Roles Of Riboflavin In The Regulation Of Reactive Oxygen Species Signaling For Pathogene Resistance And Elicitin-Induced Hypersensitive Cell Death In Plants

ParAl, as a member of elicitin family, is well known for its ability to induce hypersensitive response (HR) in tobacco leaves. However, in the present study, the HR induced by ParAl was significantly suppressed when ParAl and riboflavin were co-infiltrated, and the suppression only took place in the light but not in the dark. After the repetitions and tests, we confirmed that the HR is not simply delayed but suppressed by photoilluminated riboflavin. And subsequent experiments indicated that the real HR suppressor is hydroxyl radical (OH’), one of reactive oxygen species (ROS). Additionally, to investigate the roles and functions of riboflavin in plants, we generated the transgenic Arabidopsis plants which heterologously express riboflavin binding protein (RfBP). Against wild type, the transgenic plants have enhanced resistance to the Pst DC3000 which is due to the reduced capacity of redox homeostasis resulting from the decreased content of flavin.1. The expression, purification and function analysis of ParAlElicitins have been studied as a basic and model system in plant-pathogen interaction and signaling transduction for many years. In the present study, the ParAl, a member of elicitins, were firstly pre-expressed by prokaryotic and eukaryotic expression systems. And much more ParAl can be obtained through eukaryotic Pichia pastor is system than prokaryotic expression system. Therefore, in subsequent experiments, eukaryotic expression system was taken to express ParAl protein in large scale. After that, the Ni2+ affinity columns were applied for ParAl protein purification. The purified ParAl, which can induce HR at 6000×diluted in tobacco leaves 24 h after infiltration, displayed a clear and single band in Tricine-SDS-PAGE. In addition, the reasons about the low expression level of ParAl in prokaryote were discussed. 2. The antagonistic effect of photoexcited riboflavin on the development of HR induced by ParAlThe HR induced by ParAl was significantly suppressed when ParAl and riboflavin were co-infiltrated, and the suppression only took place in the light but not in the dark. Trypan blue staining and the time-course ion leakage measurements indicated that the photoexcited riboflavin could suppress the HR rather than simply delayed its development. Furthermore, semi-quantitative RT-PCR also confirmed the phenomenon. Riboflavin, an excellent photosensitizer, can be photolyzed under light irradiation. This character is reminiscent of that the HR suppression probably resulted from the photolytic products. However the pre-photolysis products of riboflavin failed to suppress the HR symptom in the light. This result suggested that some unstable materials formed during riboflavin photolysis suppressed the HR signaling. Moreover, the results of pre-light and the pre-dark treatment indicated that the HR suppression resulting from photoilluminated riboflavin took place in the early stage of HR process rather than in the contact recognition between ParAl and its resecptor.3. Hydroxyl radical derived from photoexcited riboflavin suppresses the development of HR induced by ParAlRiboflavin can generate varied ROS under light irradiation, so various ROS scavengers were introduced. Each scavenger and PR (ParAl+riboflavin) were co-infiltrated into leaves, and the scavengers of H2O2 and OH can restore the HR symptom. The results implied that H2O2 and/or OH’was/were involved in HR suppression. However, only the presence of OH’could suppress HR and the signal intensity of electron paramagnetic resonance (EPR) positively correlated with HR suppression, these results strongly supported that the major suppressor is OH derived from photoexcited riboflavin.4. Due to OH generation HR development is suppressed by exogenous application of high-level abscisic acid and drought stressPrevious studies have proved that exogenous application of high-level abscisic acid (ABA) and drought stress can induce the production of ROS, as well as the increase of catalytic Fe that is critical for generating OH with H2O2 through the Fenton reaction. Could the endogenetic OH suppresse the development of HR? We performed the experiments and found that exogenous application of high-level ABA and drought stress could suppresse the HR development triggered by ParAl and RS105 in tobacco leaves through the involvement of OH. Additionally, photoexcited riboflavin failed to suppress the HR triggered by HrpNEa, this probably meant that OH only could suppress the HR signaling induced by ParAl. Finally, we proposed a hypothesis about the manner in which OH performs its function in signaling.5. The transcriptome analysis of Arabidopsis thaliana expressing riboflavin binding proteinTo investigate the function of riboflavin, especially the redox regulation of riboflavin, we attempted to decrease the content of riboflavin in plant cells through heterologous expression of riboflavin binding protein (RfBP) derived from turtle. In the transgenic lines, the content of available flavins was decreased even though the total amount of flavins was increased. Riboflavin is involved in many physiological and biochemical process in living cells, therefore, we performed microarray analyses for a feasible start. The results of microarray analyses indicated that:i. Many genes involved in mitochondrial electron transport are down regulated in transgenic line; ii. Glutaredoxin family proteins participated in redox regulation are also down regulation; iii. Some genes involving proteins expression and localization are down regulation; iv. The related genes on DNA synthesis and histone expression are also down regulation in transgenic line. Additionally, the transgenic line plants have enhanced resistance to the Pst DC3000 due to the reduced capacity of redox homeostasis against wild type.