Study On The Molecular Mechanism Of Melatonin Inhibiting Arabidopsis Anthocyanin Accumulation

Melatonin(5-methoxy-N-acetyltryptamine)is an indoleamine that was initially discovered in the bovine pineal gland.It's also a well-known animal hormone.As a necessary endogenous molecule,melatonin exists in bacteria,fungi,animals,and even in plants,which has a range of physiological function.Unlike studies in mammalian cells,the role of melatonin in plant metabolism is not clear.Anthocyanin is a plant-specific flavonoid type metabolite for controlling plant fertility and protecting plants from environmental stresses.The plant hormone jasmonate also has a positive impact on anthocyanin production.Jasmonate stimulates anthocyanin biosynthesis by inducing the expression of several key anthocyanin biosynthesis genes.Here,the model plant Arabidopsis thaliana is the subject for study.We dissected the role of melatonin in plant metabolism by taking use of jasmonate to boost anthocyanin biosynthesis.Through observation,we found that melatonin alone could alter anthocyanin production and was also able to suppress jasmonate-stimulated anthocyanin production.Besides,by observing the melatonin over-producing and defective lines,we found that over-production of endogenous melatonin is also sufficient for inhibiting the jasmonate-induced anthocyanin biosynthesis.Based on these results,we performed further experiments to illuminate the mechanism.JASMONATE-ZIM-DOMAIN(JAZ)proteins are repressors of jasmonate signaling pathway.In the presence of jasmonate,JAZs can be ubiquitinated and degraded through the 26S proteasome.We found that melatonin has no effect on JAZ1 protein stability and could't suppress JA-induced JAZ2 degradation.Then we examined jasmonate-responsive marker gene(JAZ1,JAZ2,JAZ3,JAZ5,JAZ9,JAZ10)expression and other jasmonate-related physiological roles.All these characteristic jasmonate signaling features were not changed by melatonin.Furthermore,melatonin could also inhibit jasmonate-induced anthocyanin accumulation in either jazQ or jasmonate biosynthesis aos mutants.Taken together,these results demonstrate that melatonin does not antagonize jasmonate-stimulated anthocyanin production by abrogating jasmonate signaling.Meanwhile,we chose papl-D mutants to over-accumulate anthocyanin by JA and found that melatonin could not curtail the already accumulated anthocyanin in papl-D mutants.This result showed that melatonin does not elicit anthocyanin catabolism.Then we found that jasmonate-induced the transcription of almost all the key anthocyanin biosynthesis genes(PAL,C4H,4CL,CHS,CHI,F3H,F3'H DFR,LDOX,UF3G7),but that melatonin could not induce all the related genes or reverse the pattern that jasmonate-induced,which means that melatonin modulates anthocyanin biosynthesis not by gene transcription level.Finally,we supplied anthocyanin biosynthesis precursors(L-phenylalanine,cinnamate,naringenin chalcone,naringenin,dihydrokaempferol)to examine their roles in anthocyanin biosynthesis and found that only low concentrations of dihydrokaempferol could significantly rescue the melatonin effect.This result suggests that melatonin might inhibit flavonone-3-hydroxylase(F3H)activity and then reduce the production of dihydrokaempferol,which finally cut down the anthocyanin contents.Our work sheds new light on the role of melatonin in plant cell metabolism.Melatonin plays a negative role in plant anthocyanin metabolism.We also determined that melatonin does not abrogate jasmonate signaling.Through preliminary study,we proposed that melatonin acts on the anthocyanin biosynthetic pathway,most likely at the F3H-catalyzed dihydrokaempferol step.