Genetics And Transcriptional Profiling Of Riboflavin Modulation During Plant Defense Response

Riboflavin (vitamin B₂) biosynthetic and functional pathways affect plant growth, development, and defensive responses by multiple mechanisms. Riboflavin is involved in anti-oxidation and peroxidation. Both processes affect the production of reactive oxygen species (ROS) in oxidative burst. Our previous studies suggest that riboflavin is a novel elicitor of systemic acquired resistance (SAR) in plants and activates a distinct defense signaling pathway from several known ones, but the underlying molecular mechanism remains unclear. Endogenous-modulated riboflavin also triggers plant disease resistance in Arabidopsis expressing the riboflavin receptor protein encoding gene of soft-shelled turtle (Trionyx sinensis japonicus). However, the mechanism of disease resistance in transgenetic plants is still unknown. Studies in this Ph.D thesis aim at determination of signaling pathways and components in plant responses to exogenous and endogenous riboflavin, as well as the physiological processes and metabolic pathways, which are affected by endogenous riboflavin with higher levels. The results here will provide basic clues to explore the riboflavin mediated plant defense and growth pathways and crosstalk with hormonal and non-hormonal signaling pathways.1. Riboflavin-induced priming for pathogen responses in Arabidopsis requires hydrogen peroxide and NPR1Besides having a pivotal biological function as a component of coenzymes, riboflavin appears as an elicitor of systemic acquired resistance (SAR) in plants, but the underlying molecular mechanism remains unclear. SAR is associated with the ability to induce cellular defense responses more rapidly and to a greater degree than in non-induced plants, a process called "priming." Here we report that the application of riboflavin to Arabidopsis thaliana induces priming of defense responses toward infection with virulent Psedumonas syringae pv. tomato DC3000 (Pst). Induced plant resistance to the bacterial pathogen was mechanistically connected with the expression of defense response genes and cellular defensive events, including H₂O₂ burst, HCD, and callose deposition. Riboflavin treatment and inoculation of plants with Pst were neither active but both synergized to induce priming events. The defense-priming process needed NPR1 (essential regulator of systemic acquired resistance) and maintenance of H₂O₂ burst but was independent of salicylic acid (SA), jasmonic acid (JA), ethylene (ET), and abscisic acid (ABA). Our results suggest that the role of riboflavin in priming defenses is subject to signaling process distinct from the known pathways of hormone signal transduction.2. Genome-wide transcriptional profiling and metabolism analysis of transgenetic Arabidopsis plants expressing an oviparal riboflavin receptor geneWe have cloned the riboflavin receptor protein encoding gene RIR from soft-shelled turtle and transferred it into Arabidopsis and got several homozygous lines. We also constructed RIR-silencing vector, which was able to silence RIR in RIRA11 and got SiRB11, for further study on the roles of riboflavin in plant growth and defense regulation network. We used the Arabidopsis ATH1 Affymetrix chip to analyze the global transcriptional profiling. Based on the pre-hybridization and quality control results, our microarray experiments were successful and data we got was credible. Then we used the GCOS software to analyze the original data and pre-treated with robust multiarray analysis (RMA), and we got the differentially regulated genes between Col-0 and RIRA11 plants. Our choosing criterion was based on the FDR < 0.1, p < 0.05, Permutation P<0.05. Further data analysis was carried out by selecting for the genes exhibiting >2-fold differences. There were 320 genes found to be significantly up-regulated and 630 down-regulated. We then performed RT-PCR and Real-time PCR on a select group of genes and compared the results with those obtained from the array. Although the ratios from the PCR data are not the same as those in the chip, the correlation between the two groups of data are very good. Subsequently, Gene ontology analysis was carried out on the Arabidopsis Biological Research Center. To analyze the metabolism processes changed in RIRA11, we used the MapMan software to visualize the metabolic pathway assigned from the significantly regulated genes. The results suggested that there are some genes involved in secondary metabolism, transport, transcription and mitochondrial electron transport, redox homeostasis. Meanwhile, some genes involved in plant defense response, hormonal signaling pathway and protein kinase were analyzed in the text. To our great interesting, some genes in the mitochondrial electron transport chain were depressed but some ROS detoxification genes were up-regulated, suggesting that there is escalated oxidative state in RIRA11.These results provide us the insights into the effect of riboflavin on plants and explore a list of candidate genes that may act to regulate plant defense and development, hormonal and non-hormonal signaling pathways.3. Modulated riboflavin may activate the mitochondrial pathway of ROS signaling and primes