Plant growth and Treatment
Soybean seeds (Glycine max , Zhonghuang 13) were sterilized by 75% ethanol for 30 seconds and 50% sodium hypochlorite for 4 min, and washed with distilled water, and then placed on a 1% agar plate for 72 h to germination in a 28°C incubator. After 3 days of culture, the soybean seedlings were selected and cultured into growth medium with nutrient solution (vermiculite and perlite, v/v = 2:1; nitrogen-free nutrient solution 100 mg/L CaCl2, 100 mg/L KH2PO4, 50 mg/L Ferric citrate, 150 mg/L NaH2PO4, 120 mg/L MgSO4· 7H2O, 2.86 mg/L H3BO3, 2.3 mg/L MnSO4· 4H2O, 2.8 mg/L ZnSO4· 7H2O, 13 mg/L Na2MoO4· 2H2O, 2.2 mg/L CaSO4· 5H2O). The plants were grown in a controlled growth chamber with a light/dark state of 10/14 h, relative humidity of 80%, temperature of 23/25°C, and photosynthetically active radiation of 190 μmol m-2s-1.
The 7-day-old seedlings were divided into four treatment groups: Control, without rhizobia and NaHS; NaHS, with 100 μM NaHS; Q8, with rhizobia inoculation (Sinorhizobium fredii Q8 strain); Q8+NaHS, with rhizobia inoculation and 100 μM NaHS. In addition, we set three water gradients again for each group: normal water content 80%-90% (NW), moderate drought water content 50%-60% (MW), severe drought water content 20%-30% (SW). When the first true leave of soybean was fully expanded, all the seedlings of the Q8 and Q8+NaHS treatment groups were inoculated with 10 ml of rhizobia suspension (OD600= 0.5). Seedlings of the NaHS and Q8+NaHS treatment groups were added with 10 ml of 100 μM NaHS solution every 3 days until harvest. While the other two groups of seedlings were watered with double distilled water. Water contents were controlled with an electronic balance after the soybean seedlings were inoculated with rhizobia and growth for 21 days. The seedling bags were weighed every 1 or 2 days, and distilled water was used to add moisture if it was necessary. Half of the samples were dried to constant weight for dry matter determine, and the other half was immediately frozen in liquid nitrogen and stored at -80°C.