Figure Captions
Figure 1 Effects of H2S and rhizobia on plant phenotype (A) and biomass (B, C). The SPAD (D) and leaf RWC (E) of leaves by four different treatments changed under water deficiency. Control, without rhizobia or NaHS; NaHS, with 100 μM NaHS; Q8, with rhizobia inoculation; Q8+NaHS, with rhizobia inoculation and 100 μM NaHS. NW, normal moisture content 80%-90%; MW, moderate drought moisture content 50%-60%; SW, severe drought moisture content 20%-30%. Data were expressed as the mean ± SE . And columns marked with different letters indicated significant differences atP  < 0.05.
Figure 2 Effect of H2S on nodulation size (A), number of nodules (B), nodule biomass (C), endogenous hydrogen sulfide content (D), and acetylene reduction assay (E) of soybean under water deficiency. Q8, with rhizobia inoculation; Q8+NaHS, with rhizobia inoculation and 100 μM NaHS. NW, normal moisture content 80%-90%; MW, moderate drought moisture content 50%-60%; SW, severe drought moisture content 20%-30%. Data were expressed as the mean ± SE . And columns marked with different letters indicated significant differences at P  < 0.05.
Figure 3 Effects of H2S and rhizobia on photosynthetic parameters of soybean plants under water deficiency. Net photosynthetic rate (Pn, A), stomatal conductance (Gs, B), intercellular CO2 concentration (Ci, C), transpiration rate (Tr, D), stomatal limit value (Ls, E) and instantaneous moisture utilization rate (WUE, F). Control, without rhizobia or NaHS; NaHS, with 100 μM NaHS; Q8, with rhizobia inoculation; Q8+NaHS, with rhizobia inoculation and 100 μM NaHS. NW, normal moisture content 80%-90%; MW, moderate drought moisture content 50%-60%; SW, severe drought moisture content 20%-30%. Data were expressed as the mean ± SE . And columns marked with different letters indicated significant differences atP  < 0.05.
Figure 4 Effects of H2S and rhizobia on fluorescence parameters of soybean plants under water deficiency. Electronic transport ratio (ETR, A), quantum yield of PSII photochemistry (PSII, B), NPQ (C), the ratio of variable fluorescence to maximum fluorescence (Fv/Fm, D). Control, without rhizobia or NaHS; NaHS, with 100 μM NaHS; Q8, with rhizobia inoculation; Q8+NaHS, with rhizobia inoculation and 100 μM NaHS. NW, normal moisture content 80%-90%; MW, moderate drought moisture content 50%-60%; SW, severe drought moisture content 20%-30%. Data were expressed as the mean ± SE . And columns marked with different letters indicated significant differences at P  < 0.05.
Figure 5 Effects of hydrogen sulfide and rhizobia on endogenous hydrogen sulfide (A), lipid membrane peroxidation (MDA, B), hydrogen peroxide (H2O2, C), and superoxide anion (OFR, D) contents in plant leaves under water deficiency. Control, without rhizobia or NaHS; NaHS, with 100 μM NaHS; Q8, with rhizobia inoculation; Q8+NaHS, with rhizobia inoculation and 100 μM NaHS. NW, normal moisture content 80%-90%; MW, moderate drought moisture content 50%-60%; SW, severe drought moisture content 20%-30%. Data were expressed as the mean ± SE . And columns marked with different letters indicated significant differences at P  < 0.05.
Figure 6 Effects of hydrogen sulfide and rhizobia on superoxide dismutase (SOD, A) peroxidase (POD, B) catalase (CAT, C) ascorbate peroxidase (APX, D) in leaves of soybean plants under water deficiency. Control, without rhizobia or NaHS; NaHS, with 100 μM NaHS; Q8, with rhizobia inoculation; Q8+NaHS, with rhizobia inoculation and 100 μM NaHS. NW, normal moisture content 80%-90%; MW, moderate drought moisture content 50%-60%; SW, severe drought moisture content 20%-30%. Data were expressed as the mean ± SE . And columns marked with different letters indicated significant differences atP  < 0.05.
Figure 7 The effect of H2S and rhizobia inoculation on the GSH content (A), the GSSG content (B), the ratio of GSH/GSSG (C) , the AsA content (D), the DHA content (E) , the ratio of AsA/DHA(F), MDHAR activity (G), DHAR activity (H), and GR activity (I) in leaves of soybean plants under water deficiency. Control, without rhizobia or NaHS; NaHS, with 100 μM NaHS; Q8, with rhizobia inoculation; Q8+NaHS, with rhizobia inoculation and 100 μM NaHS. NW, normal moisture content 80%-90%; MW, moderate drought moisture content 50%-60%; SW, severe drought moisture content 20%-30%. Data were expressed as the mean ± SE . And columns marked with different letters indicated significant differences at P  < 0.05.
