Molecular Mechanism And Functional Study Of GmWRKY45,GmWRKY46 And Gm6PGDH1 Involved In Phosphate Starvation Response In Soybean (Glycine Max(L.)MERR.)

Phosphorus(P)is one of the essential mineral nutrients for plant growth and development.It is widely involved in a series of biological processes such as material synthesis,energy transfer and signal transduction in plants.At the same time,P is also a key component of many biological macromolecules.Plants can obtain essential phosphorus only through the uptake of inorganic phosphate(Pi)from the soil by their roots.Although the overall P content of the soil is high,most of the P is fixed by cations such as calcium,iron and aluminum or converted into organic state by soil microorganisms which are difficult to be absorbed and utilized by plant roots.Nearly half of the world’s arable land has insufficient available P,limiting crop productivity.In response to Pi starvation,plants have developed many adaptive mechanisms to improve soil P utilization efficiency,including changed root system architecture,increased root exudates,symbiosis with mycorrhizal fungi,and so on.At the molecular level,the Pi starvation response of plants is tightly regulated by a complex signal network.Although many genes have been identified that play key roles in this signaling network,the signaling molecular network in plants in response to Pi starvation is still composed of several poorly understood modules.Soybean is an important grain and oil crop and plays an important role in ensuring national food security.Soybean is a phosphorusloving crop,which needs the supply of phosphorus at all stages of growth and development.It is of great significance to further study the molecular mechanism of soybean Pi starvation response and search for key regulatory genes for cultivating new varieties of soybean with Pi starvation tolerance.In this study,soybean Pi starvation tolerance candidate genes GmWRKY45,GmWRKY46 and Gm6PGDH1 were used as the research object,soybean genome sequencing variety Williams 82 and model plant Arabidopsis thaliana columbia-0(Col-0)type were used as experimental materials.The expression of genes function characteristics and functional characteristics by using through bioinformatics analysis,real-time fluorescent quantitative PCR(RT-qPCR),the conversion of Arabidopsis,transcriptome sequencing-seq(RNA-seq),yeast one-hybrid system,yeast two-hybrid system,chromatin immune co-precipitation(ChIP),soybean transformation,soybean transformation of hairy root,virus induced gene silencing(VIGS),and other technology to research.The main results obtained are as follows:1.GmWRKY45 belong to Ⅱc group of WRKY transcription factors(TFs)family.Subcellular localization showed that GmWRKY45 was in the nucleus.The RT-qPCR results showed that GmWRKY45 was mainly expressed in the root,flower,and pod of soybean,and its transcription was induced by Pi starvation and salt stress.Vermiculite culture experiments showed that compared with wild-type(WT),the GmWRKY45-overexpressing transgenic Arabidopsis bolting not inhibited by Pi starvation,and the contents of chlorophyll and anthocyanin were lower.The results of phytagel culture experiment showed that GmWRKY45-overexpressing transgenic Arabidopsis showed significantly higher lateral root length,lateral root number,total P concentration,Pi concentration and dry and fresh weight than WT under both Pi-sufficient and Pi-deficient conditions.Further RT-qPCR analysis showed that the expression of the Pi starvation induced(PSI)genes in GmWRKY45-overexpressing transgenic Arabidopsis was enhanced compared with that of the WT under Pi-sufficient condition,but decreased under Pi-deficient condition.In addition,under salt stress,the seedling survival rate and soluble sugar content of GmWRKY45-overexpressing transgenic Arabidopsis were significantly higher than those of WT.At the same time,GmWRKY45-overexpressing transgenic Arabidopsis showed partial sterility,including partial abortion of pollen,shorter siliques,and larger seeds with reduced number.In conclusion,GmWRKY45 may be widely involved in plant response to abiotic stress and regulate plant fertility.2.GmWRKY46 belong to the Ⅲ group of the WRKY TFs family.Subcellular localization showed that GmWRKY46 was in the nucleus.The Y2 H assay showed that GmWRKY46 had transcriptional activation activity.The RT-qPCR results showed that GmWRKY46 was stably expressed in the roots of soybean at the first trifoliate stage(V1),full bloom stage(R2)and full seed(R6)stage,with a very high transcriptional abundance in the leaf of R2 and almost no expression in stem,leaf,pod,and seed of R6.Pi starvation induced GmWRKY46 transcription,especially in soybean roots.Vermiculite culture experiments showed that the GmWRKY46-overexpressing