Transcriptional Regulation Of Proanthocyanidin Biosynthesis By MYB-bHLH Complex In Chinese Bayberry(Myrica Rubra) Fruits

Proanthocyanidins(PAs)are end products of the flavonoid pathways that contribute in many ways to the growth and survival of plants.PAs affect flavor and astringency in horticultural products and also benefit human health by reducing the risk of various diseases including cancer,atherosclerosis,cardiovascular disease,and other age-related diseases induced by free radicals.Therefore,PA biosynthesis and regulation in fruits has become one of the hot research topics in plant secondary metabolism.Chinese bayberry fruit is rich in flavonoids.Previous research on flavonoid synthesis and regulation in bayberry fruit mainly focused on anthocyanin,while the types,contents and synthesis of PAs were unclear.In this study,two genes encoding key PA biosynthetic enzymes,anthocyanidin reductase(ANR)and leucoanthocyanidin reductase(LAR),MrANR and MrLAR,were isolated in bayberry fruit,and their gene functions were investigated.In order to identify putative homologues of MYB,bHLH and WD40 controlling PA synthesis in other species,transcriptomes of two cultivars of Chinese bayberry fruit with different ripening stages were compared.In addition,the transcriptional regulatory roles of the selected MYB and bHLH transcription factors were investigated using molecular biology methods.This work may provide a resource for revealing the mechanism of PA biosynthesis and regulation in Chinese bayberry fruit.The results were as follows:1.MrANR and MrLAR genes were cloned by using RT-PCR and RACE amplification from Chinese bayberry fruit,and their gene functions were investigated.The results showed that MrANR was highly expressed at the early stage of fruit development when soluble PAs accumulated at high levels.Meanwhile,the transcript abundance of both MrANR and MrLAR observed at the late stage was paralleled with the high amounts of insoluble PAs.PAs in developing Chinese bayberry fruits were comprised predominantly of epigallocatechin-3-O-gallate terminal subunits,while the extension subunits were a mixture of epigallocatechin-3-O-gallate,epigallocatechin and catechin.Recombinant MrANR protein converted cyanidin to a mixture of epicatechin and catechin and delphinidin to a mixture of epigallocatechin and gallocatechin in vitro.Recombinant MrLAR was active with leucocyanidin as substrate to produce catechin.Ectopic expression of MrANR in tobacco reduced anthocyanin levels but increased PA accumulation.The catechin and epicatechin contents in transgenic flowers overexpressed MrANR were significantly higher than those of wild-type.However,overexpression of MrLAR in tobacco led to an increase in catechin levels but had no impact on PA contents.Quantitative real time PCR revealed that the loss of anthocyanin in transgenic flowers overexpressed MrANR or MrLAR is probably attributed to decreased expression of tobacco chalcone isomerase(CHI)gene.2.Two types of Chinese bayberry varieties,’Biqi’ and ’Shuijing’,were analyzed by RNA-seq to explore the molecular mechanisms of flavonoids accumulation during fruit development and ripening.The results indicated that all genes involved in anthocyanin biosynthesis and glycosylation were identified and their expression patterns were in accordance with total anthocyanin accumulation in developing fruits of ’Biqi’ and’Shuijing’.Expression levels of LAR and ANR were down-regulated in ’Shuijing’ in agreement with the decrease in PAs.The higher expression levels of LAR and ANR in’Biqi’ may be an important reason for the higher levels of total soluble PAs as compared to’Shuijing’.Phylogenetic analysis showed that 7 MYB genes,3 bHLH genes and 3 WD40 genes could be identified as putative homologues of PA-specific regulator in other species,which deserve further research.In addition,RNA-Seq data was validated by using qRT-PCR analysis with a high correlation,suggesting that the RNA-Seq data here are credible.3.The roles of MrMYB9,MrMYB308 and MrMYBPAl in regulation of PA biosynthesis were investigated.The results indicated that MrMYB9,MrMYB308 and MrMYBPAl all localized in the nucleus and contained one R2R3 repeat and five sites conserved in PA-specific MYBs.Phylogenetic tree revealed that these three bayberry MYBs were clustered with PA-regulating MYBs in other species.MrMYB9,MrMYB308 and MrMYBPA1 could activate the promoters of MrANR and MrLAR containing the MYBCORE cis-motif and AC-rich MYB binding cis-motifs,as well as the promoters of the upstream flavonoid biosynthesis genes when co-transfected with AtEGL3.Further transient assays in tobacco leaves suggested that MrMYB9,MrMYB308 and MrMYBPA1 were associated with AtEGL3 and triggered PA production.The expression levels of MrMYB9 and MrMYBPA1 in diverse tissues were consistent with MrANR and MrLAR transcript profiles and soluble PAs contents.The higher MrANR transcript levels and soluble PAs contents in ’Biqi’ was associated with the higher expression levels of MrMYB9 and MrMYBPA1 observed at the later stages in ’Biqi’ as compared to ’Shuijing’.During fruit development in ’Shuijing’,the expression patterns of MrMYB9,MrMYB308 and MrMYBPA1 correlated with those of MrANR and MrLAR,and with the accumulation of soluble PAs.These results suggest that MrMYB9,MrMYB308 and MrMYBPAl can activate promoters of MrANR,MrLAR and the upstream flavonoid biosynthesis genes to regulate PA biosynthesis in Chinese bayberry fruit;however,their functionality may have diverged in different genotypes.4.The roles of MrEGL3,MrbHLH1 and MrbHLH2 in regulation of PA biosynthesis were investigated.The results indicated that MrEGL3,MrbHLH1 and MrbHLH2 all localized in the nucleus and contained the conserved MYB interaction region,the bHLH domain and the ACT-like domain.Phylogenetic tree showed that these three bayberry bHLHs were clustered with PA-regulating bHLHs in other species.The transcript profile of MrbHLH1 correlated with that of MrANR and MrLAR,and with the accumulation of soluble PAs in different tissues,and during fruit development in ’Shuijing’.During later stages of development,transcripts of MrbHLH1 were more abundant in ’Biqi’ compared with ’Shuijing’,consistent with the higher MrANR transcript levels and soluble PAs contents of ’Biqi’.MrEGL3,MrbHLHl and MrbHLH2 could interact with MrMYB9,MrMYB308,MrMYBPA1 and AtTT2,respectively.Coexpression of MrEGL3/MrbHLH1 with MrMYB9/MrMYB308/MrMYBPA1 activated the promoters of MrANR and MrLAR,as well as the promoters of the upstream flavonoid biosynthesis genes,and increased PA levels in tobacco leaves.However,the induction of the promoter activity of MrLAR,MrCHI,MrF3’H,MrDFR1 and MrANS,and PA levels in tobacco leaves by MrMYB9/MrMYB308/MrMYBPA1 transient expression was higher in the presence of MrbHLH1 than that in the presence of MrEGL3.Taken together,these results suggest that both MrbHLHl and MrEGL3 can be an activating partner of MrMYB9/MrMYB308/MrMYBPAl in the regulation of the PA pathway in bayberry fruit;however,the effect of MrbHLH1 is more significant.