Accepted Manuscript

Hydrogen sulphide trapeze: Environmental stress amelioration and phytohormone

crosstalk

Aditya Banerjee, Durgesh Kumar Tripathi, Aryadeep Roychoudhury

PII: S0981-9428(18)30385-1
DOI: 10.1016/j.plaphy.2018.08.028
Reference: PLAPHY 5389

To appear in: Plant Physiology and Biochemistry

Received Date: 21 May 2018

Revised Date: 11 August 2018
Accepted Date: 23 August 2018

Please cite this article as: A. Banerjee, D.K. Tripathi, A. Roychoudhury, Hydrogen sulphide trapeze:
Environmental stress amelioration and phytohormone crosstalk, Plant Physiology et Biochemistry
(2018), doi: 10.1016/j.plaphy.2018.08.028.

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 ACCEPTED MANUSCRIPT
Hydrogen sulphide trapeze: environmental stress amelioration
and phytohormone crosstalk
Aditya Banerjee1, Durgesh Kumar Tripathi2, Aryadeep
Roychoudhury1*
1

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Post Graduate Department of Biotechnology, St. Xavier’s College (Autonomous), 30,
Mother Teresa Sarani, Kolkata – 700016, West Bengal, India

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Amity Institute of Organic Agriculture, Amity University, Noida

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*
E-mail: aryadeep.rc@gmail.com

Abstract
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Hydrogen sulphide (H2S) is recognized as the third endogenous gasotransmitter in plants after
nitric oxide (NO) and carbon monoxide (CO). Though initially visualized as a toxic gaseous
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molecule, recent studies have illustrated its diverse role in regulating plant growth and
developmental physiology. H2S is also a potent inducer of osmolytes and cellular
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antioxidants of enzymatic and non-enzymatic origins. It interacts with the Ca2+ and NO
signaling pathways. Exogenous fumigation of H2S or application of the H2S donor, sodium
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hydrosulphide (NaHS) has been found to be beneficial in the amelioration of multiple abiotic
stresses like salinity, drought, temperature, hypoxia and heavy metal toxicity. H2S also
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protects stress-sensitive proteins via persulphidation of cysteine residues, prone to ROS-

mediated oxidation. It is well established that plants are highly dependent on phytohormone
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signaling during any physiological process. By virtue of the diversity of the H2S-mediated
signaling network, interactions and crosstalks of this gasotransmitter with the plant hormones
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are evident. This article presents a detailed summary regarding the role of H2S in oxidative
and environmental stress tolerance; and furthermore illustrates the reported interactions with
crucial hormones like abscisic acid, auxins, gibberellic acid, ethylene and salicylic acid under
physiologically differing circumstances.

Keywords: Hydrogen sulphide; osmolytes; antioxidants; environmental stresses; reactive

oxygen species; phytohormones; H2S-hormone interactions
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1. Introduction
The physiological diversity in plants demands dual roles of several compounds. As a result,
molecules like hydrogen sulphide (H2S), which were regarded only as toxic cellular by-
products, are currently being evaluated as potential signaling molecules during plant
responses to changing environment (Ahmad 2016). H2S strongly binds to Fe2+-containing
proteins like cytochrome oxidase, hemoglobin and myoglobin. This inhibitory association

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with crucial metabolic proteins is widely considered as the most relevant cause behind the
Permian extinction (Guo et al. 2016). Hence, it is of biological consilience that H2S is a

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primordial gas linking the physiology of plants, microbes as well as animals (Yamasaki and
Cohen 2016; Scuffi et al. 2016). The oxygen release and phosphorus uptake in rice cultivars

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like Bluebelle, Dawn, Norin 22, Saturn, Yubae and Zenith were inhibited by H2S
(Calderwood and Kopriva 2014; Hancock and Whiteman 2014). However, recent researches
support the growth promoting roles of H2S in plants. Interestingly, similar to nitric oxide

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(NO) and carbon monoxide (CO), H2S behaves like a gaseous signaling molecule at low
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cellular concentration (Yamasaki and Cohen 2016). The diverse roles of H2S in generating
plant tolerance against multiple abiotic stresses like salinity, drought, temperature, flooding
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and heavy metal toxicity has been recently signified (Li et al. 2016; Lisjak et al. 2013).
The release of H2S from compounds like sodium hydrosulphide (NaHS) and
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morpholin-4-ium 4-methoxyphenyl(morpholino) phosphinodithiolate (GYY4137) accelerated

seed germination, promoted organogenesis and regulated the advent of senescence (Li 2013;
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Wojtyla et al. 2016; Fang et al. 2014; Zhang et al. 2011). H2S has been identified as a
member of the physiological crosstalk network constituted by calcium (Ca2+), hydrogen
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peroxide (H2O2), NO and the universal stress hormone, abscisic acid (ABA) (Fotopoulos et
al. 2015; Guo et al. 2016). Li et al. (2016) has proposed H2S as a potential mediator of plant
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cross-adaptation under sub-optimal conditions.

The role of H2S in regulating plant growth and development under various external
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stimuli are partially known (Li et al. 2016; Calderwood and Kopriva 2014; Jin and Pei 2015;
Hancock and Whiteman 2014, 2016). The interactions of H2S and other important
phytohormones are not well summarized in literature. Since the physiology of plants is highly
dependent on the homeostasis between various phytohormones, it is evident that a signaling
molecule like H2S must interact with them at multiple levels. This review illustrates an in-
depth knowledge of the role of H2S in ameliorating various abiotic stresses and the crosstalk
nodes existing between the gaseous molecule and plant hormones like ABA, auxins, ethylene
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and salicylic acid. The discussion also aims at identifying the roles of H2S as a potential
signaling agent involved in the systemic establishment of plants under variable environmental
challenges.

