Riboflavin-Modulated Reactive Oxygen Species Signal In 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.ROS are essential for the hypersensitive cell death,growth,and defense against pathogens and insects,as well as many other responses to environmental stress.Besides 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.This thesis aimed at elucidating the application of riboflavin to Arabidopsis thaliana induces priming of defense responses.1.Riboflavin-induced priming for pathogen responses in Arabidopsis requires hydrogen peroxide and NPR1Here we report that induced plant resistance toward infection with virulent Pseudomonas syringae pv.tomato DC3000 was mechanistically connected with the expression of defense response genes and cellular defensive events,including H₂O₂ burst, hypersensitive cell death(HCD) and callose deposition.Riboflavin treatment and inoculation of plants with P.syringae pv.tomato DC3000 were neither active but both synergized to induce priming events.The defense-priming process needed NPRI (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.Priming of pathogen defense signal transduction by riboflavin in ArabidopsisTo explore whether the disturbed redox homeostasis also functions in the exogenous riboflavin-induced priming,we detected the ROS accumulation in plants from the beginning of riboflavin sprayed till 5 days post treatment(dpt).The results showed that riboflavin triggered ROS accumulation in plants during the period tested and the levels of ROS reach the highest at 2 hours post treatment(hpt),but restore to the original level at 5 dpt.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 P.syringae pv.tomato DC3000 inoculation, we analyzed the cellular and molecular defense responses.Catalase,which infiltrated at 30min after riboflavin treatment,nullifies the escalated oxidative state and the augmented ROS accumulation when infected by P.syringae pv.tomato DC3000. Moreover,the disease resistance is also abolished in riboflavin pretreated but catalase treated plants,which suggests that the disrupted redox homeostasis is required in riboflavin-induced priming.However,the disrupted redox homeostasis is independent of NPRI.3.The role of a long chain fatty alcohol oxidase on pathogen defense response in ArabidopsisThe Candida cloacae faol gene encodes a membrane bound,flavin-containing, hydrogen peroxide generating,long chain alcohol oxidase.This gene has not been isolated from plants or animals.The plants' resistance to pathogen,bacterial and viruses generally depends on whether the plant could identify the pathogens at an early time and start defense response.A fast response is called oxidative burst.The reactive oxygen species(ROS) generated from plant's defense response is significant to resistant pathogen intrusion.Compared with Col-0,fao3 mutant exhibits aggravating symptoms and increased bacterial propagation after P.syringae pv.tomato DC3000 infection.Then we detected the ROS levels by measuring H₂O₂ content,as well as the sensitivity of the plants to the extra oxidative stresses,generated by PQ.The results suggested that there was depressed oxidative state in fao3.The long chain fatty alcohol oxidase is related with hydrogen peroxide generating,so the change of the redox state of the mutant influenced pathogen defense response.Then we extracted the membrane protein of the wide type and mutant respectively and assayed the long chain alcohol oxidase activity. This is the first time assaying FAO activity in plants.