Plant Biology ISSN 1435-8603

RESEARCH PAPER

Exogenous melatonin-mediated regulation of K+/Na+ transport,

H+-ATPase activity and enzymatic antioxidative defence operate
through endogenous hydrogen sulphide signalling in
NaCl-stressed tomato seedling roots
M. H. Siddiqui1 , M. N. Khan2 , S. Mukherjee3, R. A. Basahi2, S. Alamri2, A. A. Al‑Amri2,
Q. D. Alsubaie2, H. M. Ali2, B. M. A. Al-Munqedhi1 & I. A. A. Almohisen4
1 Department of Botany and Microbiology, College of Science, King Saud University, Riyadh, Saudi Arabia
2 Department of Biology, College of Haql, University of Tabuk, Tabuk, Saudi Arabia
3 Department of Botany, Jangipur College, University of Kalyani, Kalyani, West Bengal, India
4 Department of Biology, Faculty of Science and Humanities, Quwayiyah, Shaqra University, Shaqra, Saudi Arabia

Keywords ABSTRACT
Hydrogen sulphide; K+/Na+ homeostasis;
melatonin; salinity; signalling. • Melatonin (Mel) and hydrogen sulphide (H2S) have emerged as potential regulators of
plant metabolism during abiotic stress. Presence of excess NaCl in the soil is one of the
Correspondence main causes of reduced crop productivity worldwide. The present investigation exami-
M. H. Siddiqui, Department of Botany and nes the role of exogenous Mel and endogenous H2S in tomato seedlings grown under
Microbiology, College of Science, King Saud NaCl stress.
University, P.O. Box 2455, Riyadh 11451, • Effect of 30 µM Mel on endogenous synthesis of H2S was examined in roots of NaCl-
Saudi Arabia. stressed (200 mM) tomato seedlings. Also, the impact of treatments on the oxidative
E-mail: mhsiddiqui@ksu.edu.sa; stress markers, transport of K+ and Na+, and activity of H+-ATPase and antioxidant
manzerhs@yahoo.co.in enzymes was assessed.
M. N. Khan, Department of Biology, College • Results show that NaCl-stressed seedlings supplemented with 30 µM Mel had increased
of Haql, University of Tabuk, Tabuk, 71491, levels of endogenous H2S through enhanced L-cysteine desulfhydrase activity. Mel in
Saudi Arabia. association with H2S overcame the deleterious effect of NaCl and induced retention of
E-mail: mo.khan@ut.edu.sa K+ that maintained a higher K+/Na+ ratio. Use of plasma membrane inhibitors and an
H2S scavenger revealed that Mel-induced regulation of K+/Na+ homeostasis in NaCl-
Editor
stressed seedling roots operates through endogenous H2S signalling. Synergistic effects
Y. Hu
of Mel and H2S also reduced the generation of ROS and oxidative destruction through
the enhanced activity of antioxidant enzymes.
Received: 31 January 2021; Accepted:
10 May 2021
• Thus, it is suggested that the protective function of Mel against NaCl stress operates
through an endogenous H2S-dependent pathway, wherein H+-ATPase-energized sec-
doi:10.1111/plb.13296 ondary active transport regulates K+/Na+ homeostasis.

2020). Changes in plant water relations result in a surge in

INTRODUCTION
Soil salinity is largely caused by the presence of NaCl in the
soil, which is also accompanied by the presence of Na2CO3
and CaSO4 (Maathuis et al. 2014). Among the various delete-
rious effects of NaCl stress in plants, ion specific stress
results in altered membrane potential and potassium (K+)
deficiency, which is accompanied by a low K+/Na+ ratio in
roots and aerial organs. Due to the similar ionic radii of
sodium (Na+) and K+ in their hydrated form, K+ influx sys-
tems fail to distinguish between these two ions, thus leading
to nonspecific entry of Na+ into the cells (Keisham et al.
2018). In order to lower the deleterious effect of Na+ toxicity
in cells, plants implement a number of mechanisms: (i)
restriction of Na+ influx in cells, (ii) vacuolar compartmen-
talization of Na+ and (iii) Na+ efflux from cells. Apart from
altering the morphological attributes of plants, salt stress is
also known to effect various primary metabolic enzymes
associated with photosynthesis and respiration (Shahid et al.
endogenous accumulation of compatible solutes which is also
accompanied by enhanced biosynthesis of phytohormones,
e.g. abscisic acid, ethylene and brassinosteroids (Shahid et al.
2020). NaCl stress results in disruption of various primary
metabolic pathways in the chloroplast, cytosol, mitochondria
and peroxisomes. Under NaCl stress the cellular system gen-
erates ROS, which at low levels function as beneficial sig-
nalling molecules in defence mechanisms (Hasanuzzaman
et al. 2020); however, ROS are phytotoxic at higher concen-
trations and cause oxidative stress (Khan et al. 2020a, 2021;
Siddiqui et al. 2020). Plants mitigate oxidative stress through
the antioxidative defence system, which is regulated by a net-
work of non-enzymatic and enzymatic components. These
include superoxide dismutase (SOD) which catalyses the con-
version of superoxide (O2• ) to hydrogen peroxide (H2O2).
Higher concentrations of H2O2 are metabolized to water and
molecular oxygen by the enzymes peroxidase (POX), catalase
(CAT), ascorbate peroxidase and glutathione reductase.