2. Insight into H2S metabolism

It has been observed that H2S exhibits cytotoxicity only at higher concentration. However, at

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lower concentration, it acts as a gaseous signaling molecule. H2S is formed in the plant cells
as an intermediate of an assimilatory sulphate reduction (Filipovic and Jovanovic 2017). The

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major enzymes responsible for maintaining H2S homeostasis are L-cysteine desulfhydrase
(LCD), D-cysteine desulfhydrase (DCD), sulphite reductase (SiR), cyanoalanine synthase

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(CAS) and cysteine synthase (Li 2013). The LCD enzyme in Arabidopsis, DES1 catalyzes
the endogenous cellular biosynthesis of H2S (Laureano-Marin et al. 2014). Fusion of the GFP
reporter gene with the DES1 promoter revealed its expression to be localized in the cytosol of

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epidermal, mesophyll and guard cells (Laureano-Marin et al. 2014). The GFP signal could be
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detected in floral tissues like sepals, petals and pistils and also in reproductive organs like
siliques (bases) and seeds in the mature Arabidopsis lines (Laureano-Marin et al. 2014).
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Importantly, the LCD/DCD equilibrium dictates the breakdown of L-/D-cysteine to

amine, pyruvate and H2S (Li 2013). These enzymes are responsive to abiotic stresses and
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hence are the production of H2S (Hancock and Whiteman 2014). The interactions between
cyanide and cysteine, catalysed by CAS, also co-yield H2S and cyanoalanine (Aroca et al.
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2017). The ferredoxin-dependent reduction of sulphite to H2S is catalyzed by SiR

(Calderwood and Kopriva 2014). During the biosynthesis of cysteine, O-acetylserine thiol
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lyase combines H2S with O-acetyl-L-serine. This enzymatic catalysis is however reversible
and hence can also degrade cysteine to H2S and O-acetyl-L-serine (Li 2015). Plants usually
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mediate stomatal release of the excess unwanted H2S into the air (Li 2015).
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3. H2S-induced protein persulphidation during oxidative stress

H2S mediates diverse signaling responses in plants. This gaseous molecule is also capable of
inducing post-translational modifications of Cys residues in macromolecular protein
complexes. Such modifications are characterized by Cys persulphidation. These are
oxidative post-translational modifications of Cys residues which include S-nitrosation, S-
sulphenylation and S-gluthionylation. Sulphurtransferases and cysteine desulphurases are

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known to catalyze the formation of persulphides. Protein modification via persulphidation
indicates that this post-translational alteration might be involved in mediating a plethora of
activities within the cell (Filipovic 2015). Aroca et al. (2017) investigated the Arabidopsis
proteome and reported at least 5% of it to be persulphidated. About 2015 proteins were found
to be endogenously persulphidated (Aroca et al. 2017). The Arabidopsis persulphidome
illustrates persulphidation of proteins involved in crucial biochemical pathways like the

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glycolytic cycle, tricarboxylic acid (TCA) cycle, Calvin-Benson cycle and also starch
biosynthesis.

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H2S exhibits ameliorative roles during multiple abiotic stresses (Li et al. 2016). We
have discussed the roles of H2S under these conditions in the next section. In continuation to

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this notion, Aroca et al. (2017) identified AUTOPHAGY-RELATED UBIQUITIN-LIKE 18a
(Atg18a) as a target of persulphidation. Such post-translational modification confers
protection to this enzyme, particularly sensitive to salinity, drought and oxidative stresses

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(Xiong et al. 2005). According to Cuevasanta et al. (2015), H2S-induced redox sulphidation is
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a conserved mechanism of protecting protein Cys residues from unwanted oxidation. In
response to prolonged oxidative stress, Cys residues are oxidized to sulfenic acids (RSOH)
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and further to sulfinic (RSO2H) and sulfonic acids (RSO3H). These conversions are
irreversible and cannot be easily reduced back to thiols if the oxidized Cys residues are
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deeply buried within protein quaternary complexes (Cuevasanta et al. 2015). Hence,
persulphidation can be portrayed as a protective mechanism during H2O2-induced oxidative
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stress (Wedmann et al. 2016). Persulphides are efficient scavengers of reactive oxygen
species (ROS) like hydrogen peroxide (H2O2), hydroxyl and superoxide radicals. On reacting
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with ROS, persulphides form RSSO3H, which are easily reduced to thiols via the action of
thioredoxins (Filipovic 2015; Inupakutika et al. 2016). The super-nucleophilic nature of
persulphides due to the maintenance of a deprotonated (RSS-) form under physiological
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conditions, have established these moieties as extensive scavengers of ROS and even reactive
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nitrogen species (RNS) (Filipovic and Jovanovic 2017).

4. Role of H2S under abiotic stress

H2S reportedly has the capacity to promote cross-adaptation against multiple environmental
stresses like salinity, drought, temperature, flooding and heavy metal toxicity (Li et al. 2016).
Like other crucial secondary messengers, the production of H2S is rapidly induced under
myriad sub-optimal environments. Such increase in endogenous H2S synthesis recharges the
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entire antioxidant machinery by regulating the osmolyte and nutrient balance within the
system. Specific heat shock proteins (HSPs) characterized as chaperones are produced to
prevent protein misfolding and aggregation (Ahmad 2016).
Interestingly, NaHS is widely considered as a potential priming agent, since it is a
H2S donor (Fig. 1). Application of NaHS results in the up regulation of H2S-anabolic genes
even under normal growth conditions (Christou et al. 2013; Zhang et al. 2010a). Due to its

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high lipophilicity, H2S transport within the cells and to other tissues is easily facilitated. Jin et
al. (2011) identified high expression of drought-responsive genes like dehydration-responsive

