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.