Plant Biology 23 (2021) 797–805 © 2021 German Society for Plant Sciences and The Royal Botanical Society of the Netherlands 797
Exogenous melatonin-mediated regulation of K+/Na+ transport
Among the various priming molecules (brassinosteroids,
salicylic acid, proline, selenium, silicon, GABA) reported to
combat plant abiotic stress (Savvides et al. 2016; Abdelaal et al.
2020; Elkelish et al. 2020; Kerchev et al. 2020; Sako et al. 2020;
Thomas et al. 2020), melatonin (Mel) and hydrogen sulphide
(H2S) have emerged as potential regulators of plant metabo-
lism for abiotic stress tolerance. Melatonin, a potent indolea-
mine, is known for its substantial effects on plants during
biotic and abiotic stress. Melatonin has many roles in plants,
e.g. abiotic stress tolerance mediated by regulation of its
biosynthesis and modulation of various metabolic pathways
(Ahn et al. 2021; Li et al. 2021a,b; Su et al. 2021; Sun et al.
2021; Wei et al. 2021). During salinity stress, Mel functions to
normalize ionic imbalances, K+ deficiency, photosynthetic effi-
ciency and redox homoeostasis (Li et al. 2012; Kaur & Bhatla
2016; Chen et al. 2017, 2020; Ahmad et al. 2020a; El-Sayed
et al. 2020). H2S functions as an important regulator of salinity,
drought and other factors associated with oxidative stress,
wherein H2S modulates antioxidative defence and signalling in
plant metabolism (Chen et al. 2013; Fang et al. 2014; Alamri
et al. 2020; Khan et al. 2020a, 2021). Recent investigations indi-
cate the role of H2S as an effective priming molecule and its
involvement in crosstalk with various biomolecules during abi-
otic stress in plants (Corpas et al. 2021; Dawood et al. 2021;
Goyal et al. 2021; Iqbal et al. 2021; Li et al. 2021a,b; Wang et al.
2021). Salinity tolerance mediated by H2S is known to be asso-
ciated with ion homoeostasis and regulation of Na+ and K+ ex-
change in plants (Christou et al. 2013; Lai et al. 2014; Deng
et al., 2016).
Although various biomolecules have been reported to
exhibit crosstalk with H2S (Corpas 2019; Corpas et al. 2019;
Corpas & Palma 2020; Khan et al. 2020a, 2021), little infor-
mation is available on the mechanism of interaction between
Mel and H2S during abiotic stress tolerance in plants.
Recent investigations have revealed the integrative role of
nitric oxide and Mel in modulation of H2S-mediated toler-
ance to iron deficiency and salinity stress (Kaya et al. 2020).
Mukherjee & Bhatla (2020) described the role of exogenous
Mel as an important regulator of endogenous H2S home-
ostasis in NaCl-stressed tomato cotyledons. Further investi-
gations are necessary to elucidate the role of Mel and H2S
in modulation of K+/Na+ homeostasis and antioxidative
defence during salinity stress.
In view of such lacunae in our understanding, the present
work aims to decipher the role of exogenous Mel and endoge-
nous H2S in the regulation of K+/Na+ homeostasis, H+-ATPase
activity and oxidative stress tolerance in roots of NaCl-stressed
tomato seedlings.
MATERIAL AND METHODS
Seed germination and treatments
Sterilized seeds of tomato (Lycopersicon esculentum Mill.) geno-
type BL-1076 were germinated in filter paper-lined Petri dishes
containing double distilled water (DDW). The Petri dishes
were kept in the dark at average day/night temperatures of 22/
13  2 °C for 8 days. After 8 days, the germinated seeds were
transferred to new filter paper-lined Petri dishes containing:
(1) DDW (Control), (2) 30 µM melatonin (Mel), (3) 30 µM
Mel +1 mM hypotaurine (Mel+HT), (4) 100 mM NaCl (NaCl),
798 Plant Biology 23 (2021) 797–805 © 2021 German Society for Plant Sciences and The Royal Botanical Society of the Netherlands
Siddiqui, Khan, Mukherjee, et al.
(5) 100 mM NaCl+ 30 µM Mel (NaCl+Mel) or (6) 100 mM
NaCl+ 30 µM Mel + 1 mM HT (NaCl+Mel+HT). The concen-
tration of Mel (30 µM) was selected from a preliminary seed
germination experiment conducted with various concentra-
tions of Mel (data not published). The seedlings were treated
for 2 days, and each treatment was replicated five times. HT
was used as a H2S scavenger. After 2 days of treatments, the
seedling roots were harvested for estimation of various charac-
teristics.
To evaluate K+ and Na+ transport mechanism, 8-day-old
seedlings, raised with DDW, were treated with 500 µM sodium
