Abstract
Hydrogen sulphide (H2S), as a new gas signal molecule, participates in
the regulation of a variety of abiotic stresses in plants. However, it
was unclear how H2S and rhizobia can together to affect the adaptation
of soybean to water deficiency. Here, the adaptation mechanism of H2S
and rhizobia in soybean to water deficiency was studied. Our results
showed that H2S and rhizobia jointly enhanced leaf chlorophyll content,
the relative water content (RWC) and caused an increase biomass in
soybean under water deficiency. Besides, under water deficiency, H2S
enhanced biomass by affecting nodule numbers and nitrogenase activity
during the growth of soybean. The expression of soybean nodulation
marker genes including early nodulin 40 (GmENOD40), ERF required for
nodulation (GmERN), and nodulation inception genes were up-regulated by
H2S and rhizobia in nodules. Moreover, the combined effect of H2S and
rhizobia were proved to affect the enzyme activities and gene expression
level of antioxidant, as well as osmotic protective substance under
water deficiency. In addition, the metabolomics results provided that
the changes of lipids and lipid-like molecules were remarkably promoted
by the combined effect of H2S and rhizobia. Thus, H2S and rhizobia
synergistically subsided the oxidative damage by increasing the
accumulation of metabolites and strengthening the antioxidant capacity
under water deficiency.
Keywords: Hydrogen sulphide
(H2S), Rhizobia, Soybean (Glycine max ),
Antioxidant defense, Osmotic adjustment, Metabolomics
Introduction
All life activities of plants are carried out with the participation of
water, and sufficient water is an important condition for plant growth
and development (Gupta et al., 2020). Water deficiency has negative
effects on plant growth and development, which further reduces plant
production (Ashraf, 2010; Parvin et al., 2019). These negative effects
are mainly due to the imbalance of water metabolism and oxidative damage
induced by water deficiency (Amrutha et al., 2019). A significant change
in plant growth, photosynthesis, biomass production, enzymatic activity,
and oxidative damage parameters has been found in plants under drought
conditions (Aref et al., 2013; Cotado et al., 2020; Husen et al., 2017;
Matos et al., 2010). Therefore, understanding the water deficiency
mechanisms of soybean is indispensable in developing drought resistant
variety to assure sustainable soybean production in water-deficiency
regions.
Water deficiency breaks the balance between the production and removal
of reactive oxygen species (ROS) in plants. Plants manifest an excessive
increment in ROS generation in response to water deficiency (Miller et
al., 2010). Enhanced ROS generation under drought produces substantial
damages to the cellular components, which causes membrane injuries,
oxidative stress, protein degradation, and enzyme inactivation (Faize et
al., 2011; Hajiboland et al., 2017; Munné-Bosch & Peñuelas, 2003). To
fight against water stress-induced oxidative damage, plants have an
efficient antioxidant defense system, which includes enzymatic system
and non-enzymatic system (Ashraf et al., 2015; Sharma et al., 2012).
Efficient enzymes such as superoxide dismutase (SOD), catalase (CAT) and
peroxidase (POD) play critical roles in removing the water
deficiency-induced excessive ROS (Anjum et al., 2017). In plants,
ascorbate-glutathione (AsA-GSH) cycle is a very crucial component of the
antioxidant defense system (Hasanuzzaman et al., 2017; Zhang et al.,
2013). The AsA-GSH cycle is depended on the activities of ascorbate
peroxidase (APX), glutathione reductase (GR), monodehydroascorbate
reductase (MDHAR), and dehydroascorbate reductase (DHAR). DHAR and MDHAR
realize the regeneration of reduced ascorbate (AsA) in this cycle. GR is
responsible for the regeneration of reduced glutathione (GSH) (Shan et
al., 2018). Moreover, plants synthesize different permeates or solutes
through metabolic activities to maintain water balance under adversity
conditions (Krasensky & Jonak, 2012). For instance, previous studies
showed the accumulation of proline and soluble sugars increase the ROS
elimination and maintain the growth of peppermint (Mentha
piperita ) and Catharanthus roseus under water deficiency
conditions (Alhaithloulet al., 2019). In addition, high levels of
glycinebetaine (GB) and soluble sugars significantly enhance water
deficiency tolerance in Spinacia oleracea seedlings (Chen et al.,
2016).