Figure 8 Effects of the addition of exogenous H2S donor NaHS and rhizobia on the contents of Sucrose (A), Fructose (B), Glucose (C), Proline (PRO, D) and Glycine betaine (GB, E) in soybean leaves under water deficiency. Control, without rhizobia or NaHS; NaHS, with 100 μM NaHS; Q8, with rhizobia inoculation; Q8+NaHS, with rhizobia inoculation and 100 μM NaHS. NW, normal moisture content 80%-90%; MW, moderate drought moisture content 50%-60%; SW, severe drought moisture content 20%-30%. Data were expressed as the mean ± SE . And columns marked with different letters indicated significant differences at P  < 0.05.
Figure 9 Effects of H2S and rhizobia on the expression level of GmCAT (A), Gm SOD1 (B), GmSOD2(C), GmPrx (D), GmGrx (E), GmBADH (F), GmSUS(G), GmUDP (H), GmFBP (I) gene in soybean leaves under water deficiency. Control, without rhizobia or NaHS; NaHS, with 100 μM NaHS; Q8, with rhizobia inoculation; Q8+NaHS, with rhizobia inoculation and 100 μM NaHS. NW, normal moisture content 80%-90%; MW, moderate drought moisture content 50%-60%; SW, severe drought moisture content 20%-30%. Data were expressed as the mean ± SE . And columns marked with different letters indicated significant differences atP  < 0.05.
Figure 10 Gene expression level of symbiotic related genes. Relative expression levels of GmENOD40 gene (A), GmERNgene (B), GmNIN1a gene (C), GmNIN2a gene (D), andGmNIN2b gene (E) are displayed in multiple line charts with symbols. Control, without rhizobia or NaHS; NaHS, with 100 μM NaHS; Q8, with rhizobia inoculation; Q8+NaHS, with rhizobia inoculation and 100 μM NaHS. NW, normal moisture content 80%-90%; MW, moderate drought moisture content 50%-60%; SW, severe drought moisture content 20%-30%. Values are means ± SE (n = 9).
Figure 11 Response of non-targeted metabolomics to water deficiency in soybean leaves. (A) OPLS-DA is used to distinguish the difference between soybean leaves Control-SW/Control-NW samples. (B) Volcano plot (difference between -log10P ANOVA and OPLS-DA load) shows the best distinguishing metabolites in Control-SW/Control-NW related to the effectiveness of water deficiency. (C) Using OPLS-DA to distinguish the sample differences of Q8-SW/Q8-NW in soybean leaves. (D) Volcano plot (difference between -log10 P ANOVAand OPLS load) shows the best distinguishing metabolites in Q8-SW/Q8-NW related to the effectiveness of water deficiency. (E) OPLS-DA is used to distinguish the sample difference of soybean leaves Q8+NaHS-SW/Q8+NaHS-NW. (F) Volcano plot (difference between -log10 P ANOVA analysis of variance and OPLS load) shows the best distinguishing metabolites related to the effectiveness of water deficiency in Q8+NaHS-SW/Q8+NaHS-NW. The red horizontal dotted line indicates the threshold P ANOVA < 0.05. The important variables that reach the threshold are marked in green (decreased when water is deficient) or red (increased when water is deficient). Control, without rhizobia or NaHS; NaHS, with 100 μM NaHS; Q8, with rhizobia inoculation; Q8+NaHS, with rhizobia inoculation and 100 μM NaHS. NW, normal moisture content 80%-90%; MW, moderate drought moisture content 50%-60%; SW, severe drought moisture content 20%-30%.
Figure 12 Response of non-targeted metabolomics to soybean leaves under different treatments under water deficiency. (A) OPLS-DA is used to distinguish the difference of soybean leaf Control-SW/Q8-SW. (B) Volcano plot (difference between -log10P ANOVA analysis of variance and OPLS-DA load) shows the best distinguishing metabolites associated with water deficiency in Control-SW/Q8-SW. (C) Using OPLS-DA to distinguish the sample differences of soybean leaves Q8-SW/Q8+NaHS-SW. (D) Volcano plot (difference between -log10 P ANOVAanalysis of variance and OPLS load) shows the best distinguishing metabolites related to water deficiency in Q8-SW/Q8+NaHS-SW. The red horizontal dotted line represents the thresholdP ANOVA <0.05. Important variables that reach the threshold are marked in green (decrease when moisture is deficient) or red (increased when moisture is deficient). Control, without rhizobia or NaHS; NaHS, with 100 μM NaHS; Q8, with rhizobia inoculation; Q8+NaHS, with rhizobia inoculation and 100 μM NaHS. NW, normal moisture content 80%-90%; MW, moderate drought moisture content 50%-60%; SW, severe drought moisture content 20%-30%.
Figure 13 Schematic diagram of the mechanisms of drought tolerance response of soybean-rhizobia symbiotic system under the regulation of hydrogen sulfide under water deficiency.
Table 1 Significantly changed metabolites between different treatments under water deficiency in soybean leaves, P ≤ 0.001.
Table 2 Changes of metabolites between different treatments under SW condition in soybean leaves, P ≤ 0.001.