orthovanadate (SOV, a plasma membrane H+-ATPase inhibi-
tor), 20 mM TEA (tetraethylammonium; K+ channel blocker)
and 50 M amiloride (Na+/H+ antiporter inhibitor) for 30 min
(Zhao et al. 2018). After 30 min, the seedlings were subjected
to 100 mM NaCl for 2 days in the presence or absence of 30 µM
Mel. After 2 days, the concentrations of K+ and Na+ were esti-
mated in roots of treated tomato seedlings (Fig. 2A–D).
Preparation of crude enzyme extract
A crude root extract for assay of enzyme activity was prepared
by homogenizing tissues (0.5 g) with three volumes (w/v) of an
ice-cold extraction buffer. The homogenate was centrifuged at
15 000 g for 20 min at 4 °C, and the supernatant used for the
enzyme assays. Protein quantification was carried out accord-
ing to Bradford (1976).
Estimation of K+ and Na+ content and plasma membrane H+-
ATPase activity
To estimate K+ and Na+ content, roots were dried for 72 h at
75 °C, ground to a fine powder and 1 g of the powder ashed in
a furnace at 500 °C then dissolved in 5 ml HCl (20%). The
concentrations of K+ and Na+ were analysed in the diluted
solution using a flame photometer.
Activity of the plasma membrane H+-ATPase (PM H+-
ATPase, EC 3.6.1.35) was measured by quantifying the produc-
tion of inorganic phosphate at 700 nm following the method of
Hejl & Koster (2004) with slight modification (Majumdar &
Kar 2018).
Estimation of L-cysteine desulfhydrase (L-DES) activity and
H2S content
The activity of L-DES (EC 4.4.1.1) was measured using the
method of Bloem et al. (2004). L-DES activity was determined
as the formation of methylene blue at 670 nm. Quantification
of H2S was performed according to Li (2015). The detailed
procedure for L-DES and H2S estimation was adopted from
Khan et al. (2017).
Determination of oxidative stress markers and associated
damage
Response of seedlings to the various oxidative stress treatments
was evaluated by measuring superoxide (O2• ) and hydrogen
peroxide (H2O2) content following Elstner & Heupel (1976)
and Velikova et al. (2000), respectively, as explained in Khan
et al. (2017). To determine H2O2 content absorbance was
recorded at 390 nm, whereas absorbance for O2• was read at
Siddiqui, Khan, Mukherjee, et al.
530 nm. The content of H2O2 and O2• was calculated based
on their respective standard curves.
Effects of the six treatments on oxidative stress-induced
damage was evaluated by estimating electrolyte leakage (ELKG)
according to Lutts et al. (1995), measured as the electrical con-
ductivity of a solution obtained after the incubation of roots in
DDW (Khan et al. 2020b). Lipid peroxidation was assessed by
estimating the concentration of thiobarbituric acid reactive
substances (TBARS) according to the method of Cakmak &
Horst (1991) as explained by Khan et al. (2017). Relative water
content (RWC) was measured according to Yamasaki & Dillen-
burg (1999) by measuring fresh weight, dry weight and turgid
weight of the roots (Khan et al. 2020b).
Assay of antioxidant enzymes
Antioxidant enzyme activity of SOD (EC 1.15.1.1) was esti-
mated by measuring its capacity to inhibit photochemical
reduction of nitro-blue tetrazolium, measured at 560 nm
(Beauchamp & Fridovich 1971). The activity of POX (EC
1.11.1.7) was determined according to Upadhyaya et al. (1985),
measured as increase in absorbance of the reaction mixture at
420 nm. CAT (EC 1.11.1.6) activity was estimated according to
Cakmak & Marschner (1992) as a decline in absorbance due to
a decline in extinction of H2O2 at 240 nm.
Statistical analysis
The data were analysed using one-way ANOVA followed by Dun-
can’s Multiple Range Test (DMRT) at P < 0.05. The values
were expressed as mean  SE of five independent replicates.
RESULTS
Melatonin-mediated regulation of K+/Na+ transport and H+-
ATPase activity in roots requires endogenous H2S signalling
Tomato seedlings (8 days old) exposed to NaCl stress
(100 mM) exhibit a significant increase in endogenous Na+
content (91.2%) associated with a reduction in K+ content
(47.9%) in comparison with their controls (Fig. 1A, B).
NaCl-stressed roots showed a marked reduction of 72.3% in
the K+/Na+ ratio accompanied by inhibition of H+-ATPase
activity (39.0%) (Fig. 1C, D). However, addition of exoge-
nous Mel (30 µM) resulted in increased retention of K+ and
