Identification And Functional Analyses Of Genes Encoding Proline Transporters In Rice(Oryza Sativa L.)

Proline(Pro)and ?-aminobutyric acid(GABA)were proved to play important roles in plant development and stress tolerance.ProTs are key members for transporting proline within plants.In this study,we identified and functional characterized the OsProT members in rice using Oryza Sativa L.var.Nipponbare.as research material.We analyzed their basic molecular characteristics and evaluated their phylogenetic relationships among different species.Properties of transport of amino acids were detected after heterologous expression of OsProTs in yeast mutant and subcellular localizations were identified by transient expression of OsProTs in protoplasts.In addition,expression of OsProT genes in different tissues and stress conditions were also determined using qRT-PCR.The results are as follows:(1)The bioinformation analyses of the basic characteristics of OsProTs showed that OsProT1,OsProT2 and OsProT3 were located on chromosomes 3,7 and 11 of rice,respectively.The proteins encoded by OsProTs are membrane-localized,and they possess 11 potential transmembrane domains except OsProT1,which only have 10.OsProTs showed higher homology to the monocotyledon such as Zea mays and Sorghum bicolor than to the dicotyledonous plants such as Arabidopsis thaliana and Lycopersicon esculentum,and OsProT2 has a relative higher homology to OsProT3.(2)Transient expression of GFP-OsProTs in plant protoplasts showed that both OsProT1 and OsProT3 are plasma membrane localized.(3)Ectopic expression of these OsProTs in yeast amino acid transport mutant elucidated that both OsProT1 and OsProT3 specifically mediate transport of Pro and GABA,while OsProT2 failed to transport any kind of amino acids.Further Kinetic analyses of Pro and GABA by OsProT1 and OsProT3 showed that the Vmax of OsProT1 and OsProT3 for Pro are 6.54±0.66 mM and 6.33±0.29 mM,the Km of OsProT1 and OsProT3 for Pro are 9.35±3.14 mM and 4.23±0.88 mM,respectively,while the Vmax of OsProT1 and OsProT3 for GABA are 6.92±0.35 mM and 6.60±0.37 mM,and the Km of OsProT1 and OsProT3 for GABA are 5.71±1.16 mM and 4.72±1.12 mM,respectively.(4)The expression pattern revealed by qRT-PCR analyses suggested that OsProT1 was preferentially expressed in leaf sheathes at vegetative growth stage and OsProT2 was mainly expressed in flag leaves.In contrast to the specifically expression pattern of OsProT1 and OsProT2,OsProT3 exhibit a relatively high expression level in many tissues including nodes,panicles and roots.These results indicated that OsProTs may have tissue specificity about their function and they work together for the distribution of proline within rice plants.(5)Further gene expression analyses of OsProTs under stresses showed that nitrogen deficiency or cadmium stress could affect the expression levels of OsProT1 and OsProT3.These data suggested that OsProTs could response to various stress signaling pathways at expression level,which may further mediates the transport of proline and even stress tolerance.Our results suggested that members of OsProTs efficiently transport Pro and GABA with low affinity,and appear to be involved in adaption to nitrogen deficiency and cadmium stress for rice plants.