H2S and rhizobia regulates nodule formation and nitrogenase activity in soybean plants under water deficiency
Symbiotic nitrogen fixation increases the available nitrogen content of plants and promotes legumes growth and development. Many small signaling molecules, such as NO, H2O2, and phytohormones have been reported to be involved in the regulation of nodulation between legumes and rhizobia (Hérouart et al., 2002; Puppo et al., 2013). In this experiment, our results showed water deficiency significantly inhibited the accumulation of nodules weight, endogenous H2S content, and nitrogenase activities (Fig. 2C, D and E), suggesting that water deficiency affected the establishment of symbiotic system between rhizobia and soybean. Besides, under SW condition, the endogenous H2S content was higher in the Q8+NaHS-treated plants than in the control treatment (Fig. 2D, E). Similarly, Chen et al. (2016) found that NaHS application enhanced the endogenous H2S content in S. oleracea leaves under drought stress. There was no significant difference in the nodule nitrogen fixation area between NaHS+Q8 and the Q8 treatments under NW and MW conditions (Fig. S1). The nodule nitrogen fixation area was notably reduced under SW condition compared with NW condition. Interestingly, the nitrogen fixation area was partially rescued in NaHS-treated nodules under SW condition (Fig. S1). These results indicated that under water deficiency condition H2S could alleviate water deficiency-induced the decrease of the nitrogenase activity. Additionally, an interesting phenomenon was found that under MW condition the nodules number was greatly increased, but nodules biomass was not obviously affected under the same condition (Fig. 2B, C). The possible reason was that water deficiency stimulated root nodulation to resist water stress. Additionally, previous studies have shown that plant can continue to nodule in the water deficiency by stimulating the early nodulation factor (Paula et al., 2016). In the present study, we investigated the expression of symbiosis-related genes in root nodules. GmENOD40 is a downstream component of NFs perception (Ferguson et al., 2010), which is expressed in peripheral cells, cortical cells, nodule primordia and developing nodules of root vascular bundles (Ferguson & Mathesius, 2014). Charon et al. (1999) reported that the change of ENOD40 expression abundance could affect nodulation, suggesting that it plays an important role in the organogenesis of nodules. In the present study, the expression abundance of GmENOD40 was up-regulated by H2S and rhizobia in soybean roots under whatever water condition (Fig. 10A). Besides, another nodulation marker genes involved in the pathway of NFs nodulation, such as GmERN , was also activated by H2S and rhizobia (Fig. 10B). NIN is essential for nodule organogenesis and nitrogen-fixing symbiosis in Medicago truncatula roots (Tatiana et al., 2015). Under any water condition, we found that the expression levels of three NIN genes were up-regulated by Q8+NaHS treatment in soybean root (Fig. 10C, D, E). Above results showed that H2S and rhizobia stimulated the expression of GmENOD40 , GmERN , and GmNIN genes under water deficiency condition in soybean roots. Moreover, the moderate water deficiency may stimulate the nodule formation in the soybean symbiosis system and help plants to adaption the water deficiency environment. Therefore, our results suggested under water deficiency condition H2S promoted nodule development and enhanced water-deficiency tolerance in the soybean-rhizobia symbiotic system.