reduced accumulation of Na+ both in the absence and pres-
ence of NaCl stress (Fig. 1A). In addition, to increase the
K+/Na+ ratio in roots, Mel treatment positively upregulated
H+-ATPase activity which was further increased in NaCl-
stressed seedlings (94.2%) than in untreated stressed seed-
lings without Mel (Fig. 1D). Furthermore, treatment with
1 mM HT (endogenous H2S scavenger) alone and in combi-
nation with Mel resulted in a marked reduction in all effects
exerted by Mel alone. Suppression of endogenous H2S by
HT increased Na+ accumulation, reduced K+ content and
inhibited H+-ATPase activity in both control and NaCl-
stressed roots. Thus, the level of endogenous H2S is crucial
for Mel-induced regulation of K+/Na+ transport and H+-
ATPase activity in roots of tomato seedlings raised in the
absence or presence of NaCl stress.
Plant Biology 23 (2021) 797–805 © 2021 German Society for Plant Sciences and The Royal Botanical Society of the Netherlands
Exogenous melatonin-mediated regulation of K+/Na+ transport
Exogenous Mel regulates activity of K+channel and H+-
ATPase-dependent K+/Na+ transport
Following treatment with 500 µM SOV (a PM H+-ATPase inhi-
bitor), 20 mM TEA (K+ channel blocker) and 50 mM amiloride
(K+/Na+ antiporter inhibitor), roots of NaCl-stressed tomato
seedlings (in absence or presence of Mel) were analysed for
Na+ and K+ content. In the presence of SOV, there was a signif-
icant reduction in K+ content (65.8%) in NaCl+Mel-treated
roots in comparison with the control ( SOV) (Fig. 2A). How-
ever, the highest K+ content was recorded with Mel under non-
stress conditions. Inhibition of K+ channels through TEA treat-
ment resulted in increased retention (46.3%) of K+ in NaCl-
stressed and Mel-treated roots than in NaCl+Mel-treated seed-
ling roots without TEA (Fig 2B). However, K+ level in the seed-
lings treated with NaCl+Mel ( TEA) was similar to NaCl-
stressed seedlings supplied with TEA alone (Fig. 2B). These
observations suggest the role of Mel in enhancing K+ content
through regulating K+ channels. Alternately, application of
SOV induced higher accumulation of endogenous Na+ in
NaCl-stressed roots both in the absence and presence of Mel
(Fig. 2C). However, unstressed seedlings treated with Mel alone
had the lowest Na+ content (Fig. 2C), suggesting the role of
Mel in reducing Na+ content through regulating H+-ATPase
activity. In congruence with the effect of SOV on Na+ content,
similar observations were recorded after amiloride treatment.
Therefore, it is evident that Mel regulates activity of K+ chan-
nels and also induces H+-ATPase energized secondary K+/Na+
transport in NaCl-stressed tomato seedling roots.
Melatonin stimulates biosynthesis of H2S in roots
Tomato seedlings raised in the presence of 100 mM NaCl had
elevated (69.5%) L-DES activity accompanied by a subsequent
increase in endogenous H2S accumulation (36.6%) in compar-
ison with the control (Fig. 3A, B). Exogenous Mel treatment
further upregulated this H2S biosynthesis enzyme (L-DES) and
increased endogenous H2S levels both in the absence and pres-
ence of NaCl stress (Fig. 3A, B). Application of HT alone or in
combination with Mel substantially reduced H2S content and
L-DES activity under non-stress and stress conditions (Fig. 3A,
B). Thus, endogenous H2S level is crucial for activity of L-DES
in roots, and exogenous Mel functions as a positive regulator
of H2S biosynthesis in roots, both with and without NaCl
stress.
Exogenous Mel and endogenous H2S alleviate osmotic stress
in NaCl-treated roots
Exposure of seedlings to NaCl stress (100 mM) resulted in a
51.7% decrease in RWC followed by a 130.0% increase in
ELKG in comparison with the control (Table 1). NaCl stress in
the growth medium also caused a two-fold increase in lipid
peroxidation in the roots. Exogenous Mel treatment signifi-
cantly improved RWC (62.3%) and reduced ELKG by 53.7%
under NaCl stress compared to stressed seedlings without Mel.
Lipid peroxidation was also reduced (47.7%) in the presence of
NaCl and Mel. Interestingly, HT treatment significantly inhib-
ited the effect of Mel on RWC, ELKG and lipid peroxidation
under both stress and non-stress conditions (Table 1). Thus,
799
Exogenous melatonin-mediated regulation of K+/Na+ transport Siddiqui, Khan, Mukherjee, et al.

