The Transcriptional Regulation In Dark And The Ubiquitylation-Mediated Degradation Of Melatonin Synthetase In Apple(Malus Zumi Mats)

Melatonin(N-acetyl-5-methoxytryptamine)is the effective scavenger of free radicals,and functions in stress response.Also,as an important bioactive reagent,it regulates plant growth and development.The synthetic pathway of melatonin has been well elucidated in plants.We firstly isolated melatonin key synthetase genes MzSNAT5,MzASMT1,MzASAMT3 and MzASMT9 from apple(Malus zumi Mats).But the molecular regulation of melatonin synthesis remains unclear.Our study contained two major pathways which regulated melatonin synthesis:one is 'transcriptional regulation',i.e.the 'increase income' pattern,which is started through upregulating the expression of melatonin synthetase genes to increase the protein levels of melatonin syhthetases.The other one is 'post-translational regulation',i.e.the 'reduce expenditure' pattern,which is achieved through decreasing the ubiquitylation-mediated degradation of melatonin synthetases to lengthen their half-life period.It has been proved that melatonin level is induced by darkness and intensive light in plants.Therefore,we conjectured that the key light/dark-responsive transcription factors could regulate the transcription of melatonin synthetase,and screened them.We found that KJ transcription factor might regulate melatonin synthesis under darkness in apple and Arabidopsis.KJ repressed the expressions of melatonin key synthetase genes and the melatonin synthesis,through directly binding to the promoters of melatonin key synthetase genes.In addition,we isolated three MzWL proteins,which were proved to have the interactions with melatonin synthetases.WL proteins bind the melatonin key synthetases and decreased their ubiquitylation-mediated degradation,resulting in stabilizing melatonin synthesis.Our research on the molecular regulation of melatonin synthesis in apple,will contribute to engineering apples with high melatonin content.The results were as follows:1.The in vitro experiments showed that the melatonin synthetases MzSNAT5,MzASMT3,AtSNAT6 and AtASMT8 could catalyze melatonin and its precursor N-acetylserotonin production.And in vivo,the melatonin contents increased in the transgenic Arabidopsis overexpressing AtSNAT6/AtASMT8 and ectopically expressing MzSNAT5/MzASMT3.Taken together,it is proved that these genes encode melatonin synthetases.2.The in vitro EMSA results proved that MzKJ could specifically bind to the promoter of MzASMT3,and AtKJ could specifically bind to the promoters of AtSNAT6 and AtASMT8.In addition,the in vivo ChIP-qPCR experiment proved that MzKJ and AtKJ could bind directly to the promoters of MzASMT3,AtSNAT6 and AtASMT8.The synthetases gene expressions and the melatonin levels are significantly higher in transgenic apple callus with RNAi interfered KJ and KJ Arabidopsis mutant,and significantly lower in transgenic apple callus/Arabidopsis overexpressing KJ.It suggested that KJ repressed the expressions of melatonin synthetase genes,through direct-binding to their promoters,and negatively regulated melatonin synthesis.3.In dark,both of the melatonin content and the expressions of its synthetase genes improved significantly in wild-type apple and Arabidopsis.The level of KJ protein decreased in dark,therefore ChIP-qPCR showed that the promoters bound with KJ decreased as well.It proved that,through decreasing the inhibition by KJ on the expressions of melatonin synthetase genes,melatonin synthesis increases in dark.4.Exogenous melatonin treatment increased melatonin content in plants and inhibited cotyledon opening.And KJ influenced cotyledon opening,through regulating melatonin synthesis under darkness.5.Three MzWL genes were cloned.And the results of BiFC and Co-IP in apple proved that MzWLs interact with MzSNAT5,MzASMTl and MzASMT9.After their binding to make a complex,the ubiquitylation-mediated degradation of melatonin syhthetases decreased significantly.Taken together,the decrease of KJ results in increased melatonin synthesis in dark and influences cotyledon opening,via decreasing its inhibition on the expressions of melatonin key synthetase genes.And WL proteins bind to melatonin key synthetases to from stable complex and decrease their ubiquitylation-mediated degradation in plants.This is the first report on the molecular mechanism of melatonin synthesis regulation in plants.