The Biosynthesis Pattern Of Anthocyanin And Flavonoid In Bud Sport 'Red Zaosu' Pear

Pear is one of the most common cultured fruit trees in China.Its fruit are enriched with flavonoid metabolites.Some kind flavonoid compounds like anthocyanins make the most contribution on red coloration on pear fruit,some other flavonoid compounds like isorhamnetin glycosides are popular diet resources because of its potential health benefits.Previously,our research group found a red fruit peel sport from ‘Zaosu'pear in 2004,and further designated and certificated at 2013 in China.Our previous research results showed that the red fruit trait and related anthocyanin pathway is not the only mutated trait in ‘Red Zaosu',other flavonoid pathways,especially for flavonols pathway also apparently changed in ‘Red Zaosu'.Therefore,this research is focused on the flavonoid biosynthesis regulatory network in the fruit of ‘Red Zaosu',and we also investigated the related mutation mechanism in ‘Red Zaosu'.The major results are the following:1.4 R2R3-MYB transcription factors(TFs)were screened as the candidate regulators of flavonoid biosynthesis in pear fruit from the pear genome because of the correlation between its expression pattern and flavonoid concentration.The expression patterns of 4 MYB TFs including PbMYB10,PbMYB10 b,PbMYB9 and PbMYB3 were highly correlated with most flavonoid biosynthetic structural genes.Moreover,the expression patterns of the 4 MYB TFs were also consistent with the flavonoid compounds concentration between ‘Zaosu'and ‘Red Zaosu'.Considering the bio-function on anthocyanin regulation of PbMYB10 has been verified in pear,PbMYB10 b,PbMYB9 and PbMYB3 were mainly focused in this research.PbMYB10 b not only positively regulated anthocyanin biosynthesis in pear fruit,but also affect proanthocyanin accumulation by up-regulating the expression of flavonoid biosynthetic structural gene PbDFR in pear fruit.PbMYB9 could not only up-regulate the biosynthesis of proanthocyanin but also the biosynthesis of anthocyanin and flavonols via promoting the expression of PbUFGT1 in pear fruit.2.The red fruit stripes and red leaf veins in ‘Red Zaosu' are resulted from vascular related anthocyanin accumulation and distribution,this atypical trait is different from other common red pear cultivars.Except for the red stripped fruit,the flowers,leaves and buds are also red coloured.The coloration model in those red organs has a common character that the red coloration areas were unevenly distributed and spatially correlated with vascular locations.3.26 candidate genes were screened as mutation co-related genes from the comparative transcriptome analysis on young leaves,mature leaves and fruitlets between ‘Zaosu'and ‘Red Zaosu'Young leaves,mature leaves and young fruits of ‘Zaosu' and ‘Red Zaosu' were selected for RNA-seq because of their typical vascular related anthocyanin.26 different expressed genes related to the mutation trait were further screened by comparative transcriptome analysis.A relative complete flavonoid biosynthesis regulatory network including HY5,the anthocyanin regulator PAP type MYBs,their target biosynthetic genes FLS and UFGT were identified among the 26 candidate genes.Moreover,the HY5 target gene early light inducible protein 1,ABA transporter in stomata ABCG40 and gibberellic oxidase GA2ox8(related to dwarf,anthocyanin,photomorphosis)were also identified in the 26 candidate genes.4.PbGA2ox8 was correlated with the mutation trait in ‘Red Zaosu'The ratio of red leaf and green leaf plantlets was near 1:1 in the F1 progeny of ‘Red Zaosu' and ‘Yuluxiang' pear.The mutation of ‘Red Zaosu' should be located in a single locus and probably not in the coding region of one gene.The expression patterns of 13 from the 26 candidate genes were linkage with the mutation trait in the F1 progeny population of ‘Red Zaosu'.Compared with ‘Zaosu',the methylation level in the promoter region of PbGA2ox8 in ‘Red Zaosu' was significantly reduced,this observation was consistent with the overexpression pattern in different organs of ‘Red Zaosu'.5.PbGA2ox8 could positively regulate anthocyanin biosynthesis in pear fruit and leaves.The anthocyanin induction by PbGA2ox8 partially depend on calcium signal and preferentially occurred on calcium enriched areas nearby vascular and fruit spots.To investigate whether PbGA2ox8 could induce the anthocyanin accumulation nearby vascular bundles,PbGA2ox8 was transiently overexpressed in fruitlets and young leaves of ‘Zaosu'pear.The results showed that overexpressed PbGA2ox8 could induce anthocyanin biosynthesis in these two organs.The anthocyanin positive regulator PbMYB10,anthocyanin biosynthetic gene PbUFGT1 and transporter PbGSTF12 were also up-regulated by PbGAox8?To be specific,the anthocyanins induced by overexpressed PbGA2ox8 was enriched nearby vascular in ‘Zaosu'leaves.We further observed that calcium was enriched in anthocyanin located areas nearby vascular or fruit spots.These results indicated that the anthocyanin accumulation in ‘Red Zaosu' fruit depent on calcium signal.Anthocyanin also cannot be accumulated nearby vasucular bundles in transient PbGA2ox8 ‘Zaosu' young leaves.Taken together,the PbGA2ox8 induced anthocyanin accumulation preferentially on calcium enriched areas nearby vascular bundles and fruit spots,and thus formed the longitudinal stripes in ‘Red Zaosu' fruit.