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element-binding 2A (DREB2A), DREB2B, C-repeat binding factor 4 (CBF4) and responsive
to dehydration 29A (Rd29A) in the Arabidopsis plants treated with 80 μM H2S. The

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Arabidopsis plants pre-treated with NaHS or overexpressing the H2S biosynthetic gene, LCD
exhibited improved tolerance to abiotic stresses like salinity, drought and chilling (Shi et al.
2015). However, the LCD knockdown plants were observed to be sensitive to these stresses

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(Shi et al. 2015). Li et al. (2016) stated that the endogenous H2S level on average increased
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by 2.0-2.5 folds across various plant species exposed to abiotic stresses when compared to the
same species growing under normal conditions.
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The involvement of H2O2 in the H2S-induced root development of Solanum

lycopersicum has been reported (Mei et al. 2017). NaHS treatment stimulated the expression
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of the NADPH oxidase, RBOH1 and promoted H2O2 formation, which further induced the
growth of lateral roots. The formation of lateral root primordial cells was thoroughly
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impaired on addition of H2O2 scavenger, dimethylthiourea or NADPH oxidase inhibitor,

diphenylene iodonium (Mei et al. 2017). The application of these inhibitors also changed the
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expression patterns of root-associated microRNAs (miRs) like miR390a and miR160. Thus,
H2S closely synchronizes with H2O2, a member of the ROS family to regulate the formation
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of lateral roots in S. lycopersicum (Mei et al. 2017).

4.1. Ameliorative roles of H2S during salt stress
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Salt stress primarily deteriorates the integrity of the photosynthetic apparatus and lowers the
photosynthetic efficiency in plants. Contents of chlorophyll and other accessory pigments are
drastically reduced in crop species exposed to salinity resulting in lowered yield and
productivity (Mbarki et al. 2018; Tang et al. 2015). Pre-treatment of wheat seeds with 0.01,
0.05, 0.09 and 0.13 mM NaHS ameliorated the salt-induced physiological injuries in the
wheat seedlings (Bao et al. 2011). Similar pre-treatment of Medicago sativa seeds induced
the activities of antioxidative enzymes, viz., superoxide dismutase (SOD), catalase (CAT),
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peroxidase, ascorbate peroxidase (APX) along with their multiple isozymes on exposure to
100 mM NaCl-stress; and also maintained a high K+/Na+ ratio (Wang et al. 2012). These
antioxidative enzymes promote effective scavenging of the ROS. The ROS are produced in
uncontrolled amounts within the plant cells on exposure to any kind of environmental
imbalance. They degrade the cell membrane via lipid peroxidation and distort the structures
of proteins and nucleic acids (Banerjee and Roychoudhury 2018a). Li et al. (2014) observed

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high K+/Na+ ratio along with elevated expression of the cell membrane-localized H+-ATPase
and Na+/H+ transporters in the NaHS-treated Arabidopsis seedlings grown under salt stress.

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Interestingly, it was also found that H2S mediates ion homeostasis in the roots of salt-exposed
Arabidopsis seedlings in a H2O2-dependent fashion. This is because the alternative

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application of the plasma membrane NADPH oxidase inhibitor, diphenylene iodonium or the
ROS scavenger, dimethylthiourea suppressed the H2S-dependent effects on the Na+/H+
antiporter (Li et al. 2014).

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4.2. Ameliorative roles of H2S during drought stress
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Drought is the most prevalent kind of abiotic stress which causes desiccation, osmotic
imbalance and drooping in widespread crops and cultivars across the globe (Banerjee and
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Roychoudhury 2017). Exogenous application of NaHS on wheat seedlings exposed to

polyethylene glycol 6000 (PEG 6000)-mediated drought stress promoted seed germination in
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a dose-dependent manner (Zhang et al. 2010b). Treatment of the seedlings with only NaHS
and not any other S2-, SO42-, SO32-, HSO4- and HSO3--containing compounds alleviated
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drought stress. This highlighted the necessity of the NaHS derivative, H2S in mitigating
drought susceptibility (Zhang et al. 2010b). The treated seeds also exhibited reduced
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lipoxygenase (LOX) activity and malondialdehyde (MDA) content along with increased
activities of APX and CAT (Zhang et al. 2010b).
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The ascorbate-glutathione (AsA-GSH) cycle is one of the most crucial antioxidant

replenishing machinery in plant cells (Banerjee and Roychoudhury 2016). Shan et al. (2011)
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reported higher activities of the enzymes belonging to the AsA-GSH cycle like APX,
glutathione reductase (GR), dehydroascorbate reductase (DHAR) in the NaHS-treated wheat
seedlings compared to the non-treated ones during drought stress. Significant increase in the
cellular accumulation of osmolytes like AsA and GSH also reduced MDA production and
electrolyte leakage in the treated plants exposed to drought (Shan et al. 2011). The overall
rate of survival was accelerated and the stomatal aperture was reduced in the NaHS-treated
Arabidopsis plants compared to the non-treated sets under drought (Jin et al. 2011). Increased
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relative water content (RWC) was observed in the Vicia faba and Impatiens walleriana plants
exogenously treated with H2S and grown under drought. The physiological improvements
imposed by H2S promoted normal plant growth under water deficient conditions (Garcia-
Mata and Lamattina 2010).
4.3. Ameliorative roles of H2S during high temperature stress
Heat stress due to high temperature is a direct effect of rapid global warming (Banerjee and

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Roychoudhury 2018b). H2S directly activates a synchronized and interactive antioxidant
network to confer protection against heat shock. The strawberry plants pre-treated with NaHS

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and exposed to 42°C exhibited increased levels of chlorophyll and a well maintained
AsA/GSH balance compared to the non-treated plants under similar conditions (Christou et

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al. 2011). High activities of CAT, MnSOD, GR and the chloroplastidic isoform of APX along
with elevated expression of genes encoding HSP70, HSP80, HSP90 and plasma membrane
intrinsic aquaporins (PIPs) were observed in the NaHS treated strawberry plants under heat

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stress (Christou et al. 2014). Similar kind of amelioration from heat-mediated injuries was
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also observed in NaHS treated seedlings of wheat and cells of tobacco (Li et al. 2014, 2015).
H2S release from NaHS significantly reduced electrolyte leakage and the production of MDA
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due to membrane lipid peroxidation (Li et al. 2012).

