Hydrogen-induced tolerance against osmotic stress in alfalfa seedlings involves ABA signaling

Abstract

Background and aims :This study investigated the detailed mechanism underlying the alleviation of osmotic stress by exogenous hydrogen (H2) in Medicago sativa.
Methods: By using biochemical and molecular approaches, the experiments were performed with the
analyses of biomass, relative water content (RWC), lipid peroxidation, abscisic acid (ABA) content, antioxidant activities, and related gene expression profiles.
Results :H2 application stimulated ABA production, which was accompanied by the regulation of ABA biosynthesis and deactivation/activation genes. Elevated H2-induced ABA synthesis was sensitive to tungstate, an inhibitor of ABA synthesis. Meanwhile, H2-alleviated osmotic stress, which was supported by the increases in biomass and RWC, and the reduction of lipid peroxidation, was impaired by the inhibition of ABA synthesis. Consistently, tungstate blocked H2-induced

Full text

REGULAR ARTICLE
Hydrogen-induced tolerance against osmotic stress
in alfalfa seedlings involves ABA signaling
Kiprotich Felix &Jiuchang Su &Rongfei Lu &
Gan Zhao &Weiti Cui &Ren Wang &Hualun Mu &
Jin Cui &Wenbiao Shen
Received: 29 December 2018 /Accepted: 4 October 2019
#Springer Nature Switzerland AG 2019
Abstract
Background and aims This study investigated the de-
tailed mechanism underlying the alleviation of osmotic
stress by exogenous hydrogen (H
2
)inMedicago sativa.
Methods By using biochemical and molecular ap-
proaches, the experiments were performed with the
analyses of biomass, relative water content (RWC), lipid
peroxidation, abscisic acid (ABA) content, antioxidant
activities, and related gene expression profiles.
Results H
2
application stimulated ABA production,
which was accompanied by the regulation of ABA
biosynthesis and deactivation/activation genes. Elevated
H
2
-induced ABA synthesis was sensitive to tungstate,
an inhibitor of ABA synthesis. Meanwhile, H
2
-alleviat-
ed osmotic stress, which was supported by the increases
in biomass and RWC, and the reduction of lipid perox-
idation, was impaired by the inhibition of ABA synthe-
sis. Consistently, tungstate blocked H
2
-induced
Plant Soil
https://doi.org/10.1007/s11104-019-04328-y
Kiprotich Felix and Jiuchang Su contributed equally to this work.
Responsible Editor: Ian Dodd.
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s11104-019-04328-y)contains
supplementary material, which is available to authorized users.
K. Felix :J. Su :R. Lu :G. Zhao :W. Cui :J. Cui :
W. Shen
College of Life Sciences, Laboratory Center of Life Sciences,
Nanjing Agricultural University, Nanjing 210095, China
K. Felix
e-mail: kiprotichfelix@yahoo.com
J. Su
e-mail: 2017216035@njau.edu.cn
R. Lu
e-mail: rongfeilu.njau@outlook.com
G. Zhao
e-mail: 2018216033@njau.edu.cn
W. Cui
e-mail: wtcui@njau.edu.cn
J. Cui
e-mail: cuijin@njau.edu.cn
J. Su :W. Shen (*)
Center of Hydrogen Science, Shanghai Jiao Tong University,
Shanghai 200240, China
e-mail: wbshenh@njau.edu.cn
R. Wang
Institute of Botany, Jiangsu Province and Chinese Academy of
Sciences, Nanjing 210014, China
e-mail: wangren@126.com
H. Mu
Shanghai Nanobubble Nano-tech Co., Ltd, Shanghai 201203,
China
e-mail: allamu@nanobubble.cn