Legumes are natural hosts of rhizobia, converting nitrogen in the
atmosphere into nitrogen available to plants, thus improving the ability
of nitrogen-fixing in plants (Janet & Sprent, 1972; Li et al., 2019;
Vitousek et al., 2002). Numerous studies have confirmed that legume
symbiosis can effectively improve the adaptation of plant to
environmental stress (Furlan et al., 2017; Liu et al., 2019). For
example, rhizobia can promote plant tolerance to salt stress by
increasing antioxidant enzyme activities, osmoregulation capacities, and
flavonoid metabolism in soybean and alfalfa (Meng et al., 2016; Qu et
al., 2016; Wang et al., 2016). Alternatively, rhizobia enhanced the low
temperature tolerance of plants by affecting N metabolism and N
absorption (Zhang et al., 2003). Moreover, we recently demonstrated that
rhizobia reinforced the Cu tolerance by increasing the activities of
antioxidant and the AsA-GSH enzymes in the soybean seedlings (Chen et
al., 2018). However, how the soybean-rhizobia symbiotic system responds
to water deficiency and the specific mechanism are still unclear.
Hydrogen sulphide (H2S), as an emerging gas signal
molecule, together with nitric oxide (NO) and carbon monoxide (CO),
plays an important role in plant growth and development (Li et al.,
2013; Li et al., 2016; Luo et al., 2020; Wang et al., 2012). For
instance, H2S can promote seed germination and root
development in Arabidopsis (Baudouin et al., 2016), participate
in the stomatal movement by mediating 8-mercaptocyclic GMP inArabidopsis (Honda et al., 2015). Chen et al. (2011) reported
that H2S promotes photosynthesis by increasing
ribulose-1, 5-bisphosphate carboxylase activity and thiol redox
modification in S. oleracea . Additionally, H2S
regulates pepper’s antioxidant system to alleviate zinc toxicity (Kaya
et al., 2018), and enhances the drought tolerance through effecting on
the biosynthesis of GB and soluble sugars in S. oleraceaseedlings (Chen et al., 2016). In our previous study, it was found that
H2S alleviates iron deficiency by promoting iron
availability and plant hormone levels in Glycine max seedlings
(Chen et al., 2020). Additionally, H2S can enhance the
establishment of the soybean-rhizobia symbiotic nitrogen fixation system
(Zou et al., 2020; Zou et al., 2019). In addition, we recently proved
that under nitrogen deficiency condition, the interaction of
H2S and rhizobia increase biomass and yield, improve the
nitrogen utilization efficiency, and affect the expression of senescence
related genes in soybean plants (Zhang et al., 2020). However, it is not
clear whether H2S and rhizobia participate in the
regulation the adaptation of soybean plants to water deficiency
environment.
The aim of this study was to investigate the antioxidant role of
H2S and rhizobia in soybean under water deficiency, and
the mechanism of how they together regulate the osmoprotectants and
differential metabolites change in response to water deficiency. In this
present study, we found that H2S and rhizobia can
improve against oxidative damage of soybean to water deficiency through
regulating symbiotic nodulation, antioxidant system, and
osmoprotectants, thus enhancing the photosynthesis and the drought
tolerance of plants. Moreover, based on non-target metabolomics methods,
we explored that H2S and rhizobia may enhance water
deficiency tolerance by changing the lipid-related metabolites compounds
in soybean leaves under water deficiency condition. Together, it was
proved that rhizobia symbiosis and H2S can jointly
improve the adaptability of water deficiency in soybean.