The close interaction of H2S with physiological regulators like proline (Pro) and Ca2+
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has been observed. Pro is a crucial compatible solute involved in equilibrating the
physiological osmotic homeostasis under control situations as well as under several
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environmental challenges (Roychoudhury et al. 2015). Li et al. (2013) reported elevated

activity of the Pro biosynthetic enzyme ∆1-pyrroline-5-carboxylate synthetase (P5CS) and
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lowered expression of the Pro catabolising gene, Pro dehydrogenase (PDH) in maize
seedlings pre-treated with NaHS. This facilitated high endogenous accumulation of Pro and
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increased viability of the seedlings under heat stress (Li et al. 2013). In another study, Li et
al. (2012) showed the synergistic responses synchronized between H2S and Ca2+ during heat
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stress in tobacco plants. The exogenous application of Ca2+ and its ionophore, A23187
significantly promoted the heat tolerance induced by NaHS. However, addition of Ca2+
chelator like ethylene glycol-bis(b-aminoethylether)-N,N,N’,N’-tetraacetic acid (EGTA) and
calmodulin antagonists like chlorpromazine and trifluoroperazine suppressed NaHS-mediated
heat responses (Li et al. 2012).
4.4. Ameliorative role of H2S during low temperature stress

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Low temperature like chilling (0-10°C) and freezing (<0°C) injure plant cells by inducing
oxidative stress and crystallization of the plasma membrane (Banerjee et al. 2017).
Exogenously treated bermudagrass exhibited freezing tolerance due to reduced electrolyte
leakage and enhanced activities of CAT, glutathione peroxidase (GPX) and GR to scavenge
the excessively produced ROS (Shi et al. 2013). Exposure of NaHS-treated Vitis vinifera
plants to 4°C resulted in the induction of cold-responsive genes like CBF3 and ICE1.

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Increased SOD activity efficiently scavenged the superoxide radicals which effectively
reduced the MDA content (Fu et al. 2013). Shi et al. (2015) observed chilling stress tolerance

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in the transgenic Arabidopsis lines overexpressing the AtLCD gene. Comparative proteomic
analyses in Lamiophlomis rotata plants, growing at very high altitudes of 4800 and 5200

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meters, revealed high activities of LCD, DCD and CAS enzymes. The plants treated
exogenously with H2S exhibited high accumulation of Pro, soluble sugars and effective ROS
and RNS scavenging by S-nitrosogluthathione reductase and other cellular antioxidants (Ma

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et al. 2015). Thus, H2S synthesis within the cell crucially promotes adaptation to cold alpine
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environments. Luo et al. (2015) observed high phenolics and P5CS-mediated Pro
accumulation along with enhanced activities of SOD, CAT, APX, GR and phenylalanine
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ammonia lyase (for phenolic production) in the fruits of banana plants fumigated with H2S
and growing in chilling conditions. The banana fruits also accumulated lower amounts of
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MDA and were normal in their physiological integrity (Luo et al. 2015). Thus, similar to heat
stress tolerance, H2S might mediate low temperature stress responses in plants by recharging
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the antioxidant machinery and accelerating the accumulation of cellular osmolytes like
soluble sugars, phenolics and especially Pro.
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4.5. Ameliorative role of H2S during hypoxia

Hypoxia is the partial deficiency of oxygen available to the tissues. Such conditions are
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prevalent during flooding or submergence. Such situations cause oxidative damages in the
susceptible plant cultivars (van Dongen and Licausi 2014). The ameliorative role of H2S has
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also been reported in the mitigation of hypoxia in plant roots. The necrosis of root tip cells in
pea seedlings exposed to hypoxic flooding was reduced in the sets treated exogenously with
H2S (Cheng et al. 2013). The membrane system in the treated plants was well protected from
ROS-induced lipid peroxidation and further damages. It was also observed that addition of
LCD inhibitor, hydroxylamine led to necrotic death in the root cells similar to that during
flooding. This clearly illustrates the potential roles of H2S in alleviating hypoxic necrosis in
the roots of flooded pea plants (Cheng et al. 2013). Interestingly, Parveen et al. (2017)
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showed the negative impacts of NaHS application on two macrophytes, Potamogeton crispus
and Myriophyllum spicatum exposed to hypoxic stress. Treatment with 0.1 and 0.5 mM
NaHS reduced CAT and APX activities. This led to increased oxidative damages in the plants
(Parveen et al. 2017). Hence, the application of H2S to alleviate hypoxia is species-dependent
and the optimum concentration of NaHS to be used requires thorough standardization.
4.6. Ameliorative role of H2S during heavy metal toxicity

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Heavy metals and metalloids (HMs) like chromium (Cr), copper (Cu), zinc (Zn), arsenic
(As), etc. are highly toxic for plant developmental growth and reproduction. Bioaccumulation

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of these harmful elements in the edible parts of plants cause ecosystem biohazard and
deteriorates the overall health of the food web (Chen et al. 2017). HM stress triggers

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uncontrolled ROS burst leading to lipid peroxidation and the oxidation of proteins and
nucleic acids. As a result, the tertiary structures of enzymes and nucleic acids are drastically
affected (Sandhi et al. 2017).

