenched by the pretreatment of NaHS, whereas individuallystrengthened by PAG or HT pretreatment, with PAG in particu-lar (Fig. 5D and E). These results indicated that endogenous H2S

D. Lai et al. / Plant Science 225 (2014) 117–129 123

Fig. 5. Effects of pretreatment with NaHS, PAG and HT on the histochemical localization of the loss of plasma membrane integrity (A) and lipid peroxidation (B), TBARSaccumulation (C), and DCF fluorescence (D) in alfalfa seedling roots upon NaCl exposure. 5-Day-old seedlings were pretreated with NaHS (100 �M), PAG (2 mM) or HT(10 mM) for 6 h, respectively, and then exposed to 0 or 175 mM NaCl for another 24 h. Afterwards, corresponding parameters were measured. Bar = 1 mm (A–D). Mean of DCFfluorescence intensity were also calculated via region of interest analysis (E). R.U.: relative unit. The sample without chemicals was regarded as the control (Con). Values arem h. Barm der is

psrwsusaticeoo

mtqNtRptb

[

eans ± SE of three independent experiments with at least three replicates for eacultiple range test. (For interpretation of the references to color in the text, the rea

roduction could modulate ROS homeostasis upon NaCl stress. Sub-equently, it was necessary to investigate the redox state of alfalfaoots, including several antioxidants and antioxidant enzymes thatere responsible for ROS scavenging. Our results revealed a con-

iderable decrease in total activity of SOD in alfalfa seedling rootspon NaCl treatment, being 26% lower than the NaCl-free controlample (Fig. 6A). The pretreatment of NaHS, however, significantlylleviated the NaCl-inhibited SOD activity, being 37% higher thanhose of NaCl treatment alone. Meanwhile, total activity of CAT wasncreased in the presence of NaCl, being 48% higher than that ofontrol sample, while pretreatment of NaHS obviously strength-ned this increasing tendency (Fig. 6B). By contrast, a pretreatmentf PAG or HT could differentially aggravated or blocked the effectsf NaCl on SOD or CAT activity.

Using real-time RT-PCR analysis, it was shown that NaCl treat-ent led to the decrease of Mn-SOD or slight induction of Cu/Zn-SOD

ranscripts in alfalfa seeding roots, both of which were subse-uently reversed or promoted by NaHS (Fig. 6C). Moreover, theaCl-induced Cu/Zn-SOD transcript was reversed by PAG or HT pre-

reatment, while Mn-SOD expression was not significantly altered.egarding to the expression of CAT, our results showed that NaHSretreatment strengthened the NaCl-triggered induction of CAT

ranscript. However, PAG or HT pretreatment brought about a slightut no significant decrease (Fig. 6D).

Because alfalfa plants contained much more hGSH than GSH18,55], a methodology using UPLC combined with fluorescence

s with different letters are significantly different at P < 0.05 according to Duncan’s referred to the web version of this article.)

detection was adopted to investigate the changes of several antiox-idants. With respect to the control samples, it was observed thattreatment of NaCl led to an obvious decrease in both GSH and hGSHcontents, as well as an increase of hGSSGh in alfalfa roots (Table 1;GSSG was not detected). However, a pretreatment with NaHS sig-nificantly diminished the effects of NaCl on the changes of GSH,hGSH and hGSSGh, further leading to the maintenance of a higherratio of hGSH/hGSSGh, which is a critical indicator for the intracel-lular redox status [18,55]. Meanwhile, our results also showed thatthe NaCl-triggered decrease of hGSH content and hGSH/hGSSGhratio were significantly aggravated by the pretreatment with PAGor HT. Comparatively, NaCl-induced the responses of the AsA pools(contents of AsA and DHA) was promoted by the pretreatment ofNaHS, with AsA in particular, further leading to the reversal of thedecreased AsA/DHA ratio. A contrasting change in AsA/DHA wasobserved when PAG or HT was individually applied. Interestingly,NaCl-induced l-cysteine content was enhanced by the pretreat-ment of NaHS, PAG or HT. Moreover, compared with NaCl-freecontrol samples, NaHS pretreatment alone did not significantlyaffect hGSSGh content, while the content of GSH, hGSH, l-cysteine(in particular) or AsA was significantly altered.

Subsequently, the total activities of several representative

enzymes involved in the recycling of GSH/hGSH and AsA poolswere analyzed, such as GR, DHAR, and MDHAR. Experimentalresults revealed that NaCl treatment produced a clear decline ofGR and DHAR activities, both of which were rescued by NaHS

124 D. Lai et al. / Plant Science 225 (2014) 117–129

Fig. 6. Effects of pretreatment with NaHS, PAG or HT on the total activities and corresponding transcripts of SOD and CAT in alfalfa seedling roots upon NaCl exposure.5-Day-old seedlings were pretreated with NaHS (100 �M), PAG (2 mM) or HT (10 mM) for 6 h, respectively, and then exposed to 0 or 175 mM NaCl for another 24 h (A, B)or 12 h (C, D). Afterwards, the total activities of SOD (A) and CAT (B) were assayed. The transcript levels of the Mn-SOD and Cu/Zn-SOD (C), and CAT (D) were analyzed byreal-time RT-PCR. The sample without chemicals was regarded as the control (Con). Values are means ± SE of three independent experiments with at least three replicatesfor each. Bars with different letters are significantly different at P < 0.05 according to Duncan’s multiple range test.

Table 1GSH, homoglutathione (hGSH and hGSSGh), l-cysteine, AsA pools (AsA and DHA) and the ratio of corresponding reduced to oxidized form (hGSH/hGSSGh, AsA/DHA) in alfalfaseedling roots. 5-Day-old seedlings were treated with 175 mM NaCl for 24 h with or without 6 h pretreatment with NaHS (100 �M), PAG (2 mM) or HT (10 mM). The samplewithout chemicals was the control (Con). Values are means ± SE of three independent experiments with at least three replicates for each. Different letters within columnsindicate significant differences (P < 0.05) according to Duncan’s multiple range test.

