Roles Of Expansins And Riboflabvin In Regulating Growthenhancement And Defense Response In Plants

Expansin proteins(EXPs) act to loose cell walls and modulate growth of the cell and plant under mediation by some hormones.It is not clear whether a hormone is specific and distinct hormones interact to regulate EXP activity.Harpins are glycine-rich, protease-sensitive,heat-stable,acidic proteins produced by Gram-negative plant pathogenic bacteria,and are required for induction of the hypersensitive response(HR) or hypersensitive cell death(HCD) in non-host plants of bacteria.Application of harpins to many plants can enhance plant growth,induce resistance to pathogens or insects and improve tolerance to environmental adversity like drought.However,what signaling processes contribute to enhanced plant growth(EPG) in plants treated with HrpN_Ea has been unclear.This study was aimed to address questions on EPG by inducing expansin genes expression involved in ethylene(ET) and gibberellin signaling pathways induced by HrpN_Ea.Riboflavin functions as a coenzyme in many physiological processes in microbes, plants and animals.It takes an important role in plant disease resistance signaling and possibly regulates plant growth signal transduction.Riboflavin is an activator of a novel signaling pathway leading to systemic resistance.Riboflavin-triggered signaling requires protein kinases and regulation by NIM1/NPR1,but does not require accumulation of salicylic acid(SA).Here,we report that riboflavin induces pathogen resistance by activating a Pto-mediated Pti cascade in tomato.1.Plant growth and expansin gene expression regulated by ethylene and gibberellin in response to a bacterial typeâ…¢effectorTo relate an ET signal with EXPs and EPG.we studied a transcriptional coincidence of ET-response genes with EXPs and EPG.ETR1 was induced by the protein and increased levels with time.EIN2 was constitutive and was enhanced quickly at 6 hours post treatment (hpt) with HrpNEa but not change evidently thereafter.The PDF1.2 and PR-3b were expressed after induction,suggesting an activation of the pathway.Consistently,LeETR1 and NtETR1,which encode ET receptor homologs in tomato and tobacco,showed to be induced by HrpN_Ea applied to 20-d plants.We found that the Arabidopsis etrl-1 mutant markedly compromised AtEXP10 expression,compared to WT,in 20-d seedlings sprayed with a HrpN_Ea solution.EPG in tomato,tobacco and rice was impaired markedly by 1-MCP, an ethylene sensitivity inhibitor,present in HrpN_Ea treatment.Growth of plants subsequent to soaking seeds in a solution of HrpN_Ea and 1-MCP was evidently decreased relative to that in treatment with only HrpN_Ea.Therefore,ET sensing is critical to the induction of EXP expression and EPG by HrpN_Ea in the four plants.We compared HrpN_Ea and GA₃ in the effects on OsEXPs,which increase expression in rice plants treated with GA₃,an important form of GA.When applied to 20-d rice plants, HrpN_Ea acted similarly as did GA₃ in inducing expression of OsEXP2,OsEXP4 and OsEXP16.However,HrpN_Ea seemed to function slower and less effectively than GA₃; Gene expression became evident earlier and expression levels also were greater in response to GA₃ vs.HrpN_Ea.In particular,OsEXP2 and OSEXP16 were expressed at high extents successively since 6 hpt with GA₃ but increased expression levels gradually with time since 12 hpt with HrpN_Ea.OsEXP4 markedly accumulated transcript at 12 hpt with HrpN_Ea or GA₃ and thereafter remained a moderate level of expression.We addressed if EXP expression and EPG require a GA signal by determining effects of the GA synthesis inhibitor pp333 on EXPs and EPG.The expression of OsEXP4 was greatly compromised when pp333 was present in HrpN_Ea treatment.In WT tomato,the expression of LeEXP2 induced by the application of HrpN_Ea was inhibited by pp333 supplied to HrpN_Ea treatment. NtEXP2 showed evident constitutive expression and was enhanced markedly to increase expression level by HrpN_Ea applied alone;pp333 eliminated the effect.In consistence, pp333 evidently impaired the effect of HrpN_Ea in promoting growth of the three plants. Based on these results,a GA signal is required for the induction of EXP expression and EPG.2.Activation of Pathogen Defense and Plant Growth Pathways by RiboflavinTo determine whether a protein kinase cascade is involved in riboflavin-induced pathogen resistance,tomato plants treated with the vitamin were tested for relevant gene expression and resistance phenotype.Tomato resistance gene Pto,its downstream protein kinase-encoding genes Pti4,Pti5,and Pti6,and defense genes PR-1a,PR-1b1,NP24,and pin2 were cloned by PCR from a Chinese variety of tomato based on previous reports. These genes were expressed,as determined by quantitative RT-PCR,in tomato plants treated with riboflavin in courses of time consistent with their function in a signaling cascade.Expression of Pti4,Pti5,and Pti6 was enhanced in plants treated with riboflavin in 12 hours post treatment.Expression of PR-1a,PR-1b1,NP24,and pin2 was induced by riboflavin to remarkable levels in 24 hours after treatment.Plants treated with riboflavin developed resistance to P.syringaes.The presence of the protein kinase inhibitor K252a in riboflavin-treated plants abolished both gene expression and development of resistance. Based on these data,we concluded that riboflavin induces pathogen resistance by activating a Pto-mediated Pti cascade in tomato.In conclusion,results obtained from studies described above have provided us with further understanding on action mechanism of growth and defense in plants responding to HrpN_Ea and riboflavin.First,plant growth enhancement was induced by HrpN_Ea concomitantly with induced expression of EXPs gene,a cell wall loose protein,and both inductions require GA and ET in plants responding to HrpN_Ea.The results show that inhibiting Arabidopsis,tomato and tobacco plants to synthesize GA or sense ET compromised EXP expression and EPG phenotypes.Second,riboflavin induces pathogen resistance by activating a Pto-mediated Pti cascade in tomato.