H2S and rhizobia synergistically increase antioxidant defense capacity of soybean leaves under water deficiency
Plants are often subjected to continuous threat from toxic ROS and lipid peroxidation. In order to cope with environment stress, plants increase their resistance by activating their antioxidant defense system (Farooq et al., 2019; Niu et al., 2012). ROS-generated oxidative injuries in plants were reflected by the MDA content, a byproduct of lipid peroxidation (Sato et al., 2011). We found that the over accumulation of MDA was observed under SW condition. Compared with the control, the Q8+NaHS-treated plants did not maintain lower levels of MDA content to some extent under water deficiency (Fig. 5B). This is contrast with the study conducted by Wang et al. (2016), who found that there is notably lower MDA content in the leaves of alfalfa with rhizobia than control seedlings. These results suggested that severe water deficiency could damage the membrane structure and lead to excessive oxidative damage in soybean leaves. Furthermore, it was reported that H2S reduced membrane peroxidation by regulating the activities of antioxidant enzymes in rice under low temperature stress (Mostofa et al., 2015), and also regulated the antioxidant system of pepper to relieve zinc toxicity (Kaya et al., 2018). In the present study, H2S and rhizobia synergistically decrease in H2O2 levels through increasing activities of antioxidants in soybean leaves under water deficiency condition (Fig. 5C). Further proof was provided by the assay of antioxidant enzyme activities in soybean leaves tissues. Our results showed that the activities of CAT and POD were markedly increased in soybean treated with H2S and rhizobia compared with the control treatment under water deficiency (Fig. 6B, C), indicating that plants maintained high levels of antioxidants activities to remove water stress induced-excess ROS. In other studies, it has been shown that the balance among POD, CAT and SOD is crucial for determining the steady-state level of H2O2 and superoxide radicals (Dong et al., 2019; Li et al., 2020). Together, these results suggested that the activities of antioxidants played a vital role in alleviating the oxidative damage of soybean under water deficiency. Although plants maintained high levels of CAT and POD activities to some extent by Q8+NaHS treatment under water deficiency, the activity of SOD did not markedly increase. Notably, this is little decrease for the OFR content to some extent for H2S and rhizobia treatment under water deficiency. Meanwhile, compared with NW condition, the content of OFR was increased by H2S and rhizobia under SW condition (Fig. 5 D). In addition, the transcript abundances of several antioxidant defense-related genes were increased by H2S and rhizobia under SW condition (Fig. 9). Importantly, GmSOD2 , which is encoded the ron-SOD2, exhibited higher expression levels in Q8+NaHS-treated plants than in the control plants under SW condition. This may be due to the fact that SOD, as an inducible enzyme, produces more superoxide anion and induces SODgene expression under water deficiency condition. These results revealed that H2S and rhizobia are more efficient in mitigating the damage caused by ROS in soybean under water deficiency.
The AsA-GSH cycle plays a vital role for plant to resist oxidative damage (Avashthi et al., 2018; Nanda & Agrawal, 2016). It is reported that Brassica napus plants can be protected from salt induced oxidative stress by regulating ASA-GSH pathway. (Hasanuzzaman et al., 2018). Furthermore, Chen et al. (2011) reported that H2S may lead to the accumulation of GSH in plant tissues. Our results showed that water deficiency altered the redox status of AsA and GSH in soybean seedlings under water deficiency condition (Fig. 7). Moreover, the mRNA abundance of 1-Cys peroxiredoxin and glutaredoxin, which are encoded byGmPrx and GmGrx , are higher in the Q8+NaHS treatment plants than control plants under SW condition. This may corroborate why the H2S and rhizobia show higher GR activity under water deficiency (Fig. 7I). Thus, the decline in the redox status levels by water deficiency is either due to the serious damage of plants. These results clearly revealed that water deficiency caused the alteration in the redox status of the cell by interfering with AsA and GSH pools in legumes. Furthermore, we found that H2S and rhizobia synergistically enhanced the AsA-GSH cycle by regulating the activities of APX, GR, and DHAR in soybean under water deficiency (Fig. 6D, 7H, I). Similar results have been reported that the application of exogenous H2S regulated the metabolism of AsA and GSH in wheat leaves by increasing the activities of APX, GR and DHAR under water stress (Shan et al., 2011). Previous studies have shown that the higher accumulation of the AsA-GSH cycle and APX activity synergistically reduced the excess production of H2O2 in cashew plants (Lima et al., 2018). Khan et al. (2017) reported that NO-induced H2S alleviated osmotic stress in wheat seedlings by enhancing the activities of APX and GR. Interestingly, the regeneration of GSH from oxidized glutathione (GSSG) is catalyzed by GR and improved the antioxidant capacity of cells (Hasanuzzaman et al., 2017). These results suggested that H2S and rhizobia synergistically triggered the up-regulation of the AsA-GSH cycle related enzymes under water deficiency, which further enhanced abiotic stress tolerance in plants.