The Regulation Of Floral Scent Metabolism By Auxin Signal And Auxin-dependent Factors In Hedychium Coronarium

Hedychium coronarium,the flowers possess the white color,elegant shape and pleasant fragrance.The blooming of H.coronarium flowers results in the emission of a large number of volatile compounds,which mainly consist of linalool,(E)-β-ocimene,1,8-cineole,(E,E)-α-farnesene as well as the benzenoids/phenylpropanoids methyl benzoate.However,the mechanisms of biosynthesis and metabolic regulation of floral fragrance remains unclear.As an important plant hormone,auxin is involved in the regulation of flower development and the biosynthesis of various secondary metabolites,but there are few studies involving the regulation of volatile secondary metabolites.In this study,the influence of auxin inhibitor p-chlorophenoxy isobutyric acid(PCIB)on the floral volatiles release of H.coronarium flowers were analyzed.The content of endogenous IAA in the petals and the emission of the volatiles during the developmental stages were measured.The Aux/IAA family as the key element in auxin signaling was identified by bioinformatics,and the key genes possible involved in floral scent production were screened out.Meanwhile we identified the genes which response to the auxin signal such as MYB and ARF and discovered their transcriptional target genes.The main results are stated below:1.After treatment of flowers with auxin inhibitor PCIB,the amount of volatiles was significantly reduced and the expression level of key bottom biosynthesis enzyme genes which involved terpene and phenylpropane pathways were also decreased.Meanwhile the contents of endogenous hormones IAA and JA(jasmonic acid)were decreased.However,the ABA(abscisic acid)content was increased.The content of endogenous IAA in the developmental process of flower petals was determined.The results showed that the content of IAA was lower in the flower bud stage,and increased gradually towards initial stage,then remained high in the blooming period.Meanwhile the release of volatiles was high in the half-open stage and reached to peak in the full bloom period.It indicated that auxin is likely the key regulated hormone which is involved in flowers scent formation and might play the role by the cross talk with other kinds of hormones.2.Through bioinformatics and using the genome data 27 Aux/IAA genes were identified in H.coronarium.The cis-element of the promoter sequences were carried out,the results indicated that Hc IAAs may respond to various hormones such as auxin,abscisic acid and ethylene.The expression levels of Aux/IAA genes were analyzed at different flower developmental stages,tissues and exogenous hormones treatment.Four key Aux/IAA(Hc IAA2,Hc IAA4,Hc IAA6,Hc IAA12)that may be involved in floral volatiles synthesis were chosen.Subcellular localization experiments were performed on these candidates and the results showed that they were all located in the nucleus.3.The expression at m RNA and protein levels of Hc IAA4 and its interacted protein Hc MYB1 were analyzed.The gene expression levels of Hc IAA4 and Hc MYB1 increased after 6 h of IAA treatment,and decreased after 2 h of PCIB treatment.The protein expression levels of both were amplified by IAA and reduced by PCIB treatment.The interaction intensity of Hc IAA4 and Hc MYB1 increased 59 % under the induction of IAA and decreased 33 % under PCIB treatment in yeast cell.Hc IAA4 as the interactor of Hc MYB1 suppressed the transcriptional activation ability to pro Hc BSMT2.Hc IAA4 might played the role as a negative interacted factor in auxin signal.4.Two novel R2R3-MYB transcription factors were cloned and named as Hc MYB2 and Hc MYB3,respectively.Quantitive Real-time PCR analysis showed that Hc MYB2 and Hc MYB3 were both flower-specific and floral developmental regulated.The expression level of Hc MYB2 and Hc MYB3 were induced by IAA and suppressed by PCIB,indicating both are the factors which involved in auxin signal response.Silencing the expression level of Hc MYB2 and Hc MYB3 by virus-induced,the total quantity of volatiles of flowers were modified.In Hc MYB2-silenced flowers,the main volatiles were decreased,such as eucalyptol,(E)-β-ocimene,1,8-cineole and methyl benzoate were down-regulated 17 %,18 %,68 % and 37 %,respectively.Moreover,the key biosynthesis genes Hc TPS3,Hc TPS5 and Hc BSMT2 were down-regulated 46 %,32 % and 57 %,respectively.In Hc MYB3-silenced flowers,only the quantity of eucalyptol was decreased into 55 %.5.Yeast one-hybrid,dual luciferase and electrophoretic mobility shift assay(EMSA)experiments confirmed that Hc MYB1 and Hc MYB2 activated the expression of methyl benzoate transferase Hc BSMT2.Additionally,Hc MYB2 activated the expression of the linalool synthase gene Hc TPS5.However,the promoters motif cannot be binded by Hc MYB3.Overexpression of Hc MYB2 in embryogenic callus,the relative expression level of Hc MYB2 in resistant buds tissues was 26 folds than control.Meanwhile the target gene Hc BSMT2 and Hc TPS5 showed 15.6 and 14.5 times higher,respectively.6.One auxin response factor Hc ARF7 was cloned.The phylogenetic tree analysis indicated that Hc ARF7 show the closest relationship with Os ARF7 which is from monocotyledonous family.Quantitive RT-PCR showed Hc ARF7 was expressed in multiple tissues;the expression of Hc ARF7 is floral developmental regulated.Yeast two-hybrid experiments showed that Hc ARF7 interacted with Hc IAA4 and Hc IAA6.Yeast one-hybrid and dual luciferase experiments confirmed that Hc ARF7 activated the expression of the(E)-β-ocimene synthase Hc TPS3.