Study on miRNAs-Mediated Seed and Stone-Hardening Regulatory Networks and the Mechanism of miRNAs’ Manipulating Gibberellin-Induced Seedless Berries in Grapevine (Vitis vinifera L.)

Abstract

A significant body of evidence supports the important roles of miRNAs in grape berry developments. However, their specific molecular functions during stone-hardening stage development remain unclear. Here, a total of 161 conserved and 85 species-specific miRNAs/miRNAs*(precursor) were identified in grape stone hardening stage berries using Solexa sequencing. Out of them, 30 VvmiRNAs were tissue-specific and identified as stone-hardening related to VvmiRNAs, whereas 52 exhibited differential expression profiles during berry development, potentially participating in modulation of development, and qRT-PCR analysis verified their expression patterns. Interestingly, high SNP variations in VvmiRNA sequences might result into generation of new VvmiRNA family members like VvmiR168, VvmiR479, VvmiR3636 families and so on. Through GO and KEGG pathway analyses, we revealed that 13 VvmiRNAs involved in the regulation of embryo development, 11 in lignin and cellulose biosynthesis, and 28 in the modulation of hormone signaling, sugar and proline metabolism. Furthermore, the target genes for novel VvmiRNAs related to berry development were validated using RLM-RACE and PPM-RACE methods, and it was revealed their cleavage sites mainly happened at the 9th-11th sites from the 5’ ends of miRNAs at their binding regions. The potential roles of these VvmiRNAs in gibberellins repressing grape stone-hardening and embryo development by potentially inducing the expression of VvmiR31-3p and VvmiR8-5p to increase the cleavage product accumulation levels of corresponding target genes like lignin biosynthesis genes, CAFFEOYL COENZYME A-3-O-METHYLTRANSFERASE (VvCCoAOMT) and DDB1-CUL4 ASSOCIATED FACTOR1 (VvDCAF1), as a potential key molecular mechanism involved in GA-induced grape seedless berry development. Based on the characterization of stone-hardening stage related to VvmiRNAs, a schematic model of miRNA-mediated grape seed and stone-hardening development was proposed in this work. This is the first report about the regulatory role of VvmiRNAs in the regulation of stone-hardening stage of grape berries, which provides valuable genetic information for the breeding of seedless grape varieties.

Identification and verification of seed development related miRNAs in kernel almond by small RNA sequencing and qPCR

“…Various studies have indicated that the expression level of miR395a is different significantly in developing fruit such as the seeds of Siberian apricot [ 17 ], pear fruit [ 18 ] and common bean seed [ 57 ]. Also, identification of known miR395 family in the stone-hardening stage of grape berries showed that involved in grape development and ripening process [ 33 ].…”

“…Dhaka and Sharma [ 25 ] identified 21 different miRNAs experimentally to regulate seed size, nutritional content, vigor, and shattering. Moreover, high throughput sequencing has been recently used for the identification and expression profiling of miRNAs in many fruit crops, such as sweet orange [ 26 ], grape [ 27 ], papaya [ 28 ], strawberry [ 29 , 30 ], olive [ 31 ], banana [ 32 ] and Grapevine [ 33 ].…”

Advances on the application of non-coding RNA in crop improvement

“…The overexpression of the PbGA20ox2 gene modified the GA biosynthetic pathway and improved GA4 synthesis, which promoted fruit set and parthenocarpic fruit improvement in pear fruits (Pyrus Bretschneider Rehd.) (Wang et al, 2020a). The PpIAA19 gene used to be concerned in the regulation of lateral root number, stem elongation, parthenocarpy, and fruit structure of tomatoes (Ding et al, 2019) and grapevines (Wang et al, 2020a).…”

“…(Wang et al, 2020a). The PpIAA19 gene used to be concerned in the regulation of lateral root number, stem elongation, parthenocarpy, and fruit structure of tomatoes (Ding et al, 2019) and grapevines (Wang et al, 2020a). The overexpression of the MaTPD1A gene in banana plants produces seedless fruits in contrast to wild-type plants (Hu et al, 2020).…”