Molecular Mechanism Of Glucose Regulating Anthocyanin Biosynthesis Of Tree Peony Cut Flower

Flower color is one of the important traits of ornamental plants. Anthocyanin is the main pigment for flower coloration of angiosperms. According to the studies of model plants, sugars not only provide metabolism substrate for anthocyanin biosynthesis, but also regulate the expression of anthocyanin biosynthetic regulatory genes and structural genes through sugar signalling. Tree peony(Paeonia suffruticosa), a Chinese traditional ornamental plant, is greatly appreciated by Chinese from ancient to now. Understanding the regulation of exogenous sugar on flower color and anthocyanin biosynthesis of tree peony cut flowers would provide theoretical foundation for the postharvest technology development of tree peony cut flowers. Cut flowers of a purple red cultivar’Luoyang Hong’of tree peony were used as the materials in this study to reveal the molecular mechanism of the involvement of glucose in improving the flower color and regulating the anthocyanin biosynthesis. What is more, gene family members of hexokinase (HXK), a key protein involved in sugar sense and signaling, were isolated in tree peony and their biological functions were further studied. The results are showed as follows:(1) The quality of flower color of cut flower is inferior to that of on-tree flower of tree peony ’Luoyang Hong’. Anthocyanins content and soluble sugar content of cut flowers are lower than those of on-tree flowers. Exogenous60,90and120g-L"1glucose enhance soluble sugar content and anthocyanins content in petals, and also improve petal color quality of cut tree peony’Luoyang Hong’ with lower lightness and higher redness and chroma.(2) The transcriptome from mixed petals of cut tree peony’Luoyang Hong’ was contructed and sequenced using the Illumina platform. A total of57,206,122raw reads were obtained. After data filtering and sequence assembly,50,829unigenes were generated. Based on the analysis of unigene information, ANS gene has one unigene, while other anthocyanin biosynthetic genes CHS, CHI, F3H, F3’H, DFR, MYB, bHLH and WD40exist in the genome of tree peony ’Luoyang Hong’ as multigene family. It is predicted that tree peony’Luoyang Hong’ probably has6HXK gene family members.(3) Based on the unigene sequences of petal transcriptome of tree peony’Luoyang Hong’,5regulatory genes and6structural genes of anthocyanin biosynthesis from tree peony were isolated. Phylogenetic tree analysis reveals that the proteins encoded by PsbHLHl, PsbHLH3, PsWD40-1, PsWD40-2and PsMYB2are probably involved in the regulation of flavonoid and anthocyanin biosynthesis in tree peony. PsCHIl is highly expressed in leaves, but other10anthocyanin biosynthetic genes are highly expressed in the flower-related tissues (petals, stamens, carpels and sepals). Low expression levels of PsbHLH3, PsWD40-1, PsWD40-2, PsMYB2, PsCHSl, PsF3H1and PsDFRl may be responsible for less anthocyanins content and color fading in cut tree peony’Luoyang Hong’.(4) Treatment of3-O-methylglucose (a kind of glucose analog which can’t be phosphorylated by hexokinase) enhances anthocyanins content and induces the expression levels of PsbHLHl, PsbHLH3, PsWD40-1, PsWD40-2, PsMYB2, PsCHS1and PsCHI1in short time, suggesting that short time improvement of osmotic pressure induces the anthocyanin biosynthesis of cut tree peony’Luoyang Hong’. Glucose signal induces the expression levels of regulatory genes PsWD40-2, PsMYB2and structural genes PsCHSl, PsCHI1, PsF3’H1. Among them, PsWD40-2, PsMYB2, PsCHSl and PsCHIl are regulated by glucose signal via HXK-independent pathway, while PsF3’H1is regulated by HXK-dependent glucose signaling pathway. Expression level of PsF3’H1shows a close relationship to the anthocyanins content of cut tree peony ’Luoyang Hong’, so PsF3’H1is regarded as the key gene involved in the regulation of glucose on the anthocyanin biosynthesis of cut tree peony’Luoyang Hong’. Treatment of mannose, known to be phosphorylated by HXK, constantly up-regulated the expression of several anthocyanin biosynthetic genes, down-regulated the expression of PsF3’H1and PsANSl, and greatly inhibited anthocyanin accumulation in petals.(5) Anthocyanins content of cut tree peony ’Luoyang Hong’ with the treatment of the same molar concentration sucrose is lower than that of glucose-treated cut flowers. The expression levels of PsWD40-1, PsWD40-2, PsCHSl, PsF3H1, PsDFR1and PsANS1in sucrose-treated cut tree peony flowers are significantly higher than those in glucose-treated cut tree peony flowers, while the expression levels of PsCHIl and PsF3’H1in sucrose-treated cut tree peony flowers are lower than those in glucose-treated cut tree peony flowers.(6) Full-lenth cDNA sequences of PsHXK1and PsHXK2, the homologous genes of HXK gene involved in hexose sense and siganlling, were isolated from tree peony in this present study. Based on sequence analysis and phylogenetic tree analysis, it is predicted that PsHXK1and PsHXK2have the catalytic function of hexose phosphorylation. PsHXK1is highly expressed in the petals, while PsHXK1is highly expressed in the stamens. It is also predicted that the expression levels of PsHXK1and PsHXK2are probably associated with the hexoses content in the petals of tree peony’Luoyang Hong’. Overexpression of PsHXK1and PsHXK2in Arabidopsis thaliana reveals that PsHXK1and PsHXK2have the ability of sensing and transducting sugar signal and overexpression of PsHXK1and PsHXK2enhance anthocyanins content of Arabidopsis under high concentration of glucose condition through the up-regulation of the expression of several anthocyanin biosynthetic genes.The above results indicated that, besides osmotic effect and metabolic effect, glucose promotion of anthocyanin accumulation is mainly attributed to the regulation of glucose signalling. Furthermore, PsHXK1and PsHXK2play essential roles in the regulation of glucose on anthocyanin biosynthesis. Above conclusions should contribute to a better understanding of molecular mechanism of glucose regulating anthocyanin biosynthesis in cut tree peony, and would provide theoretical foundation for the postharvest technology development of cut tree peony.