Involvement Of Hexokinase Genes Of Tree Peony Cut Flower In The Regulation Of Anthocyanin Biosynthesis By Glucose And Ethylene

Tree peony(Paeonia suffruticosa Andr.)has been widely accepted as cut flowers for its commercial value by people at home and abroad.However,up till now,relative research on postharvest techniques and application is rarely reported.Flower color is one of the most important postharvest quality of cut flowers,and anthocyanin is the pnmary pigment in higher plants.Research on model plant not only revealed that sugar could interact with phytohormone like ethylene as a signal molecule,but also identified the significant roles of hexokinase(HXK)in sugar signaling.Our group have elucidated in previous research on tree peony cut flowers that in the senescence term,glucose could interact with ethylene signaling at several sites in a HXK-dependent way,and thus impair the effects of ethylene on senescence of cut flowers;while in terms of flower color,exogenous glucose could promote the biosynthesis of anthocyanin,by contrast,exogenous ethylene inhibits anthocyanin biosynthesis and combined supplying of glucose and ethylene weakens the inhibition by ethylene.Therefore,it would contribute to dissecting the crosstalk between glucose and ethylene signaling and illustrating the mechanism of how anthocyanin biosynthesis is affected by glucose and ethylene to study on the roles of HXK in this process,and thus laying a theoretical foundation for developing preservation techniques.In this study,'Luoyang Hong',a cultivar sensitive to ethylene,was chosen to isolate HXK homologous genes.The PsHXKs were analysed based on bioinformatics and expression patterns,and their roles in regulating anthocyanin biosynthesis by glucose and ethylene signaling are explained by means of transgenosis.The principle experimental results are as follows:(1)Based on the petal transcriptome of tree peony 'Luoyang Hong',a HXK homologous gene,PsHKL1,was isolated.Combined with the other two homologous genes,PsHXK1 and PsHXK2,the predicted protein sequences and expression patterns of the three PsHXKs genes were analysed.Bioinformatics analysis showed that the predicted protein sequence of all the PsHXKs contain similar structural domain and conserved sequences related to sugar-binding in Arabidopsis AtHXKs.Among the three,the sequence of PsHKL1 has many substituted sites of amino acids in these conserved sequences,while in PsHXK1 and PsHXK2,these conserved sequences share high similarity with those of AtHXKl,the identified glucose sensor.It is predicted that PsHXK1 and PsHXK2 might belong to type B HXK,like AtHXK1.Besides,in the phylogenetic tree,both PsHXK1 and PsHXK2 cluster together with AtHXK1.Thereby,it is speculated that PsHXK1 and PsHXK2 might execute signaling functions.Results of RT-PCR showed that during the process of postharvest development,expression levels of all three genes rise at first and then drop,which is similar to patterns of a*and C*values and anthocyanin content;exogenous glucose evidently boost the expression of PsHXK1 and PsHXK2 but PsHKL1;the transcript abundance of all three are elevated by exogenous ethylene,while 1-MCP(1-methylcyclopropene)restrain the influence.Besides,the expression of PsHXK1 is hypersensitive to exogenous glucose,and although the expression level of PsHXK1 is relatively lower,it exhibits the most notable changes in response of ethylene.Based on the significant regulatory functions of glucose and ethylene in anthocyanin biosynthesis,it is worthy to make further study on the roles of PsHXK1 and PsHXK2 in the regulation of anthocyanin biosynthesis by glucose and ethylene signaling.(2)Overexpression of PsHXKs in Arabidopsis reveals that:compared to wild-type plants,transgenetic plants show more sensitivity to exogenous glucose,ethylene and combined supplying of glucose and ethylene in respects of root length,anthocyanin content and expression levels of relative genes involved in anthocyanin biosynthesis,ethylene biosynthesis and ethylene signaling;with combined treating of glucose and ethylene,the anthocyanin contents and most genes involved in anthocyanin biosynthesis of transgenetic plants are observably lower than those with independent glucose supplying,and the negative regulator gene,AtMYBL2,shows an oppositive expression pattern,while in wild-type plants,there is little difference between these treatments;Accordingly,compared to wild-type,the expression levels of AtACS7,AtEIN3 and AtEIL1 in transgenetic plants are evidently higher under combined supplying of glucose and ethylene than those with glucose.Therefore,glucose could interact with ethylene signaling at the sites of AtEIN3 and AtEIL1 and thus regulating anthocyanin biosynthesis at transcriptional level.This crosstalk occurs at least partly in a PsHXKs-dependent way.