antioxidant defense. Molecular evidence revealed that
miR528 was down-regulated by H
2
,showinganegative
correlation with its target gene POD2.Whentungstate
was added together, the decreased miR528 and in-
creased POD2 transcripts were respectively blocked.
Transcriptional factor genes involved in ABA signaling,
including MYB102,MYC2,andABF/AREB2, were dif-
ferentially upregulated by H
2
, but further impaired by
the co-incubation with tungstate.
Conclusions Collectively, our results suggested the pos-
sible role of ABA signaling in exogenous H
2
-mediated
tolerance against osmotic stress in alfalfa.
Keywords ABA .Antioxidant defense .Hydrogen .
miRNA .Transcriptional factors .Osmotic stress
Introduction
Water deficit stress is a common adverse environmental
condition that has damaging effects on plant metabolic
processes. These include the reduction in nutrient up-
take, stomatal aperture, and photosynthetic assimilates,
thus causing crop losses (Neumann 2008). In response
to stress, plants employ various mechanisms against
osmotic stress, including metabolism regulation and
morphological changes. Often, the osmoticum polyeth-
ylene glycol (PEG6000) is used to mimic osmotic stress
in scientific research (Srinath and Jabeen 2013), even
though soil drying induces both water and mechanical
stress (Jin et al. 2013a).
The best-identified trigger of drought tolerance is
phytohormone abscisic acid (ABA) (Hu et al. 2006;
Lu et al. 2009). Evidence revealed that zeaxanthin
epoxidase (ZEP; also named as ABA1), 9-cis
epoxycarotenoid dioxygenase (NCED), MoCo
sulfurase (ABA2), aldehyde oxidase (AAO3), β-D-glu-
cosidase (βG1), and ABA glucosyltransferase (AOG),
regulate ABA biosynthesis and deactivation/activation
(Fujii 2014). The expression of these genes is induced
either by exogenous ABA or osmotic stress (Iuchi et al.
2001; Xiong et al. 2001,2002;SeoandKoshiba2002;
Xiong and Zhu 2003; Tan et al. 2003).
Although reactive oxygen species (ROS) are consid-
ered to be critical signaling molecules in sensing stress-
es, their toxic effects, including protein denaturation,
nucleotides degradation, and lipids peroxidation (Hu
et al. 2008;Milleretal.2010; Vaseem et al. 2017),
especially under osmotic stress conditions (Jiang and
Zhang 2001,2002), were discovered. The tight regula-
tion is therefore needed to balance ROS production, and
to scavenge and preserve cellular redox poise (Bailly
et al. 2008;Milleretal.2010). It is well established that
antioxidant defense against ROS includes the enzymatic
system, such as superoxide dismutase (SOD), ascorbate
peroxidase (APX), guaiacol peroxidase (POD), and cat-
alase (CAT) (Foyer and Noctor 2005). Above antioxi-
dant enzymes are regulated at transcriptional and post-
transcriptional levels (Carrington and Ambros 2003;
Bartel 2004). For example, previous results revealed
that drought in maize seedlings downregulated
miR528, while its target transcript POD was upregulated
(Wei et al. 2009). MiR398 targets two closely related
Cu/Zn SODs (CSD1 and CSD2) that are involved in the
detoxification of oxidative stress (Mittler 2002;Wei
et al. 2009).
Besides, the targets of microRNAs (miRNAs) in-
clude transcriptional factors (TFs) (Guo et al. 2005).
It is well-known that TFs represent the critical mo-
lecular switches composing the regulation of plant
developmental processes in response to a variety of
stresses (Joshi et al. 2016). A large number of genes
in the plant genome (up to 10%) possibly encode TFs
(Franco-Zorrilla et al. 2014), which were classified
into different families, such as ABA-responsive ele-
ment (ABRE)-binding proteins (AREB),
DEHYDRATION-RESPONSIVE ELEMENT
BINDING (DREB), myeloblastosis (MYB), NAM
(NO APICAL MERISTEM), ATAF1/2
(ARABIDOPSIS TRANSCRIPTION ACTIVATING
FACTOR1/2), and CUC2 (CUP-SHAPED
COTYLEDON2) (NAC), and the BASIC LEUCINE
ZIPPER (bZIP). Above classification is according to
their distinct DNA-binding domain structure
(Golldack et al. 2011; Cai et al. 2017). The modula-
tion of above TFs genes in response to osmotic stress
is via ABA-dependent or -independent pathway
(Todaka et al. 2012).
Hydrogen gas (H
2
) is an odorless, colorless, tasteless,
and flammable gas. Although several reports discovered
H
2
evolution and uptake in illuminated leaves and ger-
minating seeds about fifty years ago (Renwick et al.
1964), there is little evidence about the specific mecha-
nism of its biosynthesis and even physiological roles in
plants (Dong et al. 2003). Since the direct application of
H
2
gas is not appropriate in practice and may be dan-
gerous due to its inflammable and explosive feature
(Zheng et al. 2011), hydrogen-rich water (HRW)
Plant Soil