30 30
Na+ content
+
(A) K co n ten t (B)
25 a 25
a b

mg g-1 dry weight

d r y w e ig h t

20 b bc 20
c
15 d d de 15
e
f
-1

f fg
mg g

10 10
g

5 5

0 Na Na Control Na Na 0
C o n tr o l M el HT M e l+ H T N aC l Cl Cl Mel HT Mel+HT NaCl Cl+ Cl+
T rea tm en ts
+M +M
Treatments Me Me
el e l+ l l +H
H T
T

4 40
(C) H+-ATPase activity (D)

-1
3 30

mmol Pi mg protein h
a
a b
K+/Na+

-1
2 b 20
d
e ef g
cd c
1 e 10
f
g
0 Na Na 0
Control Mel HT Mel+HT NaCl Na Na Control Mel HT Mel+HT NaCl Cl+ Cl+
Cl+ Cl+
Me Me Treatments Me Me
Treatments l l+H l l+H
T T

Fig. 1. Effect of Mel and NaCl on K+ and Na+ concentration and H+-ATPase activity in roots of tomato seedlings. (A) K+ content, (B) Na+ content, (C) K+/Na+
ratio, (D) H+-ATPase activity. Double deionized water (control), 30 lM melatonin (Mel), 1 mM hypotaurine (HT: H2S scavenger), 100 mM NaCl. Values are mean
of five independent replicates, bars indicate SE. Bars with different letters indicate that differences were statistically significant at P < 0.05 (DMRT).

exogenous Mel and endogenous H2S synergistically alleviate

DISCUSSION
osmotic stress in NaCl-stressed tomato seedling roots.
Recent investigations have revealed the integrative role of Mel
and H2S signalling in modulating abiotic stress tolerance in
Exogenous Mel-mediated elevated antioxidative defence
plants (Kaya et al. 2020; Mukherjee & Bhatla 2020). The pre-
(enzymatic) operates through a H2S-dependent pathway
sent work provides evidence of exogenous Mel-mediated regu-
The effect of NaCl stress on ROS levels in tomato seedling roots lation of K+/Na+ homeostasis and elevation of antioxidative
was analysed by measuring O2• and H2O2 content. These ROS defences through endogenous H2S signalling in roots of NaCl-
were significantly higher in NaCl-stressed roots in comparison stressed tomato seedlings. Furthermore, exogenous Mel upreg-
with the control. Mel treatment resulted in a marked reduction ulates L-DES-dependent H2S biosynthesis in roots subjected to
in the O2• and H2O2 content under stress and non-stress con- NaCl stress. Analysis of K+ and Na+ content in plant tissues
ditions (Table 1). However, the level of ROS increased with the indicates the extent of salinity stress, where K+ deficiency
addition of HT under both stressed and non-stressed condi- results in impaired plant metabolism (Khan et al. 2012;
tions in the presence or absence of Mel (Table 1). Ansch€ utz et al. 2014; Demidchik et al. 2014; Zhao et al. 2018).
Exposure of seedlings to NaCl stress positively upregulated In the present work, presence of Mel influenced endogenous
the activity of SOD (30.7%), POX (45.1%) and CAT (16.4%), K+ and Na+ content in roots of tomato seedlings grown in the
especially in the presence of exogenous Mel (Table 2). How- absence or presence of NaCl stress. Interestingly, HT (endoge-
ever, HT treatment reversed the effects of Mel and decreased nous H2S scavenger) treatment revealed that Mel-induced reg-
activity of the antioxidant enzymes. A similar trend for the ulation of K+/Na+ homeostasis in NaCl-stressed roots operates
modulation of enzyme activity was observed for HT and through endogenous H2S signalling. Furthermore, the incre-
Mel+HT treatments in control seedlings. Thus, exogenous Mel ment in K+ content is positively correlated with Mel and H2S-
and endogenous H2S jointly regulate ROS level and induce dependent elevation of H+-ATPase activity in both the absence
enzymatic antioxidative defence. and presence of NaCl stress. Increased Na+ accumulation

