Molecular Cloning And Characterization Of Flavonol Synthase Gene(FLS) In Freesia Hybrida

Flavonoids are well-known natural products which are widely distributed in plants.As the most abundant flavonoids,flavonol has attracted increasing attentions due to its versatile physiological functions in plants and significant health-promoting properties,including UV protection,modulation of flower color,signaling and antioxidant activity.In the past several decades,works achieved on model plants broadened our knowledges on flavonol biosynthesis.However,more studies especially in monocot plants and flowers are needed to fill the gaps remain in our understanding of the flavonol biosynthesis and regulation mechanism.Freesia,a monocotyledonous genus of herbaceous perennial flowering plants,is native to southern Africa and then cultivated worldwide because of its colorful flowers and sweet fragrance.Early studies on Freesia suggested that it had the potential to be a model system for investigating of flavonol biosynthesis in monocot plants.In this study,6 flavonol synthase-like genes of Freesia hybrida have been cloned and characterized through a serial of bioinformatic and genetic methods.The main results were summarized as follows:1.FhFLSs were cloned and analyzed.Amino acid sequence of At FLS was used as bait probe during the in situ TBLASTN search of transcriptomic database of Freesia hybrida.Consequently,six putative sequences were isolated and predicted as flavonol synthase genes encoding proteins having the HXD motif for ligating ferrous iron and the RXS motif to bind2-oxoglutarate(2OG).A phylogenetic tree was further generated by the neighbor-joining method,and the results showed that only FhFLS1,FhFLS2 and FhFLS3 clustered within a subgroup containing FLS proteins from other plants,which suggested that FhFLS1,FhFLS2 and FhFLS3 were most likely to be the bona fide FLS genes.2.Expression patterns of FhFLSsTo examine whether the expression patterns of the potential FhFLS genes coincided with flavonol accumulation in flower developmental stages and various tissues,their expression levels were investigated temporally and spatially.Similar expression patterns of FhFLS1 and FhFLS3 were observed among the five flower development stages.They were highly expressed in flower buds with non-pigmented tepals,decreased gradually with the development of flowers and minimized when the flowers fully opened,showing an expression pattern synchronous to the flavonol accumulation.Comparatively,the expression of FhFLS2 initiated from green bud,peaked in the flower buds with pale-red tepals,and fluctuated during flower development.As for the expression patterns of FhFLSs genes in various plant tissues,FhFLS1 and FhFLS3 were dominantly expressed in torus,while FhFLS2 was observed in other tissues.It could be deduced that the duplicated FhFLSs genes might function divergently in flavonol biosynthesis in Freesia hybrida.3.Heterologous expression of FhFLSs in tobacco plantsIn order to further investigate the potential roles of FhFLSs in flavonol biosynthesis in planta,they were introduced into tobacco cultivar K326 under the control of 35 S promoter.Phenotypic observation showed that there were slight loss of color in transgenic flowers overexpressing FhFLS1、FhFLS2 and FhFLS3.HPLC analysis further revealed that the amounts of anthocyanins in transgenic plants decreased,while the amounts of flavonols increased.These results demonstrated that FhFLS1,FhFLS2 and FhFLS3 could catalyze dihydroflaonols to flavonols in planta.4.In vitro catalytic activities of FhFLSsTo further confirm the enzymatic properties of FhFLS1,FhFLS2 and FhFLS3,substrate specificity studies were performed using the purified recombinant proteins extracted from E.coli BL21 system.Consequently,the purified FhFLS1、FhFLS2 and FhFLS3 could convert dihydrokaempferol and dihydroquercetin to kaempferol and quercetin,respectively,whereas only FhFLS2 could utilize dihydromyricetin as substrate to catalyze the formation of myricetin in vitro,suggesting that the accumulation of kaempferol and quercetin in Freesia hybrida might result from the enzymatic properties of FhFLS1,FhFLS2 and FhFLS3.To our knowledge,this is the first report of the characterization of flavonol synthase gene family in Freesia hybrdia,and the results will not only contribute to the study of flavonol biosynthetic regulation,but also provide new candidate genes in crop improvement.