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The pea seedlings treated with NaHS exhibited efficient alleviation of As-mediated
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injuries due to the maintenance of high endogenous cysteine (Cys) level (Singh et al. 2015).
The excess Cys moieties were possibly channelized to form phytochelatins (PCs) which
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scavenged the toxic As within the cells (Banerjee and Roychoudhury 2018a). The activities
of the enzymes participating in the AsA-GSH cycle were also enhanced after NaHS treatment
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during As stress (Singh et al. 2015). In a very recent study, Li et al. (2018) studied the
potential effects of molybdenum sulphide (MoS2) on rice plants. Interestingly, in spite of
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being a heavy metal-sulphide, MoS2 conferred growth enhancing effects by increasing the
root/shoot length, biomass and chlorophyll content index. The expression of the genes
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encoding aquaporins was also up regulated. MoS2 promoted overall rice growth by inducing
photosynthesis, metabolism, cellular division and elongation and the assimilation of nitrogen
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sources (Tan et al. 2011; Zhang et al. 2015). Increase in the basal level production of H2S by
the application of MoS2 can also be involved in accelerating the physiological development
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in treated rice seedlings (Li et al. 2018).

In another experiment, wheat seeds pre-treated with NaHS germinated better under Cr
stress due to higher activities of amylase, esterase and antioxidative enzymes like APX, SOD,
CAT and GPX. The LOX activity along with H2O2 and MDA content were significantly
reduced (Zhang et al. 2010c). Similar alleviatory effects were also observed in case of the
wheat plants pre-treated with NaHS and growing under Cu stress (Zhang et al. 2008). Liu et
al. (2016) reported that H2S reversed the Zn-induced growth inhibition in the seedlings of
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Solanum nigrum. The increase in free cytosolic content of Zn2+ suppressed the Zn2+ uptake
and down regulated the expression of genes like zinc-regulated transporter (ZRT), iron-
regulated transporter (IRT)-like protein (ZIP) and natural resistance associated macrophage
protein (NRAMP), all associated with Zn homeostasis. The treated seedlings also exhibited
high accumulation of metallothioneins, which efficiently chelated and scavenged the excess
Zn2+ causing the overall toxicity (Liu et al. 2016). In another study, it was reported that H2S

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significantly reversed citrate secretion and activated the antioxidant system in the NaHS-
treated Hordeum vulgare seedlings exposed to high concentrations of the non-essential
element, aluminium (Al3+) (Chen et al. 2013). Zhang et al. (2010d) also highlighted similar

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ameliorative effects of NaHS application in the wheat seedlings exposed to Al stress.

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5. Interaction between H2S and phytohormones
Hormones are the most important signaling molecules in plants and any potential signaling
molecule eventually interacts with them in order to trigger any downstream response

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(Banerjee and Roychoudhury c, d; Jin et al. 2011; Cheng et al. 2013) (Fig. 2). This area of
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research is a rapidly developing field, since a well defined interactome of H2S with other
phytohormones can present novel signaling nodes which can be genetically targeted for
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yielding multiple stress-tolerant traits in the susceptible cultivars.

5.1. Interaction between H2S and abscisic acid
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Abscisic acid (ABA) is the universal stress hormone in plants, known to regulate almost all
kinds of environmental stresses ranging from salinity, desiccation, oxidative stress and even
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some HM toxicity (Roychoudhury and Banerjee 2017). H2S, being a novel plant
gasotransmitter for cross-adaptation, must be interacting with the ABA signalosome.
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Consistent with this prediction, Aroca et al. (2017) observed reduced persulphidation of ABA
receptors, PYRABACTIN RESISTANCE 1 (PYR1) and PYRABACTIN RESISTANCE
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LIKE 1 (PYL1) in the Arabidopsis knockout mutants of DES1. NaHS pre-treatment triggered
the biosynthesis and accumulation of ABA; and also up regulated the expression of ABA-
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responsive genes in the leaves of wheat plants exposed to drought stress (Ma et al. 2016).
However, the genes involved in both biosynthesis and catabolism of ABA were up regulated
in the roots of these treated plants. H2S accumulation due to NaHS pre-treatment also
promoted ABA-mediated signaling by inducing the genes encoding ABA receptors during
drought. As a result, the treated plants accumulated lower amounts of MDA and H2O2 due to
efficient activation of the antioxidant machinery via ABA-dependent pathway (Ma et al.
2016). Cao et al. (2014) reported the requirement of sulphur-containing compounds in the
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steady production of ABA. The Arabidopsis mutants of SULPHATE TRANSPORTER 3;1
(SULTR3;1) exhibited reduced activity of aldehyde oxidase and ABA level. The
transcriptional regulation of ABA3 and 9-cis-epoxycarotenoid dioxygenase 3 (NCED3) in
Arabidopsis plants exposed to salt stress was found to be co-mediated by S-metabolism and
ABA synthesis (Cao et al. 2014).
Stomatal closure during various environmental abnormalities has been known to be

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regulated by ABA. Interestingly, along with ABA, H2S was also found to co-regulate
stomatal movement in the epidermal strips of Vicia faba, Arabidopsis and Impatiens

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walleriana (Garcia-Mata and Lamattina 2010). It was also observed that exogenous
application of NaHS promoted stomatal closure in an ABA-dependent pathway. This was

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because, treatment with glibenclamide (inhibitor of ATP-binding cassette transporter) or an
H2S scavenger or any inhibitor of H2S biosynthesis partially blocked stomatal closure even
under normal endogenous ABA levels. H2S ameliorated drought-induced injuries by

