Molecular Cloning And Characterization Of BHLH Regulator Genes Related To Flavonoid Biosynthesis In Freesia Hybrida

The improvement of flower color has always been the focus of the botanist and horticulturalist.Flavonoid,as a kind of secondary metabolites in plants,is very important for flower color.The flavonoid biosynthesis is regulated by a complex consisting of MYB,bHLH,and WD40 proteins.The bHLH related in flavonoid biosynthesis seem to be quite complicated though a few bHLHs has been characterized especially in dicots.Freesia hybrida,a monocotyledonous genus in the Iridaceae consisting of 15 species,mainly distributes in southern Africa.The flower color diversity covers all the color ranges from white to red,yellow,pink and purple.Two bHLHs named FhGL3 L and FhTT8 L were characterized from Freesia.The results were detailed as follows.1.Cloning and bioinformatics analysis of FhGL3 L and FhTT8LBased on the transcriptome sequencing,two candidate bHLHs regulating the flavoniod pathway were revealed.The 2394-bp and 2440-bp full-length cDNA s were isolated by PCR from Freesia full-blooming flower cDNAs.The 2394-bp full-length FhTT8 L contained a putative opening reading frame(ORF)of 2094-bp that encoded a peptide of 697 amino acids,and the 2440-bp full-length FhGL3 L contained a putative ORF of 2061-bp that encoded a peptide of 686 amino acids,respectively.The full-length protein sequence alignment of FhTT8L/FhGL3 L and its homologs revealed the signatures of bHLH proteins,especially the bHLH domains involved in the formation of homo-or heterodimers with other bHLH proteins or DNA recognition 2.Expression pattern of FhGL3 L and FhTT8LExpression of FhTT8 L and FhGL3 L was analyzed by RT-qPCR at different stages of Freesia flower development.Overall,transcripts of FhTT8 L and FhGL3 L could be detected in all examined samples at different expression levels.Furthermore,both FhTT8 L and FhGL3 L could also be found in vegetative organs,which indicated that they might involved in different biological developments in different organs.3.Ectopic expression of FhGL3 L and FhTT8 L in ArabidopsisThe FhGL3 L were transferred into Arabidopsis of Col background under the control of cauliflower mosaic virus 35 S promoter.Results showed that FhGL3 L enhanced accumulation of anthocyanins.However,trichome formation on leaf lamina and hypocotyl was restricted.Several structural genes in the anthocyanin biosynthetic pathway in Arabidopsis were characterized by RT-qPCR,while AtGL2 was revealed severely down-expression.The Arabidopsis leaves protoplasts transiently expressing FhTT8 L and control plasmids were used to extract total RNA and synthesize cDNA templates,respectively.The FhTT8 L over-expressed in Arabidopsis protoplasts also mainly induced higher transcripts of genes related in flavonoid biosynthesis.4.Function mechanism of FhGL3 L and FhTT8LSeveral plasmids were constructed and used in Arabidopsis protoplast transfection system.FhGL3 L showed strong interactions with AtPAP1,AtTT2 and AtGL1.Either AtPAP1 or AtTT2 could interact with FhTT8 L.No interaction was detected between FhTT8 L and AtGL1,however.We concluded from these results that FhTT8 L participated in regulation of anthocyanin biosynthesis and proanthocyanidin accumulation by interacting with AtPAP1 and AtTT2,while FhGL3 L was involved in the regulation of anthocyanin accumulation,proanthocyanin biosynthesis and trichome formation by interacting with AtPAP1,AtTT2 and AtGL1,respectively.Later experiments revealed that FhGL3 L induced anthocyanin biosynthesis and inhibited trichome formation by inhibiting the formation of the active AtGL1-AtGL3-AtTTG1 complex.5.Function of FhGL3 L and FhTT8 L domainsA serises of FhGL3 L and FhTT8 L protein derivatives lacking the N-terminal region or C-terminal region,FhGL3 C,FhGL3N,FhTT8 LC and FhTT8 LN,were used to conduct the transiently transformation experiments.The results strongly supported that the N-terminal region was indispensable for the interaction with MYB partners while the C-terminal region played important roles in dimerization.Above all,two bHLH proteins named FhGL3 L and FhTT8 L were studied by comprehensive use of bioinformatics,biochemistry,genetics and molecular biology.The research may not only provide some experimental data for flavoniod biosynthesis but also some genes that may be used to modify flower color later.