Toward Dissection Of Riboflavin-Receptor Signaling In Regulation Of Floral Transition And Defense Response In Arabidopsis

Riboflavin (vitamin B₂) functions in many reactions with its derivatives ravin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), which are coenzyme of many enzymes. Riboflavin participates in many physiological processes, particularly those characterized by oxidation and reduction, producing reactive oxygen intermediates (ROIs). ROIs are essential for the hypersensitive cell death, growth, and defense against pathogens and insects, as well as many other responses to environmental stress. Study has revealed that exogenously application of riboflavin could accelerate growth, elevate yield, enhance drought tolerance and resistance to pathogens by a novel disease resistance pathway. An assumption is brought up that alteration of riboflavin content in plants will impact complicated processes, and plant defense and growth are regulated in consequence. This provides us with a particular insight into studying cross-talk between riboflavin signaling and the well-known basal defense pathway in plant growth and defense regulating network.Our lab has cloned the riboflavin receptor protein encoding gene MR from soft-shelled turtle (Trionyx sinensis japonicus) and transferred it into Arabidopsis resulting several homozygous lines. Compared to Col-0, RIRA contains higher endogenous riboflavin, FMN and FAD than those in Col-0. RIRA also grows better and flowers earlier. And besides, RIRA exhibits alleviative symptoms and reduced bacterial propagation after Pseudomonas syringae pv. tomato DC3000 infection.The gene encoding RIR(riboflavin receptor protein) in soft-shelled turtle was obtained by polymerase chain reaction (PCR) technique, and the PCR product was 729 bp in size. RIR and pET30a (+) were double digested by BamHâ… and Hindâ…¢restriction enzymes. The purified target gene and vector fragments were ligated resulting pRIR, which was transformed into cells of the Escherichia. coli strain BL21 (DE3) pLySs. The bacterium was induced by isopropyl-Beta-D-thiogalactopyranoside (IPTG) and the extracted total protein were loaded onto sodium dodecyl sulphate polyacrylamide gel electrophoresis(SDS-PAGE), approximately 34 kD exogenous protein was observed on the SDS-PAGE. In vitro, RIR could quench the fluorescence of riboflavin, indicating the bio-activity of the receptor protein. These results provide a good basis for further study on the role of RIR in modulating riboflavin contents and regulating plant growth and development.We explored the genetic background in R/RA18 to provide foundation for the further research. RIRA18 was crossed back to Col-0 as the male parent. The statistics showed that segregation ratio of Km resistance in second filial generation (F₂) was 3:1, indicating solo inherited factor according to the law of segregation. Simultaneously, the flanking sequence of T-DNA insertion in the transgenic line was amplified using inverse PCR strategy. The product was a single band and showed high similarity to the spacer between 18S and 25S ribosome DNA in the second chromosome in Arabidopsis. Both results confirm single transgene copy in RIRA18 and cancel the doubt that some gene T-DNA inserted in mutated and raised the changes. By the methods of gene transferring and cross, RIRA18 was separately integrated with mutants in basal defense pathways, such as etr 1-1, ein2;aux1-7, jar1-1, abi1-1, npr1-1. All the crossbreeds were identified following their F₁ and F₂ generations subjected to corresponding screening on special plates and accessorial judgment from molecular characteristic and appearance. These experiments afford pivotal plant materials to dissect the cross-talk between riboflavin pathway and the basal defense pathways.To validate the early flowering phenotype, Real Time PCR method 2^{-ΔΔCt} was adopted to detect the expression difference of several floral transition genes by time course way within 24h and defense related genes in RNA samples extracted from RIRA and Col-0. The results indicate that transcripts of photoreceptors PHYA, PHYB, CRY1, CRY2 are up-regulated, the components of central oscillates TOC, CCA change dramatically. The downstream effect genes FT and SOC accumulate higher in Col-0 than RIRA18 which don't coincide with early flowering phenotype observed in RIRA18. These datas implicate clues for further MR functions studying and critical factor searching. The information of defense related genes come from the chip experiment, quantitative results confirm their up-regulation in chip. These molecular outcomes are consistent with enhanced disease resistance in RIRA.