DkMiR397 Regulates Proanthocyanidin Biosynthesis Via Negative Modulating DkLAC2 In Chinese PCNA Persimmon

Persimmon fruits accumulate a large amount of proanthocyanidin(PAs),which makes the sensation of astringent.Proanthocyanidin(PAs)are the polymers of flavan3-ols stored in plant vacuoles under laccase activation.A laccase gene,DkLAC2,is putatively involved in PAs biosynthesis and regulated by microRNA(DkmiR397)in persimmon.However,the polymerization of PAs in association with miRNA397 still needs to be explored in persimmon.Here,we identified pre-DkmiR397 and its target gene DkLAC2 in ’Eshi 1’ persimmon.Histochemical staining with GUS and dual luciferase assay both confirmed DkmiR397-DkLAC2 binding after co-transformation in tobacco leaves.Diverse expression patterns of DkLAC2 and DkmiR397 were exhibited during persimmon fruit development stages.Moreover,a contrasting expression pattern was also observed after the combined DkLAC2-miR397 transformation in persimmon leaves,suggesting that DkmiR397 might be a negative regulator of DkLAC2.Similarly,the transient transformation of DkmiR397 in persimmon fruit discs in vitro also reduced PA accumulation by repressing DkLAC2,whereas the up-regulation of DkLAC2 increased the accumulation of PAs by short tandem target mimic STTM-miR397.A similar expression pattern was observed when overexpressing of DkLAC2 in Arabidopsis wild type(WT)and overexpression of DkLAC2,DkmiR397 in persimmon leaf callus.Our results revealed the role of DkmiR397 repressed the expression of DkLAC2 concerning PA biosynthesis and providing a potential target for the manipulation of PAs metabolism in persimmon.The main results are as follows:Full-length of pre-DkmiR397 and its target gene DkLAC2 were isolated through transcriptome database and small RNA libraries of ’Eshi 1’(C-PCNA)persimmon(15,20 WAB).Phylogenetic analysis revealed that the candidate target gene DkLAC2 was related to AtLAC15.Quantitative real-time PCR analysis of DkLAC2 and DkmiR397 showed contrasting divergent expression patterns in(C-PCNA)fruit development stages.Those expression patterns presented a negative correlation.Subcellular localization assays revealed that DkLAC2 was localized in the vacuole using laser scanning confocal microscopy technology,which is consistent with the function to promote procyanidin precursor polymerization in a vacuole.Sequencing of the RNA ligase-mediated 5’RACE PCR clones indicated that DkmiR397 has unique cleavage sites in their corresponding targeted sequences at the 10-1 lnt sites separately.Histochemical staining of GUS and dual-luciferase(LUC)assay like wisely validate DkmiR397 targeting DkLAC2 applied co-transformation technology in tobacco leaves.Based on Agrobacterium-mediated injection infiltration,the DkLAC2 and DkmiR397 were expressed transiently in ’Eshi 1’ persimmon leaves in vivo and fruit discs in vitro.Stable transgenic transformation of ’Gongcheng Shuishi’(PCA genotype)persimmon was performed as defined with slight modifications,including reduced concentrations of zeatin and NAA Further,in persimmon leaf callus DkLAC2,DkLAC2-i,DkmiR397 overexpression,and STTM-miR397 comprised of the previous effects.To further verify the function of 35S:DkLAC2 in Arabidopsis,we obtain a similar result in T1 generation as before.Brownish pigment in the DkLAC2 seed color is similar to the WT seed.The PA content in seed coats expressing DkLAC2 was significantly higher than that of WT in seed coats.These results indicate that the DkLAC2 are functionally involved in PA biosynthesis.In conclusion,the DkmiR397 translational represses the expression of DkLAC2 for modulating PAs biosynthesis in ’Eshi 1’(C-PCNA).The results provide a scientific basis for elucidating the mechanism of PAs metabolic regulation in persimmon.