| Arbuscular mycorrhiza(AM)fungi expand the root absorption area through a large number of external hyphae to enhance the absorption of water and nutrients by plants,and improve drought tolerance.Many scholars have studied the physiological and biochemical mechanisms of the drought-tolerant effects,but the underlying molecular mechanisms are still unclear.The 14-3-3 protein is a protein target that plays a key role in plant biotic and abiotic stress.In this study,we investigated the effects of Rhizophagus intraradices on drought tolerance physiological indicators and 14-3-3 protein family genes expression in Populus cathayana,explored the effects of AM fungi and phosphorus levels on reactive oxygen species homeostasis,phosphorus metabolism and sugar metabolism with the expression of 14-3-3genes under drought stress,and explored the molecular mechanisms of AM fungi to enhance drought tolerance in P.cathayana.The main findings are as follows:1.Effects of AM fungi on drought-tolerant indicators and 14-3-3 genes expression of P.cathayanaUnder mild drought(45%～50% of field water capacity)and extreme drought(20%～25%of field water capacity)conditions,drought tolerance indicators such as root activity,biomass,photosynthesis,antioxidant enzyme activity,soluble sugars and phosphorus content of P.cathayana inoculated with AM fungi were all improved compared with non-inoculation.Under mild drought stress,mycorrhizal colonization was higher,AM fungi promoted plant height and root activity,and significantly increased the root biomass,the root-to-shoot biomass ratio,net photosynthetic rate,transpiration rate,antioxidant enzyme activity,soluble sugars(leaves)and phosphorus content.Similarly,the expression of 14-3-3 protein gene Pc GRF10 was most affected by mycorrhizal induction in roots and leaves under mild drought,while Pc GRF11 was only induced by drought in roots.The expression levels of Pc GRF10 and Pc GRF11 were significantly and positively correlated with superoxide dismutase(SOD),peroxidase(POD)and soluble sugar content.This indicates that AM fungi could improve photosynthesis,belowground biomass,nutrient absorption,antioxidant enzyme activity and14-3-3 protein genes expression,and may regulate the expression of genes encoding antioxidant and osmotic regulation to improve antioxidant enzyme system and osmotic regulation substance content and enhance drought tolerance of P.cathayana,with the strongest effect under mild drought.2.Effects of AM fungi and phosphorus addition on the drought-tolerant reactive oxygen species homeostasis and 14-3-3 genes expression of P.cathayanaP.cathayana were inoculated with AM fungi at four different phosphorus levels(P0: 0mg,P50: 50 mg,P100: 100 mg and P150: 150 mg)under drought stress(30%～35% of field water capacity).Compared with non-inoculation,AM fungi significantly increased plant height and root activity,especially P0(47.56% and 93.52%)and P50(39.00% and 40.93%);increased aboveground and total biomass significantly at P50(60.44% and 35.57%)and belowground biomass only at P50(6.76%);enhanced CAT activity in leaves(P0: 266.47%and P50: 204.29%)and roots(P50: 172.15% and P100: 81.55%)and POD activity in roots(P100: 51.38%);significantly increased proline content in leaves(P100: 502.85%)and roots(P0: 222.35% and P50: 60.80%).The results showed that P150 had a negative effect on growth and AM fungi had a batter mycorrhizal effect at P50 level under drought stress.14-3-3 protein genes Pc GRF10(P0 and P100)and Pc GRF11(P50)were most induced by mycorrhiza;Pc GRF10 and Pc GRF11 were significantly positively correlated with reactive oxygen balance regulators(POD,CAT and proline in leaves and SOD,POD,CAT,proline and root activity in roots),and may regulate the expression of stress-related genes to improve antioxidant system,osmotic accumulation and root activity to maintain reactive oxygen species homeostasis.3.Effects of AM fungi and phosphorus addition on the drought-tolerant phosphorus metabolism and 14-3-3 genes expression of P.cathayanaP.cathayana were inoculated with AM fungi at four different phosphorus levels under drought stress.Compared with non-inoculation,AM