Treatment GSH hGSH hGSSGh hGSH/hGSSGh l-Cysteine AsA DHA AsA/DHA(nmol g−1 FW) (nmol g−1 FW) (nmol g−1 FW) (�g g−1 FW)

Con 11.0 ± 0.2c 192.3 ± 4.9b 72.8 ± 2.6e 2.63 14.6 ± 1.1d 52.4 ± 1.7e 17.3 ± 0.6c 3.03NaHS → Con 7.5 ± 0.5d 206.9 ± 9.5a 78.7 ± 2.6de 2.62 47.0 ± 2.6a 65.3 ± 0.8c 20.6 ± 0.7bc 3.17NaCl 6.9 ± 0.1d 164.7 ± 9.3d 136.6 ± 4.4a 1.20 23.1 ± 8.2c 72.4 ± 3.0b 25.3 ± 1.0ab 2.86NaHS → NaCl 14.1 ± 0.5a 178.0 ± 1.9c 113.0 ± 6.5b 1.58 31.5 ± 0.2b 92.1 ± 4.1a 28.8 ± 2.4a 3.20

1

8

p(Nb(

enoNstwo

3

o

PAG → NaCl 14.2 ± 0.1a 91.8 ± 6.2f 90.5 ± 9.4cd 1.0HT → NaCl 12.2 ± 0.6b 105.9 ± 4.9e 98.5 ± 4.3c 1.0

retreatment, but further differentially inhibited by PAG or HTFig. 7A and B). However, the activity of MDHAR was elevated uponaCl stress, and this inducible tendency was further strengthenedy NaHS, but reversed to a lower level by PAG or HT pretreatmentFig. 7C).

The transcripts encoding above-mentioned enzymes were alsoxamined. It was observed that the NaCl treatment produced a sig-ificant up-regulation of GR1 transcript, while expression of GR2r MDHAR was not altered, all of which were up-regulated by theaHS pretreatment (Fig. 7D and F). MDHAR transcript was further

uppressed by PAG or HT pretreatment. Upon NaCl stress, however,he transcript abundance of DHAR was declined significantly, whichas reversed and elevated to a higher level by the administration

f NaHS (Fig. 7E).

.6. Maintenance of ion homeostasis

To test the effect of NaHS on element contents in root tissuesf alfalfa seedlings, Inductively Coupled Plasma-Optical Emission

34.1 ± 1.8b 66.5 ± 2.2c 23.9 ± 5.4ab 2.7834.6 ± 1.7b 61.1 ± 1.4d 26.8 ± 3.0a 2.28

Spectrometer (ICP-OES) assay was performed. As shown in Fig. 8Aand B, compared with those of NaCl-treated alone samples, theexposure of alfalfa seedlings to NaHS pretreatment led to a remark-able increase of K+ content in root parts, a slight but no significantincrease of Na+ level was also observed. However, pretreatmentof PAG or HT had no significant impact on both Na+ and K+ levelsin alfalfa seedling roots. In addition, the sub-cellular distributionof element was examined with a scanning electron microscopeequipped with an energy-dispersive X-ray detector. In compari-son with those of salt-stressed alone samples, NaHS pretreatmentresulted in the enhancement of K/Na ratio in stellar cells but not incortical cells of root tissues (Fig. 8C).

To further characterize the effect of H2S on the modulationof K+ homeostasis in alfalfa seedling roots upon salt stress, thenon-invasive micro-test technology (NMT) was adopted. Similar

to previous studies [8–10], acute NaCl stress led to a large K+ effluxin the mature zone of alfalfa seedling roots (Fig. 9A). However,a 6-h pretreatment of alfalfa seeding roots with NaHS (100 �M)significantly reduced the NaCl-induced transient K+ efflux, further

D. Lai et al. / Plant Science 225 (2014) 117–129 125

Fig. 7. Effects of pretreatment with NaHS, PAG or HT on the total activities and corresponding transcripts of GR, DHAR, and MDHAR in alfalfa seedling roots upon NaCle HT (1( R (C) wa ontrolr are sig

rK

itTtSnbasaw(NowN

xposure. 5-Day-old seedlings were pretreated with NaHS (100 �M), PAG (2 mM) orA–C) or 12 h (D–F). Afterwards, the total activities of GR (A), DHAR (B), and MDHAnalyzed by real-time RT-PCR. The sample without chemicals was regarded as the ceplicates for each. Within each identified enzyme/gene, bars with different letters

esulting in a 49% decrease in the magnitude of NaCl-induced total+ efflux (Fig. 9B).

The NaCl-triggered K+ efflux out of cells is a result of NaCl-nduced membrane depolarization, leading to the activation ofhe depolarization-activated outward-rectifying K channels [11].herefore, we further examined the transcript that encoding pro-ein potential responsible for K+ efflux. The observed results of theKOR expression, which encodes an outward rectifying K+ chan-el involved in the release of K+ to the xylem, was up-regulatedy salt stress (Fig. 9C). This induced tendency, which was in par-llel with the electron-physiological data (Fig. 9A and B), wasignificantly impaired by NaHS pretreatment, while not markedlyffected by PAG or HT pretreatment (Fig. 9C). These expression dataere also in accordance with the K/Na result observed in cortex

Fig. 8C). Additionally, compared with that of control sample, the

aHS-pretreatment alone also caused significant down-regulationf SKOR transcript. Meanwhile, the transcript abundance of IRKas also examined (Supp. Fig. S3). By contrast, it was found thataHS pretreatment had no significant impact on the NaCl-induced

0 mM) for 6 h, respectively, and then exposed to 0 or 175 mM NaCl for another 24 here assayed. The transcript levels of the GR1/2 (D), DHAR (E) and MDHAR (F) were

(Con). Values are means ± SE of three independent experiments with at least threenificantly different at P < 0.05 according to Duncan’s multiple range test.

decrease of IRK level. Combined with the electrical-physiologicaldata, there results further indicated that NaHS-maintained K+

homeostasis upon NaCl stress might mainly attributed to its pre-vention of K+ loss but not K+ uptake.

