Functional Analysis Of GmBRI1and GmCPD And Investigation Of Their Roles In Soybean Flowering

The soaring demand for more plant-derived products makes a new green revolutionessential. For enhancing yields which remains the eternal theme of the greenrevolution, modulation of the overall development statue of the crop is required.Many researches have highlighted the important role of phytohormones in plantdevelopment and architecture that underlie high yield. Among the phytohormones, thepotential of brassinosteroids (BRs) in agriculture has been recognized, as there issubstantial evidence that the genetic modification of BR signaling and biosynthesispathway can create a plant with ideal plant type and maturity period that providesgreat yield potential. Brassinosteroids (BRs) are a group of steroidal phytohormoneswith essential roles in plant growth and development; especially, its impacts on plantarchitecture, production and florescence have raised the considerable concern. As animportant industrial crop providing oil and protein worldwide, soybean is limited onthe output by lodging and the inadaptable maturity period. Conventional breeding wasthe common means of improving plant type and regulating the florescence in the past,but molecular techniques currently provide a broader space for creation, as a range ofgenes involved in the BR signaling and biosynthesis pathway have been identified.However, although great progress has been made in other species, evidence insoybean is limited. The current study is the first time that the BRI1and CPD genewere isolated and characterized in soybean. Our data provide a foundation for futurestudies into the regulation of soybean production by BR signaling. The successfuldeciphering of the GmBRI1and GmCPD genes will contribute toward generating newsoybean germplasm.In this study, we cloned and characterized one cDNA (GmBRI1) corresponding tothe BRI gene and four cDNA (GmCPD) that are homologous to the CPD gene. Wepresent sequence, structural and functional evidence that the deduced GmBRI1andfour GmCPD genes have significant homology with AtBRI1and AtCPD, and possessmultiple functions in plant development progress. The expression patterns of these five BR related genes have also been investigated and discussed in the floweringinduction system and the GmFT2a transgenic plants in soybean which will increaseour understanding of the relationship between the GmBRI1and GmCPD genes andsoybean flowering pathway to further benefit production. The main results arementioned as follow:1. A brassinosteroid insensitive gene homologous with AtBRI1and other BRIs wasisolated from Glycine max and designated as GmBRI1. A bioinformatic analysisrevealed that GmBRI1shares a conserved kinase domain and25tandemleucine-rich repeats (LRRs) that are characteristic of a BR receptor for BRreception and reaction and bear a striking similarity in protein tertiary structure toAtBRI1. The transformation of GmBRI1into the Arabidopsis dwarf mutant bri1-5restored the phenotype, especially regarding pod size and plant height.Additionally, this complementation is a consequence of a restored BR signalingpathway demonstrated in the light/dark analysis, root inhibition assay andBR-response gene expression. Therefore, GmBRI1functions as a BR receptor toalter BR-mediated signaling and is valuable for improving plant architecture andenhancing the yield of soybean.2. Four cDNA homologous with Arabidopsis CPD were isolated from Glycine max,named GmCPD1, GmCPD2, GmCPD3and GmCPD4respectively. Aphylogenetic analysis suggested that the four GmCPD are all closest in homologyto CPDs from dicotyledons, especially leguminous plants. A sequence analysisindicated that all the GmCPD proteins contain the proline-rich domain,dioxygen-binding domain, steroid-binding domain and heme-binding domainwhich are conserved among plant CPDs and are the characterization of thecytochrome P450family. The GmCPD proteins are strikingly similar to AtCPD insequence and structure. Additionally, the transformation of four GmCPD genesinto the Arabidopsis cpd-91mutant respectively can restore the phenotype and thiscomplementation is a consequence of a restored BR biosynthesis pathway. Theseresults support these four GmCPD genes all as novel CPD gene and displayimportant roles in root development, leaf expansion and plant type architecture,3. The relative expression level of GmBRI1and four GmCPD genes in differentsoybean tissues/organs and responding to exogenous BR were analyzed. GmBRI1transcripts were more abundant in soybean rapidly growing tissues, such ashypocotyls and cotyledons; GmCPD1, GmCPD3and GmCPD4are mainly expressed in leaves and cotyledons; whereas GmCPD2display a lower level ineach tissue but mainly expresses in young pods; GmBRI1and four GmCPD genesall exhibit a rapid and efficient response to exogenous BR treatment with aupregulation of GmBRI1and downregulation of all GmCPD genes.4. The potential roles of GmBRI1and GmCPDs in flowering regulation wereinvestigated. First, GmCPDs expression complemented the late floweringphenotype of Arabidopsis mutants deficient in CPD. It is therefore clear thatGmCPDs are associated with flowering. Moreover, We detected the expressionpatterns of GmBRI1and four GmCPD genes in flowering reversion system ofsoybean. The flowering reversion occurs in soybean variety Zigongdongdou whentransferred into long day condition after a13-day short day treatment. We detectedthe transcript level of GmBRI1and four GmCPD genes in the flowering reversiontreatment described above and the treatment of continued long day and short day.The results are interesting that the expression levels of GmCPDs are underphotoperiod control in Zigongdongdou and show a sudden peak upon floralmeristem initiation. Further, GmBRI1and GmCPDs expression in soybeanvarieties with different photoperiod sensitivities was detected. The results showthe increased GmBRI1and GmCPDs expression in the leaves and cotyledons ofphotoperiod-insensitive early-maturity soybean, it is clear that GmBRI1andGmCPDs contribute to flowering development and are essential in the early stagesof flowering regulation.5. The GmFT2a transgenic soybean plants were achieved and confirmed bymolecularidentification. Among them, T0-2plant appears an advanced floweringtime and a high level of GmFT2a transcription. The next generation, T1-2, inheritsthe high expression of GmFT2a gene but have the same flowering time as thenon-transgenic plants. In these transgenic background with upregulated GmFT2aexpression, GmBRI1and four GmCPD genes perform the same expressionpattern: no matter florescence is promoted or unchanged, the expression trend ofGmBRI1and four GmCPD genes presents a positive correlation with that ofGmFT2a gene, suggesting a relationship between these BR relative genes andGmFT2a...