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increasing the relative water content (RWC). It promoted stomatal closure by regulating the
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ABA-dependent pathway and the ABC transporters localized in the guard cells (Garcia-Mata
and Lamattina 2010). In another interesting study, Papanatsiou et al. (2015) used two-
electrode voltage clamp measurements for observing the effects of H2S on the K+ channels of
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the guard cells. It was found that in Nicotiana tabacum, H2S selectively inactivated the
inward-rectifying K+ channels in the guard cells. Similar phenomenon occurs after the ABA-
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induced spike in the cytosolic concentration of Ca2+. As a result, the plasma membrane
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remains de-polarized, the pH rises and this activates the outward-rectifying K+ channels to
promote K+ efflux. Further changes in the cellular turgor and osmotic pressure in the guard
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cells lead to stomatal closure (Schroeder et al. 2001). Thus, H2S uniquely regulates the K+
transport within guard cells separately and in conjunction with ABA, ultimately to trigger
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stomatal closure during desiccating conditions (Papanatsiou et al. 2015).

Jin et al. (2013) reported that though H2S application decreased the stomatal diameter
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in the aba3 mutants, ABA alone could not induce stomatal closure in the lcd mutants of
Arabidopsis. The H2S biosynthetic enzyme, DES1 has been observed to mediate a crosstalk
between NO and H2S in the ABA-dependent signaling responses in the guard cells (Scuffi et
al. 2014). It was further shown that ABA could not induce stomatal closure in des1
Arabidopsis mutants. However, exogenous H2S could close the stomata in the
pyr1/pyl1/pyl2/pyl4 quadruple ABA receptor mutants (Scuffi et al. 2014). This along with the
previously discussed data (Papanatsiou et al. 2015) suggests that H2S might act either
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downstream of ABA reception or as a separate guard cell gasotransmitter. The functional
protein phosphatase 2C, ABA INSENSITIVE 1 (ABI1: a negative regulator of ABA
signaling) is however essential for H2S mediated stomatal closure in Arabidopsis (Scuffi et al.
2014).
5.2. Interaction between H2S and auxins
Auxin is a major plant growth regulator critically controlling diverse physiological and

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developmental processes like apical dominance, development of phyllotaxis, embryogenesis,
induction of lateral and main roots, tropism and vascular differentiation (Woodward and

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Bartel 2005). These processes are dependent on the establishment of an auxin gradient due to
polar auxin transport (PAT) from the aerial to the basal tissues (Normanly 2010). Such PAT

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is inhibited by high concentrations of H2S, leading to altered root structures. H2S changed the
expression of several ACTIN-BINDING PROTEINS (ABPs) and reduced the percentage of
occupancy of the F-acting bundles in Arabidopsis roots (Jia et al. 2015). Since the

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distribution of the PINFORMED (PIN) proteins responsible for mediating PAT is dependent
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on ABPs, it was significantly altered under the influence of H2S (Jia et al. 2015). Thus, the
report indicated towards a ‘tightly regulated intertwined signaling network’ existing among
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auxins, H2S and ABPs. Recently, Zhang et al. (2017) investigated the effects of NaHS
treatment on the growth of primary roots in Arabidopsis seedlings. High cellular
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accumulation of H2S triggered the production of NO, activated the MITOGEN-ACTIVATED

PROTEIN KINASE 6 (MPK6) and induced ROS accumulation. These severely inhibited the
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growth of the primary root system. It was also observed that NO was generated due to the
H2S-induced accumulation of ROS. The activated MAPK6 regulated the parallel production
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of H2S-induced NO (Zhang et al. 2017). Thus, the H2S-mediated repression of root growth in
Arabidopsis due to altered PAT is simultaneously controlled by interactive communications
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between H2S and NO. However, optimum levels of H2S have been observed to induce the
formation of lateral roots via potential interactions with NO, CO and the auxin, indole-3-
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acetic acid (IAA). IAA was also found to promote H2S biosynthesis by regulating the
expression of LCD (Li et al. 2014). Zhang et al. (2009) observed rapid increases in the
cellular levels of H2S, IAA and NO in the shoot tips of sweet potato (Ipomoea batatas)
seedlings treated with exogenous NaHS. H2S was found to regulate upstream to the IAA and
NO signaling pathways during the induction of adventitious root formation in Salix
matsudana and Glycine max (Zhang et al. 2009). Application of IAA transport inhibitor,
naphthylphthalamic acid and NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-
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tetramethylimidazoline-1-oxyl-3-oxide alleviated the H2S-dependent root formation
phenotype (Zhang et al. 2009). Fang et al. (2014) also highlighted the synergistic roles of H2S
and auxins during the lateral root formation in NaHS treated Solanum lycopersicum
seedlings. Depletion in the cellular auxin level significantly suppressed the activity of the H2S
anabolic enzyme, DES. Treatment of the plants with the auxin, naphthalene acetic acid
(NAA) increased DES1 expression, DES activity and also the H2S concentration. These

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together stimulated the growth of lateral roots (Fang et al. 2014). Application of the H2S
scavenger, hypotaurine reduced lateral root growth. This inhibition was reversed only by

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plant treatment with NaHS and not NAA. Lateral root growth co-ordinated by the
synchronization of H2S and NAA was also characterized by the up regulation of specific cell