4. Discussion

It becomes increasing evident that an exogenous administrationof some chemicals is able to reduce the adverse effects of abioticstresses, and subsequent possess great significance from both the-oretical and practical perspectives [39,44,56]. H2S, a water-solublecolorless molecule with foul odor of rotten eggs, is recently receiv-ing increasing attentions as a potential bio-regulator involved inmodulation of multiple physiological processes [24–29]. By exoge-

nously using H2S donor NaHS or GYY4137, numerous resultsdemonstrated that exogenous H2S participates in plant adaptiveresponses against multiple abiotic stresses, as well as growth anddevelopment processes [29,53]. For example, NaHS could induce

126 D. Lai et al. / Plant Science

Fig. 8. H2S regulates ion homeostasis. 5-Day-old seedlings were pretreated withor without NaHS (100 �M), PAG (2 mM) or HT (10 mM) for 6 h, respectively, andthen exposed to 0 or 175 mM NaCl for another 120 h. After that, seedling roots werecollected, and contents of K and Na element were determined (A and B). Meanwhile,K/Na ratio in the cortex or stele cells of root tips was also measured by electron probeX-ray microanalysis (C). The sample without chemicals was regarded as the control(ra

larm[iet

potHcfp

m

Con). Values are means ± SE of three independent experiments with at least threeeplicates for each. Bars with different letters are significantly different at P < 0.05ccording to Duncan’s multiple range test. ND: none detected.

ong-lasting priming effects and tolerance to subsequent salinitynd non-ionic osmotic stress in strawberry plants [31]. Previousesults also found that NaHS treatment promoted alfalfa seed ger-ination and alleviated salinity damage involving NO pathway

39]. However, little information was known about the physiolog-cal significance of endogenous H2S production and its-associatednzymatic source(s), which were involved in the defense strategieso counteract the threat imposed by salt and oxidative stresses.

In this paper, our results provide clear evidence to show thatromotion of endogenous H2S levels alleviates, but a decreasef its levels aggravates NaCl-triggered toxic symptoms, fur-her supporting the involvement of l-DES-related endogenous2S homeostasis in the regulation of alfalfa salt tolerance. Thisonclusion is based on following pieces of results obtained

rom pharmacological, biochemical, molecular, and electron-hysiological approaches.

First, it was observed that NaCl exposure elicited approxi-ately dose- and time-dependent increases in total activity of

225 (2014) 117–129

l-DES (Fig. 1C and E), a key enzyme of H2S biosynthesis in higherplants [32,35,38], while total activity of d-DES was not significantlyaltered (Fig. 1F), the former of which in turn leading to the increaseof endogenous H2S production (Fig. 1B and D). We also noticedthat above endogenous H2S induction apparently preceded theinhibition of root elongation upon NaCl exposure (Fig. 1A). By con-trast, no significant changes in endogenous H2S production wasobserved in Arabidopsis roots upon the exposure of 100 mM NaClfor 24 h [57], while prolonged exposure to salinity stress (7 days,100 mM) greatly enhanced H2S concentration in strawberry leavesby approximately 2-folds [31]. These results revealed the differ-ential dynamic changes of endogenous H2S metabolism, and wefurther suggested that these differences might be related to dif-ferent plant species and detection time point(s), as well as theconcentrations of NaCl used in the experiments.

Second, the pretreatment of 100 �M NaHS, a well-known H2Sdonor [28,53,54], could not only mimic the NaCl-triggered promo-tion of endogenous H2S level within a physiological range (Fig. 3A),but also resulted in the alleviated symptoms of salinity stress, interms of the morphologic observation (Fig. 4A) and inhibition ofroot elongation (Fig. 2A) as well as the enhancement of TBARSaccumulation (Fig. 2B). The observation of histochemical stainingin lipid peroxidation, plasma integrity and ROS production is alsoin parallel with this notion (Fig. 5A, B, D and E).

Third, we also noticed that the inhibition of endogenous H2Srelease via the administration of PAG, an efficient inhibitor of l-DES,the key enzyme of H2S synthesis (Supp. Fig. S1) or HT, a scavengerof endogenous H2S (Supp. Fig. S2), could exacerbate NaCl-triggered toxic responses (except the changes of TBARS contents;Figs. 4 and 5). Above results concluded that l-DES-associatedendogenous H2S homeostasis exerts a protective function againstNaCl toxicity. The beneficial behavior of l-DES-dependent endoge-nous H2S involved in heat and drought stress has also been reportedin maize and Arabidopsis [29,30]. The expression pattern of Ara-bidopsis l-DES was similar to the drought-inducible genes upondehydration, resulting in the induction of H2S production andthereafter enhancement of plant responses against drought [58]Combined with our results, l-DES-associated H2S production mightbe regarded as an emerging bio-regulator to play protective rolesagainst variety of abiotic stresses in several plant species.