in vitro experiments is used to mimic the functions of
endogenous H
2
in plants. By using this approach, relat-
ed results showed that H
2
could act as a significant
gaseous molecule with numerous biological functions
in plant responses against oxidative stress triggered by
drought, salinity, and paraquat exposure in alfalfa and
Arabidopsis (Xie et al. 2012,2014; Jin et al. 2013b,
2016). It was further suggested that exogenous H
2
might
enhance cold tolerance and tolerance against aluminum
in plants, at least partially, by the restoration of redox
homeostasis via modulating the expression of miRNAs
like miR398 and miR528 (Xu et al. 2017a,2017b). The
induction of lateral root and adventitious rooting was
also discovered (Lin et al. 2014; Cao et al. 2017).
Interestingly, previous results (Xie et al. 2014;Jin
et al. 2016) demonstrated that exogenously applied
ABA increased endogenous H
2
production under the
normal growth conditions and upon drought stress. By
manipulating endogenous nitric oxide (NO) level with
its synthetic inhibitor and scavengers, the requirement of
NO in hydrogen-alleviated osmotic stress was con-
firmed (Su et al. 2018). Since NO was found to be
involved in brassinosteroid-induced ABA biosynthesis
and -alleviated oxidative stress in maize leaves (Zhang
et al. 2011), the detailed molecular mechanism underly-
ing the functions of H
2
in the enhancement of plant
tolerance against osmotic stress, especially the possible
crosstalk between H
2
and ABA, remains to be fully
elucidated.
This study aims to address how hydrogen gas
enables alfalfa seedlings to cope with osmotic stress.
H
2
application increased endogenous ABA metabo-
lism and antioxidants defense in alfalfa seedlings.
Afterward, it led to the reestablishment of redox
balance. Gene expression analyses of miRNAs, TFs,
and antioxidant genes supported H
2
action during
osmotic stress, which was differentially abolished
by the addition of ABA synthetic inhibitor tungstate
(Jiang and Zhang 2002; Liu et al. 2012). The inhibi-
tion in ABA synthesis triggered by tungstate was
confirmed in our experimental conditions. The alle-
viation of osmotic stress by H
2
was blocked as well.
Together, our results support the idea that ABA, at
least partially, operates downstream of H
2
alleviating
osmotic stress through the reestablishment of redox
balance. Combined with the previous results (Xie
et al. 2014;Jinetal.2016), it was further deduced
that H
2
-triggered ABA synthesis could be an ampli-
ficationloopinH
2
signaling.
Materials and methods
Plant material and growth conditions
Alfalfa (Medicago sativa L. cv. Victoria) seeds were
surface-sterilized with 5% NaClO for 10 min, and rinsed
extensively in distilled water then germinated for 1 day
at 25 °C in the darkness. Uniform seedlings were select-
ed and transferred to the plastic chambers and cultured
in quarter-strength Hoagland’s solution: 0.97 mM
Ca(NO
3
)
2
,0.66mMKNO
3
, 0.5 mM MgSO
4
,25μM
KH
2
PO
4
,12.5μMFe-EDTA,0.12μMH
3
BO
3
,
0.14 μM ZnSO
4
,0.23μMMnSO
4
,0.26μM CuSO
4
,
and 0.04 μMNa
2
MoO
4
. The pH was adjusted to 6.0.
Seedlings were grown in the illuminating incubator
(14 h light with a light intensity of 200 mol m
−2
s
−1
,
25 ± 1 °C, and 10 h dark, 23 ± 1 °C). Five-day-old
seedlings were then incubated in quarter-strength
Hoagland’s solution with or without 0.39 mM H
2
,
0.1 mM ABA, 1 mM tungstate (Tu; an inhibitor of
ABA synthesis; Jiang and Zhang 2002; Liu et al.
2012), alone or their combinations for 12 h, then ex-
posed to 20% (w/v) polyethylene glycol (PEG; MW
6000; osmotic potential −0.5 MPa) stress for the indi-
cated time points. The sample without chemicals was
the control (Con). The pH of both nutrient medium and
treatment solutions was adjusted to 6.0. After various
treatments, plants were photographed, and shoot tissues
were sampled for use immediately or flash-frozen in
liquid nitrogen, and stored at −80 °C for further analysis.
Preparation of hydrogen-rich water (HRW)
Purified hydrogen gas (99.99%, v/v) generated from a
hydrogen gas generator (SHC-300; Saikesaisi Hydrogen
Energy Co., Ltd. Shandong, China) was bubbled into
1000 ml quarter-strength Hoagland’s solution (pH 6.0,
25 °C) at a rate of 150 ml min
−1
for 60 min (Jin et al.
2013b). Then, the corresponding HRW was immediate-
ly diluted to the required 50% saturation (v/v), which
contains 0.39 mM H
2
for at least 12 h, determined by
gas chorography (Su et al. 2018).
Analysis of water status and phenotypic analysis
Water status of tissues, measured as relative water con-
tent (RWC), was determined as previous method
(García-Mata and Lamattina 2001). Fresh weight
(FW), dry weight (DW), and root elongation were
Plant Soil

