Mechanism Of Arbuscular Mycorrhizal Fungi Rhizophagus Irregularis Enhancing The Drought Tolerance Of Populus Simonii × P. Nigra

Populus simonii×P.nigra is a hybrid poplar of P.simonii and P.nigra,which is fast-growing and productive,and has excellent material,important ecological and economic values.It is widely planted in northern China,but drought stress has restricted the growth of P.simonii×P.nigra and forestry development.Arbuscular mycorrhiza（AM）fungi form symbiotic relationships with trees to enhance their drought tolerance.However,the effect of AM fungus on drought tolerance of P.simonii×P.nigra is not known.In this study,AM fungus Rhizophagus irregularis was inoculated on potted P.simonii×P.nigra seedlings under drought stress and the effects of AM fungus on the physiological growth,physiologi-cal damage,non-structural carbon metabolism and invertase（INV）and mitogen activated protein kinase（MAPK）genes expressions of P.simonii×P.nigra were investigated,using bioinformatics methods and quantitative real-time PCR（qRT-PCR）technology.The mech-anism of AM fungus improving drought tolerance of P.simonii×P.nigra was revealed from physiological and molecular levels.The main results are as follows:1.Effect of AM fungus on growth of P.simonii×P.nigra under drought stressDrought stress significantly inhibited seedlings growth,but the inhibition was allevi-ated by AM fungus inoculation.The plant height and fresh weight of roots of mycorrhizal P.simonii×P.nigra increased by 79.11%and 99.56%,respectively,the ratio of root to shoot decreased by 45.29%,and relative water content and chlorophyll content of leaves also in-creased significantly（P<0.05）.Under drought stress,AM fungus promoted photosynthesis of P.simonii×P.nigra and enhanced the net photosynthetic rate,stomatal conductance,and transpiration rate by 137.79%,195.81%and 278.75%,respectively;AM fungus improved nutrient uptake of P.simonii×P.nigra and increased the K content in roots and P content in leaves and roots by 54.88%,132.77%and 175.86%,respectively.The results indicated that AM fungus had a significant promoting effect on the growth of P.simonii×P.nigra under drought stress and effectively improved photosynthesis and nutrient element accumulation.2.Effect of AM fungus on physiological indicators of resistance of P.simonii×P.nigra under drought stressUnder drought stress,the levels of malondialdehyde（MDA）,H₂O₂,and proline accu-mulated in mycorrhizal P.simonii×P.nigra were lower than those of non-mycorrhizal P.simonii×P.nigra,indicating that inoculation with AM fungus reduced drought-induced oxidative damage,while non-mycorrhizal P.simonii×P.nigra was more sensitive to drought.Under drought stress,inoculation with AM fungus caused lower superoxide dis-mutase activity in both leaves and roots of mycorrhizal P.simonii×P.nigra than in non-mycorrhizal P.simonii×P.nigra,which was associated with lower levels of reactive oxygen in mycorrhizal P.simonii×P.nigra.Catalase（CAT）activity in leaves of mycorrhi-zal P.simonii×P.nigra was higher than that of non-mycorrhizal P.simonii×P.nigra,in-dicating that inoculation with AM fungus enhanced the ability of H₂O₂ scavenging in leaves.However,CAT activity in the roots of mycorrhizal P.simonii×P.nigra was lower than that of non-mycorrhizal P.simonii×P.nigra,which was related to the low level of H₂O₂ accu-mulation in the roots of mycorrhizal P.simonii×P.nigra.3.Effects of AM fungus on non-structural carbon metabolism of P.simonii×P.nigra under drought stressUnder drought stress,inoculation with AM fungus increased soluble sugar concentra-tion in leaves and roots by 24.02%and 16.42%,respectively;glucose concentration in leaves and roots by 42.71%and 44.39%,respectively;and fructose concentration in leaves and roots by 17.86%and 55.16%,respectively.Sucrose concentration in leaves decreased by 20.73%and sucrose concentration in roots increased by 30.82%.Meanwhile,under drought stress,AM fungus increased cell wall invertase and alkaline/neutral invertase activ-ities in leaves,sucrose synthase（catabolic direction）and amylase activities in roots of P.simonii×P.nigra,altering the accumulation and distribution of non-structural carbohy-drates and the activities of enzymes related to carbon metabolism.It was shown that my-corrhizal P.simonii×P.nigra has higher level of hexose accumulation and stronger osmo-regulation ability.The accumulation of hexose in mycorrhizal P.simonii×P.nigra roots was not dependent on the invertase activity but was attributed to high sugar transport activ-ity and cleavage of sucrose synthase in roots.4.Effects of AM fungus on the expressions of INV genes of P.simonii×P.nigra under drought stressThe 16 PsnINVs were identified in the P.simonii×P.nigra genome,and both drought stress and inoculation with AM fungus affected the expressions of PsnINVs.The differen-tially expressed genes in leaves responding to drought stress and inoculation were screened.Among them,the expressions of PsnA/NINV1,PsnA/NINV2,PsnA/NINV3 and PsnA/NINV5were positively correlated with photosynthesis,and the expressions of PsnA/NINV6,PsnA/NINV10 and PsnA/NINV12 were positively correlated with sugar content.Under drought stress,AM fungus promoted the synthesis of alkaline/neutral invertase in chloro-plasts and improved photosynthesis by up-regulating the expressions of PsnA/NINV1,PsnA/NINV2,PsnA/NINV3 and PsnA/NINV5 in leaves;promoted the synthesis of alka-line/neutral invertase in cytoplasm and enhanced osmotic regulation ability by up-regulating the expressions of PsnA/NINV6,PsnA/NINV10 and PsnA/NINV12 in leaves,improving the drought tolerance.In addition,AM fungus induced the expressions of PsnA/NINV1,PsnA/NINV4,PsnA/NINV7,PsnA/NINV8,and PsnA/NINV12 involved in mycorrhizal colo-nization or root development.However,under drought stress,the expression of most PsnINVs in mycorrhizal P.simonii×P.nigra roots decreased,which may be due to the negative feedback effect of high sugar concentration.5.Effects of AM fungus on the expressions of MAPK genes of P.simonii×P.nigra under drought stressThe 17 PsnMAPKs were identified in the P.simonii×P.nigra genome,and both drought stress and inoculation with AM fungus affected the expressions of PsnMAPKs.The higher photosynthesis of mycorrhizal P.simonii×P.nigra was associated with AM fun-gus-induced PsnMAPKs.The PsnMAPK7-2 and PsnMAPK20-2 were screened in leaves in response to drought stress and inoculation treatment.The PsnMAPK7-2,PsnMAPK16-1,and PsnMAPK19-2 were screened in roots in response to drought stress and inoculation treatment.Their expressions were significantly positively correlated with MDA concentra-tion and negatively correlated with biomass indexes,negatively regulating the drought tol-erance and growth of P.simonii×P.nigra.AM fungus reduced the drought sensitivity of P.simonii×P.nigra and alleviated physiological damage by down-regulating the expressions of these genes in leaves and roots.Under drought stress,inoculation with AM fungus in-duced the expressions of PsnMAPK7-1,PsnMAPK19-1,and PsnMAPK20-2 in roots,which could enhance the drought tolerance of P.simonii×P.nigra by activating MYB transcrip-tion factor.The above studies elucidate the mechanism by which AM fungi improve the drought tolerance of P.simonii×P.nigra by improving physiological growth,alleviating physio-logical damage,promoting non-structural carbon metabolism and regulating the expressions of INV and MAPK genes.