pathogen defense response in transgenetic Arabidopsis plantsCompared with Col-0 and SiRB11 plants, RIRA11 contains higher endogenous riboflavin than that in Col-0 and SiRB11. Moreover, RIRA11 exhibits alleviative symptoms and reduced bacterial propagation after DC3000 infection, but the underlying mechanisms are unclear. Based on the microarray data, we deduced that the inhibition of the mitochondrial electron transport chain may result in the escalated oxidative state in RIRA11.Then we detected the ROS levels by DAB and DCFH-DA staining and H₂O₂ content measuring, as well as the sensitivity of the plants to the extra oxidative stresses, generated by Paraquat (PQ), the results suggested that there was escalated oxidative state in RIRA11. Combined with catalase, riboflavin, and DC3000 inoculation, we analyzed the cellular and molecular defense responses. Thus, our results suggested that riboflavin may inactive the mitochondrial electron transport chain resulting in the escalated oxidative state and shifted the plants to the primed state, which plays a pivotal role in augmented defense response in RIRA11 when infected by DC3000. Endogenous riboflavin-induced priming is the mechanism of disease resistance in RIRA11. Then we analyzed the signaling pathways required in endogenous riboflavin-induced priming using the mutants npr1-1,etr1-1,jar1-,and abi1-1, each of which has the RIR gene by the methods of gene transferring and crossing with RIRA11. The results show that endogenous riboflavin-induced priming requires NPR1, but is independent of ET, JA and ABA signaling pathways. 4. Priming of pathogen defense signal transduction by riboflavin in ArabidopsisOur previously studies suggest that the disturbed redox homeostasis plays a pivotal role in endogenous riboflavin-mediated priming. To explore whether it also functions in the exogenous riboflavin-induced priming, we detected the ROS accumulation in plants from the beginning of riboflavin sprayed till 5 dpt. The results showed that riboflavin trigger ROS accumulation in plants during the period tested and the levels of ROS reach the highest at 2 hpt, but restore to the original level at 5dpt. Meanwhile, the riboflavin-treated plants are more sensitive to the extra oxidative stresses, generated by Paraquat, suggesting that the plants have escalated oxidative state. Combined with catalase and DC3000 inoculation, we analyzed the cellular and molecular defense responses. Catalse, which infiltrated at 30min after riboflavin treatment, nullifies the escalated oxidative state and the augmented ROS accumulation when infected by Pst. Moreover, the disease resistance is also abolished in riboflavin pretreated but catalase treated plants, which suggest that the disrupted redox homeostasis is required in riboflavin-induced priming. However, the disrupted redox homeostasis is independent of NPR1. Based on the microarray data, we then analyzed the two genes involved in mitochondrial electron transport with Real-time PCR technology. Both of the genes were depressed during the course of riboflavin treatment. Therefore, the results here suggest that disrupted redox homeostasis plays a pivotal role in riboflavin-induced priming and may connect with functional inhabitation of mitochondria.5. Practical use of defense priming by harpin in riceHpaG_10-42, which generated from truncating the Xanthomonas oryzae pv. oryzicola HpaG_Xooc protein, is active in priming defense responses in rice. When 6μg/ml HpaG_10-42 was applied once to seedling nursery and three times to transplanting fields of indica and japonica rice varieties, bacterial blight caused by X. oryzae pv. oryzae and panicle blast caused by Magnaporthe grisea were both less severe, relative to controls. Meanwhile, when HpaG_10-42 applied once at the four stages of rice growth, the effects on decreasing disease severities were also significant. HpaG_10-42 treatment was similar to local agronomic measures, including use of chemicals, in decreasing disease severities of 9 rice varieties. Hence, results here provide an example for effective use of beneficial pathogen defense priming agents to control diseases in the staple food crop. Conclusive remarksResults described above have provided us with further understanding on the mechanisms of riboflavin-mediated pathogen defense in plants. Firstly, priming is an important cellular mechanism in SAR by riboflavin and requires hydrogen peroxide and intact NPR1. Secondly, disrupted redox homeostasis induced by riboflavin, which is independent of NPR1, plays a pivotal role in shifting the plants to primed state. Thirdly, Endogenous riboflavin dose affect many physiological processes, such as secondary metabolism, transport, transcription, redox metabolism, as well as hormonal and non-hormonal signaling pathways, which provide us the cues to explore the mechanisms of riboflavin mediated plant defense and development, the crosstalk between riboflavin signaling with hormonal and non-hormonal signaling pathways. Fourthly, riboflavin may inactivate mitochondrial electron transport, resulting in ROS accumulation and disrupted redox homeostasis in plants. Fifthly, HpaG_10-42, a selected fragment of HpaG_Xooc, could prime disease resistance of rice effectively in fields.