measured as previously described (Xie et al. 2016;Xu
et al. 2017b).
Determination of thiobarbituric acid reactive substances
(TBARS) and ABA content
Lipid peroxidation was estimated by measuring the
amount of TBARS using an extinction coefficient of
155 mM
−1
cm
−1
and expressed as nmol g
−1
dry weight
(DW) (Wang et al. 2012;Xuetal.2017b).
The extraction and purification of ABA were carried
out following the previous method (Guinn et al. 1986).
ABA contents were analyzed by high performance liq-
uid chromatography (HPLC) (Shimadzu, D-2000,
Hitachi Ltd., Tokyo, Japan) using ultraviolet detector,
and C18 column. Pure ABA (Sigma-Aldrich, St Louis,
MO, USA) was used as a standard. ABA was identified
and quantified by retention time.
Enzymatic activities assays
Fresh samples (about 0.3 g) were homogenized in 5 ml
of 50 mM cold phosphate buffer (pH 7.0), containing
1 mM EDTA and 1% (w/v) polyvinylpolypyrrolidone
(PVP) for superoxide dismutase (SOD, EC 1.15.1.1),
catalase (CAT, EC 1.11.1.6), and guaiacol peroxidase
(POD, EC 1.11.1.7) activity assays, or the combinations
with 1 mM ascorbic acid (ASA) in the case of ascorbate
peroxidase (APX, EC 1.11.1.11) determination. The
homogenates were centrifuged at 15000 gfor 20 min
at 4 °C, and the supernatants were used for assays of
enzymatic activity.
Total SOD activity was assayed according to the
previous method (Beauchamp and Fridovich 1971),
and one unit of SOD (U) was defined as the amount of
crude enzyme extract required to inhibit the reduction
rate of nitroblue tetrazolium (NBT) by 50%. The CAT
activity was analyzed by monitoring the consumption of
H
2
O
2
(extinction coefficient 39.4 mM
−1
cm
−1
)at
240 nm for at least 2 min (Xu et al. 2017b). POD activity
was determined by measuring the oxidation of guaiacol
(extinction coefficient 26.6 mM
−1
cm
−1
) at 470 nm (Han
et al. 2008). APX activity was determined by monitor-
ing the decrease at 290 nm (extinction coefficient
2.8 mM
−1
cm
−1
) (Xu et al. 2017b). Protein was deter-
mined by the method previously described (Bradford
1976), using bovine serum albumin (BSA) as a
standard.
Quantitative reverse transcription polymerase chain
reaction (qPCR) analysis
Total RNA in samples was extracted by using Trizol
reagent (Invitrogen, Gaithersburg, MD, USA), and then
digested with RNase-free DNase to eliminate genomic
DNA contamination. First-strand cDNA was synthe-
sized with oligo(dT) primers using SuperScript™re-
verse transcriptase (Invitrogen, Carlsbad, CA, USA).
qPCR was performed using a Mastercycler® ep realplex
real-time PCR system (Eppendorf, Hamburg, Germany)
with SYBR® Premix Ex Taq™(TaKaRa Bio Inc., Da-
lian, China). The accession numbers (GenBank/
miRBase) and oligonucleotide primers were shown in
Supplemental Table S1. Melting curves were analyzed
at the dissociation step to examine the specificity of
amplification. Relative expression levels were presented
as values, relative to that of the corresponding control
samples, after normalization with Actin2 and ELF2 tran-
script levels (Gu et al. 2017; Mei et al. 2017). Three
independent experiments with three replicates were ob-
tained for each data.
Statistical analysis
Results were expressed as the means ± SE of three
independent experiments with at least three replicates
for each. Statistical analysis was performed using SPSS
17.0 software. Data was analyzed by one-way analysis
of variance (ANOVA) followed by Duncan’smultiple
range tests, and Pvalues <0.05 were considered as
statistically significant.
Results
The beneficial role of H
2
against osmotic stress was
blocked by the ABA synthetic inhibitor
To assess the role of ABA in H
2
-alleviated osmotic stress,
tungstate (1 mM; the inhibitor of ABA synthesis) was
used together with H
2
and ABA (as a positive control),
followed by PEG stress. Similar to the response of ABA,
H
2
was able to alleviate osmotic stress (Fig. 1a). This was
further correlated with the alleviation of seedling growth
inhibition, including the changes in fresh weight and dry
weight (Fig. 1b), relative water content (Fig. 1c), and the
root elongation (Fig. 1d). A correlation analysis between
fresh weight and relative water content further revealed
Plant Soil

that H
2
promoted plant tolerance against osmotic stress.
Above H
2
responses were differentially blocked by the
addition of 1 mM tungstate. Comparatively, weaker or no
significant changes were observed when ABA was
supplemented with or without tungstate, followed by
stress. Above results thus indicated the possible link
between ABA and H
2
in the alleviation of osmotic stress
in alfalfa seedlings.
Fig. 1 Exogenous H
2
-alleviated osmotic stress was sensitive to
the inhibitor of ABA synthesis. Five-day-old alfalfa seedlings
were grown in nutrient solutions containing 0.39 mM H
2
,
0.1 mM ABA, 1 mM tungstate (Tu; an inhibitor of ABA synthe-
sis), alone or their combinations for 12 h, then exposed to 20%
PEG for an additional 48 h. Afterward, the representative pheno-
type (a), freshweight and dry weight (b), the relative water content
in shoot parts (c), and root elongation (d) were provided or
measured. The plants without chemicals were regarded as the
control sample (Con)
Plant Soil

