| The biological nitrogen fixation(BNF)of legumes provides a large amount of green and environmentally friendly nitrogen for agricultural production and plays an important role in sustainable agriculture.The nitrogen-fixing nodules development and nitrogen-fixation process require the high level of phosphorus(P).Therefore,the regulation of phosphate(Pi)uptake and homeostasis in nodules will inevitably affects the BNF efficiency.G3PT(Glycerol-3-phosphate transporter)exerts its important function as a sugar-phosphate/anion anti-porter in P transport.However,the role of G3 PTs involvement in regulating Pi uptake and allocation in nodules has not been widely reported.Here,we used soybean as plant materials to analyze GmG3 PTs family genes through.bioinformatics related methods.On this basis,GmG3PT3 which significantly induced by low phosphorus in nodules was screened and identified for further studies.In order to further clarify the functions of GmG3PT3 in soybean BNF,the transgenic soybean plants with overexpression and RNA interference(RNAi)of GmG3PT3 were grown in hydroponics under low phosphate(25 μM Pi)/high phosphate(250 μM Pi)conditions and in field,respectively.The findings are shown as follows:(1)Bioinformatics results indicated that there are totally 6 glycerol-3-phosphate transporter in soybean.GmG3 PTs were classified into three distinct groups based on phylogenetic and gene structure analysis,and the class I includes GmG3PT1 and GmG3PT6;class II contains GmG3PT2 and GmG3PT5 and class III consisted of GmG3PT3 and GmG3PT4.The gene structure was highly similar,and the sequence similarity of the coding region exceeds 90% within the same class,respectively.(2)GmG3PTs expression patterns analysis indicated that GmG3 PTs were differentially expressed in six tissues including roots,stems,leaves,flowers,pods and seeds.Class I(GmG3PT1 and GmG3PT6)and II(GmG3PT2 and GmG3PT5)genes were predominantly expressed in pods and highly induced by Pi starvation.While class III(GmG3PT3 and GmG3PT4)genes,were dramatically up-regulated in all tested tissues by low P deficiency.Among them,GmG3PT3 had highest expression level in nodules and maintained the relative high expression levels in different developmental stages of nodules when compared with other GmG3 PTs family numbers.The results indicated that GmG3PT3 may play an important role in soybean nodulation and Pi homeostasis in nodules.(3)The results of subcellular and tissue localization showed that the GmG3PT3 located in the vacuole membrane,and mainly expressed in vascular bundle,cortex and infection zone in nodules.(4)Results from Pi concentration measurement showed that overexpression of GmG3PT3 promoted the Pi transport from cortex into fixation zone of nodules,but RNAi of GmG3PT3 resulted in accumulation of Pi in cortex and Pi concentration was decreased in fixation zone when compared to wild type.suggesting that altered the expression of GmG3PT3 might affect Pi allocation in nodules.(5)Results of hydroponics and field experiments showed that overexpression of GmG3PT3 significantly increased nodule number,nodule dry weight,and thus enhanced nitrogen(N)and P content,biomass as well as yield;RNAi of GmG3PT3 showed that significantly reduced nodule number,nodule dry weight,and thus decreased N and P content,biomass as well as yield when compared to wild type.(6)3.5 h after inoculation with rhizobium induced the expression of GmG3PT3 in soybean and RNAi of GmG3PT3 innocated with rhizobium carring GFP significantly reduced the infection threads when compared with wild type plants.All above,GmG3PT3 encodes a tonoplast localized sugar-phosphate/anion anti-porter,which mainly expressed in vascular bundle,cortex and infection zone of nodules.GmG3PT3 participated in Pi transport from nodule cortex to fixation zone,and therefore affected Pi allocation in nodules.Altering the expression of GmG3PT3 significantly affected the nodule number and nitrogenase activity,N and P content as well as biomass,and subsequently affected the soybean yield.Our study partiallyrecovered the physio-molecular mechanisms of phosphorus regulating biological N fixation in soybean,and thus provides the candidate genes for genetic improvements for soybean nutrient efficiency. |
