Funtional Analysis And Post-translational Regulation Of Phosphate Transporter Genes GmPT5/7 In Soybean

Soybean[Glycine max?L.?Merr.]is one of the most world widely grown leguminous crops,and could form nodules through symbiosis with rhizobia for biological nitrogen fixation?BNF?,which acts as a central player in agro-ecosystems.Due to the high temperature and intensive rainy conditions in tropical and subtropical regions,nitrogen?N?and phosphorus?P?availability are quite low in most soils,which have become the main limiting factors on agricultural production in these regions.In agricultural practices,rhizobium inoculation has been used for more than 100 years to improve yield of legume crops,and the promotion of P fertilization on N efficiency has also existed in soybean cultivation,while the underlying mechanism is unclear.With the development of modern biology,the molecular mechanisms of recognization between rhizobia and host legume roots for initiation,organogenesis and development of nodules have been making great progress.Nodule development and the process of BNF need a lot of nutrients?especially P?,however,understandings of nutrient entry?e.g.P?into nodules are still limited.Here,we combined physiological,molecular approaches from greenhouse to field,to analyze the functions and post-translational regulations of the two important gene,Gm PT5 and Gm PT7,which might control P entry in soybean nodules.The main results are shown as follows:?1?GmPT7 is involved in direct phosphate?Pi?uptake from rhizosphere and Pi translocation from host cells to bacteroids.Expression pattern analysis showed that GmPT7highly expressed in nodules,which was significantly up-regulated by P deficiency.Yeast heterologous complementary and subcellular location assays demonstrated that GmPT7was a dual-affinity Pi transporter localized on plasma membrane.Tissue localization showed that GmPT7 was localized on nodule cortex and infected cells.In vitro assays showed that overexpression/knockdown of GmPT7 exhibited higher/lower[³³P]Pi accumulation in nodules and bacteroid compared to that of the empty vector control plants,respectively.Transgenic soybean composite plants and whole transgenic soybean plants overexpressing/knockdown of Gm PT7 exhibited enhancement/inhibitation of soybean nodulation,plant N/P content and plant biomass.?2?The combination of GmPT5 and GmPT7 controls most of the Pi acquisition in soybean nodules.Double suppression of GmPT5 and GmPT7 in the composite transgenic lines resulted in nearly complete elimination of nodulation and reduced bacteroid in infected cells,thus,decreased plant N/P content and soybean biomass.These results illustrated that the combination of GmPT5 and GmPT7 played a critical role in Pi entry into soybean nodules.?3?GmPT5/GmPT7 affected the dominant Pi source for nodules,and thus influenced soybean N₂ fixation and yield.Overexpressing/knockdown of GmPT5 or GmPT7 soybeans inoculated with rhizobia exhibited increase/decrease of soybean effective pod numbers,seed numbers and yields under hydroponics conditions.In two years'field trails,alteration of GmPT5 or GmPT7 expression indeed significantly affected soybean yield.?4?Besides nodule Pi entry,there might be other fuctions of GmPT5 to increase yield.Expression pattern analysis showed that GmPT5 also highly expressed in flowers,which was significantly up-regulated by low P.Tissue localization showed that GmPT5 was localized on nodule,leafstalk and calyx.Immunostaining showed that GmPT5 was localized on the plasma membrane of calyx.Overexpressing/knockdown of GmPT5soybeans without rhizobium inoculation exhibited increase/decrease of soybean biomass,flower numbers,effective pod numbers,seed numbers and yields under hydroponics conditions.The results indicated that GmPT5 maybe affect Pi homeostasis in soybean flowers,and therefore,affect flowering time,flower number and effective pod number,and thus,yield in soybean.?5?Besides nodule Pi entry,GmPT7 might also function in other pathways to increase soybean yield.Expression pattern analysis showed that GmPT7 expressed in leaves,flowers,roots and nodules.Tissue localization showed that GmPT7 was localized on leaf,nodule,flower,pod,pod wall and seed coat.Immunostaining showed that GmPT7 was localized on the vascular tissue of roots and the plasma membrane of flowers.Knockdown of GmPT7 soybeans without rhizobium inoculation exhibited early flowering under hydroponics conditions,and overexpressing/knockdown of GmPT7 exhibited increase/decrease of effective pod numbers,seed numbers and yields.The results indicated that GmPT7 maybe affect plant Pi homeostasis,and thus,affect yield in soybean.?6?Analysis of the Pi transporter traffic facilitator?PHF?family have been done in the present study.In total,there were 2 members in the soybean PHF family,GmPHF1 and GmPHF2,and both of them had the conserved WD40 domains of PHF1 proteins according to bioinformatics analysis.Phylogenetic analysis revealed that the two PHFs could be categorized into one subgroup.These two PHF genes shared similar temporal and spatial expression patterns in leaves,stem,root tip,roots,nodules and flowers,with the highest expression in roots,and the expression was up-regulated by Pi stravation.Furthemore,the expression of GmPHF1 and GmPHF2 was affected in different degrees by different abiotic stresses,including potassium?K?and iron?Fe?deficiencies,cold and hot stresses.?7?GmPHF1 and GmPHF2 has been functionally analyzed currently.Subcellular location assays showed that GmPHF1/2 localized on the endoplasmic reticulumv?ER?.Yeast two-hybrid and Bimolecular Fluorescence Complementation?BiFC?showed that there were interactions between GmPHF1/GmPHF2 and GmPT5/GmPT7 in yeast cells and plants.Overexpressing of GmPHF2 increased numbers and fresh weight of nodules,N/P content,plant biomass and yield in soybean under both low P and high P conditions.Knockdown of GmPHF2 decreased plant P content and yield in soybean under high P conditions.Above all,our study analyzed the functions of two soybean Pi transporter genes,GmPT5/GmPT7,and PHF gene GmPHF2 using transgenic soybean materials through modern molecular biological approaches.The results showed that GmPT5 and GmPT7controlled most of the Pi entry in soybean nodules,and played critical roles in organogenesis and development of nodules.The expression of GmPT5,GmPT7 or GmPHF2 had great influence on soybean yield.This study revealed the physiological and molecular mechanisms of Pi entry in soybean nodules,which not only provides the scientific basis and candidate genes for genetic improvements on N and P efficiency in soybean,but also generates several new soybean materials coordinated with N and P efficiency.