It is well-known that high concentrations of H2S exhibited tox-icity response, such as binding to cytochrome c oxidase, furtherleading to the inhibition of mitochondrial electron transport chain[59]. In our study, the modulation of alfalfa salt tolerance wasdirectly attributed to the changes in H2S homeostasis. In con-trast to the moderate induction of total l-DES activity and H2Sreleasing triggered by NaCl up to 200 mM, a 300 mM NaCl treat-ment resulted in the greatest induction in total l-DES activityand H2S releasing (Fig. 1B and C), whereas the maximum inhi-bition of root growth subsequently occurred (Fig. 1A). However,it should be noted that the negative effects of NaCl (300 mM)were not entirely due to the overproduction of H2S, since 175 mMNaCl-triggered negative symptoms could only be partially rescuedby NaHS (Figs. 2 and 4). Comparatively, the administration of ahigh dose of NaHS (1000 �M) also brought about toxicity response(Fig. 2). This observation strongly suggested that the protectiverole of endogenous H2S may be restricted to a rather narrow range[59], and the maintenance of endogenous H2S homeostasis is crit-ical for alfalfa salt tolerance. One possible explanation might beattributed to that H2S at high levels might lead to the ROS over-production, due to its biding cytochrome c oxidase [59], which inturn leading to the accumulation of TBARS. Similar behaviors of

NaHS have been reported in the regulation of flower senescence,showing that low-dose of NaHS prolonged flowering lifespan whilehigh-dose led to toxicity response [60]. Comparatively, positiveeffects of H2S at higher concentration (more than 60 nmol g−1 FW)

D. Lai et al. / Plant Science 225 (2014) 117–129 127

Fig. 9. H2S regulates K+ efflux and SKOR transcripts in alfalfa seedling roots upon salt stress. 5-Day-old alfalfa seedlings were pretreated with or without NaHS (100 �M), atime-course analysis of transient K+ efflux was measured at the mature zone upon 175 mM NaCl (A), and average K extrusion during 20 min of NaCl stress was also detected(B). Meanwhile, 5-day-old seedlings were pretreated with or without NaHS (100 �M), PAG (2 mM) or HT (10 mM) for 6 h, respectively, and then exposed to 0 or 175 mMN etectem three ra nt at P

hdfivesHsrlsen

a[saiiHccpbt

caom

aCl for another 12 h. Afterwards, SKOR expression in alfalfa seedling roots were deans ± SE of 6 seedlings (A and B) or three independent experiments with at least

t P < 0.05 according to t-test (B). Bars with different letters are significantly differe

as also been reported in Arabidopsis [57], suggesting that theouble-edged sword behavior of H2S (the deleterious and bene-cial effects) is related to different plant species. Different plantarieties, which showed different sensitivities to salinity, mightxhibit increased or reduced H2S level when experienced salttress. Meanwhile, its might be related to the dynamic changes of2S metabolism, as well as its sub-cellular distribution and corre-

ponding diffusion rate. Regardless of this point, our experimentalesults indicated the maintenance of a suitable level of intracellu-ar H2S might play vital role in conferring salt tolerance in alfalfaeedlings. This double-edge sword behavior of l-DES-associatedndogenous H2S production should be further investigated in theear future.

As a H2S donor, NaHS has been widely used for exogenouslypplied in solutions to examine the beneficial behavior of H2S31,53,58]. However, this solution still contains Na+ and otherulfur-containing components [39,51,52]. To test whether themeliorative effect of NaHS on NaCl-dependent seedling growthnhibition is via the production of H2S or HS−, the effects of var-ous sulfur-containing components pretreatment on endogenous

2S content and seedling root elongation were investigated. Inontrast to the behaviors of NaHS, pretreatment with several sulfur-ontaining components failed in the induction of endogenous H2Sroduction and the alleviation of root elongation inhibition causedy NaCl exposure (Fig. 3). Therefore, our results further clarifiedhat NaHS-associated responses are H2S-specific.

Plant cells have evolved antioxidant enzymatic system to

ope with salt stress-induced oxidative damage in plants, suchs SOD and CAT involving in cellular detoxification of ROSverproduction [5,15–17]. Consequently, it is noteworthy to deter-ine the effects of the alternation of l-DES-related endogenous

d (C). The sample without chemicals was regarded as the control (Con). Values areeplicates for each (C). Asterisk indicates significantly different between treatments

< 0.05 according to Duncan’s multiple range test (C).

H2S production on these representative enzymes in salt stressedalfalfa seedlings. Our study illustrated that NaHS was able toincrease endogenous H2S production (Fig. 3B), and subsequentlyalleviate oxidative damage in NaCl-treated alfalfa plants by reestab-lishment of redox homeostasis (Figs. 5–7). These cytoprotectivebehaviors of H2S homeostasis were supported by the observa-tion that NaHS could differentially activate/up-regulate, while PAGor HT could suppress/down-regulate, representative antioxidantenzymes, including SOD and CAT, or corresponding transcripts(Fig. 6), both of which are correlated with the phenotypes of plantsalt stress [1,4]. Above findings are consistent with the previousresults, showing that NaHS was able to improve longevity of cutflowers by the activation of antioxidant enzymes, such as CAT andSOD, thus leading to the decrease of oxidative damage to cells [60].

