Functional Characterization Of AtALMT12 In Cl~- Transport In Arabidopsis Thaliana And Cloning And Expression Analysis Of PcALMT12 In Pugionium Cornutum

Chloride ion(Cl~-),as the main toxic anion in saline soil,poses a serious threat to crop production.The mechanisms for most Cl~--sensitive plants to reduce Cl~-toxicity mainly include reducing net Cl~-uptake by roots,increasing vacuolar Cl~-compartmentation,decreasing net xylem loading of Cl~-.Increasing xylem unloading of Cl~-is an important approach to decrease net xylem loading of Cl~-,however,the protein involved in Cl~-xylem unloading has not been identified.It is of great significance to identify the key proteins and further explore their functions for enhancing salt tolerance of plants and increasing yield of forages and crops.AtALMT12(Aluminum-activated malate transporter 12),which locates at the plasma membrane,mediates Cl~-influx at the cellular level.Its encoding gene is highly expressed in root stelar cells in Cl~--sensitive plant Arabidopsis thaliana.Therefore,it can be speculated that AtALMT12may be a candidate protein involved in xylem unloading of Cl~-in A.thaliana,but there is no relevant experimental evidences yet.Besides,the homologous genes related to salt tolerance in different plants may display different responsive patterns to salt stress.Pugionium cornutum is a typical species with strong Cl~--tolerance;different from Cl~--sensitive plant A.thaliana,it can accumulate a large amount of Cl~-in shoots as a beneficial osmolyte to resist salt and drought stresses.The analysis on the expression pattern ofALMT12 homologous gene in P.cornutum under salt stress may lay an important theoretical foundation for elucidating the difference in the mechanisms underlying Cl~-transport between Cl~--tolerant and Cl~--sensitive species.In this study,in order to explore function of AtALMT12 in Cl~-xylem unloading in A.thaliana,the tissue location of AtALMT12 and its expression pattern under salt treatment were analyzed by GUS staining and q RT-PCR,the differences of ion accumulation and salt tolerance among wide-type seedling,atalmt12 mutants,tissue-specific complementation lines were analyzed.Meanwhile,theALMT12 homologous gene was cloned from the Cl~--tolerance species P.cornutum,and its expression patterns,tissue and subcellular localization were analyzed by q RT-PCR,in situ PCR and transient expression in tobacco,respectively.The main results can be summarized as follows:1.AtALMT12 was mainly expressed both in stomatal guard cells and root stelar cells,and its expression level in roots was significantly induced by salt treatments.2.The mutation of AtALMT12 caused a significant increase in Cl~-concentration in shoots under salt treatments,resulting in a decrease in salt tolerance.The stele-specific complementation of AtALMT12 significantly reduced shoot Cl~-content in atalmt12mutant,and restored the salt tolerance of atalmt12 to the same level as wide-type seedling;while the stomata-specific complement lines held the same shoot Cl~-content and Cl~--sensitivity with atalmt12 mutant,indicating that the increase in shoot Cl~-content and the decrease in salt tolerance of atalmt12 mutant were related to the functional impairment in root stele.It can be concluded that AtALMT12 is involved in xylem unloading of Cl~-in A.thaliana roots under salt stress,which plays an important role in salt tolerance of A.thaliana.3.The functional loss of AtALMT12 resulted in the up-regulated expression of At NRT1.8 involved in NO3~-xylem unloading in root,causing a significant reduction in shoot NO3~-content under salt stress;meanwhile,the expression of At SKOR,a functional gene related to xylem loading of K~+,was decreased in root,thus affecting K~+accumulation in shoot under KCl treatment.4.The full length of the cDNA of PcALMT12 was 2115 bp,which contained an open reading frame of 1683 bp,and encoded 560 amino acids.PcALMT12 was expressed specifically in root stele of P.cornutum,and the encoded protein located at the plasma membrane.5.The expression level of PcALMT12 in roots was significantly inhibited under salt treatment,which was contrary to the expression pattern of AtALMT12 in Cl~--sensitive A.thaliana responding to salt treatment.The results showed thatALMT12genes of Cl~--tolerance and Cl~--sensitive species displayed different responsive pattern to salt treatments.All above results revealed the function ofALMT12 in Cl~-xylem unloading in roots of Cl~--sensitive A.thaliana,and the responsive pattern ofALMT12 homologous gene in Cl~--tolerant species P.cornutum to salt stress.The results would lay an important foundation for subsequent studies on plant Cl~-tolerance mechanisms as well as the genetic improvement of stress tolerance in forages and crops.