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cycle genes like cyclin dependent kinases CDKA;1 and CYCA2; and the down regulation of
the cyclin dependent kinase inhibitor, KRP2 in the tomato plants (Fang et al. 2014). Such
regulation of gene expression promotes cellular division and organogenesis in plants (Atkins
and Cross 2018).
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5.3. Interaction between H2S and gibberellic acid
Like auxin, gibberellic acids (GAs) are important plant growth regulating hormones involved
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in promoting seed germination and internodal elongation. The antioxidative effects of H2S on
the aleurone layers were reported in the Hordeum barley grains imbibed in 0.25 mM NaHS
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solution (Zhang et al. 2015). H2S accumulation effectively induced the activities of SOD,
CAT, APX and guaiacol peroxidase (POD); and suppressed the LOX activity in the aleurone
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layers. These enzymes scavenged superoxide radicals, H2O2 and reduced MDA
accumulation. H2S also delayed programmed cell death (PCD) in the GA-treated aleurone
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layers. The production of α-amylase was accelerated by H2S regardless of the presence or
absence of GA (Zhang et al. 2015). Interestingly, Xie et al. (2014) reported that GA down
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regulates the expression of LCD, suppresses H2S production and further accelerates PCD in
the aleurone layer cells of Triticum aestivum. The exogenous application of NaHS alleviated
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GA-triggered PCD by increasing the endogenous content of GSH. The NaHS-mediated

amelioration of aleurone cell apoptosis was mediated via HEME OXYGENASE-1 (HO-1)
and was suppressed in the presence of l-buthionine-sulfoximine, a selective inhibitor of GSH
synthesis (Xie et al. 2014). Thus, H2S accumulation prevents PCD in GA-treated aleurone
layer cells in association with GSH homeostasis and HO-1 expression.
5.4. Interaction between H2S and ethylene

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The gaseous phytohormone ethylene regulates almost every stage of plant development.
Especially climacteric fruits like tomato, banana, apple, mango, etc. exhibit a burst of
ethylene production during ripening. This leads to degradation of starch, chlorophyll, cell
wall component and accumulation of carotenoids along with the generation of aroma (Gray et
al. 1992; Johnson and Ecker 1998). Ge et al. (2017) investigated the effects of H2S on
ethylene- mediated ripening of Musa acuminata fruits. It was observed that the application of

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1 mM NaHS solution reversed the ethylene-induced peel yellowing and fruit softening due to
reduced polygalacturonase activity in banana. Combined treatment of NaHS and ethephon

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(ethylene donor) solutions promoted the activities of antioxidants and scavenged the ROS.
Chlorophyll degradation was delayed and the content of total phenolics and flavonoids

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increased (Ge et al. 2017). H2S also suppressed the expression of ethylene biosynthetic genes
like 1-aminocyclopropane-1-carboxylate synthase 1 (ACS1), ACS2, 1-aminocyclopropane-1-
carboxylate oxidase 1 (ACO1) and pectate lyase; and up regulated the expression of genes

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encoding ethylene receptors, ETHYLENE RECEPTOR (ETR), ETHYLENE RESPONSE
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SENSOR1 (ERS1) and ERS2 (Ge et al. 2017). Thus, H2S acts antagonistically with ethylene
to delay ROS accumulation, ripening and hence senescence in banana fruits. In another study,
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the effects of H2S on the metabolic homeostasis of postharvest banana fruits exposed to
chilling stress were documented (Li et al. 2016). Exogenous fumigation of the banana fruits
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with H2S reduced ethylene production, MDA accumulation, ATP degradation and cold-
induced injuries. The activities of H+-ATPase, Ca2+-ATPase, cytochrome c oxidase and
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succinate dehydrogenase were all enhanced in response to H2S treatment (Li et al. 2016).
Thus, H2S maintains the overall energy charge by activating the cellular metabolism and
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delaying senescence in banana fruits exposed to chilling temperatures.

5.5. Interaction between H2S and salicylic acid
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Salicylic acid (SA) is a multifunctional growth regulator with diverse roles in biotic as well
as abiotic stress tolerance. It is mainly synthesized via phenylalanine in the cytoplasm
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(Roychoudhury et al. 2016). SA treatment induced the activities of SOD and CAT in barley
seedlings exposed to heavy metals like Zn2+, Cu2+, Mn2+, Cd2+, Hg2+ and Pb2+ (Song et al.
2013). SA application significantly induced DES activity followed by H2S production in Zea
mays seedlings exposed to heat stress (Li et al. 2015). NaHS treatment also enhanced the
stress-ameliorative effects of SA by triggering the activities of SOD, APX, GR, CAT, POD
and increasing the contents of AsA, GSH, Pro, betaine and trehalose in the maize seedlings
exposed to heat stress (Li 2015). However, NaHS treatment did not significantly stimulate
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SA production. Application of inhibitors of SA synthesis like paclobutrazol (PAC) and 2-
aminoindan-2-phosphonic acid (AIP) also did not affect NAHS-induced heat tolerance (Li et
al. 2015). These facts indicate that H2S acts synergistically yet independent of SA to confer
heat stress tolerance in maize seedlings.

6. Conclusion

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In spite of being considered as a toxic gaseous molecule, recent research has validated the
growth promoting and physiological significance of H2S in plants (Jin and Pei 2015).

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However, the beneficiary effects of H2S are conferred only when used at optimum
concentrations and in a species-specific pattern. H2S interacts with other secondary

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messengers like Ca2+, H2O2 and NO to stimulate growth under sub-optimal conditions.
Oxidative stress accelerates the unregulated production of ROS which oxidize membrane
systems, nucleic acids and even target Cys residues in proteins. H2S activates the entire