Changes in ABA concentration in response to H
2
and tungstate
To confirm above hypothesis, we measured the ABA
concentrations in shoot tissue of alfalfa plants. As ex-
pected, compared to the control plants, the ABA con-
centrations were increased in response to PEG stress for
both 12 and 24 h (Fig. 2a). Meanwhile, supplemented
H
2
and ABA obviously increased endogenous ABA
production under stressed and non-stressed conditions.
In the presence of tungstate at 1 mM, above H
2
-induced
ABA production was significantly blocked, which was
different from the response of exogenously applied
ABA. The decreased ABA concentration was also
observed when tungstate was applied alone. Above
results indicated that 1 mM tungstate is effective in the
inhibition of ABA synthesis, at least under our experi-
mental conditions. Combined with the corresponding
phenotypic parameters (Fig. 1), we deduced that the
beneficial roles of H
2
in the alleviation of osmotic stress
might be dependent on ABA.
H
2
-alleviated lipid peroxidation was sensitive
to tungstate
To further assess the molecular mechanism of H
2
governing the alleviation of osmotic stress, TBARS pro-
duction, an indicator of lipid peroxidation, was investigat-
ed. As expected, the increased TBARS level triggered by
osmotic stress was obviously alleviated by H
2
,whichwas
reversed by the addition of tungstate (Fig. 2b). However,
the combination of tungstate and ABA in the stressed
condition brought about a contrasting response. These
results obtained from the above suggested that H
2
-allevi-
ated lipid peroxidation was, at least partially, in ABA-
dependent fashion, although the possibility of ABA-
independent pathway could not be easily ruled out. Con-
sistently, we noticed that H
2
alone did not influence lipid
peroxidation, in comparison with the control plants.
Changes in ABA metabolism gene expression
ABA metabolism gene expression was increased by
osmotic stress (Fujii 2014). To confirm the role of
ABA in above H
2
responses, several major genes re-
sponsible for ABA biosynthesis (ABA2 and AAO3)and
deactivation/activation (AOG and βG1) were analyzed
(a simple ABA metabolism pathway was shown in
supplemental Fig. S1). As expected, the transcripts of
above ABA biosynthesis genes (in particular) and
deactivation/activation genes were elevated after osmot-
ic stress with (especially) or without ABA supplemen-
tation (Fig. 3). Subsequent results revealed that in the
presence of osmotic stress, the expression of ABA2 was
significantly enhanced, but AOG and βG1 were obvi-
ously inhibited, by H
2
pretreatment. However, AAO3
transcripts were similarly induced by PEG regardless of
H
2
. By contrast, the addition of tungstate was able to
differentially reverse the effects of H
2
on the transcript
levels of ABA2,AAO3,AOG,andβG1 under the
stressed conditions. The up-regulation of ABA2 and
AAO3 mRNA was observed by H
2
alone. We also
discovered the similar changes in AOG and βG1
Fig. 2 Changes in ABA and TBARS contents. Five-day-old
alfalfa seedlings were grown in nutrient solutions containing
0.39 mM H
2
, 0.1 mM ABA, 1 mM tungstate (Tu; an inhibitor of
ABA synthesis), alone or their combinations for 12 h, then ex-
posed to 20% PEG. Afterward, the shoot tissues were collected at
12 and 24 h for the determination of ABA content using HPLC (a),
or at 48 h for TBARS content (b). The plants without chemicals
were regarded as the control sample (Con)
Plant Soil

transcripts under the identical treatments, suggesting
that deactivation/activation of ABA might be not impor-
tant factor(s) responsible for H
2
-triggered ABA produc-
tion in osmotic stress. Consequently, the ability of H
2
to
trigger ABA metabolism genes further strengthens its
reliance on ABA under osmotic stress.
Changes in antioxidant enzyme activities, transcripts
of antioxidant genes, and miR528
To further evaluate the contribution of antioxidant de-
fence in above H
2
responses, the SOD, POD, APX, and
CAT enzymatic activities and their transcripts were an-
alyzed. As anticipated, in shoot parts of alfalfa plants,
PEG exposure led to significant decreases in above
mentioned antioxidant enzyme activities (Fig. 4). Treat-
ment with H
2
significantly blocked the decreases in the
activities of these antioxidant enzymes induced by os-
motic stress. Further experiments revealed that the co-
incubation with tungstate differentially blocked the en-
hancement in the activities of antioxidant enzymes trig-
gered by H
2
. Meanwhile in the stressed conditions, no
significant differences were observed in ABA-treated
plants either in the presence or absence of tungstate.
Comparatively, molecular evidence revealed that chang-
es in CuZnSOD,APX1,CAT, and POD2 transcripts
exhibited the similar tendencies (except MnSOD;
Fig. 5a-d).
Subsequently, miR528 transcript was investigated
(Fig. 5e), and compared with POD2 mRNA (Fig. 5d),
to assess their roles in osmotic stress tolerance elicited
by H
2
. The expression levels of miR528 were upregu-
lated upon osmotic stress, compared to the control sam-
ple. The addition of H
2
or ABA further obviously
Fig. 3 Changes in ABA biosynthesis and deactivation/activation
gene expression. Five-day-old alfalfa seedlings were grown in
nutrient solutions containing 0.39 mM H
2
, 0.1 mM ABA, 1 mM
tungstate (Tu; an inhibitor of ABA synthesis), alone or their
combinations for 12 h, and then exposed to 20% PEG. Afterwards,
the shoot tissues were collected at 24 h for determining ABA2 (a),
AAO3 (b), AOG (c), and βG1 (d) gene expression analyzed by
qPCR, normalized against two internal reference genes Actin2 and
ELF2, which were stably expressed. The plants without chemicals
were regarded as the control sample (Con)
Plant Soil

