A Study On Mechanisms Of Arbuscular Mycorrhizal Fungus Regulating Drought Resistance And Phosphorus Metabolism Of Lycium Barbarum Under Water Stress

In this paper,Lycium barbarum L.and Rhizophagus irregularis were chosen as experimental materials.By means of real-time quantitative PCR(q PCR),COnsensusDegenerate Hybrid Oligonucleotide Primer(CODE-HOP)based homology cloning,RapidAmplification of CDNA Ends(RACE),yeast mutant complementation,protein transient expression in tobacco and laser scanning confocal microscopy,this research analyzed the effects of arbuscular mycorrhizal(AM)fungus on phosphorus level,water status,photosynthesis and sugar level of L.barbarum under different degree of water stress,studied the response of AM-induced and non-AM-induced phosphate transporter genes expression to the phosphate uptake and redistribution of L.barbarum under water stress,focusing on the phosphorus metabolism in the "carbon-phosphorus exchange" of AM symbiosis,revealed the mechanism of arbuscular mycorrhizal fungus influencing drought resistance and phosphorus metabolism of L.barbarum under water stress.The main results and conclusions are as follows:1.Response of AM fungal community in rhizosphere of L.barbarum to phosphate treatment in mycelium compartmentBy using the rhizosphere soil of L.barbarum for enrichment of AM fungi in the selfdeveloped layered culture pot,and supplied phosphate treatment in mycelium compartment of the layered culture pot to detect whether the AM fungal community in L.barbarum roots is affected.Results showed that AM fungi were sporulated in vast numbers in high-phosphate mycelium compartment,R.irregularis was the dominant strain by the means of single-spore PCR.Low-phosphate treatment in mycelium compartment increased the species richness of AM fungi in L.barbarum roots.These indicated that the high-phosphate treatment in mycelium compartment was suitable for propagation of AM fungi,L.barbarum would take the "the more,the better" strategy to recruit AM fungi under low-phosphate treatment in mycelium compartment.2.Effects of AM fungus on water,photosynthesis and sugar levels of L.barbarum under water stressBy treating AM plants and control plants of L.barbarum with different degree of water stress in summer(average temperature: 30°C),we investigated leaf water status,aquaporin expression in roots,leaf temperature,leaf gas exchange and chlorophyll fluorescence parameters as well as sugar levels of leaves and roots.Results showed that AM plants increased Gs and Tr significantly under normal water condition and moderate water stress,with reducing RWC and leaf temperature,comparing with control plants.AM plants increased the transcript level of Lb PIP2-1,Lb TIP3-1 of L.barbarum and Rir-AQP2 of R.irregularis in roots under different degree of water stress,comparing with control plants.The sugar levels of AM plants under normal water condition was similar to that of control plants under moderate water stress.Severe water stress inhibited the photochemical reaction of leaves in control plants,not affect that of AM plants.These results indicated that AM plants displayed an obvious superiority over control plants at coping with moderate water stress in the hot environment;AM plants maintained normal photochemical processes under severe water stress,while sugar levels were affected strongly.3.Effects of AM fungus on phosphorus performance of L.barbarum under water stressBy measuring the growth,tissue phosphorus content,leaf anthocyanin content and root acid phosphatase activity of AM plants and control plants under different degree of water stress,results showed that severe water stress reduced leave biomass of AM plants and control plants,severe water stress increased anthocyanin content of control leaves.Compared with control plants,AM plants increased the biomass of roots and stems,increased phosphorus content of the whole plant,decreased the anthocyanin content of leaves under different degree of water stress.These suggested that water stress indirectly leads to phosphorus(P)deficiency in plants,and supply of phosphate(Pi)by AM fungus to plants was adequate.4.Cloning of phosphate transporter genes from L.barbarum and their promoter regionsBy the homology cloning strategy and High-tail PCR,Lb PT1-Lb PT5 and Lb PT7 from PHT1 family in L.barbarum were cloned,their promoter regions were amplified.Six phosphate transporters had the structural features of PHT1 family protein.The cis-elements analysis showed that promoter regions of six PHT1 genes were similar to that of other homologous PHT1 genes,having the same cis-element,P1 BS,PHO,MYCS,etc.In combination with phylogenetic analysis of Lb PTs and cis-element analysis of their promoter regions,we speculated that Lb PT3,Lb PT4 and Lb PT5 were AM-induced.These results indicated that the phosphate transporter genes of L.barbarum had the same evolutionary pattern as other Solanaceae plants,such as Lycopersicon esculentum,Solanum tuberosum,Nicotiana tabacum,etc.5.Yeast complementary analysis and subcellular localization of phosphate transporters from L.barbarumBy carrying out functional verification and subcellular localization test of six phosphate transporters,we found that Lb PT1,Lb PT3 and Lb PT7 had the Pi transport activity and Pi sense function of the high affinity phosphate transporter PHO84 of yeast,Lb PT1 may not have the Pi sense function of PHO84;Lb PT1,Lb PT3 and Lb PT7 were localized in yeast cell membrane,Lb PT2 was centralized at several points in yeast cell membrane,while Lb PT4 and Lb PT5 were not located in yeast cell membrane;Lb PT1,Lb PT2,Lb PT4,Lb PT5 and Lb PT7 were localized in plasma membrane of tobacco leaf cell,Lb PT3 was localized in plasma membrane of tobacco root cell.These results indicated that the Pi transport activity of Lb PT1,Lb PT3 and Lb PT7 were attributed to their correct localization in yeast cells,six phosphate transporters were localized in plasma membrane of tobacco cell,the correct localization of Lb PT3 might need posttranslational modifications in root.6.Effects of AM fungus on the expression of phosphate transporter genes from L.barbarum under water stressBy detecting the transcriptional response of Lb PTs to AM symbiosis,phosphate(Pi)treatment and the different degree of water stress,results showed that Lb PT3,Lb PT4 and Lb PT5 were induced in AM roots under low Pi treatment;Lb PT1,Lb PT2 and Lb PT7 were expressed in leaves and roots,the transcript level of which were enhanced in senescent leaves;All six were inhibited under high Pi treatment;moderate water stress increased the transcript levels of Lb PT1,Lb PT2 and Lb PT7 in leaves and roots of control plants,water stress increased the transcript level of Lb PT1 in roots of AM plants;Besides,water stress did not affect the transcript level of Lb PT3,Lb PT4 and Lb PT5.These results suggested that L.barbarum had three AMinduced phosphate transporter genes(Lb PT3,Lb PT4 and Lb PT5),similar to other Solanaceae plants;Pi deficiency induced by moderate water stress increased the transcript level of Lb PT1,Lb PT2 and Lb PT7,to make the three perform Pi uptake in roots and Pi redistribution in shoots;the mycorrhizal Pi uptake pathway was high-efficiency under water stress,satisfying the requirement of root system,with up-regulation of Lb PT1 in AM roots to transfer Pi from roots to aerial parts under water stress.7.Regulation of Lb PT7 from L.barbarum by ethyleneBy supplying ethylene treatment to AM roots and control roots under low/high Pi conditions,this study investigated the AM colonization and the transcript level of Lb PT7 under these treatments,results showed that ethylene reduced the colonization rate of arbuscule by 55.3% under low Pi condition,increased the colonization rate of arbuscule by 3.6 times under high Pi condition;meanwhile,ethylene upregulated Lb PT7 under low Pi condition,not under high Pi condition.These results suggested that ethylene would enhance the direct Pi uptake by upregulating Lb PT7 and weaken the mycorrhizal Pi uptake under low Pi condition;ethylene would restore the mycorrhizal Pi uptake pathway under high Pi condition.