Characterization of Vv-miR156: Vv-SPL pairs involved in the modulation of grape berry development and ripening.

SPL is a plant-specific transcription factor family. Many researchers reported that SPL members targeted by miR156s could play crucial roles in the modulation of plant growth and development. Although there are similar reports on grapes, till now little is known about grape berry development and ripening. To gain more insight into how grape miR156s (Vv-miR156s) modulated the above given processes of grape berries by mediating their target gene Vv-SPLs, here we identified the precise sequences of Vv-miR156s in 'Giant Rose' grape berries, predicted their potential targets, and revealed that the matching degree of various Vv-miR156: Vv-SPL pairs exhibited some discrepancy, implying the divergence of their interaction. Subsequently, we also discovered similar motifs such as ABRE, CGTCA and ERE, which are more specific to berry development and ripening, within the promoters of both Vv-MIR156s and Vv-SPLs. With berry development and ripening, meanwhile, Vv-miR156a, b/c/d, e and f/g/i exhibited an overall increasing expression trend, while their targets showed opposite trends at the corresponding stages. Additionally, exogenous ABA and NAA application promoted or curbed the expression of Vv-miR156s to some extent, before grape berry ripening stage. The cleavage products, sites and frequencies of Vv-miR156a, b/c/d, e, f/g/i and their respective targets (Vv-SPL2, 9, 10, 16) during grape berry development and ripening process were validated by our developed PPM-RACE and modified RLM-RACE together with qRT-PCR, which demonstrated that Vv-miR156s can be involved in the modulation of grape berry development and ripening process by mediating the expression of Vv-SPL2, 9, 10, 16. Our findings lay an important foundation for further recognizing their functions in grape berries, and enrich the knowledge of the regulatory mechanism of miRNA-mediated grape berry development and ripening.

Study on Expression Modes and Cleavage Role of miR156b/c/d and its Target Gene Vv-SPL9 During the Whole Growth Stage of Grapevine.

miR156 regulates the expression of its target SPL (PROMOTER BINDING-LIKE) genes during flower and fruit development, diverse developmental stage transitions, especially from vegetative to reproductive growth phases, by cleaving the target mRNA SPL of one plant-specific transcription factor. However, systematic reports on grapevine have yet to be presented. Here, the precise sequence of miR156 (vvi-miR156b/c/d) in grapevine "Takatsuma" was cloned with a previously cloned grapevine SPL (Vv-SPL9). Expression profiles in 18 grapevine tissues were identified through stem-loop RT-PCR. The interaction mode between vvi-miR156b/c/d and Vv-SPL9 was further validated by detecting the cleavage site and cleavage products of 3'- and 5'-ends via an integrated approach of 5'-RLM-RACE (RNA ligase-mediated 5'-rapid amplification of cDNA ends), 3'-PPM-RACE (poly(A) polymerase-mediated 3'-rapid amplification of cDNA ends), and qRT-PCR (real time reverse transcriptase-polymerase chain reaction). The variation in their cleavage roles in the whole growth stage of grapevine was also systematically investigated. Results showed that vvi-miR156b/c/d exhibited typical temporal-spatial-specific expression levels. The expression levels were higher in vegetative organs, such as leaf, than in reproductive organs, such as tendrils, flowers, and berries. A significant variation was observed during vegetative-to-reproductive transition. The expression patterns of Vv-SPL9 showed the opposite trends with those of vvi-miR156b. We confirmed that the cleavage site was at the 10th site of vvi-miR156b/c/d complementary to Vv-SPL9 in "Takatsuma" grapevine. We also identified the temporal-spatial variation of the cleavage products. This variation can indicate the regulatory function of miR156 on SPL in grapevines. Our findings provide further insights into the functions of vvi-miR156b/c/d and its target Vv-SPL9, and also help enrich our knowledge of small RNA-mediated regulation in grapevine.

Determination of the precise sequences of computationally predicted miRNAs in Citrus reticulata by miR-RACE and characterization of the related target genes using RLM-RACE.

MicroRNAs play vital roles in various biological and metabolic processes by regulating the expression of their target genes in model plants. Since there are limited reports on miRNAs in Citrus reticulata (Crt-miRNAs), the determination of precise sequences of miRNAs is essential to further analyze the functions of miRNAs in Citrus reticulata. Here, miR-RACE, a recently developed technique for determination of the potential miRNAs computationally, was employed to identify the precise sequences of Crt-miRNAs. Tissue- and development-specific expression of nine miRNAs were identified by quantitative RT-PCR in the leaves, stems, flowers and fruits Subsequently, 10 potential target genes were predicated for the eight Crt-miRNAs, most of which were transcription factors and disease resistance proteins. Four target genes were experimentally validated by Poly (A) polymerase-mediated 3' rapid amplification of cDNA ends and RNA ligase-mediated 5' rapid amplification of cDNA ends (PPM-RACE and RLM-RACE). Our findings showed that regulatory miRNAs in C. reticulata may play a key role in regulating growth, development, and response to disease. Future work is required to study the functions of miRNAs and their targets of C. reticulata.

Characterization of regulatory mechanism of Poncirus trifoliata microRNAs on their target genes with an integrated strategy of newly developed PPM-RACE and RLM-RACE.

MicroRNAs (miRNAs) play an important role in post-transcriptional gene regulation that involved various biological and metabolic processes. Many extensive studies have been done in model plant species, to discover miRNAs' regulating expression of their target genes and analyze their functions. But, the function of Poncirus trifoliata miRNAs has not been properly investigated. In this study, we employed the RNA ligase-mediated 5' rapid amplification of cDNA ends (RLM-RACE) and the newly developed method called poly (A) polymerase-mediated 3' rapid amplification of cDNA ends (PPM-RACE), which mapped the cleavage site of target mRNAs and detected expression patterns of cleaved fragments that could in turn indicate the regulatory functions of the miRNAs on their target genes. Furthermore, the spatiotemporal expression levels of target genes were analyzed by qRT-PCR, with exhibiting different expression trends from their corresponding miRNAs, thus indicating the cleavage mode of miRNAs on their target genes. The expression patterns of miRNAs, their target mRNAs and cleaved target mRNAs in different organs of juvenile and adult trifoliate orange were studied. The results showed that the expression of miRNAs and their target mRNAs was in a trade-off trend. When the miRNA expression was high, its corresponding target mRNA expression was low, while the cleaved target mRNA expression was high; when the miRNA expression was low, its target mRNA expression was high, while the expression of cleaved target mRNAs follows that of the miRNA. The validation of the cleavage site of target mRNAs and the detection of expression patterns of cleaved fragments can further broaden the knowledge of small RNA-mediated regulation in P. trifoliate.