The Molecular Mechanism Study Of Salicylic Acid And Methyl Salicylic Acid Regulation Of Pollen Tube Growth In Arabidopsis

Salicylic acid(SA)is an important phytohormone in plants involved in regulation of plant defense,growth and development.Although it is much clear in its role of plant defense,the mechanism of SA in plant growth remains unknown.Rapid tip growth,as the extreme form of polar growth,is a common mode of cell expansion.In the model tip growth system,Arabidopsis pollen tubes,several small molecule hormones regulate their elongation,but how these rapidly-diffusing molecules control extremely localized growth remains mysterious.Here we study the effects and molecular mechanism of salicylic acid and methyl salicylic acid regulation of pollen tube growth in Arabidopsis.We showed that the inter-convertible salicylic acid(SA)and methylated SA(MeSA),well-characterized for their roles in plant defense,antagonistically regualte Arabidopsis pollen tip growth with SA being inhibitory and MeSA stimulatory.By analyzing pollen tube phenotype of mutant nprl,npr3,npr4 and npr3/npr4,we found that the effect of SA and MeSA was independent of known NPR3/NPR4 SA receptor-mediated signaling pathways,which implied that some other SA receptors regulating growth are located in pollen tube in Arabidopsis.Stable transgenetic overexpression of NahG was made to verify the effects of SA and MeSA on pollen tube growth.NahG pollen tube length was longer than wild type and SA-induced inhibition of pollen tube growth is suppressed by SA hydroxylase NahG.These results not only provided some evidence that endogenous SA inhibited pollen tube growth but also further proved that SA inhibited pollen tube growth in vitro.In order to understand the mechanism of SA and MeSA in regulation pollen tube tip growth,we observed several different important pathways in regulation pollen tube growth,such as clathrin-mediated endocytosis and exocytosis,alteration of methyl-pectin and demethyl-pectin in cell wall,calcium and actin concentration,Rop activity.Our results showed that SA inhibited clathrin-mediated endocytosis in pollen tubes associated with an increased accumulation of less stretchable demethylated pectin in the apical wall,whereas MeSA did the opposite.By employing active ROP1 maker CRIB4-GFP,the results showed that SA and MeSA alter the apical activation of ROP1 GTPase,a key regulator of tip growth in pollen tubes with SA promotion ROP1 activity while MeSA did the opposite.To verify this result,the amount of REN1,a negative regulator of ROP1,was observed by using stable transgenic plant line Lat52:REN1/ren1-1 The results was consistent with CRIB4-GFP.Analysis of GFP-ROP1 and renl-3 pollen tube phenotype with SA or MeSA treatment also supported the conclusion above that SA promoted ROP1 activty while MeSA did the opposite.However,neither SA nor MeSA affected exocytosis pathway,calcium,and actin concentration.Both MeSA methylesterase(MES6)and SA methyltransferase(BSMT1)that catalyze the interconversion between SA and MeSA are localized to the apical region of pollen tubes with MES6 location to the pollen tube germination site as well as the pollen tube tip,mimicking that of the active ICR1/RIP1 and BSMT1 enriching in the apical region similar to the distribution of exocytosis vesicles,indicative of their tip-localized production and consistent with their effects on the apical cellular activites.Furthermore,through forward genetic method,we found that a T-DNA insertional mutant mes6-1,which converted MeSA to SA,grew longer than Col-0 and was hypo sensitive to SA,while being hypersensitive to MeSA.The mes6-1 mutant reduced tip-localized CRIB4-GFP signal and plasma membrane over-accumulation of GFP-ROPI,thus suppressing the ROP1 over-expression and renl-3 phenotype and this inhibition can be re-captured by SA or MeSA treatment.In this study,we first time found that SA and MeSA antagonistically regulate Arabidopsis pollen tip growth though CME pathway and affecting ROP1 activity,which is independent of the known NPR3/NPR4 pathway.Furthermore,the pollen tube tip localization of MES6 and BSMT1 suggested that local generation of SA/MeSA signal can regulate polarized cell growth,providing a novel mechanism of cell polarity control apart from the one involving protein and mRNA polarization.