800 Plant Biology 23 (2021) 797–805 © 2021 German Society for Plant Sciences and The Royal Botanical Society of the Netherlands
Siddiqui, Khan, Mukherjee, et al. Exogenous melatonin-mediated regulation of K+/Na+ transport

40 50
(A) K+ content Mel K
+
co n ten t M el (B)
-SOV -T E A
+SOV +TEA 40
30

d r y w e ig h t
mg g-1 dry weight

a a 30

20 b b

-1
c

mg g
cd 20

c
10 d e e
10

0 0
NaCl Treatments NaCl+Mel N aC l T rea tm en ts N a C l+ M e l

50 60
(C) Na+ content Mel Na+ content Mel (D)
-SOV -Amiloride
+Amiloride 50
40 +SOV

mg g-1 dry weight

40
mg g-1 dry weight

30 a a
b b
30
c
20 d c
d 20

10 e e 10

0 0
NaCl Treatments NaCl+Mel NaCl Treatments NaCl+Mel

Fig. 2. Effect of inhibitors on K+ and Na+ content in the absence or presence of Mel in NaCl-stressed tomato roots. Effect of SOV (A) and TEA (B) on K+ con-
tent, effect of SOV (C) and amiloride (D) on Na+ content. 30 lM melatonin (Mel), 100 mM NaCl, 500 lM sodium orthovanadate (SOV: PM H+-ATPase inhibitor),
20 mM tetraethylammonium chloride (TEA, K+ channel blocker), 50 lM amiloride (Na+/H+ antiporter inhibitor). Values are mean of five independent replicates,
bars indicate SE. Bars with different letters indicate statistically significant differences at P < 0.05 (DMRT).

40 140
(A) L-cysteine desulfhydrase (L-DES) activity Hydrogen sulfide (H2S) content (B)
120
n mol g-1 protein min-1

30 a 100
n mol g-1 dry weight

a
b
b 80
20 c bc
d d 60
e fg ef
40
10
20
fg f e
0 Na Na Control Mel Na Na 0
Control Mel HT Mel+HT NaCl Cl+ Cl+ HT Mel+HT NaCl Cl+ Cl+
Me Me Me Me
Treatments l l+H Treatments l l+H
T T

Fig. 3. Hydrogen sulphide (H2S) biosynthesis in tomato seedling roots treated with 30 lM Mel and 100 mM NaCl. (A) L-cysteine desulphydrase (L-DES) activity
and (B) hydrogen sulphide (H2S) content. Double deionized water (control), 30 lM melatonin (Mel), 1 mM hypotaurine (HT: H2S scavenger), 100 mM NaCl
(NaCl). Values are mean of five independent replicates, bars indicate SE. Bars with different letters indicate statistically significant differences at P < 0.05
(DMRT).

Plant Biology 23 (2021) 797–805 © 2021 German Society for Plant Sciences and The Royal Botanical Society of the Netherlands 801
Exogenous melatonin-mediated regulation of K+/Na+ transport Siddiqui, Khan, Mukherjee, et al.

Table 1. Effect of melatonin and NaCl on ROS production and associated damage. Relative water content (RWC, %), electrolyte leakage (ELKG, %), lipid per-
oxidation (nmol
TBARS
g 1
DW), superoxide content (O2• , µmol
g 1
DW),and hydrogen peroxide content (H2O2, µmol
g 1
DW).

Parameters

Treatments RWC ELKG Lipid peroxidation O2• content H2O2 content

Control 88.40  3.65 ab

12.48  2.47ef
14.37  2.61 ef
9.51  0.98 ef
78.29  3.62ef
Mel 91.20  4.18a 8.29  1.65g 10.65  1.35g 7.63  1.69g 71.57  2.91gh
HT 53.72  1.76e 19.36  1.71c 21.54  1.72c 15.50  0.51c 88.20  5.30cd
Mel+HT 62.80  2.51cd 17.52  2.34cd 19.72  1.95cd 13.66  0.73d 91.74  3.67c
NaCl 42.74  4.22f 28.71  2.67b 29.52  2.89b 21.32  1.18ab 142.90  9.42b
NaCl+Mel 69.35  3.57c 13.29  1.55e 15.43  1.57e 10.41  0.87e 84.23  7.31de
NaCl+Mel+HT 25.37  2.36g 34.20  2.86a 36.29  2.48a 23.20  1.89a 176.85  8.29a

Values are mean  SE of five independent replicates. Values with different letters within a column indicate statistically significant differences at P < 0.05
(DMRT). Double deionized water (control), 30 µM melatonin (Mel), 100 mM NaCl (NaCl), 1 mM hypotaurine (HT: H2S scavenger).

