Functional Analysis And Molecular Mechanism Study Of Key Phosphorus Efficiency Genes GmERF1 And GmPT8 In Soybean

Phosphorus(P)is one of the essential nutrient elements for plant growth and development.In plant cells,P concentrations are typically greater than 10 m M,while there is usually less than 10 μM P available in the soil solution,creating the need for active transport of P across the plasma membrane by specialized transporters.Soybean is an important grain and oil crop and has a greater demand for P than other crops.Low phosphorus(LP)stress will seriously affect soybean plant growth,flower bud differentiation,seed development and root nodulation formation,and ultimately affect soybean yield and quality.At present,the low content of available P in soil has become an important factor limiting the development of soybean production.Therefore,it is an important way to increase soybean yield and promote agricultural sustainable development to explore the genetic potential of soybean and cultivate soybean varieties with high P efficiency(P uptake and use efficiency).Ethylene is a stress hormone involved in a variety of stress responses.LP can induce plant roots to produce ethylene,thus improving plant’s ability to obtain P by changing root morphology and enhancing plant’s adaptability to LP stress.P uptake and transportation in plants are mainly mediated by P transporter genes of the Pht1 family on the plasma membrane.Therefore,it is of great significance to study the molecular mechanism of ethylene response factor and P transporter regulating P efficiency in soybean.Two candidate genes related to P efficiency,ethylene response factor GmERF1 and P transporter GmPT8,were obtained by combining genetic mapping and transcriptomic analysis in the laboratory.On this basis,a series of physiological and biochemical methods were used to study the biological functions of two genes and the molecular mechanism of regulating LP in soybean,including soybean hairy root transformation,gene editing,yeast two-hybrid,yeast one-hybrid and dual luciferase(Dual-LUC)assay.Specific research results are as follows:(1)Based on the whole genome resequencing data of 559 core soybean accessions,we sequenced the SNPs in the genomic region(about 7 kb)of GmERF1 and carried out local scanning for candidate gene associations.The results showed that a total of 5 SNPs with significant associations were identified in the promoter region of GmERF1.Five highly linked SNPs were identified in the promoter region of GmERF1 by analyzing the candidate gene association of GmERF1 in 559 genomic regions(about 7 kb)of soybean materials.The GmERF1 gene contained three haplotypes(Hap1 ~ Hap3)in 559 soybean accessions,among which Hap1 was the most excellent haplotype and had the highest frequency in wild soybeans,but Hap1 gradually disappeared during domestication.These results further prove that GmERF1 may be a candidate gene for regulating P efficiency in soybean.(2)The stable knockout strain of GmERF1 was obtained by gene editing technology,and the plants of wild-type and knockout of GmERF1 were treated with normal phosphorus(NP)and LP hydroponic.It was found that the knockout of GmERF1 promoted the root development of soybean,including total root length,root volume,root surface area and number of root tips compared with wild-type plants.The biomass and phosphorus absorption efficiency(PAE)were also significantly increased.The determination of ethylene precursor 1-aminocyclopropane-1-carboxylic acid(ACC)showed that the ACC content in the leaves and roots of GmERF1 strain was increased,especially the ACC content in the roots was twice as high as that in the wild-type strain.These results indicate that GmERF1 can affect the root development of soybean by regulating ACC content and improve PAE.(3)Using GmERF1 protein as bait,the soybean root c DNA library was screened by yeast two-hybrid technique.It was found that GmERF1 could interact with GmWRKY6.The interaction between GmERF1 and GmWRKY6 was further confirmed by one to one verification of yeast two-hybrid,bimolecular fluorescence complementation(Bi FC)and luciferase complementation imaging(LCI)assay.RT-q PCR analysis showed that GmWRKY6 was induced by LP stress,and was positively correlated with GmERF1 at the transcription level,which was involved in the root response to LP stress.Further,yeast one-hybrid and Dual-LUC detection system were used to explore the regulatory relationship between GmERF1 and GmWRKY6 on the downstream target genes.The results showed that the luciferase activities of GmPT5,GmPT7 and GmPT8 slightly decreased when GmWRKY6 was expressed.However,the co-expression of GmERF1 and GmWRKY6 was seriously impaired,suggesting that GmPT5,GmPT7 and GmPT8 are potential targets of GmWRKY6.These results indicate that GmERF1 and GmWRKY6 can jointly inhibit the expression of a series of downstream target genes such as GmPTs in the P metabolic pathway through interaction,and ultimately affect the P utilization efficiency of plants under LP stress.(4)The knockout strain of GmPT8 gene was obtained by gene editing technology and field phenotypic identification was conducted.The results showed that compared with the wild type,the knockout strain of GmPT8 showed a semi-dwarfing phenotype,with a significant reduction in height and hundred-seed weight of 45% and 30%,respectively,indicating that the knockout of GmPT8 significantly affected the growth and development of soybean and yield related traits.Further promoter sequence analysis revealed that the promoter region of GmPT8 have some LP stress regulatory elements.The GmPT8 promoter region was transformed into hairy roots and treated with NP and LP.The results showed root GUS staining was significantly different and was strongly expressed under LP condition,indicating that the GmPT8 promoter was induced by LP stress.Then,it was identified by yeast one-hybrid that WRKY family proteins GmWRKY21 and ERF family proteins GmERF4 could bind to the promoter of GmPT8.The presence of GmWRKY21 increased the luciferase signal significantly by Dual-LUC assay.These results suggest that GmWRKY21 can enhance the promoter activity of GmPT8 and thus activate GmPT8 transcription.(5)Tissue expression analysis showed that GmWRKY21 had the highest expression level in the root,and had a similar expression pattern to GmPT8.The overexpression and interference plants of GmWRKY21 were obtained by Agrobacterium rhizogenes mediated transgenic hairy root transformation experiment of soybean.RT-q PCR analysis showed that the expression of GmPT8 was higher than that of the control after overexpression of GmWRKY21,while the expression of GmPT8 was lower than that of the control after interference.These results indicated that GmWRKY21 affected the expression of GmPT8.In addition,the phenotypic identification results showed that when GmWRKY21 was overexpressed,the plants showed better growth under LP conditions than the control,specifically,the total root length increased,the root volume and root surface area increased,the number of branches increased,the root dry weight and the shoot dry weight increased significantly,and the PAE also increased significantly.These results suggest that GmWRKY21 may act as a positive regulator in response to LP stress and play an important role in soybean plant growth.