reduced the transcript levels of miR528,comparedtothe
PEG-treated alone. Above response of H
2
, other than
that of ABA, was significantly blocked by tungstate, in
stressed conditions. Interestingly, above changes in
miR528 displayed a negative correlation with its target
gene, POD2 (Fig. 5d). These results indicated the re-
quirement of ABA in the induction of antioxidant de-
fence elicited by H
2
in the stressed conditions, at least
partly.
Expression profiles of transcriptional factors associated
with osmotic stress
TFs have been reported to respond to osmotic stress via
ABA-dependent or -independent pathways (Joshi et al.
2016). The gene expression of some representative TFs
involved in ABA signaling in response to H
2
,ABA,and
tungstate application was studied. As expected, osmotic
stress resulted in the significant decreases in MYB102,
MYC2,andABF/AREB2 in alfalfa seedling shoots, com-
pared to the untreated control samples (Fig. 6). The
addition of exogenous H
2
upregulated the above tran-
scripts compared to the PEG-treated alone samples. By
contrast, the inclusion of tungstate in above H
2
treat-
ment impaired MYB102,MYC2,andABF/AREB2 tran-
scripts. Furthermore, we observed that exogenously ap-
plied ABA followed by osmotic stress significantly
increased above mentioned gene expression, which
were not altered by the co-incubation with tungstate.
Discussion
During the recent years, the interaction between H
2
and
phytohormones has attracted a lot of attention. In our
previous work (Xie et al. 2014; Jin et al. 2016), we
Fig. 4 Changes in antioxidant enzyme activities. Five-day-old
alfalfa seedlings were grown in nutrient solutions containing
0.39 mM H
2
, 0.1 mM ABA, 1 mM tungstate (Tu; an inhibitor of
ABA synthesis), alone or their combinations for 12 h, then
exposed to 20% PEG. The shoot tissues were collected at 24 h
for determining SOD (a), POD (b), APX (c), and CAT (d)activ-
ities. The plants without chemicals were regarded as the control
sample (Con)
Plant Soil

discovered that ABA and osmotic stress significantly
induced H
2
production in both alfalfa and Arabidopsis
plants, and the role of H
2
in alfalfa stomata sensitivity to
ABA was partly associated with its effect on the modi-
fication of leaf apoplastic pH (Jin et al. 2016). Here, by
using inhibitor (tungstate, an inhibitor of ABA
biosynthesis; Jiang and Zhang 2002; Liu et al. 2012)
test and molecular approach, we discovered that H
2
could increase ABA synthesis (Fig. 2a). Importantly,
an increase in the concentration of ABA might be crit-
ical for exogenous H
2
-mediated plant adaptive re-
sponses to osmotic stress via the upregulation of tran-
scriptional factor genes and antioxidant defense, and
above study extended the former results (Fig. 7;Xie
et al. 2014; Jin et al. 2016;Suetal.2018).
Collectively, we further deduced that osmotic
stress-triggered ABA might increase H
2
production
(Xie et al. 2014; Jin et al. 2016), which enhances
Fig. 5 Changesin antioxidant gene expression and miRNA. Five-
day-old alfalfa seedlings were grown in nutrient solutions contain-
ing 0.39 mM H
2
, 0.1 mM ABA, 1 mM tungstate (Tu; an inhibitor
of ABA synthesis), alone or their combinations for 12 h, then
exposed to 20% PEG. The shoot tissues were collected at 12 h for
CuZnSOD and MnSOD (a), APX1 (b), CAT (c), POD2 (d), and
miR528 (e) gene expression analyzed by qPCR. The plants with-
out chemicals were regarded as the control sample (Con)
Plant Soil

