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.