On the other hand, GSH/hGSH and AsA are other versatilecompounds of plant antioxidant system [15]. In comparison withmaize [61], strawberry [31], and Arabidopsis [57], hGSH is moreabundant than GSH in alfalfa plants (approximate 17-fold in ourstudy) [18]. Moreover, although the antioxidant defence systemhas been reported to be modulated by NaHS under salt stress[31,39,61], the regulatory role of endogenous H2S on hGSH needsto be further elucidated in legumes. In the present study, it wasfound that endogenous H2S could regulate the AsA-GSH/hGSHrecycling pathway under salt stress, thus maintaining redox buffer-ing [62]. For example, compared with samples treated withNaCl alone, NaHS could increase the total enzymatic activitiesof GR, DHAR and MDHAR, and the abundance of correspond-

ing transcripts, which are responsible for the GSH/hGSH and AsAmetabolism (Fig. 7). Contents of GSH, hGSH and AsA pools werecorrelated with the changes of the enzymatic activities and theexpression of these genes, further leading to the restoration of

1 ience

hNvsretrbAIdoec(bStateve

psrwnKtNsdutt(nNritKdfibtehcietvalpm

5

n

[

[

[

[

[

[

[

[

28 D. Lai et al. / Plant Sc

GSH/hGSSGh and AsA/DHA ratios (Table 1). By contrast, theaCl-triggered decrease of hGSH/hGSSGh ratio was further aggra-ated by the administration of PAG or HT. Together, above resultstated that the AsA-GSH metabolism, regulated by the AsA-GSHecycling-associated enzymes, was strengthened by the elevatedndogenous H2S (Fig. 7), and could further partially preventinghe NaCl-triggered oxidative injury observed in alfalfa seedlingoots (Figs. 2B and 5). Consistent with our results, it has alsoeen reported that NaHS induced the accumulation of GSH andsA contents in wheat seedling leaves under water stress [62].

nterestingly, it was observed that the above-mentioned transcriptata, especially Cu/Zn-SOD, GR1/2 and MDHAR (Figs. 6C and 7D, F),nly showed partially consistent with corresponding tendencies ofnzyme activities. Two explanations exist. First, these discrepan-ies might be due to the differential detection time of transcripts12 h) and enzyme activities (24 h), respectively. Second, H2S haseen identified to regulate biological functions through protein-sulfhydration [63,64]. Therefore, besides the regulation at theranscriptional level, the existence of other modifications suchs at post-translation level, could not be excluded. Regardless ofhis point, our results confirm the evidence that the activation ofndogenous H2S-triggered antioxidant defence might be a uni-ersal strategy for plants adaptation response against multiplenvironmental stimuli [27–31,52,54,58].

Maintenance of K/Na homeostasis is another key component oflant salt tolerance [4,9]. Meanwhile, the critical role of K+ homeo-tasis in plant salt tolerance has also been proposed [8,10]. Ouresults further showed that the biological beneficial role of NaHSas due to its specifically retained K+ (Fig. 8B), whereas Na+ wasot significantly affected (Fig. 8A), thus alleviating the decrease of/Na ratio in stellar cells (Fig. 8C). In Arabidopsis, NaHS was found

o maintain a lower Na+/K+ ratio by diminishing the NaCl-induceda+ percentage via regulating plasma membrane Na+/H+ antiporter

ystem [57], indicating that alfalfa and Arabidopsis might adoptifferent H2S-related mechanisms to maintain ion homeostasispon NaCl stress. Our electron-physiological experiment revealedhat the administration of NaHS effectively prevented the NaCl-riggered K+ efflux in the mature zone of alfalfa seedling rootsFig. 9A and B), suggesting the biological significance of endoge-ous H2S in the maintenance of K+ homeostasis. Accordingly, theaCl-triggered up-regulation of SKOR, which encodes an outwardly

ectifying K+ channel located in the roots [12], was significantlympaired by NaHS (Fig. 9C). Combined with the changes of IRKranscripts (Supp. Fig. S3), our results implied the H2S-restored+ efflux might be partially related to the SKOR channel, and thiseduction needs further genetic characterization. Considering theact that the cytosolic K+ concentration is much more high thann the extracellular space [65], another possible explanation mighte due to the H2S-maintained plasma membrane integrity (Fig. 5A),hus preventing the K+ efflux across the plasma membrane to somextent. However, it was found that the pretreatment of PAG or HTad no significant suppression on the NaCl-induced decrease of Kontent and induction of SKOR transcript (Figs. 8B and 9C), furtherndicating other complementary and redundant mechanism(s) ofndogenous H2S-prevented K+ loss. Recently, it has been reportedhat salicylic acid improves salinity tolerance in Arabidopsis by pre-enting salt-induced K+ loss via a GORK channel [66]. Taking intoccount the fact that GORK gene has not been cloned in alfalfa, ateast under our experimental conditions, we cannot exclude theossibility that GORK channel and transport-independent systemsight be involved in this process.

. Conclusion

Taken together, the present study revealed that the mainte-ance of endogenous H2S homeostasis is critical for improving

[

225 (2014) 117–129

salt tolerance of alfalfa seedlings, which might be, at leastpartially, associated with the total activity of l-DES. Our studyfurther illustrated the participation of endogenous H2S homeo-stasis in the regulation of salinity toxicity in alfalfa seedlings by(1) activation of antioxidant signaling pathway to maintain theredox balance, including the improvement of hGSH/hGSSGh inparticular, (2) preventing the NaCl-induced K+ efflux across theplasma membrane.

Acknowledgements

This work was financially supported by the National NaturalScience Foundation of China (J1210056 and J1310015), the Funda-mental Research Funds for the Central Universities (KYTZ201402),and the Priority Academic Program Development of Jiangsu HigherEducation Institutions (PAPD). We also thank Dr Evan Evans fromthe University of Tasmania, Australia for his kind help in editing themanuscript.

Appendix A. Supplementary data

Supplementary data associated with this article can befound, in the online version, at http://dx.doi.org/10.1016/j.plantsci.2014.06.006.

References

[1] J.K. Zhu, Plant salt tolerance, Trends Plant Sci. 6 (2001) 66–71.[2] Y. Xie, S. Xu, B. Han, M. Wu, X. Yuan, Y. Han, Q. Gu, D. Xu, Q. Yang, W. Shen,

Evidence of Arabidopsis salt acclimation induced by up-regulation of HY1 andthe regulatory role of RbohD-derived reactive oxygen species synthesis, PlantJ. 66 (2011) 280–292.