transgenic Arabidopsis plants bolting were not inhibited by Pi starvation compared with the WT.The results of phytagel culture experiment showed that overexpression of GmWRKY46 significantly improved primary root length,lateral root length,lateral root number,fresh weight,and Pi concentration of transgenic Arabidopsis under Pi-deficient condition,moreover,the lateral root length and number of transgenic Arabidopsis under Pi-sufficient condition were also significantly higher than that of WT.Genes expression of GmWRKY46-overexpressing transgenic Arabidopsis and WT plants under three different Pi levels were compared by RNA-seq.The results showed that the expression levels of many genes involved in energy metabolism,stress response and plant hormone signaling were altered in the GmWRKY46-overexpressing Arabidopsis.Among these differentially expressed genes(DEGs),one gene AtAED1 encoded a family of proteins known as eukaryotic aspartate proteinases(APs)was screened and its function was studied.The results of vermiculite culture showed that the bolting of AtAED1-overexpressing transgenic Arabidopsis plants was not inhibited by Pi starvation compared with the WT,and the plant fresh weight and the Pi concentration of shoots were significantly higher under the Pi-deficient condition than those of the WT.YIH and ChIP-qPCR assays showed that GmWRKY46 could directly bind to W-box motif on AtAED1 promoter in vitro and in vivo.Therefore,GmWRKY46 may enhanced Pi starvation tolerance of transgenic Arabidopsis by activating target gene AtAED1.Furthermore,overexpression of GmWRKY46 in soybean hair roots significantly increased the total P concentration of transgenic composite plants under Pi-deficient condition,and enhanced hairy roots development.The above results showed that GmWRKY46 enhanced the tolerance of transgenic plants to Pi starvation and root development,and may be a positive regulator of Pi starvation tolerance in soybean.3.Gm6PGDH1 belong to soybean 6-phosphogluconate dehydrogenases(6PGDH),and its prokaryotic expression product had dehydrogenase activity in vitro.Gm6PGDH1 was expressed in all tissues of soybean,and the highest expression was found in the root at the R2 stage,and it was strongly induced by Pi starvation.Subcellular localization showed that Gm6PGDH1 was in the cytosolic.The results of a heterogeneous transformation system showed that the overexpression of Gm6PGDH1 also enhanced the fresh weight,primary root length,lateral root length and Pi concentration of transgenic Arabidopsis under Pi-deficient condition,while the content of ROS was lower than that of WT.The results of soybean hair roots genetic transformation experiment showed that the overexpression of Gm6PGDH1 significantly increased the dry weight and total P concentration of transgenic hairy roots under Pi-deficient condition,and weakened some GmPAPs genes response to Pi starvation.The results of soybean genetic transformation experiment showed that the root length and number of root tips of Gm6PGDH1-overexpressing transgenic soybean were significantly higher than those of WT,while the chlorophyll content was lower than that of WT under Pideficient condition.The root activity test showed that the root activity of Gm6PGDH1-overexpressing transgenic soybean was higher than that of WT under Pi-deficient condition.In addition,Gm6PGDH1-overexpressing transgenic soybean have lower levels of reactive oxygen species(ROS)level and higher antioxidant enzyme(SOD,POD and APX)activities when compared with WT under Pi-deficient condition.At the same time,the expression of some oxidative stress related genes decreased in response to Pi starvation compared with WT under Pi-deficient condition.These results suggested that the overexpression of Gm6PGDH1 may weakened the damage of Pi starvation to ROS homeostasis in the root tip of transgenic soybean by regulating the antioxidant system.The results of virus-induced gene silencing(VIGS)indicated that silencing Gm6PGDH1 did not affect the dry weight and total P concentration of silenced soybean plants under Pi-deficient condition,suggesting that it might have functional redundancy in soybean.The above results suggested that Gm6PGDH1 may enhanced the tolerance of soybean to Pi starvation by improving root development and regulating antioxidant system,and the positive effect of Gm6PGDH1 on Pi starvation was not limited to soybean.In conclusion,three PSI genes GmWRKY45,GmWRKY46 and Gm6PGDH1 were identified and cloned in this study.They played an important role in the response to Pi starvation in soybean.These results provide a basis for understanding the signal molecular regulation network of Pi starvation in plants and improving the molecular genetics and breeding of Pi starvation tolerance in soybean.