Transcriptomics Analysis Of Soybean Nodules Responsive To Phosphorus Deficiency And GmSPX5 Functions In Regulating Nodule Growth

Soybean is an important grain and oil crop in China.As a typical legume,soybean can interact with rhizobia and form a new organ,nodule.Symbiotic nitrogen fixation（SNF）occurs in nodules and thus facilitates atmosphere nitrogen to be reduced into ammonia,which provides a sustainable source of nitrogen for plants.Therefore,SNF is very important for agricultural production and environmental protection.Phosphorus（P）is an essential macronutrient for plant growth and development.It is not only a major component of essential structural molecules such as nucleic acids and proteins,but also participates in many important biochemical processes such as energy conversion and enzyme activity regulation.However,due to physicochemical properties of phosphate（Pi）fertilizers,they are easily fixed by soil particles,and thus lead to relative low availability on soils.It has been documented that nodules have developed a special set of physiological and molecular mechanisms in adaption to P deficiency,such as high capability to maintain Pi homeostasis in nodules.However,genome wide analysis of gene expression profiles in soybean nodules responsive to P deficiency,and the functions of the key regulator remain largely unkown.In this study,several physiological mechanisms underlying soybean nodules adaptation to P deficiency were studied.Subsequently,global gene expression profiles of soybean nodules responsive to P deficiency were analyzed.Finally,functions of a Pi starvation up-regulated gene,GmSPX5 were characterized to be involved in regulating soybean nodule growth.The main results are shown as follows:（1）Phosphorus deficiency significantly inhibited soybean growth and nitrogen-fixtion ability in soybean nodules,mainly reflected by significant decreases of nodule size,biomass,and nitrogenase activity.Although Pi concentration in nodules significantly decreased by P deficiency,the decrease was significantly less than that in leaves and roots,indicating that nodules had a strong ability to maintain the Pi homeostasis.Howerer,P deficiency increased the total amino acid concentration of nodules,especially for asparagine,which was increased by 36%.Similarly,acid phosphatase activity in root nodules were also significantly increased by P deficiency,mainly due to increased activity of original acid phosphatase isozymes,and induced new acid phosphatase isoenzymes.（2）The RNA-seq technique was used to analyze global changes of gene expression profiles in soybean nodules responsive to P deficiency.A total of 2,989 differentially expressed genes（DEGs）were identified in soybean nodules,including 1,914 up-regulated genes and 1,075 down-regulated genes by P deficiency.Functions of DEGs were mainly involved in nutrient acquisition and translocation,transcriptional regulation,metabolic pathways and other biological processes.Among them,29 genes might be involved in Pi homeostasis maintenance in soybean nodules,such as 9 high affinity Pi transporter genes,12 purple acid phosphatase genes and 8 SPX genes.（3）A P deficiency up-regulated gene,GmSPX5 was selected for further studies.Expression patterns of GmSPX5 were analyzed through both qRT-PCR and GUS staining of P_GmSPX5:GUS transgenic soybean plants.It was found that expression levels of GmSPX5were significantly increased in nodules after 15 d of P deficiency.Moreover,GmSPX5 was mainly expressed in the infected cells in soybean nodules.Meanwhile,GmSPX5 was found to be colocalized in the nucleus and cytoplasm through subcellular localization analysis in tobacco leaves.（4）The yeast two-hybrid system was conducted to identify the target proteins interacted with GmSPX5.Interactions were firstly examined between GmSPX5 and five GmPHR members in soybean,including GmPHR25 functions in controlling Pi homeostasis in soybean,and four GmPHR members（i.e.,GmPHR9,17,22,32）exhibiting high homology with AtPHR1 in Arabidopsis（Arabidopsis thaliana）.The results showed that interaction between of GmSPX5 and GmPHR members was not observed.Meanwhile,using GmSPX5 as the bait protein,a soybean full-length cDNA library was screened.A total of eight proteins were found to be interacted with GmSPX5,including two transcription factors,GmNF-YC4 and GmJAZ.（5）Functions of GmSPX5 were further examined in soybean transgenic lines with GmSPX5 overexpression and suppression.It was found that GmSPX5 overexpression significantly increased nodule number,the density of infected cells within soybean nodules,and thus enhanced plant growth as reflected by a significant increase of biomass,nitrogen and phosphorus content under normal P conditions.Furthermore,it was observed that overexpressing GmSPX5 significantly increased soybean grain number and grain dry weight in field experiments.Subsequently,RNA-seq was used to analyze changes of global gene expression profiles of nodules between wild type and the transgenic line with GmSPX5overexpression.The results showed that overexpressing GmSPX5 led to increased transcripts of 131 genes in soybean nodules,in which a set of genes were involved in the asparagine synthesis and lipid transport.The results strongly suggested that GmSPX5 might affect soybean nodule growth and development by regulating asparagine synthesis and lipid transport.In summary,genome wide analysis of gene expression patterns was conducted in soybean nodules,suggesting complex molecular mechanisms underlying soybean nodules adaptation to P deficiency.Subsequently,functional characterization of a Pi starvation responsive gene,GmSPX5 suggested that GmSPX5 could regulate asparagine synthesis and lipid transport,and thus enhance soybean nodule growth.Results from the study provide theoretical basis,candidate genes and transgenic resources for development of cultivars with both high nitrogen and phosphorus efficiency.