Studies On Gene Identification Of SPX Family,Functional Characterization Of BnaSPX1s And Regulatory Mechanism Of BnaC3.SPX3 In Brassica Napus

Allotetraploid rapeseed(Brassica napus),one of the major oil crops in China,has high P(phosphorus)requirement for its optimal seed yield and quality.Howerer,in the middle and lower valley of the Yongtze River in South China,the largest cultivated region for rapeseed has low soil available P concentration that is deficient or severely deficient for rapeseed growth.Hence,it is of important theoretical and practical significance to study the mechanisms of P effieiency for the genetic improvement of P nutrition.The importance of SPX domain-encoding proteins to phosphate(Pi)homeostasis and signaling pathways has been well-documented in rice and Arabidopsis,however,few related studies were found in other species.In this study,we identified all SPX domain-containing genes in the B.napus genome and analysed the sequence characteristics and their responses to Pi starvation,and then,the candidate genes,BnaA2.SPX1 and BnaC3.SPX1 were selected for further research to analyze their potential roles in Pi signaling.We also used a Pi-starvation specially induced gene,BnaC3.SPX3,to study the molecular mechanism underlying plant transcriptional responses to Pi starvation.The main results were as follows:1.Genome-wide analysis of SPX domain-containing in Brassica napus.A total of 69 SPX domain-containing genes(BnaSPXs)were identified in Brassica napus.Out of them,67 genes were unevenly distributed across the 18 chromosomes,and the majority genes were located on the chromosome terminal arms associated with high rates of recombination.The number of SPX genes on a single B.napus chromosome ranges from 1(A4)to 11(A9).The phylogenetic tree constructed using SPX protein sequences from B.napus and Arabidopsis indicated that SPX proteins were classified into4 distinct subfamilies: SPX(11),SPX-EXS(43),SPX-MFS(8),and SPX-RING(7).Gene structure and conserved motif analyses further validated the classification.A cis-element analysis indicated that 2-4 P1 BS elements(GNATATNC)were enriched in the promoter of SPX subfamily genes except BnaSPX4 s.2.The expression patterns of BnaSPX genes in response to Pi deficiencyA total of 50 BnaSPX genes were detected in the RNA-seq data of shoots and/or roots at the seedling stage in response to Pi starvation.Among them,all SPX subfamily genes except BnaSPX4 s were significantly up-regulated by Pi stress both in the shoot and root.A further qRT-PCR analysis indicated that the induction was durative during Pistarvation and reversible upon resupply of Pi.The diverse expression profiles between duplicated BnaSPX genes across different tissues under Pi deficiency indicated that they had functional differentiation during the long-term evolution.3.Functional analysis of BnaA2.SPX1 and BnaC3.SPX1 in response to Pi deficiencyBnaA2.SPX1 and BnaC3.SPX1 were both PSI genes,and located in the nucleus,but they shared different tissue expression patterns in response to Pi stress.Phenotypic performances of pBnaA2.SPX1::BnaA2.SPX1 and pBnaC3.SPX1::BnaC3.SPX1 transgenic Arabidopsis under HP(1000 ?M)and LP(50 ?M Pi)indicated that the biomass and total Pi concentration of BnaA2.SPX1 transgenic plants were decreased under Pi deficiency condition.A further analysis of PSI genes in BnaA2.SPX1 transgenic lines indicated that BnaA2.SPX1 is a negative regulator of Pi signaling.Howerer,the pBnaC3.SPX1::BnaC3.SPX1 transgenic Arabidopsis has no obvious phenotypic changes relative to Col-0.4.Regulatory mechanism analysis of BnaC3.SPX3 geneThe previous results in our lab indicated that BnaC3.SPX3 was a Pi-stravation specially induced gene,and unannotated Pi-responsive cis-acting elements might exist in the promoter region between-746 and-326.In silico analysis showed that there are four P1BS-like elements existed in this region,i.e.,P1BS-like1(CTATATAC,-337/-330),P1BS-like2(GTTTATAC,-387/-380),P1BS-like3(ACATATGC,-672/-665)and P1BS-like4(GGGTATGC,-708/-701),so we designated this fragment as LZ(Like Zone).Deletion and mutation analysis of LZ indicated the structure containing the proximal two P1BS-like and its frame sequence was necessary and sufficient to mediate independent Pi-starvation responsiveness.Consistent with the full-length promoter of BnaC3.SPX3,the induction of this DNA sequence by Pi starvation was rapid,durative and reversible,furthermore,we detailed the expression pattern of this DNA sequence in response to Pi stress across a range of tissues using pLZ5?::GUS transgenic Arabidopsis,revealing the GUS activity was detected in tissues including shoot,root,flower and pod.The two P1BS-like elements and its frame sequence present in the LZ5? structure were necessary and sufficient for being enhancer of P1 BS.Dual luciferase assay demonstrated that PHR1,PHL1(PHR1-like1),PHL2(PHR1-like2)and PHL3(PHR1-like3),four members of the MYB-CC family,specifically bind to this DNA sequence and activate the transcription of this structure.In summary,we performed the first comprehensive research on the evolution and theresponses to Pi stress of SPX domain-containing genes throughout the B.napus genome,providing a solid foundation for further functional studies of BnaSPX genes.The function diversity between two paralogous BnaSPX1 s genes in Arabidopsis may result from their different promoters,demonstrating the function differentiations in polyploidy during the long time evolution.The structure of proximal P1BS-like/P1BS-like with its frame sequence confered the independent responsiveness to Pi stress.At the same time,the structure was the “propellant” to P1 BS,in starting its more efficient response to Pi deficiency.