DkmiR397 Regulates Proanthocyanidin Biosynthesis via Negative Modulating DkLAC2 in Chinese PCNA Persimmon

Abstract

Persimmon fruits accumulate a large amount of proanthocyanidins (PAs), which makes an astringent sensation. Proanthocyanidins (PAs) are the polymers of flavan-3-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 that the role of DkmiR397 repressed the expression of DkLAC2 concerning PA biosynthesis, providing a potential target for the manipulation of PAs metabolism in persimmon.

Keywords: laccase; microRNA; persimmon; polymerization; tannin.

Figure 1

Proanthocyanidins and DkmiR397 with DkLAC2 variation in three persimmon genotypes fruits. (A) Changes in soluble PAs content. (B) Changes in insoluble PAs content. (C) Changes in total PAs content. (D) Expression variation in DkmiR397. (E) Expression variation in DkLAC2. The ‘Eshi 1’ (C-PCNA) persimmon fruits from 2.5 WAB to 27.5 WAB were used for determining the PAs content based on the tannin printing technique (F,G). Error bars indicate SE from three biological replicates (n = 3).

Figure 2

Identification and characterization of DkmiR397 and DkLAC2. (A) Phylogenetic tree of DkLAC2 homologs. The protein names from various plant species are labeled on the branches. Bootstrap replicates 3000 were used to calculate the bootstrap values. (B) Subcellular localization of the DkLAC2 proteins. (C) Target validation for DkmiR397 by RLM-5′ RACE. The cleavage sites are indicated by arrows, and the values represent the frequency of accurately sequenced clones. (D) GUS (histochemical β-glucuronidase) was a co-expression of DkmiR397 and its target genes in tobacco leaves via Agrobacterium tumefaciens-mediated transformation. (E) The interaction between DkmiR397 and its target genes was demonstrated using a Dual-LUC assay. Renilla (REN) luciferase activity was used to standardize relative LUC activity. Error bars indicate SEs from four biological replicates (n = 4). Asterisks above the bars represent values determined to be significantly different from the control by the Student’s t-test (* p < 0.05).

Figure 3

Transient expression of DkmiR397 and DkLAC2 in ‘Eshi 1’ (C-PCNA) persimmon leaves in vivo. (A) Evaluation of DkmiR397, DkLAC2, and PA biosynthesis pathway gene transcript levels, as well as the related fluctuation in PA content, after transient overexpression of pre-miR397 at ten days after agroinfiltration. (B) Evaluation of DkmiR397, DkLAC2, and PA biosynthesis pathway gene transcript levels, and the related fluctuation in PA content, after transient of STTM397 at ten days after agroinfiltration. (C) Evaluation of DkLAC2-OE, and PA biosynthesis pathway gene transcript levels, and the related fluctuation in PA content, after transient overexpression of DkLAC2 at ten days after agroinfiltration (D) Evaluation of DkLAC2-i, and PA biosynthesis pathway gene transcript levels, and the related fluctuation in PA content, after transient silencing of DkLAC2-i at ten days after agroinfiltration. F3′H: flavonoid 3′-hydroxylase; F3′5′H: flavonoid 3′5′-hydroxylase; DFR: dihydroflavonol 4-reductase; ANS: anthocyanidin synthase; ANR: anthocyanidin reductase; LAR: leucoanthocyanidin reductase; GST: glutathione S-transferase; MATE: multi-drug and toxic compound extrusion transporter. Errors bars indicate SEs from three biological replicates (n = 3). Asterisks above the bars indicate values determined by Student’s t-test to be significantly different from the control (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 4

Transient expression of DkmiR397, DkLAC2 in vitro, and DMACA staining technique in ‘Eshi 1’ (C-PCNA) persimmon. Relative expression and PAs concentration by Folin-Ciocalteu after transformation with (A) DkmiR397, (B) STTM-DkmiR397, (C) DkLAC2-OE, (D) DkLAC2-I, and (E) DMACA staining of fruit discs of ‘Eshi 1’ was performed 3 days after infection. CK, blank control; pMDC32, empty vector. Error bars indicate SEs from three biological replicates (n = 3). Asterisks above the bars indicate values determined by Student’s t-test to be significantly different from the control (* p < 0.05, ** p < 0.01); N.S: non-significant.

Figure 5

Overexpression of Pre-miR397, STTM-miR397, DkLAC2-OE, and DkLAC2-i with a stable transformation system in ‘Gongcheng Shuishi’ persimmon. Quantitative measurement of transcript level of genes in the PAs biosynthesis pathway and PAs contents variation in ‘Gongcheng Shuishi’ persimmon transgenic lines of expressing by (A) DkmiR397, (B) STTM397, (C) DkLAC2, and (D) DkLAC2-i. (E) The DMACA staining of regenerated transgenic lines leaves of DkmiR397-OE, DkSTTM-397, DkLAC2-OE, and DkLAC2-i. CHI: chalcone isomerase; F3′H: flavonoid 3′-hydroxylase; F3′5′H: flavonoid 3′5′-hydroxylase; ANS: anthocyanidin synthase; ANR: anthocyanidin reductase; LAR: leucoanthocyanidin reductase; GST: glutathione S-transferase; MATE: multi-drug and toxic compound extrusion transporter. Errors bars indicate SEs from three biological replicates (n =3). Asterisks above the bars indicate values determined by Student’s t-test to be significantly different from the control (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 6

Functional analysis of DkLAC2 via transformation in Arabidopsis. (A) Semi-quantification of DkLAC2 in WT Arabidopsis and transgenic lines. (B) Seed coat pigmentation before and after staining colorations by DMACA reactions comparison between wild-type Arabidopsis seeds and T1 Arabidopsis transgenic lines derived from DkLAC2 transformation. Bars = 250 µm. (C) PAs content of WT and 2 × 35S:DkLAC2 Arabidopsis transgenic plants seeds. (D) HPLC analysis of PAs monomers in Arabidopsis transgenic line compared to WT. Data represent the mean of three biological replicates, and error bars represent the standard deviation of three replicates (n = 3). Asterisks above the bars indicate values determined by Student’s t-test to be significantly different from the control (** p < 0.01, *** p < 0.001).

Figure 7

A working model of DkmiR397 and DkLAC2 regulates PA biosynthesis in C-PCNA persimmon fruits. PAL: phenylalanine ammonia-lyase; 4CL: 4-coumarate: coenzyme A ligase; CHS: chalcone synthase; F3′5′H: flavanone 3′5′-hydroxylase; DFR: dihydroflavonol 4-reductase; LAR: leucoanthocyanidin reductase; ANS: anthocyanidin synthase; ANR: anthocyanidin reductase; GST: glutathione S-transferase; MATE: multi-drug and toxic compound extrusion transporter; CA: catechin; EC: epicatechin; ECG: epicatechin-3-O-gallate; EGC: epigallocatechin; EGCG: epigallocatechin 3-O-gallate.