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antioxidant machinery and the cellular accumulation of non-enzymatic antioxidants is
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triggered. The antioxidant enzymes are efficiently activated to scavenge ROS. As a result,
less MDA accumulates within NaHS treated plants. H2S also stimulates persulphidation of
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the Cys residues, which are highly prone to cellular oxidation. Thus, the proteins remain
protected during prevailing oxidative stress (Aroca et al. 2017). H2S also mediates multiple
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interactions with plant hormones like ABA, auxin, gibberellic acid, ethylene and SA. H2S
acts downstream of ABA reception to activate the outward-rectifying K+ channels and
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induces stomatal closure during desiccation (Papanatsiou et al. 2015). Though high levels of
H2S were found to inhibit PAT, its optimum concentration regulated lateral root growth in an
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auxin- and NO-dependent manner (Jia et al. 2015; Li et al. 2014). H2S stimulated endogenous
production of GSH and other antioxidants to delay PCD in the GA-treated aleurone layer
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cells (Xie et al. 2014). Over-ripening and senescence in banana fruits were delayed via H2S-
mediated repression in ethylene biosynthesis (Ge et al. 2017). H2S and SA acted
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synergistically, yet independently to activate the antioxidant machinery and generate heat
stress tolerance in the susceptible maize cultivars (Li et al. 2015). Thus, H2S has been
correctly popularized as a gasotransmitter capable of inducing cross-adaptation in plants (Li
et al. 2016). It mainly replenishes the reservoir of antioxidants, necessary to quench the toxic
ROS produced under any stress.

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7. Future perspectives
Though the interactions and crosstalks between H2S and phytohormones have been
well investigated, yet the complete picture is partially revealed. Future perspectives should
include the scientific generation of a blueprint encompassing the H2S-mediated signaling
network. It would be beneficial in understanding the signaling hierarchy amongst the global
plant signaling web. The associations between H2S and the cell replicating phytohormone,

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cytokinin are yet to be elucidated. Complete physiological, biochemical and molecular data
regarding these unknown interactions would definitely help in the identification of a

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molecular target which can be genetically manoeuvred to yield stress tolerance. RNASeq
studies need to be performed to identify the transcriptome during NaHS treatment under

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various stimuli. Identification of the alterations in gene expression from such investigations
can also help in elucidating the H2S-phytohormone crosstalk nodes.

Acknowledgements
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Financial support from Council of Scientific and Industrial Research (CSIR), Government of
India through the research grant [38(1387)/14/EMR-II] to Dr. Aryadeep Roychoudhury is
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gratefully acknowledged. The authors thank University Grants Commission, Government of

India for providing Junior Research Fellowship to Mr. Aditya Banerjee.
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Figure legends

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Fig. 1. Priming with sodium hydrosulphide (NaHS) triggers endogenous accumulation of
hydrogen sulphide (H2S). This gasotransmitter promotes cross-adaptation to multiple
abiotic stresses like extremes of temperature, salinity, drought, hypoxia and heavy
metal toxicity by replenishing the cellular reservoir of antioxidants. High activities
of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX),
guaiacol peroxidase (POD), glutathione reductase (GR) and specific heat shock

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proteins (HSPs) have been observed under such circumstances. H2S also induces the
accumulation of non-enzymatic antioxidants like ascorbate (AsA), glutathione

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(GSH), phenolics, flavonoids; and that of compatible solutes like betaine and proline
(Pro). As a result, the entire antioxidative machinery is recharged. The plant can

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better tolerate environmental abnormalities due to efficient scavenging of the toxic
reactive oxygen species (ROS). H2S production helps in maintaining a high relative
water content (RWC) and K+/Na+ ratio during stress. Due to suppressed activity of

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lipoxygenase (LOX) and reduced levels of ROS, the lipid membranes are protected
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from peroxidation. Thus, the malondialdehyde (MDA) content is also lowered. H2S
also promotes persulphidation (-SSH) of protein cysteine residues which are prone
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to oxidation. In absence of oxidative stress, this protective post-translational

modification is removed in the presence of thioredoxins.
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Fig. 2. The potential interactions between hydrogen sulphide (H2S) and phytohormones.
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H2S induces the expression of 9-cis-epoxycarotenoid dioxygenase 3 (NCED3) and

abscisic acid 3 (ABA3). This accelerates the biosynthesis of ABA. The H2S anabolic
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enzyme L-cysteine desulfhydrase (LCD/DES1) mediates a crosstalk between H2S,

nitric oxide (NO) and ABA. In a less understood synchronized fashion, ABA and
H2S inhibit the inward-rectifying K+ channels (KIN) and stimulate the outward-
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rectifying K+ exporters (KOUT) during desiccation. This promotes guard cell

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flaccidity and stomatal closure. H2S also mediates persulphidation of the ABA
receptor, PYRABACTIN RESISTANCE/PYRABACTIN RESISTANCE LIKE
(PYR/PYL) involved in the transduction of ABA-dependent signals. In a synergistic
crosstalk, H2S and SA act independently to promote tolerance against heat stress.
High concentrations of H2S are toxic for the cell as it inhibits normal distribution of
the PINFORMED (PIN) transporters and interferes with polar auxin transport.
However, at optimum concentrations, both indole-3-acetic acid and 1-naphthalene
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acetic acid (NAA) stimulated the development of lateral roots. NAA application also
elevated the activity of DES1. H2S increased the endogenous glutathione (GSH)
content and triggered the expression of heme oxygenase-1 (HO-1) to ameliorate
gibberellic acid (GA)-induced programmed cell death (PCD) of the cells in the
aleurone layer. The expression of ethylene biosynthetic genes like 1-
aminocyclopropane-1-carboxylate synthase 1 (ACS1), ACS2 and 1-

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aminocyclopropane-1-carboxylate oxidase 1 (ACO1) were suppressed in the post-
harvested banana fruits fumigated with H2S. This delayed over-ripening and

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senescence even under chilling temperatures.

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• H2S mediates crucial signaling during oxidative and other environmental stress
• H2S induces the antioxidant machinery
• The molecule acts as a gasotransmitter
• It confers plant tolerance against multiple abiotic stresses

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AB drafted the overall manuscript. AR supervised the entire work, critically analyzed the
manuscript and made modifications. DRK provided his valuable suggestions during the drafting
of the manuscript.

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