Characterizaion ANR Pathway Of Proanthocyanidin Biosynthesis In Vitis Bellula And Gossypium Hirsutum

Proanthocyanidins (PAs) also known as condensed tannins, are oligomers orpolymers of flavan-3-ols. They widely distribute in plant kingdom and play anti-stressroles to help plants against UV-demage and herbivores. PAs also have many nutritivebenefits to our human due to their powerful antioxidative activities. In the past twodecades, there are many works focused on PAs biosynthesis at model plantsArabidopsis and Medicago, although these works gain a great achievement, but studiesof PAs biosynthesis in economic crops are still limited.In this study, we reported characterization of PAs formation and anthocyanidinreductase (ANR) homolog genes from two species Vitis bellula and Gossypiumhirsutum. Phytochemical analysis showed the formation of (-)-epicatechin,(±)-catechin,(-)-epigallocatechin and (-)-gallocatechin during the entire leave development of V.bellula, those four PAs monomers also had been detected in leave, stem, root, anddeveloping fiber of G. hirsutum. Butanol-HCl boiling of crude extraction of PAs fromthese two species produced pelargonidin, cyanidin and delphinidin, indicating that PAsof V. bellula and G. hirsutum include diverse structures of extension unit.Two ANR cDNA homologs (named as VbANR and GhANR, respectively) werecloned from leave of V. bellula and G. hirsutum by RT-PCR. These two homologs wereexpressed in E. coli to obtain recombinant fusion proteins. In the presence of NADPH,the recombinant enzymes Trx-VbANR and Trx-GhANR converted cyanidin,delphinidin and pelargonidin into their corresponding (-)-epicatechin and (-)-catechin,(-)-epigallocatechin and (-)-gallocatechin, and (-)-epiafzelechin and (-)-afzelechin,respectively. In addition, Trx-VbANR had ability to catalyze anthocyanidin substratesto the third products at pH7.5reaction system. The sub-cellar localization of VbANRshowed that ANRs were cytosolic protein. Meanwhile, virus induced gene silencing(VIGS) of GhANR led to significant increase of anthocyanins but decrease of PAs.Ectopic expression of VbANR and GhANR in Arabidopsis ban mutant reconstructed theANR pathway and leading to PAs biosynthesis in seeds.In a word, our data demonstrated that proanthocyanidins of V. bellula and G.hirsutum had constructive diversity, VbANR and GhANR were key genes duringbiosynthesis of flavan-3-ols and PAs in V. bellula and G. hirsutum. Charaterization ofGhANR and VbANR indicated the existence of ANR pathway in these two plants. Theseresearch results provided some information to improve PAs for nutrient grapes andanti-pest cottons breeding by metabolic engineering.