Roles Of Riboflavin And Riboflavin-Binding Protein On Pathogen Resistance And Abiotic Stress Tolerance In Arabidopsis

Riboflavin is the precursor of FMN and FAD, which play key roles in plant development and growth. They are required for many metabolism reactions such as mitochondrial electron transport, photosynthesis, fatty acid degradation and redox regulation. As for the redox homeostasis modulation, FAD is an indispensable component of many ROS-scavenging enzymes, such as glutathione reductase, monodehydrogen ascorbate reductase, and NADPH-dependent thiol reductase. Interestingly, FAD or FMN is also required for many ROS-generating enzymes, such as the NADPH-oxidase, glycolate oxidase and certain amine oxidases. In addition, riboflavin can generate singlet oxygen in the presence of light in vitro or in vivo.Reactive oxygen species (ROS) is an unavoidable toxic by-product of plant metabolism under favorable or adverse conditions. These ROS, such as superoxide anion, hydrogen peroxide, hydroxyl radical and singlet oxygen, also can function as signaling molecules in plant defense against pathogen attack and abiotic stress challenge. For example, H2O2is required for the hypersensitive response (HR) in pathogen resistance. In addition, the early increase of ROS content also plays an "alarm" role in plant for response the abiotic stress challenge.In vitro experiment indicates that riboflavin is an important "elicitor" in plant pathogen resistance. In addition, the in vivo experiment (up-or down-regulate endogenous riboflavin content in plant) also shows riboflavin has a close correlation with pathogen resistance. In our experiment, a riboflavin-binding protein encoding gene TsRfBP was introduced into Arabidopsis thaliana Col-0and acquired transgenic plant with low free riboflavin availability. Riboflavin-binding protein was expressed in animal cells and there is no report of similar gene or protein in plant species.Recently, riboflavin was applied on plant and acquired significant enhancement of pathogen defense capacity. For example, foliar spraying of riboflavin solution can confer Arabidopsis enhanced both biotrophic and necrotrophic pathogen resistance by trigger a noval signaling pathway. As for this broad-spectrum pathogen resistance, a "priming" hypothesis was proposed by our laboratory. Thus, we want to kow whether foliar application of riboflavin can also trigger abiotic stress tolerance.1. Ectopic expression of riboflavin-binding protein gene TsRfBP incuces Pst DC3000resistance but impairs Pcc in transgenic Arabidopsis thalianaHere, the TsRfBP was further identified in the transgenic plant K11with Northern blot and immunolocalization techniques. Then, we compared the pathogen resistance between the Col-0and the transgenic plant K11(carried riboflavin-binding protein) in two growth time points (20-day and40-day old). In compared with the Col-0, enhanced Pst DC3000(Pseudomonas syringae pv tomato DC3000, a biotrophic or facultative pathogen) and impaired Pcc (Pectobacter carotovora subsp. carotovora, a necrotrophic pathogen) resistance were observed in K11plant. Higher content of H2O2accumulation and severe cell death were monitored in K11over Col-0in the vegetative growth stages. In addition, accelerated senescence (or more matured growth state) was observed in K11over Col-0under our growth conditions. As for this interesting phenomenon, an "ARR-like" or Age-Related Response like mechanism was proposed to illustrate these two different trophic pathogen response, although more detailed experimental evidence was required to support this hypothesis.2. Ectopic expression of riboflavin-binding protein gene TsRfBP paradoxically enhances both plant growth and drought tolerance in transgenic Arabidopsis thalianaIn the second chapter, we compared the antioxidant response of the Col-0and the transgenic plant K11under drought stress exposure. Under favorable conditions, higher accumulation of H2O2and TBARS, lower values of redox ratio were observed in K11over Col-0. However, enhanced drought tolerance was monitored in the K11over the Col-0coupled with higher accumulation of soluble sugar and free proline content. In consistent with these, lower decrease of antioxidant enzyme (SOD, CAT, APX and GR) activities and higher redox homeostasis were measured in K11over Col-0under stress exposure. In addition, faster growth rate, accelerated flowering and enhanced senescence are the basic features of "drought escape" strategy adopted by certain plant under gradual enhancement of drought stress. Here, we proposed a "stress escape" concept to illustrate the low riboflavin-mediated "both plant growth and drought tolerance enhancement" phenomenon observed in the K11plant over its Col-0counterparts. This work may help us to improve crop plant with higher growth rate and greater drought tolerance.3. Exogenous riboflavin fails to induce salt stress tolerance in Arabidopsis thalianaIn the chapter Ⅲ, Arabidopsis thaliana were pretreated with different concentration (0,20μM,200μM and2mM) of riboflavin solution and their abiotic stress tolerance (such as salt, drought, and shade) were compared in this experiment. Reduced salt and drought tolerance were observed in the riboflavin-pretreated plants compared with the control (0μM riboflavin treated group). In line with these, enhanced oxidative damage, decreased antioxidant enzyme activities and redox ratio values were measured in the riboflavin-pretreated plant than these in the control Arabidopsis under salt conditions. Expression level of stress-related genes (MSD1, CAT1, APX1et al.) was down-regulated in the riboflavin-treated leaves over the control. In the discussion, we considered the riboflavin-mediated ROS production play a key role in the impaired abiotic stress tolerance observed in this experiment. In addition, it suggests that this abiotic stress tolerance impairement should be considered in the pathogen resistance with exogenous riboflavin application.In conclusion, riboflavin-binding protein mediated pathogen response and drought tolerance give us a noval insight of the roles of riboflavin in environmental stress response of plant. The exogenous riboflavin fails to induce abiotic stress tolerance indicates the riboflavin-mediated ROS (singlet oxygen) should play a different role in environmental stress challenge (including pathogen and salt stress et al. resistance).