Effect of Drought and AMF inoculation on plant growth
Mycorrhizal colonization was not found in the roots of uninoculated seedlings. The mycorrhizal colonization rate of the AMF-inoculated seedlings was 81.56%±0.10% under WW treatment and 64.84%±0.12% under DS treatment, respectively. DS treatment significantly inhibited the growth of seedlings, but the AMF inoculation promoted plant growth, which was reflected in the height of the plant increased by 79.11%. In the case of WW treatment, AMF inoculation also increased the height of plants by 25.43% (Table 1). Under WW treatment, AMF inoculation significantly promoted the fresh weight of shoots, fresh weight of roots and total biomass, which increased by 45.59%, 29.96%, and 39.97%, respectively (Table 1). Under DS treatment, AMF inoculation dramatically increased fresh weight of roots by 99.56% but had no significant effect on fresh weight of shoots and total biomass. Notably, DS treatment increased the root-shoot ratio, but inoculation with AMF reduced the root-shoot ratio by 24.00%.
Table 1. Effects of drought and R. intraradices on growth of P. simonii×P. nigra seedlings
Treatments
|
AM colonization rate (%)
|
Plant height(cm)
|
Fresh Weight of Shoot (g)
|
Fresh Weight of Root (g)
|
Root/Shoot
|
Total Biomass (g)
|
WW-NM
|
—
|
33.23±2.06b
|
21.33±2.90b
|
11.95±1.20b
|
0.57±0.06c
|
33.28±3.79b
|
WW-AM
|
81.56±0.10a
|
41.68±0.62a
|
31.06±3.35a
|
15.53±2.09a
|
0.50±0.02c
|
46.58±5.38a
|
DS-NM
|
—
|
3.95±0.42d
|
1.47±0.3c
|
2.86±0.21d
|
2.00±0.32a
|
4.33±0.48c
|
DS-AM
|
64.84±0.12b
|
7.08±0.39c
|
3.78±0.35c
|
5.71±0.29c
|
1.52±0.06b
|
9.49±0.64c
|
Note: WW=well-watered, DS=drought stress. NM=nonmycorrhizal seedling, AM=mycorrhizal seedlings. Data (means ± SD, n = 4) followed by different letters in the column indicate significant differences (P < 0.05) between treatments.
Effect of Drought and AMF inoculation on Photosynthesis
Compared with WW treatment, drought stress significantly decreased Pn, gs and Tr of seedlings. AMF-inoculation significantly enhanced Pn, gs, and Tr by 34.66%, 78.36%, and 47.36%, respectively, under well-watered conditions. Under drought conditions, AMF inoculation also significantly enhanced Pn, gs, and Tr by 137.79%, 195.81%, and 278.75%, respectively, compared with NM seedlings. However,there was no significant difference in the Ci among the four treatments (Table 2).
Table 2. Effects of drought and R. irregularis on photosynthetic parameters of P. simonii×P. nigra seedlings.
Treatments
|
Pn (µmol/m2 per second)
|
gs (µmol/m2 per second)
|
Ci (µmol/mol)
|
Tr (mmol/m2 per second)
|
WW-NM
|
8.35±0.79b
|
70±0.01b
|
241.56±10.84a
|
1.70±0.11b
|
WW-AM
|
11.25±1.95a
|
120±0.04a
|
250.57±29.10a
|
2.51±0.74a
|
DS-NM
|
2.95±0.47c
|
20±0.00c
|
219.77±26.33a
|
0.65±0.07c
|
DS-AM
|
7.02±0.98b
|
60±0.00b
|
225.57±15.98a
|
1.56±0.28b
|
Note: WW=well-watered, DS=drought stress. NM=nonmycorrhizal seedling, AM=mycorrhizal seedlings. Data (means ± SD, n = 4) followed by different letters in the column indicate significant differences (P < 0.05) between treatments.
Effect of Drought and AMF inoculation on MDA and H2O2 concentrations
Compared with WW treatment, the MDA concentrations in leaves and roots were found to be significantly increased under DS treatment. Under WW treatment, no matter whether it was inoculated or not, there was no significant difference in MDA concentration in leaves and roots. Whereas under DS treatment, AMF inoculation obviously decreased MDA concentration by 23.56% in leaves, and 47.2% in roots (Figure 1). Under the two water treatments, there was no significant difference in the concentration of H2O2 in the leaves of AM and NM seedlings, but the concentration of H2O2 in the leaves of NM seedlings was dramatically increased by 32.97% under DS treatment, while the concentration of H2O2 in the leaves of AM seedlings was also increased by DS treatment, but it did not reach a significant level. Under WW treatment, there was no significant difference in the concentration of H2O2 in the roots of the NM and AM seedlings. However, under DS treatment, the concentration of H2O2 in the roots of NM seedlings was dramatically increased by 45.41%. Although DS treatment also increased the concentration of H2O2 in the roots of AM seedlings, it did not reach a significant level (Figure 2).
