Characterization And Functional Analysis Of Flavonoid Biosynthesis Pathway Key Genes In Apocynum Venetum And Apocynum Hendersonii

Flavonoids are important secondary metabolites imparting biological effects to many plants ranging from vegetables to fruits,cereals to herbs,including Apocynum venetum and its close related specie A.hendersonii.A.venetum has a good record as a medicinal plant and stimulant as tea due to its rich flavonoid contents.Several types of research were carried out on the flavonoid contents of the two,with very few focusing on the flavonoid biosynthesis key genes.Two of such key genes are downstream flavanone-3-hydroxylase(F3H)and upstream flavonoid-3-O-glucosyltransferase(UGTs).While the F3 H functions in the hydroxylation process,the UGT catalyzes the transfer of sugar moieties from activated donor molecules to specific acceptor molecules.The flavonoid modification,including hydroxylation and glycosylation,is the reason for its higher diversity.Flavonols and anthocyanidins are two common types of flavonoids that are often glycosylated,which is significant in the flavonoids’ stability,bioavailability and solubility.A paucity of gene sequence resources has largely restricted in-depth research into the molecular study of Apocynum specie.Chromosome assembly and higher quality genome assembly facilitate comparative studies of the evolution of significant traits in the plant.The study’s objectives were tailored to identify the key genes involved in flavonoid biosynthesis pathways and,with genomic,transcriptome and experimental data,unravel the natural variation in the accumulation of flavonoids in the two species.We reported the draft genome and chromosome structure of A.venetum and A.hendersonii,which is imperative in providing valuable resources for elucidating flavonoid biosynthesis and related genes,which may aid in the improvement of the species and enhancing economic values.The overall result of the genome analysis revealed more similarities than differences between the two species.The most significant difference between the two species was due to the DNA mediated copy and paste Class II transposable elements(TEs),which are comparably higher in A.hendersonii,having the highest rate of 3.49%(total number of 30,140)than in A.venetum which have 2.96%(total number of 26,716).Among these miniature inverted-repeat TEs(MITEs),rolling-circle DNA transposons(Helitron),self-synthesizing DNA transposons(Mavericks)and terminal inverted repeats(TIRs)were the highest.Except for Helitron,which has a comparably higher rate and length in A.venetum,all the other Class II elements presented more significant expansion in the genome of A.hendersonii.Insertion of TE in the coding region of structural genes may disrupt normal gene function and generate null mutant allele as established in morning glory resulting in morphological changes and diversity in flower colour.TE insertion may also similarly occur in regulatory elements imparting novel properties as MITE miniature ping(m Ping)was reported to have produced a new regulatory framework for salt,cold and dehydration stresses in rice.Due to the pleiotropic role of the flavonoid biosynthesis pathway in plants,any single mutation may lead to phenotypic differences.Thus the TEs might have impacted the two species,resulting in phenotypic variation,growth characteristics and environmental adaptation,which will require further investigation.Structural genes,including the key flavonoid biosynthesis genes such as F3 H,and UGTs,among others,are regulated by a large network of transcription factors.MYB is the most diverse TFs with an established role in flavonoid biosynthesis.We used genomic and transcriptome data to investigate MYB regulatory genes in A.venetum and A.hendersonii.A total of 163 candidates comprised 55 1R,101 R2R3,6 R1R2R3,and 1 4R repeats for A.venetum and 162(56 1R,99 R2R3,6 R1R2R3 and 1 4R repeats)for A.hendersonii were obtained.R2R3,the most functional class of the MYB and the most studied was selected for further characterization and renamed Av MYB1 – Av MYB101(Ah MYB1 – Ah MYB99)based on their positional order on chromosomes.Phylogenetic analysis revealed MYB TFs from Arabidopsis thaliana with established regulatory function in the flavonoid anthocyanin biosynthesis such as At MYB75 and At MYB90 At MYB113,and At MYB114 to have exhibited higher homologies with Av/Ah-MYB42/43/44/45.We compared transcript levels of A.venetum and A.hendersonii to evaluate the expression pattern of these four MYBs.The result showed significant differences in the transcript levels between the two species.The expression of one of the candidate genes,MYB42,was higher in stems and leaves of A.venetum than in A.hendersonii,consistent with the phenotypic data of both species.We constructed a phylogenetic tree of these four and their homolog in Arabidopsis,Apple and maize to explore specific homology relationships.The results showed that they were highly homologous to the anthocyanin activators At MYB75 and At PAP2.Therefore,we can infer that Av/Ah-MYB42 is the transcriptional activator of anthocyanins in the two species.For Av MYB43,Av MYB44 and Av MYB45,the expression pattern is opposite to the evolutionary relationship,and we speculate that the function change is due to mutation of individual locus,which needs further verification.F3H genes from A.venetum and A.hendersonii were isolated,and preliminary evaluation using cluster analysis revealed 100% homologies between the duos.Subsequently,the gene was cloned in the p BI121 overexpression vector and introduced into the Apocynum species and Arabidopsis thaliana via Agrobacterium-mediated transformation.Overexpression of the Av/Ah-F3 H in Arabidopsis enhanced flavonoid biosynthesis.Total flavonoids,total tannins and anthocyanins contents were comparably higher in the transgenic lines than in the wild type.Total tannins contents in one of the three transgenic lines(Lines 6)evaluated more than doubled that of the wild type.This finding indicated that overexpression of F3 H has tremendous potential in improving flavonoid biosynthesis and accumulation and may be exploited for molecular breeding tailored to enhancing flavonoid accumulation.Gene cloning provides tools to study the expression profiles of several genes and was successful in many plants.Though many flavonoid biosynthesis enzymes have been isolated and characterized,sequential modification such as glycosylation is relatively not explored.Studying the expression and function of the UGT gene has tremendous significance for the understanding of flavonoid synthesis and accumulation mechanism.We selected 8 UGTs(UGTs1,2…8)from both A.hendersonii and A.venetum genomic data for prokaryotic expression and characterization.A preliminary investigation of the UGTs sequence showed 100% similarity between Ah UGT1/Av UGT1,Ah UGT4/Av UGT4,and Ah UGT7/Av UGT7.There exists a non-sense single nucleotide polymorphism(SNP)between Ah UGT6/Av UGT6 at the 114 th position(GGU/GGC)of the nucleotide sequences,each coding for glycine.Whereas Ah UGT3/Av UGT3 have a sense SNP at the 1144 th nucleotide sequence position(CGG/UGG)coding for arginine and tryptophan,respectively.There were remarkable differences ranging from SNP to the absence of nucleotide fragments in the remaining UGTs except for UGT10,which is not found in A.venetum.Phylogenetic analysis placed the UGTs into different clades based on homology.UGTs1 and UGTs6 from both species belong to the flavonoid 3-O-glycosyltransferases(3GT)clade,whereas UGTs2 and UGTs4 from both species as well as Av UGT8 belong to flavonoid glycoside glycosyltransferases(GGT).It is worthy to note that Av UGT8 and Ah UGT8 fall into different clades,with the latter being in flavonoid-C-glycosyltransferases(CGT)together with UGTs5 from both species and Ah UGT10.Enzyme kinetic analysis showed distinctive differences within and between the sets of UGTs in both species.For example,the deletion difference in C-terminal results in the catalytic efficiency of Av UGT6 for apigenin substrates is significantly higher than that of AhUGT6.