Cloning And Functional Analysis Of A Citrate Transporter AhFRDLl Gene In Peanut

Aluminum(Al)and iron(Fe)are the first and second abundant metal elements in the earth's crust.However,these metal elements have different effects on plant growth:Al is toxic to plant growth at acidic soil condition which contains low pH value,whereas iron is an essential micronutrient for plant growth and development.Therefore,plants have evolved different mechanisms to acquire Fe and to cope with Al stresses.Citrate transporter gene which belongs to MATE family is the key factor involves in Al detoxification or Fe translocation.Currently,some of them are involved in Al detoxification by citrate exudation from root tip;the others mediate secretion of citrate into xylem which involved in the long-distance transport of Fe.Although HvMATE has the two biological functions only in Al tolerance barley varieties,so it is not universal in barley.We report here the isolation and characterization of a citrate transporter in peanut(Arachis hypogaea L.),which is proposed to be involved in the translocation of Fe from root to shoot,and Al-induced secretion of citrate in plant.The electrophysiological,gene location and RNAi in peanut hair root transformation studies showed that:1.AhFRDL1 gene was obtained from peanuts through homologous cloning.Through the phylogenetic analysis,it was found that this gene belongs to the MATE family,and its encoding protein contains 12 transmembrane domains.Subcellular localization of tobacco leaf epidermal cells showed that the gene encodes a plasma membrane protein.AhFRDL1 was expressed in Xenopus oocytes,which significantly promoted the extracellular secretion of citrate.Indicating that AhFRDL1 encodes a citrate transporter.2.The expression of AhFRDL1 was up-regulated in roots and shoots of peanut for 7 days' Fe deficiency.By in situ hybridization and GUS staining,it was found that this gene was mainly expressed in the phloem of stem and new leaf,pericycle of root.Under Fe deficiency,the AhFRDL1 expression in the root is extended from pericycle to the endodermis.In the peanut root silencing of AhFRDL1 significantly inhibited the xylem citrate concentration and the active iron in new leaf,and the new leaf appeared more severe Fe deficiency induced chlorosis symptom.These results indicate that the citrate transporter AhFRDL1 secreted citrate into xylem in the peanut root to promote the transport of Fe to the shoot.It provided the molecular and physiological evidences that AhFRDL1 played an important role in promoting the transfer of Fe from root to shoot in peanuts.3.AhFRDL1 was apparently induced by Al toxicity in peanut roots.The expression level of AhFRDLl was significantly increased when the root tip was treated with 80 ?M Al concentration for 24 h,and the concentration of citrate secreted by root was significantly higher than that of control.The site of expression of AhFRDL1 gene was induced by Al in root,and the AhFRDL1 expression in the root is extended from the stele to the entire cross section of the root tip.Electrophysiological studies indicated that the activity of AhFRDL1 protein is induced by external Al induction.Therefore,as a citrate transport gene,AhFRDL1 is induced by Al at root tips to promote root secretion of citrate,significantly improving the ability of peanut root to resist Al toxicity.4.The AhFRDL1 gene was significantly affected by Fe deficiency and Al toxicity in the peanut varieties associated with Fe efficient(LH11)and Al resistance(LH14,DBS),resulting in an increase in the concentration of citrate in xylem and secreted by roots.It improved the physiological and ecological adaptability of peanuts in different environments.In summary,AhFRDL1 gene has the biological functions of Fe translocation and Al tolerance,and the realization of the two functions may be regulated by Fe deficiency and Al specific induced transcription factors which were found in the promoter region of AhFRDL1.And the regulated expression of this gene mainly occurs in the Fe efficient or Al resistance varieties.This research provides theoretical basis and technical support for cultivating excellent resistant peanut varieties.