Functional Characterization Of GmPR10.1 And GmACP1 During Soybean(Glycine Max) Nodulation

Symbiosis between legume species and compatible rhizobium could convert gaseous N2 into nitrogen nutrient form to support plant growth,which efficiently decreased the dependent on environmental nitrogen nutrient,and reduced the green house gas emission during chemical fertilizer production.Soybean seeds contain 20%fat and 40%protein respectively,makes it one of the most important energy crop in the world.In this sense the research on soybean biosymbiosis is critical for the ecological environment protection and modern agriculture development.With the availability of soybean reference genome sequence and application of high-throughput approaches,molecular biology of soybean biosymbiosis encountered great chance and challenge.Up to date,soybean nodule formation process has been analyzed systematically by transcriptomic,proteomic and metabolomic study,and consequently plenty of genes,proteins and metabolites has been identified to be regulated when rhizobium infect soybean root hair,but largely functionally unknown.In this study,we analyzed biological function of two soybean gene,pathogenesis-related protein 10 and acyl carrier protein,during soybean rhizobium infection and nodule formation,with the purpose to uncover soybean-rhizobium interaction at the level of molecular mechanism and provide theoretical basis and gene resource for soybean genetic improvement.1.In this study,19 candidate genes were selected based on previously performed proteomic study.Of which,Glyma12g01970.1 and Glyma18g47950.1 were found to significantly reduce soybean nodule number when they were down-regulated.These two genes encode soybean pathogenesis-related protein 10(GmPR10.1)and soybean acyl carrier protein(GmACP1)respectively.There were 43 GmPR10s and 21 GmACPs coding gene in soybean genome.Evolutionary analysis showed that GmPR10s could be phylogenetically clustered into three groups.P-loop motif of the group in which GmPR10.1 located was the most conserved.Synonymous substitution rate analysis revealed GmPR10s had undergone two rounds of whole genome duplications in soybean,and the earliest divergence time was prior to that of Rosales and Asterales.GmACPs could also be clustered into three groups,and was found to be originated prior to Gymnospermae and Angyospermae divergence time.Ka/Ks analysis suggested a purify selection were occoured during evolutionary process of both GmPR10s and GmACPs.2.GmPR10.1 is distinct from other GmPR10s phylogenetically.GmPR10.1-GFP fusion protein localized GmPR10.1 to the cytoplasm of the tobacco epidermis cell.GmPR10.1 was expressed constitutively in all tissues tested,namely soybean root,stem,leaves,flower,and shoot apical meristem,and a relatively higher expression level was observed in root.GmPR10.1 was found to be induced by Bradyrhizobium japonicum(6hpi-48hpi),and reduced significantly at 16 days after inoculation.Additionally exogenous application of cytokinin could induce the expression of GmPR10.1.Biochemical analysis of GmPR10.1 showed an in vitro RNase activity,but no binding activity was observed to the ligand tested(trans-zeatin,kinetin and daidzein).Gene silencing of GmPR10.1 mediated by RNAi reduced nodulation number on transgenic root significantly.On the contrary,nodule number increased significantly by over expression of GmPR10.1.All these data indicated that GmPR10.1 played critical role in soybean nodulation by promoting nodule initiation during the process of rhizobium infection,which is absolutely distinct from traditional PR10.3.GmACP1 is a soybean acyl carrier protein containing a conserved phosphopantetheinyl transferase binding site(DSL motif),clustered with ACPs from legume species.GmACP1 was constitutively expressed in soybean root,stem,leaves,flower and shoot apical meristem,and relatively abundant in root.And also induced by rhizobium infection.RNAi-mediated gene silencing of GmACPl reduced soybean nodulation efficiency,but did not make any change in nodule infection zone.Over expression of GmACPl did not obtained opposite phenotype compared with GmACP1-RNAi.In comparison with control fatty acid composition in GmACP1-RNAi tissue had not been altered significantly.GmACPl was a plastidial protein confirmed experimentally by GmACPl-GFP fusion protein expressed in tobacco epidermis cell,which is consistent with the subcellular compartment of other plant ACPs involved in fatty acid synthesis.Taking together GmACPl was critical for soybean nodulation and could regulate nodule formation by some mechanism other than involved in fatty acid synthesis.