ABA accumulation, thus forming an amplification
loop in osmotic stress tolerance signaling (Fig. 7).
Similarly, the existence of positive amplification
loops in ROS signaling has previously been discov-
ered (Zhang et al. 2006). Certainly, we can not ex-
clude the possibility that tungstate (also regarded as
the inhibitor of nitrate reductase; Tossi et al. 2009;
Xiong et al. 2012) used in the present study, may not
specifically target ABA. However, combined with
the changes in ABA metabolism (Figs. 2a,3), our
results collectively point to the fact that there might
be existing, not only a linear pathway, but also a
crosstalk between H
2
and ABA (synthesis and/or
sensitivity) in higher tissue water status and biomass
when plants are exposed to osmotic stress.
Experimental evidence supported the involvement of
H
2
in plant tolerance against abiotic stresses, including
salinity (Xie et al. 2012), paraquat exposure (Jin et al.
2013b), cadmium stress (Cui et al. 2013), and mercury
stress (Cui et al. 2014). The mechanism underlying
above H
2
in plants is largely associated with the en-
hancement of antioxidant defence. Meanwhile, ample
evidence confirmed that ABA plays vital roles in plant
tolerance to stresses (Hu et al. 2006;Luetal.2009). To
better characterize the mechanism of H
2
governing plant
tolerance against osmotic stress, alfalfa plants were
treated with tungstate since it can block the formation
of ABA from abscisic aldehyde by impairing abscisic
aldehyde oxidase (Hansen and Grossmann 2000) and its
encoding gene (AAO3;Fig.3b). First, the results
Fig. 6 Gene expression of transcriptional factors involved in
ABA signaling. Five-day-old alfalfa seedlings were grown in
nutrient solutions containing 0.39 mM H
2
, 0.1 mM ABA, 1 mM
tungstate (Tu; an inhibitor of ABA synthesis), alone or their
combinations for 12 h, then exposed to 20% PEG. The shoot
tissues were collected at 12 h. Afterward, gene profiles of
MYB102 (a), MYC2 (b), and ABF/AREB2 (c), were analyzed by
qPCR, and normalized against two internal reference genes Actin2
and EIF2, which were stably expressed. The plants without
chemicals were regarded as the control sample (Con)
Plant Soil

presented here demonstrated that upon osmotic stress, a
higher level of ABA induced by H
2
(Fig. 2a), correlates
with the enhancement of plant tolerance against osmotic
stress (Fig. 1). Combined with results shown in Fig. 2a
and Fig. 3, we confirmed that H
2
increased the leaf ABA
concentration by upregulating the ABA biosynthesis
gene (especially ABA2) expression. This finding is crit-
ical because one of the best-identified triggers of the
cascade of drought signaling is ABA (Iuchi et al. 2001;
Seo and Koshiba 2002;Huetal.2006;Luetal.2009).
In view of the changes in ABA levels (Fig. 2a), we
deduced that ABA metabolism is influenced by H
2
.
These behaviors might be because, under osmotic stress,
H
2
requires endogenous ABA action to alleviate stress.
Similar beneficial responses of ABA, when exogenous-
ly applied, were thus provided as positive controls. For
example, when the ABA concentration in vivo is high in
response to exogenous H
2
or ABA, osmotic stress-
triggered seedling growth inhibition and the loss of
water content were differentially improved (Fig. 1). By
contrast, when ABA synthesis is inhibited by tungstate
(Fig. 2a), osmotic stress-triggered growth stunt and the
loss of water content were blocked (Fig. 1). In agree-
ment with these results, the beneficial roles of H
2
were
also impaired by the co-incubation with tungstate. These
data thus supported the hypothesis that ABA might
mediate H
2
-induced plant tolerance against osmotic
stress.
Ample evidence portrays that avoidance of oxidative
stress and reestablishment of redox homeostasis are
essential determinants of alleviating osmotic stress in
plants (Jiang and Zhang 2002; Mittler et al. 2004).
Meanwhile, the ability of a plant to regulate a series of
genes, including antioxidant defence genes, that further
alter plant physiology and morphology, gives it the
ability to avoid or escape osmotic stress (Jiang and
Zhang 2002;Luetal.2009). Our further experiment
revealed that mimicking the effects of ABA, H
2
was
able to alleviate PEG-triggered lipid peroxidation in
shoots of alfalfa seedlings (Fig. 2b), both of which were
supported by the enhanced activities of SOD, POD,
APX, and CAT (Fig. 4), and the upregulation of corre-
sponding transcripts (Fig. 5). These may be beneficial
for the improvement of alfalfa seedling growth under
osmotic stress, since the over-expression of Arabidopsis
APX gene in tobacco chloroplast could enhance plant
tolerance against water deficit and salinity stress
(Badawi et al. 2004). Consistently, tobacco plants over-
expressing a maize E3 ubiquitin ligase gene exhibited
drought tolerance by regulating antioxidant defence and
stomatal closure (Liu et al. 2013). However, the inclu-
sion of the ABA synthetic inhibitor tungstate reversed
this alleviating effect given by H
2
(Fig. 2b). Changes in
enzymatic activities (Fig. 4) and gene transcripts (Fig. 5)
showed the similar tendencies. These results of Fig. 2b
and Fig. 4revealed that the decrease of TBARS con-
centration was attributed to the increased antioxidant
enzyme activity induced by H
2
in a ABA-dependent
fashion. Additionally, ABA-alleviated lipid peroxida-
tion might be partially used to explain the reason why
H
2
alleviates osmotic stress.
MicroRNAs (miRNAs), including miR528 and
miR398, have been implicated in plant tolerance against
osmotic stress, particularly in the regulation of ABA
(Wang et al. 2011;Dingetal.2013). Previous results
showed that osmotic stress modulated miR528 tran-
scripts in maize (Wei et al. 2009)andM. truncatula
Fig. 7 Model summarizing the interaction of H
2
and ABA in
plant tolerance against osmotic stress. Exogenous H
2
could in-
crease ABA synthesis, and an increase in the concentration of
ABA might be critical for exogenous H
2
-mediated plant adaptive
responses to osmotic stress (This study). H
2
accumulates in re-
sponse to ABA (Xie et al. 2014) and drought stress (Jin et al.
2016), thus forming an amplification loop in osmotic stress toler-
ance signaling. The role of nitrate reductase-dependent nitric oxide
is also suggested (Su et al. 2018). Additionally, the involvement of
ABA-independent pathway is not easily ruled out, and illustrated
with dash line. The above pathways might be mediated by mod-
ulating gene expression of miRNA, TFs, and antioxidant defence
Plant Soil

