Arbuscular Mycorrhizal Fungi (AMF) Alleviates Salt Stress In Black Locust

In the present study,we analyzed effects of salt stress and AMF?Rhizophagus irregularis?inoculation on water status,photosynthesis,ion homeostasis and antioxidant capacity in black locust?Robinia pseudoacacia?.We explored physiology,biochemistry and molecular mechanisms underlying the improved salt tolerance of black locust by AMF.The main results are as follows:1.Effects of AMF on growth and expression of aquaporin genes in black locust subjected to salt stressThe response of biomass production,roots morphology,leaf relative water content and the expression of six aquaporin genes to three levels?0,100 and 200 mM?of salinity and arbuscular mycorrhizal?AM?symbiosis were examined.The results showed that salinity significantly reduced the leaf relative water content,the biomass production,as well as the root volume,surface area and tip and fork numbers in both non-mycorrhizal?NM?and AM plants.AMF significantly improved the biomass production and the root volume,surface area,tip and fork numbers at three salinity levels.AM plants also displayed a significant increase in the leaf relative water content and the total root length under severe salinity stress?200mM NaCl?compared with NM plants.Furthermore,salinity downregulated the leaf expression of RpPIP1;1,RpPIP2;1,RpTIP1;1 and RpTIP1;3 and the root expression of RpPIP1;3,RpPIP2;1,RpTIP1;1,RpTIP1;3 and RpTIP2;1 in NM plants.In AM plants,the expression of RpPIP2;1 and RpTIP1;3 in leaves and the expression of RpTIP1;3 in roots were downregulated,while the expression of RpPIP1;1 in leaves and the expression of RpPIP1;3,RpPIP2;1 and RpTIP1;1 in roots were upregulated with increasing salinity.In addition,AMF upregulated the leaf expression of RpPIP1;1 and RpPIP1;3 under moderate salinity stress?100 mM NaCl?,and the root expression of RpPIP1;1,RpPIP1;3,RpPIP2;1and RpTIP1;1 under severe salinity stress.The results indicated beneficial effects of AMF on the growth performance of black locust exposed to salt stress and suggested that AMF could improve the water status of black locust through regulation of the expression of aquaporin genes.2.Effects of AMF on photosynthesis and expression of genes associated with photosynthesis in black locust subjected to salt stressThe regulation of chlorophyll content,net photosynthesis rate,gas exchange parameters,water use efficiency,photochemistry efficiency of photosystem II?PSII?and the expression of four genes?RppsbA,RppsbD,RprbcL and RprbcS?associated with photosynthesis by AM symbiosis were investigated in black locust.The results showed that salinity significantly reduced the net photosynthesis rate,the stomatal conductance,the transpiration rate and the expression of RppsbA,RppsbD and RprbcL in both NM and AM plants.In addition,in NM plants,the maximum quantum efficiency of PSII photochemistry?Fv/Fm?,PSII operating efficiency??PSII?,PSII maximum efficiency?Fv?/Fm??and photochemical quenching coefficient?qP?decreased with increasing salinity,while salinity had no significant effects on these parameters in AM plants.Under stress condition,AMF significantly improved the contents of chlorophyll a and b,the net photosynthesis rate,Fv/Fm,?PSII,Fv?/Fm?and the expression of RppsbA,but evidently reduced the intercellular CO₂ concentration.AM plants also had a higher qP but a lower non-photochemical quenching?NPQ?than NM plants under severe salinity stress.The results indicated that AM plants had a less stressed PSII,a higher photochemistry efficiency of PSII and a greater ability to drive electron transport in the PSII reaction center,which increased the photosynthetic capacity in these plants.3.Effects of AMF on nutrients uptake,ion homeostasis and expression of genes coding for membrane transport proteins in black locust subjected to salt stressThe effects of AM symbiosis on contents of macronutrients,micronutrients and Na,the K/Na ratio,the leaf/root Na ratio as well as the expression of four genes?RpSOS1,RpNHX1,RpHKT1and RpSKOR?coding for membrane transport proteins involved in K/Na homeostasis were determined.The results showed that salinity significantly increased the leaf and root Na content and the leaf/root Na ratio in both NM and AM plants.But the K/Na ratio was reduced by salinity in both NM and AM plants.AMF significantly reduced the leaf Na content under stress condition and decreased the leaf/root Na ratio under severe salinity stress.AM plants also showed an increase in the ratio of K/Na in leaves at 0 and 100 mM NaCl.In addition,AMF increased the contents of macronutrients?N,P,K and Mg?and micronutrients?Mn,Cu and Zn?,depending on plant tissue and soil salinity.In both NM and AM plants,salinty downregulated the leaf expression of RpSKOR,but upregulated the root expression of RpSOS1.Under stress condition,AMF upregulated the leaf expression of RpSKOR and the root expression of RpSKOR and RpSOS1.Under severe salinity stress,the root expression of RpHKT1 was also upregulated by AMF.The results indicated that AMF could improve the nutrient uptake,and that AMF could reduce the accumulation and the root-to-shoot allocation of Na through regulation of the expression of genes encoding membrane transport proteins,which contributed to the improved ion homeostasis in black locust.4.Effects of AMF on ROS scavenging ability and expression of genes encoding antioxidative enzyme in black locust subjected to salt stressThe effects of AM symbiosis on contents of H₂O₂,MDA,ascorbate and glutathione,and activities of SOD,POD,CAT,APX,MDHAR,DHAR and GR in leaves and roots were examined.The leaf relative electrolyte leakage?REL?was also determined.The results showed that salinity significantly increased the leaf REL and the contents of H₂O₂ and MDA in leaves and roots of both NM and AM plants.AM plants showed a significant decrease in the leaf REL under stress condition,and a less accumulation of H₂O₂ and MDA in leaves under severe salinity stress.No significant differences in the root MDA content were observed between NM and AM plants.The H₂O₂ content in roots of AM plants was significantly higher than that in NM plants under stress condition.In leaves,AMF increased the activities of SOD,CAT,APX,MDHAR,DHAR and GR as well as the contents of ascorbate?total and oxidized?and glutathione?total,reduced and oxidized?.In roots,AM plants had higher activities of SOD and POD,but lower activities of APX and MDHAR,as well as contents of ascorbate?total,reduced and oxidized?and total glutathione.In addition,AMF upregulated the leaf expression of RpCu/Zn-SOD,RpMn-SOD,RpFe-SOD,RpAPX2and RpGR under severe salinity stress,but downregulated the root expression of RpCu/Zn-SOD at 0 and 100 mM NaCl.The results indicated that the positive effects of AMF on antioxidant capacity were more evident in leaves than in roots and that AMF improved the ROS scavenging ability in leaves through upregulating the expression of genes encoding antioxidative enzymes and through increasing the activities of most antioxidative enzymes as well as the contents of ascorbate and glutathione.