Table 2. Effect of melatonin and NaCl on the activity of antioxidant It is evident from the data that NaCl stress (100 mM) causes
enzymes. Superoxide dismutase (SOD, mg 1
protein
min 1), peroxidase osmotic and ionic stress and reduces RWC, K+/Na+ ratio and
(POX, mg 1
protein
min 1) and catalase (CAT, mg 1
protein
min 1) activity. increases ELKG. Osmotic stress affects cell wall integrity, apo-
Parameters
plast pH and enzyme activity associated with various metabolic
processes (Liu et al. 2010; Garcıa-Morales et al. 2018; Darko
Treatments SOD POX CAT et al. 2019). However, plants attain osmotic stress tolerance
primarily through intracellular K+/Na+ homeostasis, and K+
Control 26.72  2.47 cd
9.38  0.59 d
121.38  5.69cd
plays a substantial role in osmotic adjustment (Marschner
Mel 29.58  1.92c 11.27  1.32c 129.72  3.25bc
1995). Exogenous Mel, in association with endogenous H2S,
HT 18.72  1.13fg 5.33  0.76fg 76.53  2.88fg
enhanced H+-ATPase activity, which created an H+ gradient
Mel+HT 21.30  2.06e 5.87  1.15f 82.30  3.51ef
NaCl 34.93  2.84b 13.61  2.05b 141.28  4.72b
and coincided with a significant increase in K+ influx and
NaCl+ Mel 45.10  3.17a 17.47  1.82a 168.42  4.03a
maintenance of a higher intracellular K+ concentration
NaCl+Mel+HT 19.04  1.22f 7.19  1.29e 88.71  5.19e (Fig. 1A). Furthermore, Mel also modulates activity of K+
channels (Liu et al. 2020). Therefore, NaCl-stressed plants trea-
Values are mean  SE of five independent replicates. Values with different ted with Mel can maintain optimum concentrations of intra-
letters within the column indicate are statistically significant at P < 0.05 cellular K+, which combat osmotic stress (Marschner 1995)
(DMRT. [Double deionized water (control), 30 µM melatonin (Mel), 100 mM and maintain ionic homeostasis, as seen in the higher K+/Na+
NaCl (NaCl), 1 mM hypotaurine (HT: H2S scavenger). ratio (Fig. 1C), RWC (Table 1) and reduced ELKG (Table 1)
(Khan et al. 2021). However, addition of HT (H2S scavenger)
during NaCl stress (NaCl+Mel+HT) abolished the mitigating
associated with inhibition of Na+/H+ antiporters (amiloride effects of Mel, suggesting that the endogenous H2S level is cru-
treatment) and inhibition of H+-ATPase activity (SOV treat- cial for Mel-induced recovery of osmotic homeostasis, RWC
ment) therefore suggest proton-energized secondary active and reduced ELKG. Exogenous application of sodium hydro-
transport mechanisms operate during Mel-induced K+/Na+ sulphide (NaHS, a H2S donor) and Mel have been shown to be
homeostasis. Furthermore, as evident from TEA treatment, effective as priming molecules to reduce ELKG in tomato seed-
Mel also modulates the activity of K+ channels (Liu et al. lings subjected to 120 mM NaCl stress, wherein both roots and
2020). Thus, exogenous Mel and endogenous H2S exert syner- cotyledons exhibited a similar response (Mukherjee & Bhatla
gistic effects in regulating K+/Na+ homeostasis through H+- 2020). Thus, exogenous Mel instigates endogenous H2S sig-
ATPase-energized secondary active transport mechanisms. Mel nalling, which alleviates osmotic stress by maintaining K+/Na+
induces energy turnover and enhances intracellular ATP level homeostasis associated with restoration of optimal RWC and
in salt-stressed roots, which improves ion homeostasis (Yu reduced ELKG.
et al. 2018). Mel promotes Na+ efflux in roots subjected to In this context, Mel-induced upregulation of K+/Na+ home-
salinity stress (Li et al. 2010) and also modulates the expression ostasis during NaCl stress is also accompanied by elevated L-
of inward-rectifying K+ channels during salinity stress, thus DES activity and concomitant increased accumulation of
increasing levels of endogenous K+ (Liu et al. 2020). Salinity endogenous H2S (Fig. 3A, B). L-DES activity and endogenous
stress is known to reduce H+-ATPase activity which, upon H2S levels are known to be crucial in modulating salinity stress
treatment with Mel and H2S, might create an H+ gradient that tolerance in plants (Kaya & Ashraf 2019; Mukherjee & Bhatla
is followed by influx of K+ (Li et al. 2014; Deng et al. 2016; 2020; Khan et al. 2021). L-DES is a cytosolic enzyme induced
Zhao et al. 2018). In the present work, Mel-mediated regula- by intracellular cysteine and H2S levels in plants (Khan et al.
tion of secondary active transport and ion homoeostasis oper- 2018; Khan et al. 2020a, 2021; Kaya & Ashraf 2019; Mukherjee
ate through positive regulation of endogenous H2S signalling & Bhatla 2020). A surge in endogenous H2S biosynthesis is evi-
in roots of tomato seedlings. NaCl-stressed tomato seedlings dent in Mel-treated tomato roots irrespective of the presence
had reduced K+/Na+ ratio, which was improved upon treat- or absence of NaCl stress (present work). However, a remark-
ment with Mel. able decrease in H2S content in seedlings treated with HT alone

