Functional Analysis Of Phosphate Transporter Genes MtPT5 And MtPT6 In Medicago Truncatula

Phosphorus is one of the essential macronutrients required by plant growth and development,but the phosphate concentration in soil is usually lower than that plants need.Phosphate resources are finite and diminishing rapidly because of the huge need in global agriculture;thus,it is important to improve phosphate acquisition efficiency and phosphate utilization efficiency in plants.Recently,the study of high-affinity phosphate transporter genes(PHT1 family)is adorable,because these genes play a key role in uptaking and translocating phosphate in plants.Moreover,microbio-plant symbiosis is essential for sustainable development of agriculture and ecosystem.Some studies have suggested that PHT1 play an important role in symbiosis between arbuscular mycorrhizal fungi and plants,and the symbiosis can improve phosphate acquisition of plants from soil.However,it is little to know the roles of PHT1 in symbiosis between rhizobium and plants.In this study,the expression patterns of PT1 family members in response to low phosphate stress were analyzed in Medicago truncatula.MtPT5 and MtPT6 genes,encoding phosphate transporters in Medicago truncatula were cloned and overexpressed in alfalfa and Arabidopsis mutants of pht1.1 and pht1.4,while the MtPT6 promoter-UdiA gene fusion was expessed in the roots of Medicago truncatula.The functions of these two genes were characterized according to the transgenic plants.The results are as following:1.According to the BLAST in NCBI,the PHT1 family is composed of 11 members but not 5 members reported previously in Medicago truncatula.In normal and low phosphate conditions,7 genes of PHT1 family were available by qRT-PCR.Most of these genes are expressed in roots,and all genes almost induced by low phosphate stress except MtPT7 in nodule.The expression of MtPT5 and MtPT6 genes was relatively high in nodules and induced by low phosphate stress.2.The phylogenetic analysis of amino acids revealed that MtPT6 was not closely related to other members of PHT1 family in Medicago truncatula,revealing that its functions may be different to others.3.The relationship between phosphate concentration and MtPT5 and MtPT6 expression levels in nodules was further analyzed.The phosphate concentration reduced fastest at the first three days,and then the reduction rate became lower and lower.The expression level of MtPT5 was higher and higher during the reduction of phosphate concentration,while the expression level of MtPT6 was signicantly higher than that before low phosphate stress in spite of the fluctuation.These results suggested that these two genes were likely related to phosphate homeostasis of nodules.4.The vectors of MtPT5 and MtPT6 genes for subcellular localization were successfully constructed,and subcellular localization analysis in tobacco revealed that the two genes were expressed in cellular membrane.5.The heterologous expression of MtPT6 in yeast mutant EY1707 could contribute to the yeast growth,revealing that the protein coded by MtPT6 had the founction of phosphate transporter.6.The overexpression of MtPT5 and MtPT6 in Arabidopsis mutants of pht1.1 and pht1.4 made seedlings more sensitive to arsenate treatment,and the phosphate content in pht1.1 was higher in high phosphate conditions by expressing MtPT6.These results suggest that the two genes can promote phosphate acquisition efficiency of plants.7.The analysis of promoter sequences of MtPT5 and MtPT6 revealed that both promoters contain the elements involved in the response to phosphate starvation and rootmotiftapox1 motifs.The fusion expression of MtPT5 and MtPT6 promoters-UdiA gene in Medicago truncatula suggested that the expression of two genes was induced by phosphate starvation.MtPT5 was mainly expressed in root tips,nodule tissues and vascular tissues of younger roots but not older roots.MtPT5 was also expressed in cortex strongly under low phosphate stress.MtPT6 was mainly expressed in vascular tissue and tips,and it was also expressed in cortex under low phosphate stress.In nodule,MtPT6 was also mainly expressed in vascular tissue and induced by low phosphate stress.These results suggest that these two genes not only play an important role in phosphate uptake and translocation but also are involved in phosphate homeostasis maintainance in nodules under low phosphate conditions.8.The MtPT6 gene was successfully transformed into alfalfa by Agrobacterium tumefaciens in this experiment,and the transgenic alfalfa plants were obtained through tissue culture,which provides the new material for alfalfa breeding.In conclusion,the expression patterns of PT1 family members were characterized successfully in various tissues of Medicago truncatula under normal and low phosphate conditions.MtPT5 and MtPT6 genes were highly expressed in nodules and responded to low phosphate stress.They could improve phosphate uptake in transgenic Arabidopsis.Moreover,MtPT5 and MtPT6 genes might prompt the translocation from roots to nodules to maintain phosphate homeostasis of nodules under low phosphate.