[3] I.S. Bhandal, C.P. Malik, Potassium estimation, uptake, and its role in the physi-ology and metabolism of flowering plants, Int. Rev. Cytol. 110 (1988) 205–245.

[4] R. Munns, M. Tester, Mechanisms of salinity tolerance, Annu. Rev. Plant Biol.59 (2008) 651–681.

[5] R.P. Jacoby, N.L. Taylor, A.H. Millar, The role of mitochondrial respiration insalinity tolerance, Trends Plant Sci. 16 (2011) 614–623.

[6] Y. Xie, Y. Mao, D. Lai, W. Zhang, T. Zheng, W. Shen, Roles of NIA/NR/NOA1-dependent nitric oxide production and HY1 expression in the modulation ofArabidopsis salt tolerance, J. Exp. Bot. 64 (2013) 3045–3060.

[7] J.K. Zhu, Regulation of ion homeostasis under salt stress, Curr. Opin. Plant Biol.6 (2003) 441–445.

[8] T.A. Cuin, S. Shabala, Exogenously supplied compatible solutes rapidly ame-liorate NaCl-induced potassium efflux from barley roots, Plant Cell Physiol. 46(2005) 1924–1933.

[9] Z. Chen, I.I. Pottosin, T.A. Cuin, A.T. Fuglsang, M. Tester, D. Jha, I. Zepeda-Jazo, M.Zhou, M.G. Palmgren, I.A. Newman, S. Shabala, Root plasma membrane trans-porters controlling K+/Na+ homeostasis in salt–stressed barley, Plant Physiol.145 (2007) 1714–1725.

10] T.A. Cuin, S.A. Betts, R. Chalmandrier, S. Shabala, A root’s ability to retain K+

correlates with salt tolerance in wheat, J. Exp. Bot. 59 (2008) 2697–2706.11] R.E. Hirsch, B.D. Lewis, E.P. Spalding, M.R. Sussman, A role for the AKT1 potas-

sium channel in plant nutrition, Science 280 (1998) 918–921.12] F. Gaymard, G. Pilot, B. Lacombe, D. Bouchez, D. Bruneau, J. Boucherez, N.

Michaux-Ferrière, J.B. Thibaud, H. Sentenac, Identification and disruption ofa plant shaker-like outward channel involved in K+ release into the xylem sap,Cell 94 (1998) 647–655.

13] E. Hosy, A. Vavasseur, K. Mouline, I. Dreyer, F. Gaymard, F. Porée, J. Boucherez,A. Lebaudy, D. Bouchez, A.A. Very, T. Simonneau, J.B. Thibaud, H. Sentenac, TheArabidopsis outward K+ channel GORK is involved in regulation of stomatalmovements and plant transpiration, Proc. Natl. Acad. Sci. U. S. A. 100 (2003)5549–5554.

14] U. Deinlein, A.B. Stephan, T. Horie, W. Luo, G. Xu, J.I. Schroeder, Plant salt-tolerance mechanisms, Trends Plant Sci. (2014), http://dx.doi.org/10.1016/j.tplants.2014.02.001.

15] R. Mittler, Oxidative stress, antioxidants and stress tolerance, Trends Plant Sci.7 (2002) 405–410.

16] V. Arbona, V. Flors, J. Jacas, P. García-Agustín, A. Gómez-Cadenas, Enzymatic andnon-enzymatic antioxidant responses of Carrizo citrange, a salt-sensitive citrusrootstock, to different levels of salinity, Plant Cell Physiol. 44 (2003) 388–394.

17] N. Sreenivasulu, B. Grimm, U. Wobus, W. Weschke, Differential response of

antioxidant compounds to salinity stress in salt-tolerant and salt-sensitiveseedlings of foxtail millet (Setaria italica), Physiol. Plant 109 (2000) 435–442.

18] M.A. Matamoros, J.F. Moran, I. Iturbe-Ormaetxe, M.C. Rubio, M. Becana, Glu-tathione and homoglutathione synthesis in legume root nodules, Plant Physiol.121 (1999) 879–888.

ience 2

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

D. Lai et al. / Plant Sc

19] C. Ortega-Villasante, R. Rellán-Alvarez, F.F. Del Campo, R.O. Carpena-Ruiz, L.E.Hernández, Cellular damage induced by cadmium and mercury in Medicagosativa, J. Exp. Bot. 418 (2005) 2239–2251.

20] C.Q. Chen, H. Xin, Y.Z. Zhu, Hydrogen sulfide: third gaseous transmitter, but withgreat pharmacological potential, Acta Pharmacol. Sin. 28 (2007) 1709–1716.

21] R. Wang, The gasotransmitter role of hydrogen sulfide, Antioxid. Redox. Signal.5 (2003) 493–501.

22] C.A. Wagner, Hydrogen sulfide: a new gaseous signal molecule and blood pres-sure regulator, J. Nephrol. 22 (2009) 173–176.

23] B.H. Tan, P.T. Wong, J.S. Bian, Hydrogen sulfide: a novel signaling molecule inthe central nervous system, Neurochem. Int. 56 (2010) 3–10.

24] S. Fiorucci, E. Antonelli, E. Distrutti, G. Rizzo, A. Mencarelli, S. Orlandi, R.Zanardo, B. Renga, M. Di Sante, A. Morelli, G. Cirino, J.L. Wallace, Inhibitionof hydrogen sulfide generation contributes to gastric injury caused by anti-inflammatory nonsteroidal drugs, Gastroenterology 129 (2005) 1210–1224.

25] G. Yang, L. Wu, B. Jiang, W. Yang, J. Qi, K. Cao, Q. Meng, A.K. Mustafa, W. Mu, S.Zhang, S.H. Snyder, R. Wang, H2S as a physiologic vasorelaxant: hypertensionin mice with deletion of cystathionine �–lyase, Science 322 (2008) 587–590.

