Cloning And Functional Analyses Of Three Genes Essentially Involved In Plant Riboflavin Synthesis In Relation With Redox

Riboflavin (vitamin B₂) participates in many physiological processes, particularly those characteristic of oxidation and reduction. The vitamin is phosphorated and combined with nucleotides to form flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), important components of the respiratory electron-transmission chain, which affects antioxidation through the role of vitamin C. FMN and FAD often serve as coenzyme of many enzymes (flavoproteins), which control many physiological reactions, and many aspects of responses to environmental stress. As an important component, riboflavin can only be synthesized in plants and microbes. The two enzymes, lumazine synthase and riboflavin synthase, which are encoded by the gene of LS and RS, catalyze the last two steps in the biosynthesis of riboflavin in organisms. The two genes of many bacteria and yeasts have been cloned, but there are only few reports on plants and even no relative research on rice. It has been reported that the cos1 mutant (ls mutation) restores the coil-related phenotypes, including defects in jasmonate acid sensitivity, senescence and plant defense responses in Arabidopsis, but there are no research about the functions on growth, disease resistance, active oxygen and hypersensitive cell death of the two genes of rice by so far.The genes encoding lumazine synthase and riboflavin synthase in rice, named as OsLS and OsRS, respectively, are first cloned in this study. The two genes OsLS and OsRS were cloned by a polymerase chain reaction protocol. Phylogenetic relationships between OsLS or OsRS and homologues from other plant species were evaluated, indicating a particular relevance of the gene in plants. OsLS and OsRS protein was produced by recombinant Escherichia coli cells. When applied to tobacco leaves, however, neither the OsLS nor the OsRS protein could cause hypersensitive response. Thus, OsLS protein and OsRS protein may fail to execute effects on plants by activating programmed cell death mechanisms. Moreover, certain softwares were used to analyse subcellular localization and three-dimensional structure of the two proteins.The cloned genes were inserted into the plant transformation vector pBI121. After confirming the correct orientation of OsLS and OsRS in the vector, the recombinant unit was introduced into tobacco plants. Transgenic lines in T0 generations were obtained based on screening seeds on MS medium with kanamycin and molecular analyses of the plants. Southern blot hybridization and PCR analysis suggested that both of the transgenes were integrated into chromosomes of tobacco. RT-PCR analyses indicated that the two genes were expressed in transgenic plants.The endogenous flavins, including free riboflavin, FMN and FAD, determined by HPLC, in transgenic lines of OsLS and OsRS were much higher than those in the control plants. Moreover, transgenic plants were different from control plants in several morphological properties, including plant height, rate of seeds germination and so on. These results provide an insight into the role of modulating riboflavin contents in plant growth and development regulation.Thioredoxins play important roles in diverse bioprocesses of plants, animals, and microbes. A hypothesized tobacco (Nicotiana tobacum) gene encoding a thioredoxin h-like (NtTRX-hl) protein has been deposited in GenBank, but the bioactivity of the protein is unclear. Here, we report cloning of the NtTRX-hl gene and functional studies of the NtTRX-hl protein. The gene was cloned by a polymerase chain reaction protocol. Phylogenetic relationships between NtTRX-hl and homologues from other plant species were evaluated, indicating a particular relevance of the gene in plants. NtTRX-hl protein was produced by recombinant Escherichia coli cells. In an assay using dithiothreitol and insulin as substances, NtTRX-hl showed evident enzymatic activity. When applied to tobacco leaves, however, the protein did not cause hypersensitive response. Thus, NtTRX-hl may not execute effects on plants by activating programmed cell death mechanisms. Subcellular localization and three-dimensional structure of NtTRX-hl were predicted, indicating that the protein may be situated at different cellular compartments depending on processes it affects. Our results shed light on further dissection of NtTRX-hl functions in plants.