| Riboflavin (vitamin B2) biosynthetic and functional pathways affect plant growth, development, and defensive responses by multiple mechanisms. Endogenous-modulated riboflavin also triggers plant disease resistance in Arabidopsis expressing the riboflavin receptor protein encoding gene from soft-shelled turtle(Trionyx sinensis japonicus). However, the mechanism of disease resistance in transgenic plants is still unknown. Studies in this Ph.D thesis aim at determination of signaling pathways and components in plant responses to endogenous riboflavin, as well as the physiological processes and metabolic pathways, which are affected by endogenous riboflavin with higher levels. Biotic stress and abiotic stresses such as osmotic, oxidative, and salinity affect normal growth and development in plants. Our objective is identifying new genes involved in biotic stress and abiotic stress.1. Riboflavin modulation of hydrogen peroxide signaling that regulates Arabidopsis resistance to a bacterial pathogenRiboflavin (RIB) mediates multiple bioprocesses through flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). Here we show that RIB plays an initial role in hydrogen peroxide (H2O2) signaling that regulates pathogen defense in Arabidopsis {Arabidopsis thaliana). We transformed Arabidopsis with TsRIR, a RIB receptor gene from soft-shelled turtle (Trionyx sinensis japonicus), and characterized the RIR-expressing Arabidopsis (RIRA) line RIRA11. Resistance to a bacterial pathogen was promoted coincidently with increased contents of RIB, decreased contents of FMN and FAD in RIRA11 compared to wild-type (Col-0) plant during vegetative growth. Transcriptomics analysis revealed that 950 genes assorted in various cellular processes were altered in RIRA11. Significantly down-regulated profiles included 13 genes critically functional in the mitochondrial electron transport chain (METC), indicating a facilitated production of H2O2 via METC. The transcriptomics information was confirmed by RT-PCR and Real-Time PCR. Silencing the riboflavin receptor protein (RIR) in transgenic plant (RIRA) restored WT characters. Our results uncover a novel signaling contrail:the modulation of RIB reduces FMN and FAD contents and thus depresses METC flux but promotes peroxidation; these changes in METC result in H2O2 burst, which in turn stimulates defense response.2. A riboflavin receptor protein modulates the floral transition in ArabidopsisPlants flower in response to specific signals. Hydrogen peroxide from flavin-dependent redox mediates many bioprocesses but is unclear for effects on the floral transition. In early-flowering Arabidopsis plants expressing turtle riboflavin receptor protein, the protein localizes to chloroplasts, alters flavin form conversion, and represses the mitochondrial electron transport chain. Cellular hydrogen peroxide is thus elevated to promote the function of the FT protein in activating API, an integrator of the floral transition pathways. Comparing wild-type or silencing Arabidopsis, the transcription levels of genes involved in flowering were increased in over-expression riboflavin receptor protein Arabidopsis (RIRA). RIRA plants show early flowering phenotype. Our results demonstrate a molecular basis by which H2O2 promotes flower development.3. The circadian clock regulates riboflavin responses in ArabidopsisThe circadian clock plays a pervasive role in the temporal regulation of plant physiology, environmental responsiveness, and development. In contrast, the riboflavin play a similarly far-reaching role in the spatial regulation of plant growth and development. Here we present work that the circadian clock regulates riboflavin signal transduction. Using transgenic Arabidopsis plants (RIRA) which ectopic express riboflavin receptor protein from turtle (TsRIR), we found that endogenous riboflavin levels showed circadian rhythms in both wild-type and RIRA. RIRA plants with high concentration of endogenous riboflavin affected the circadian input and central genes expression pattern. By RT-PCR assay, we verified that riboflavin-mediate transcriptional response are circadian regulated. Exogenous riboflavin can induce priming of defense toward infection by Pseudomonas syringae pv. tomato DC3000 (Pst), but the clock controls plant sensitivity to applied riboflavin. Thus the circadian clock regulates some riboflavin response. 4. Functional characterization of Arabidopsis RIR-like protein (AtRIR) in abiotic stressesAbiotic stresses such as osmotic, oxidative, and salinity affect normal growth and development in plants. The production and accumulation of reactive oxygen species (ROS) cause oxidative stress under these abiotic conditions. AtRIR (At5G27830) was identified as a unknown function protein that contain no previously defined domains or motifs, a putative riboflavin receptor protein (RIR) like protein. AtRIR transcripts are found in all major organs including root, leaf and flower by RT-PCR and AtRIR promoter controlled GUS activity. The AtRIR T-DNA insertion mutant (Atrir) showed a higher sensitivity to osmotic, oxidative and salinity stress than the wild-type (Col-0), as revealed by root growth length. Over expression of AtRIR protein in Arabidopsis (AtRIR-OE) was found to enhance of plants to osmotic, oxidative stress. AtRIR-OE reduces the content of ROS and transcripts of oxidative responsive genes (APX1, FSD1) under salt, oxidant, osmotic and abscisic acid treatment. The induced expression of stress-responsive genes (COR47, RD29B, RD29A) was more sensitive to abiotic stress and exogenous ABA in AtRIR-OE than in the wild-type. This indicates that AtRIR modulates the abiotic stress response via the regulation of oxidative stress response.5. Mutant in Arabidopsis RAP2.6L transcription factor enhancing plant defense against Pseudomonas syringaeA common feature of plant defense responses is the transcriptional regulation of a large number of genes upon pathogen infection or treatment with pathogen elicitors. A large body of evidence suggests that plant ERF transcription factors are involved in plant defense including transcriptional regulation of plant host genes in response to pathogen infection. However, there is only limited information about the roles of specific ERF DNA-binding transcription factors in plant defense. We analyzed the role of the RAP2.6L transcription factor from Arabidopsis in plant defense against the bacterial pathogen Pseudomonas syringae. RAP2.6L translational fusion with green fluorescent protein is localized to the nucleus. RAP2.6L expression is responsive to general environmental stress. Analysis of Pst DC3000-induced RAP2.6L in the defense signaling mutants abi1-1,jar1-1, ein2-1 and npr1-1 further indicated that this gene is positively regulated by the salicylic acid (SA) and jasmonic acid (JA) signaling pathway and negatively regulated by ethylene signaling pathway. T-DNA insertion mutants for RAP2.6L reduced growth of Pst DC3000 and displayed reduced disease symptom severity as compared to wild-type plants. The rap2.6L mutant plants also displayed increased expression of the SA-regulated PR1 and PR2 genes after the pathogen infection. Based on analysis of T-DNA insertion mutants, stress-induced RAP2.6L functions as a negative regulator of SA-mediated defense responses to P. syringae. RAP2.6L was a transcriptional activator and was able to activate the expression of genes involved in plant defense.Conclusive remarksData obtained from studies in RIR (riboflavin receptor protein) transgenic Arabidopsis have provided us the further understanding on the mechanisms of riboflavin mediated pathogen defense and floral transition in Arabidopsis. Firstly, RIR transgenic Arabidopsis increased riboflavin content in leaves affecting many physiological processes, which provide us the cues to explore the mechanisms of riboflavin mediated plant defense and development. Secondly, the modulation of RIB reduces FMN and FAD contents and thus depresses METC flux, these changes in METC result in H2O2 burst, which in turn stimulates defense response. Thirdly, cellular hydrogen peroxide is elevated to promote flowering in RIR transgenic Arabidopsis and the circadian clock regulates some riboflavin response in wild type Arabidopsis. Fourthly, we have identified Arabidopsis AtRIR function in abiotic stresses. Fifthly, we have identified a new transcription factor which regulates plane defense in Arabidopsis. |