Effect of Drought and AMF inoculation on Osmotic adjustment substances
Compared with WW treatment, the proline concentrations in leaves and roots were found to be enhanced obviously under DS treatment. Under WW treatment, AMF inoculation had no significant effect on proline concentration. Whereas under DS treatment, AMF inoculation obviously decreased proline concentration by 27.42% in leaves, and 52.24% in roots (Figure 3). Under WW treatment, AMF inoculation had no significant effect on soluble sugar concentration. It is worth noting that AMF inoculation dramatically reduced soluble sugar concentration in leaves by 20.75% under DS treatment, compared with NM seedlings. Contrary to the result in the leaves, AMF inoculation dramatically enhanced soluble sugar concentration by 129.28% under DS treatment (Figure 4).
Effect of Drought and AMF inoculation on Antioxidant Enzymes Activities
DS treatment significantly enhanced SOD activity in leaves, but SOD activity in leaves of AM seedlings was lower versus NM seedlings under both water conditions. Under WW treatment, there was no significant difference in SOD activity in leaves between NM and AM seedlings. DS treatment enhanced SOD activity in roots, but SOD activity of AM seedlings was also lower versus NM seedlings (Figure 5A). Under WW treatment, there was no significant difference in CAT activity in leaves between NM and AM seedlings. DS treatment significantly enhanced CAT activity in leaves of AM seedlings but had no significant effect on NM seedlings. Notably, CAT activity in roots of AM seedlings was dramatically higher than NM seedlings under WW treatment. DS treatment significantly enhanced CAT activity in roots of NM seedlings but had no significant effect on AM seedlings (Figure 5B).
Effect of Drought and AMF inoculation on expressions of MAPK genes
According to the homology with MAPKs of A. thaliana and rice, the MAPKs of P. simonii×P. nigra (PsnMAPKs) were divided into four groups: A, B, C, and D (Figure 6). Phylogenetic analysis showed that PsnMAPK20-1 belonged to group A, PsnMAPK7-1 and PsnMAPK7-2 belonged to group C, and other PsnMAPKs were attributed to group D, without genes belonged to group B. The notable PsnMAPKs in leaves were PsnMAPK7-1 and PsnMAPK20-1, belonging to group C and A. The effects of DS treatment on the expressions of them were significantly different between NM and AM seedlings. The expressions of these two genes dramatically increased in NM seedling leaves under DS treatment, but PsnMAPK7-1 was down-regulated in AM seedling leaves. Moreover, DS treatment did not affect the expression of PsnMAPK20-1 in AM seedling leaves to a significant level. The PsnMAPKs of group D in NM and AM seedling leaves were down regulated by DS treatment. In addition, the effects of inoculation on the expression of PsnMAPK7-1 in leaves were also significantly different between both water treatment. Under WW treatment, AMF inoculation increased the expression of PsnMAPK7-1 in leaves, but under DS treatment, AMF inoculation decreased the expression of PsnMAPK7-1. However, the expression of PsnMAPK20-1 in leaves was down-regulated by AMF inoculation under both water treatments. Additionally, the expressions of several PsnMAPKs in group D increased dramatically after AMF inoculation under WW treatment, including PsnMAPK19-1, PsnMAPK20-2, PsnMAPK12, PsnMAPK17 (Figure 7A).
In the present study, we observed that more PsnMAPKs were affected by DS treatment in roots versus leaves. DS treatment increased the expressions of all PsnMAPKs in roots of NM seedlings except PsnMAPK15-2 and PsnMAPK20-1. Among these MAPKs, PsnMAPK7-1 and PsnMAPK7-2 in group C performed different responses to drought between NM and AM roots. DS treatment increased the expressions of PsnMAPK7-1 and PsnMAPK7-2 in NM roots but decreased them in AM roots. The expressions of PsnMAPK15-2, PsnMAPK7-1, PsnMAPK3-2, PsnMAPK12 and PsnMAPK17 were dramatically increased by AMF inoculation under WW treatment. However, under DS treatment, except PsnMAPK15-2 and PsnMAPK3-2, the expressions of other MAPKs were decreased by AMF inoculation. Interestingly, the effect of DS treatment on the expressions of PsnMAPK15-2 were obviously different between NM and AM roots. DS treatment dramatically reduced the expression of PsnMAPK15-2 in AM roots but did not significantly affect the expression in NM roots (Figure 7B).