fungi increased the phosphorus content of leaves,roots,and rhizosphere soil and the available phosphorus content of soil at four phosphorus levels,especially significantly increased at P100(248.10%,343.69%,113.45%and 46.84%)and P150(276.76%,492.30%,76.56% and 55.65%);increased the alkaline phosphatase activity in the rhizosphere soil but decreased in the leaves and roots;increased the phytase activity in root and rhizosphere soil;increased the content of phytic acid in leaves(P100)and roots(except P50);increased chlorophyll content decreased anthocyanin content.The results showed that P.cathayana had the strongest demand and stimulation for phosphorus at low phosphorus(P0)and high phosphorus(P150)levels under drought stress,but AM fungi may have the best mycorrhizal effect on drought-tolerant phosphorus metabolism at P100 level.The 14-3-3 genes Pc GRF10 and Pc GRF11 are positively correlated with regulatory factors for phosphorus metabolism(phytase and carotenoids in leaves,phytase,acid and alkaline phosphatase in roots).At the same time,the activities of phosphatase and phytase in rhizosphere soil were increased by mycorrhizal induction under drought stress.The 14-3-3proteins may regulate the expression of genes encoding phosphatase and phytase synthesis and secretion and promote production and secretion of phosphatase and phytase by roots into rhizosphere soil to promote the release of soil organic phosphorus and regulate phosphorus metabolism.4.Effects of AM fungi and phosphorus addition on the drought-tolerant sugar metabolism and 14-3-3 genes expression of P.cathayanaP.cathayana were inoculated with AM fungi at four different phosphorus levels under drought stress.Compared with non-inoculation,AM fungi in P.cathayana leaves increased sucrose and starch content(P100: 64.30% and 51.74%)at all phosphorus levels;increased the activities of sucrose synthase(P150: 11.67%)and sucrose phosphate synthase(P100: 42.10%);decreased the activity of acid and alkaline invertase,amylase(except P50)and starch phosphorylase(except P0).AM fungi in P.cathayana roots increased the contents of reducing sugar(P100: 7.58%)and soluble sugar(P100: 47.89%),sucrose(P100: 71.95%),and starch(P100: 72.65% and P150: 39.03%);increased the activity of sucrose synthase(P100: 41.28%),sucrose phosphate synthase(P100: 47.75%),amylase(except P150),starch phosphorylase(P0:34.42% and P100: 7.79%),and acid and alkaline invertase(P100: 13.51% and 15.06%)at all phosphorus levels.The results showed that AM fungi had the best mycorrhizal effect on drought-tolerant sugar metabolism at the P100 level under drought stress.The expression levels of 14-3-3 protein genes Pc GRF10 and Pc GRF11 were significantly positively correlated with sucrose synthase,sucrose phosphate synthase,sucrose and starch phosphorylase in leaves,reducing sugar,soluble sugar,sucrose,sucrose phosphate synthase,acid invertase,alkaline invertase,amylase,and starch phosphorylase in roots,and negatively correlated with starch content in leaves and roots,It was speculated that Pc GRF10 and Pc GRF11 may regulate the expression of genes encoding sucrose synthesis and secretion,and then induce sucrose and starch synthesized from leaves to be transported to roots through phloem to participate in osmotic regulation under drought stress.In summary,this study elucidates that the 14-3-3 gene induced by mycorrhizal fungi mediates drought-tolerance mechanism of P.cathayana may regulate the expression of genes encoding phosphatase and phytase synthesis and secretion and promote production and secretion of phosphatase and phytase by roots into rhizosphere soil to promote the release of soil organic phosphorus and regulate phosphorus metabolism;regulate the expression of genes encoding sucrose synthesis and secretion,and then induce sucrose and starch synthesized from leaves to be transported to roots through phloem to participate in osmotic regulation;regulate the expression of stress-related genes to improve antioxidant system,osmotic accumulation and root activity to maintain reactive oxygen species homeostasis. |
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