Characterization Of Proanthocyanidin Precursor Transport Related Gene DkGSTF1 In Persimmon

Persimmon proanthocyanidins(PAs),also known as condensed tannins,are abundant in persimmon fruits.PAs are important secondary metabolites in persimmon fruits and leaves,and cause sensation of ‘astringency’ after eating persimmon fruits.Persimmon proanthocyanidins are long-chain polymers and their building precursor units(PAs monomers)are produced in the cytoplasm,then transported into vacuoles.That is to say,the transmembrane transport mechanism is involved from the production site to the storage site.In this study,11 differentially expressed genes annotated as glutathione S-transferase were screened out from the transcriptome database of C-PCNA ‘Eshi 1’ fruits at 10 and 20 weeks after bloom.The expression pattern of DkGSTF1 was highly consistent with the variation of PAs content.This research focus on the function characterization of DkGSTF1 in persimmon.The main results are as following.1.The full length of DkGSTF1 gene was 654 bp after RACE amplification.DkGSTF1 was predicted to encode 217 amino acids.Sequence alignment indicated that typical glutathione binding sites in DkGSTF1 N-terminal domain and conservative hydrophobic substrate binding sites in C-terminal domain.2.After DkGSTF1 was transiently expressed in persimmon fruit disc,the content of proanthocyanidins increased significantly after overexpression,and decreased after RNAi.Similarly,transient overexpression of DKGSTF1 in persimmon leaves resulted in the proanthocyanidin content increasing.Meanwhile,the expression of PAs biosynthesis structural genes exhibited significant increase,especially for DKANR.The concentration of C,EC and ECG presented a remarkable increment after transient overexpression in leaves by HPLC.3.Subcellular localization of DkGSTF1 was carried out in Nicotiana tabacum L.leaves.DkGSTF1 was visualized in the nucleus and tonoplast after transformation by using a confocal laser scanning microscope.This suggested DkGSTF1 might be participate in the transporting and accumulation of monomers.4.The promoter of DkGSTF1 was amplified sequenced as 1524 bp from ‘Eshi 1’.Bioinformatics analysis predicted cis-acting elements in the promoterand MYB transcription factor related to PAs biosynthesis.DkGSTF1 promoter was shown to drive GUS expression in Nicotiana tabacum L.leaves.5.Through heterologous expression of DkGSTF1 in Arabidopsis tt19 mutant,complementing transormants seeds showed a wild-type brown color.This implied that DkGSTF1 can complement the phenotype of Arabidopsis tt19 and promote to the PAs accumulation in the transformed mutants.In conclusion,the expression pattern of DkGSTF1 was consistent with the change trend of proanthocyanidins content in different development stages of persimmon fruit.DkGSTF1 may selectively transport C,EC and ECG.This research revealed that DkGSTF1 mediate PAs monomers transporting and accumulation in persimmon.These findings can provide a new insight on elucidating molecular mechanism on PAs biosynthesis in persimmon.