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.