(Wang et al. 2011).Also,miRNAshavebeenreportedto
be involved in plant response to oxidative stress and
regulating ROS balance (Zhu et al. 2011; Ma et al.
2015). In this study, we observed that miR528 was
increased by osmotic stress in alfalfa seedlings, which
was sensitive to the administration with H
2
(Fig. 5e),
showing a negative correlation with its target gene
POD2 (Fig. 5d; Wei et al. 2009). The reversing effects
were observed when tungstate was added together.
Combined with the changes in endogenous ABA levels
(Fig. 2a), above results suggested that in the presence of
PEG stress, H
2
-regulated the expression of miRNAs
was associated with ABA, and similar down-
regulation of miR398 elicited by H
2
have been discov-
ered in rice plants upon cold stress (Xu et al. 2017b)and
aluminum stress (Xu et al. 2017a).
Transcriptional factors contain two distinct domains,
a DNA-binding domain and a transcriptional activation/
repression domain, that regulate diverse cellular pro-
cesses through controlling the transcriptional rates of
target genes (Guo et al. 2005; Golldack et al. 2011;
Todaka et al. 2012; Franco-Zorrilla et al. 2014; Joshi
et al. 2016; Cai et al. 2017). In this study, osmotic stress
downregulated the expression of three transcriptional
factor genes, including MYB102,MYC2,andABF/
AREB2 (Fig. 6). By contrast, the addition of H
2
and
ABA reversed above changes. Contrasting results were
observed when tungstate was added together with H
2
,
suggesting that these transcriptional factors might be
targeted by H
2
in an ABA-dependent fashion.
On the other hand, ample evidence revealed that
osmotic stress-responsive gene expression is regulated
by ABA-dependent and ABA-independent pathways
(Yoshida et al. 2014). Meanwhile, ABA-independent
cold- and drought-responsive gene expression is respec-
tively modulated by CBF/DREB1 and DREB2 proteins
(Liu et al. 1998; Haake et al. 2002). In our experimental
conditions, PEG-induced alfalfa DREB1 transcripts
were intensified by H
2
(supplemental Fig. S2). Since
transgenic soybean overexpressing alfalfa DREB1 gene
displayed drought tolerance (Li et al. 2017), we further
deduced that H
2
-triggered tolerance against osmotic
stress might be partially mediated by ABA-
independent pathway. Certainly, this possibility should
be carefully investigated.
In response to abiotic stress, plants normally rely on a
complex network of signaling transduction pathways.
Combined with the previous results (Xie et al. 2014;Jin
et al. 2016;Suetal.2018), our data support the model
(Fig. 7) involving ABA signaling during H
2
-enhanced
plant response to counterbalance the deleterious effects
of osmotic stress, and H
2
-triggered ABA synthesis
might be an amplification loop in H
2
signaling. The
participation of representative miRNAs, TFs, and anti-
oxidant defense were also suggested. Certainly, the role
of ABA-independent pathway is not easily ruled out.
Conclusions
In summary, our physiological and molecular data dem-
onstrate that upon osmotic stress, H
2
could trigger an
increase in ABA production, thus maintaining cell ho-
meostasis and attenuate osmotic stress-derived redox
imbalance and seedling growth inhibition. Further ge-
netic evidence should be provided to confirm above
results, which will provide insights into H
2
signaling
and regulation of its bioactivity in agriculture, including
the enhancement of crop production in osmotic stress
environment.
Acknowledgments This research was supported by Foshan Ag-
riculture Science and Technology Project (Foshan City Budget
No. 140, 2019.), the Fundamental Research Funds for the Central
Universities (Grant number KJQN201640), the National Natural
Science Foundation of China (31371546), and the Funding from
Center of Hydrogen Science, Shanghai Jiao Tong University,
China.
Availability of data and materials All relevant data are within
this article and its supporting information files.
Author contributions Conception and design of the study: WS.
Acquisition of data for the study: KF, JS, GZ, WC, and HM.
Analysis of data for the work: KF, JS, and GZ. Interpretation of
data for the work: KF, JS, LR, GZ, RW, HM, JC, and WS. All
authors read and approved the final manuscript.
Compliance with ethical standards
Conflict of interest The authors declare no conflict of interest.
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