26] Y. Kimura, R. Dargusch, D. Schubert, H. Kimura, Hydrogen sulfide protects HT22neuronal cells from oxidative stress, Antioxid. Redox. Signal. 8 (2006) 661–670.

27] H. Zhang, L.Y. Hu, K.D. Hu, Y.D. He, S.H. Wang, J.P. Luo, Hydrogen sulfide pro-motes wheat seed germination and alleviates oxidative damage against copperstress, J. Integr. Plant Biol. 50 (2008) 1518–1529.

28] H. Zhang, Z.Q. Tan, L.Y. Hu, S.H. Wang, J.P. Luo, R.L. Jones, Hydrogen sulfidealleviates aluminum toxicity in germinating wheat seedlings, J. Integr. PlantBiol. 52 (2010) 556–567.

29] Z.G. Li, M. Gong, H. Xie, L. Yang, J. Li, Hydrogen sulfide donor sodiumhydrosulfide-induced heat tolerance in tobacco (Nicotiana tabacum L.) suspen-sion cultured cells and involvement of Ca2+ and calmodulin, Plant Sci. 185–186(2012) 185–189.

30] J. Shen, T. Xing, H. Yuan, Z. Liu, Z. Jin, L. Zhang, Y. Pei, Hydrogen sulfide improvesdrought tolerance in Arabidopsis thaliana by microRNA expressions, PLoS ONE8 (2013) e77047.

31] A. Christou, G.A. Manganaris, I. Papadopoulos, V. Fotopoulos, Hydrogen sul-fide induces systemic tolerance to salinity and non-ionic osmotic stress instrawberry plants through modification of reactive species biosynthesis andtranscriptional regulation of multiple defence pathways, J. Exp. Bot. 64 (2013)1953–1966.

32] J. Papenbrock, A. Riemenschneider, A. Kamp, H.N. Schulz-Vogt, A. Schmidt,Characterization of cysteine-degrading and H2S-releasing enzymes of higherplants-from the field to the test tube and back, Plant Biol. (Stuttg.) 9 (2007)582–588.

33] C. García-Mata, L. Lamattina, Gasotransmitters are emerging as new guard cellsignalling molecules and regulators of leaf gas exchange, Plant Sci. 201–202(2013) 66–73.

34] A. Riemenschneider, R. Wegele, A. Schmidt, J. Papenbrock, Isolation and char-acterization of a d-cysteine desulfhydrase protein from Arabidopsis thaliana,FEBS J. 272 (2005) 1291–1304.

35] C. Álvarez, I. García, I. Moreno, M.E. Pérez-Pérez, J.L. Crespo, L.C. Romero, C.Gotor, Cysteine-generated sulfide in the cytosol negatively regulates autophagyand modulates the transcriptional profile in Arabidopsis, Plant Cell 24 (2012)4621–4634.

36] H. Zhang, H. Jiao, C.X. Jiang, S.H. Wang, Z.J. Wei, J.P. Luo, R.L. Jones, Hydrogensulfide protects soybean seedlings against drought-induced oxidative stress,Acta. Physiol. Plant 32 (2010) 849–857.

37] Y. Xie, D. Lai, Y. Mao, W. Zhang, W. Shen, R. Guan, Molecular cloning, char-acterization, and expression analysis of a novel gene encoding l-cysteinedesulfhydrase from Brassica napus, Mol. Biotechnol. 54 (2013) 737–746.

38] C. Álvarez, I. García, L.C. Romero, C. Gotor, Mitochondrial sulfide detoxifica-tion requires a functional isoform O-acetylserine (thiol) lyase C in Arabidopsisthaliana, Mol. Plant 5 (2012) 1217–1226.

39] Y. Wang, L. Li, W. Cui, S. Xu, W. Shen, R. Wang, Hydrogen sulfide enhances alfalfa(Medicago sativa) tolerance against salinity during seed germination by nitricoxide pathway, Plant Soil 351 (2012) 107–119.

40] M.M. Bradford, A rapid and sensitive method for the quantitation of micro-gram quantities of protein utilizing the principle of protein–dye binding, Anal.Biochem. 72 (1976) 248–254.

41] Y. Xie, D. Xu, W. Cui, W. Shen, Mutation of Arabidopsis HY1 causes UV-C

hypersensitivity by impairing carotenoid and flavonoid biosynthesis and thedown-regulation of antioxidant defence, J. Exp. Bot. 63 (2012) 3869–3883.

42] Y. Han, J. Zhang, X. Chen, Z. Gao, W. Xuan, S. Xu, X. Ding, W. Shen, Carbon monox-ide alleviates cadmium-induced oxidative damage by modulating glutathionemetabolism in the roots of Medicago sativa, New Phytol. 177 (2008) 155–166.

[

25 (2014) 117–129 129

43] G.L. Wu, J. Cui, L. Tao, H. Yang, Fluroxypyr triggers oxidative damage by pro-ducing superoxide and hydrogen peroxide in rice (Oryza sativa), Ecotoxicology19 (2010) 124–132.

44] Y. Xie, T. Ling, Y. Han, K. Liu, Q. Zheng, L. Huang, X. Yuan, Z. He, B. Hu, L. Fang,Z. Shen, Q. Yang, W. Shen, Carbon monoxide enhances salt tolerance by nitricoxide-mediated maintenance of ion homeostasis and up-regulation of antioxi-dant defence in wheat seedling roots, Plant Cell. Environ. 31 (2008) 1864–1881.

