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.

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