MicroRNAs (miRNAs) have been shown to play key roles in regulating gene expression at post-transcriptional level, but miRNAs associated with natural deastringency of Chinese pollination-constant nonastringent persimmon (CPCNA) have never been identified. In this study, two small RNA libraries established using ‘Eshi No. 1’ persimmon (Diospyros kaki Thunb.; CPCNA) fruits collected at 15 and 20 weeks after flowering (WAF) were sequenced through Solexa platform in order to identify miRNAs involved in deastringency of persimmon. A total of 6,258,487 and 7,634,169 reads were generated for the libraries at 15 and 20 WAF, respectively. Based on sequence similarity and hairpin structure prediction, 236 known miRNAs belonging to 65 miRNA families and 33 novel miRNAs were identified using persimmon transcriptome data. Sixty one of the characterized miRNAs exhibited pronounced difference in the expression levels between 15 and 20 WAF, 17 up-regulated and 44 down-regulated. Expression profiles of 12 conserved and 10 novel miRNAs were validated by stem loop qRT-PCR. A total of 198 target genes were predicted for the differentially expressed miRNAs, including several genes that have been reported to be implicated in proanthocyanidins (PAs, or called tannin) accumulation. In addition, two transcription factors, a GRF and a bHLH, were experimentally confirmed as the targets of dka-miR396 and dka-miR395, respectively. Taken together, the present data unraveled several important miRNAs in persimmon. Among them, miR395p-3p and miR858b may regulate bHLH and MYB, respectively, which are influenced by SPL under the control of miR156j-5p and in turn regulate the structural genes involved in PA biosynthesis. In addition, dka-miR396g and miR2911a may regulate their target genes associated with glucosylation and insolubilization of tannin precursors. All of these miRNAs might play key roles in the regulation of (de)astringency in persimmon fruits under normal development conditions.

The persimmon Diospyros kaki Thunb. is an important commercial and deciduous fruit tree. The fruits have proanthocyanidin (PA) content of >25% of the dry weight and are astringent. PAs cause astringency that is often undesirable for human consumption; thus, the removal of astringency is an important practice in the persimmon industry. Soluble PAs can be converted to insoluble PAs by enclosing the fruit in a polyethylene bag containing diluted ethanol. The genomic resource development of the persimmon is delayed because of its large and complex genome. Second-generation sequencing is an efficient technique for generating huge sequences that can represent a large number of genes and their expression levels.

Persimmon proanthocyanidin (PA) biosynthesis is controlled by structural genes and regulated by transcription factors (TFs). MicroRNAs are a key factor involved in regulating gene expression at the posttranscriptional level whose functions in persimmon PA biosynthesis are poorly understood. Here, we identified a microRNA, miR858b, that putatively targets two R2R3-MYB TFs,DkMYB19andDkMYB20.DkMYB19,DkMYB20, and miR858b showed divergent expression patterns during fruit development, and the interaction between miR858b andDkMYB19orDkMYB20was experimentally validated by 5′ RNA ligase-mediated RACE, LUC enzyme activity analysis, and GFP signal detection. The DkMYB19 localized to the nucleus as well as the cytoplasm and DkMYB20 localized to the nucleus. The overexpression of miR858b led to the downregulation ofDkMYB19andDkMYB20, which reduced the content of PA, whereas a reduction in miR858b activity upregulatedDkMYB19andDkMYB20, resulting in a high content of PA in leaves transiently expressing a small tandem target mimic construct for blocking miR858 (STTM858b)in vivo. The transient transformation of miR858b in fruit discsin vitroalso reduced the content of PA, while the content of PA increased under the transient transformation of fruit discs with STTM858b,DkMYB19, orDkMYB20. A similar phenomenon was observed upon the overexpression of miR858b in wild-type (WT)ArabidopsisandDkMYB19orDkMYB20in persimmon leaf calli. These findings suggested that miR858b repressed the expression ofDkMYB19andDkMYB20, which contributed to the PA accumulation in persimmon.

MicroRNAs (miRNAs) are endogenous noncoding small RNAs containing 21–24 nucleotides (nt) that regulate gene expression precisely and efficiently at the posttranscriptional level through the negative regulation of target messenger RNA (mRNA) expression, such as translational inhibition or degradation. Likewise, as a controlling element, miRNA itself is regulated by a variety of factors when performing its basic purposes, such as SNP detection, miRNA purging, methylation, and the circadian clock in model crops. In current years, miRNA-mediated controls have been intensely investigated in horticultural plants, leading to the discovery of numerous novel mechanisms that exhibit significantly greater mechanistic complexity and distinctive regulatory properties than those explored in model species. In fruit crops, miRNAs play a crucial role corresponding to various biological, metabolic functions and environmental challenges, including growth, expansion, response to biotic and abiotic stress, signaling of growth hormones, and the regulation of secondary product metabolism. In this study, we appraisal the current improvement of small regulatory RNA research in fruit crops, emphasizing miRNA mechanisms and their correlation with key trait rule. Considering that miRNAs engaged in the regulation of all aspects of fruit tree life activities, we focus here on their biosynthesis, target genes, function and regulatory network, as well as the mechanistic connection among them, to provide a theoretic base and breakthrough for upcoming exploration on miRNAs in fruit plants.