45] L. Zhao, F. Zhang, J. Guo, Y. Yang, B. Li, L. Zhang, Nitric oxide functions as a signalin salt resistance in the calluses from two ecotypes of reed, Plant Physiol. 134(2004) 849–857.

46] J. Sun, S. Chen, S. Dai, R. Wang, N. Li, X. Shen, X. Zhou, C. Lu, X. Zheng, Z. Hu, Z.Zhang, J. Song, Y. Xu, NaCl-induced alternations of cellular and tissue ion fluxesin roots of salt-resistant and salt-sensitive poplar species, Plant Physiol. 149(2009) 1141–1153.

47] W. Cui, L. Li, Z. Gao, H. Wu, Y. Xie, W. Shen, Haem oxygenase-1 is involved insalicylic acid-induced alleviation of oxidative stress due to cadmium stress inMedicago sativa, J. Exp. Bot. 63 (2012) 5521–5534.

48] G. Queval, G. Noctor, A plate reader method for the measurement of NAD,NADP, glutathione, and ascorbate in tissue extracts: application to redoxprofiling during Arabidopsis rosette development, Anal. Biochem. 363 (2007)58–69.

49] Y. Law, S.A. Charles, B. Halliwell, Glutathione and ascorbic acid in spinach(Spinacia oleracea) chloroplasts, Biochem. J. 210 (1983) 899–903.

50] W. Zhao, J. Zhang, Y. Lu, R. Wang, The vasorelaxant effect of H2S as a novelendogenous gaseous KATP channel opener, EMBO J. 20 (2001) 6008–6016.

51] R. Hosoki, N. Matsuki, H. Kimura, The possible role of hydrogen sulfide as anendogenous smooth muscle relaxant in synergy with nitric oxide, Biochem.Biophys. Res. Commun. 237 (1997) 527–531.

52] L. Li, Y. Wang, W. Shen, Roles of hydrogen sulfide and nitric oxide in the allevia-tion of cadmium-induced oxidative damage in alfalfa seedling roots, Biometals25 (2012) 617–631.

53] C. García-Mata, L. Lamattina, Hydrogen sulphide, a novel gasotransmitterinvolved in guard cell signaling, New Phytol. 188 (2010) 977–984.

54] Z.G. Li, S.Z. Yang, W.B. Long, G.X. Yang, Z.Z. Shen, Hydrogen sulphide may bea novel downstream signal molecule in nitric oxide-induced heat tolerance ofmaize (Zea mays L.) seedlings, Plant Cell Environ. 36 (2013) 1564–1572.

55] M.H. Cruz de Carvalho, J. Brunet, J. Bazin, I. Kranner, A.d’. Arcy-Lameta, Y.Zuily-Fodil, D. Contour-Ansel, Homoglutathione synthetase and glutathionesynthetase in drought-stressed cowpea leaves: expression patterns and accu-mulation of low-molecular-weight thiols, J. Plant Physiol. 167 (2010) 480–487.

56] Q. Hayat, S. Hayat, M. Irfan, A. Ahmad, Effect of exogenous salicylic acid underchanging environment: a review, Environ. Exp. Bot. 68 (2010) 14–25.

57] J. Li, H. Jia, J. Wang, Q. Cao, Z. Wen, Hydrogen sulfide is involved in maintainingion homeostasis via regulating plasma membrane Na+/H+ antiporter system inthe hydrogen peroxide-dependent manner in salt-stress Arabidopsis thalianaroot, Protoplasma (2014), http://dx.doi.org/10.1007/s00709-013-0592-x.

58] Z. Jin, J. Shen, Z. Qiao, G. Yang, R. Wang, Y. Pei, Hydrogen sulfide improvesdrought resistance in Arabidopsis thaliana, Biochem. Biophys. Res. Commun.414 (2011) 481–486.

59] D. Mancardi, C. Penna, A. Merlino, P. Del Soldato, D.A. Wink, P. Pagliaro, Physi-ological and pharmacological features of the novel gasotransmitter: hydrogensulfide, Biochim. Biophys. Acta 1787 (2009) 864–872.

60] H. Zhang, S.L. Hu, Z.J. Zhang, L.Y. Hu, C.X. Jiang, Hydrogen sulfide acts as aregulator of flower senescence in plants, Postharvest Biol. Technol. 60 (2011)251–257.

61] C. Shan, H. Liu, L. Zhao, X. Wang, Effects of exogenous hydrogen sulfide on theredox states of ascorbate and glutathione in maize leaves under salt stress, Biol.Plant. 58 (2014) 169–173.

62] C. Shan, S. Zhang, D. Li, Y. Zhao, X. Tian, X. Zhao, Y. Wu, X. Wei, R. Liu, Effectsof exogenous hydrogen sulfide on the ascorbate and glutathione metabolismin wheat seedlings leaves under water stress, Acta. Physiol. Plant 33 (2011)2533–2540.

63] Z.Y. Wu, G. Gruber, J. Bian, Hydrogen sulfide regulates cardiac function via S-sulfhydration of adenylyl cyclase, Nitric Oxide 27 (2012) S29.

64] A.K. Mustafa, M.M. Gadalla, N. Sen, S. Kim, W. Mu, S.K. Gazi, R.K. Barrow, G. Yang,R. Yang, S.H. Snyder, H2S signals through protein S-sulfhydration, Sci. Signal. 2(2009) ra72.

65] A.A. Véry, H. Sentenac, Molecular mechanisms and regulation of K+ transport

in higher plants, Annu. Rev. Plant Biol. 54 (2003) 575–603.

66] M. Jayakannan, J. Bose, O. Babourina, Z. Rengel, S. Shabala, Salicylic acidimproves salinity tolerance in Arabidopsis by restoring membrane potentialand preventing salt-induced K+ loss via a GORK channel, J. Exp. Bot. 64 (2013)2255–2268.