802 Plant Biology 23 (2021) 797–805 © 2021 German Society for Plant Sciences and The Royal Botanical Society of the Netherlands
Siddiqui, Khan, Mukherjee, et al. Exogenous melatonin-mediated regulation of K+/Na+ transport

or HT+Mel suggests HT-induced scavenging of H2S. This indi-
cates that H2S homoeostasis functions as a down-stream com-
ponent to Mel signalling in roots. Earlier evidence from
tomato cotyledons found possible catabolic regulation of H2S
in the presence of combined treatment with Mel and NaCl
stress (Mukherjee & Bhatla 2020). Here, we found Mel-
induced positive regulation of endogenous H2S biosynthesis in
roots grown in the presence or absence of NaCl stress.
Exposure of roots to 100 mM NaCl resulted in oxidative
stress, as reflected by increases in O2• and H2O2 content
(Table 1). Deleterious effects of these ROS were confirmed
in higher ELKG and lipid peroxidation (Table 1) (Khan
et al. 2021); however, a concomitant increase in activity of
antioxidant enzymes (SOD, POX and CAT) was also noticed
(Table 2). The seedlings struggled to counter NaCl-induced
oxidative stress and, in spite of increased activity of antioxi-
dant enzymes, their capacity for ROS scavenging was insuffi-
cient. Addition of Mel to the NaCl-stressed incubation
medium significantly reduced levels of O2• and H2O2
(Table 1). The decrease in ROS by Mel was positively corre-
lated with elevated activity of SOD, POX and CAT
(Table 2). Interestingly, reversal of the effect of Mel by HT
treatment suggests that endogenous H2S levels are crucial
for Mel-induced elevation of antioxidative defence in NaCl-
stressed tomato seedling roots. Recent reports confirm the
beneficial role of Mel and H2S in reducing oxidative stress
and eliciting antioxidative defence in plants (Ahmad et al.
2020a,b; Alamri et al. 2020; Buttar et al. 2020; Chen et al.
2020; Rather et al. 2020; Siddiqui et al. 2020; Khan et al.
2021). Our work provides additional evidence of the
involvement of endogenous H2S in Mel-induced modulation
of antioxidative defence in NaCl-stressed roots. These two
biomolecules exert synergistic effects, reducing oxidative
stress through modulation of ROS generation and regulation
of SOD, POX and CAT activity.
In summary, Mel signalling during NaCl stress in tomato
seedling roots operates through an endogenous H2S-dependent
pathway wherein H+-ATPase-energized secondary active trans-
port regulates K+-Na+ homoeostasis. Furthermore, increased
L-DES activity and higher accumulation of endogenous H2S
during Mel treatment appear to induce enzymatic antioxidative
defences. Decreased ROS, improved RWC and higher retention
of K+ in NaCl-stressed tomato seedling roots suggest a syner-
gistic role of Mel and H2S in modulating NaCl stress tolerance.
Our results provide a basis for future investigations on Mel and
H2S as two synergistic abiotic stress priming molecules of agro-
nomic importance.
ACKNOWLEDGEMENTS
The authors extend their appreciation to the Dean of Scientific
Research at King Saud University for funding this work
through Research Group No. RG-1441-438.
CONFLICT OF INTEREST
The authors declare that they have no known competing finan-
cial or personal interests.

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