Spatio-temporal expression of miRNA159 family members and their GAMYB target gene during the modulation of gibberellin-induced grapevine parthenocarpy.

Grapevine, Vitis vinifera, is an important economic fruit crop that is highly sensitive to gibberellin (GA), and the exogenous application of GA can efficiently induce grapevine parthenocarpy. However, the molecular mechanisms underlying this process remain elusive. In this study, morphological changes during flower development in response to GA treatments were examined in the 'Zuijinxiang' cultivar. To obtain insights into the roles of miRNA159s in GA-induced grapevine parthenocarpy, VvmiR159a, VvmiR159b, VvmiR159c, and their target gene VvGAMYB were isolated, sequenced and characterized. Spatial-temporal expression analyses showed that VvmiR159c exhibited the highest expression levels at 4 d before flowering, followed by a gradual decrease, while VvGAMYB displayed an opposite pattern of expression with the lowest expression at the corresponding stage in response to GA treatment. A cleavage interaction between VvmiR159s and VvGAMYB and variations of their cleavage roles were confirmed in grapevine floral development. In addition, the potential roles of VvmiR159s in GA signaling were investigated through DELLA-protein repressors, indicating that GA-DELLA (SLR1)-VvmiR159c-VvGAMYB is the key signaling regulatory module in grapevine. Our findings provide novel insights into the GA-responsive roles of VvmiR159s in modulating grapevine floral development, which have important implications for the molecular breeding of high-quality seedless grapevine berry.

Characterization of grapevine microR164 and its target genes.

MicroRNAs (miRNAs) are an extensive class of newly identified small RNAs that regulate gene expression at post-transcription level by mRNA cleavage or translation. In our study, we used qRT-PCR and found that Vv-miR164 is expression in grapevine leaves, stems, tendrils, inflorescences, flowers and fruits. In addition, two potential target genes for Vv-miR164 were also found and verified by PPM-RACE and RLM-RACE. The results not only maps the cleavage site of the target mRNA but allowed for detection the expression pattern of cleaved fragments that can indicate the regulatory function of this miRNA on its target genes. These target genes were explored by qRT-PCR where some exhibited different expression patterns from their corresponding miRNA, indicating the cleavage mode of the miRNA on its target genes. The efficient and powerful approach used in this study can help in further understanding of how miRNAs cleaved their target mRNAs. Results from this study prove the importance of Vv-miR164 in regulating development and growth of grapes, and adds to the existing knowledge of small RNA-mediated regulation in grapes.

Isolation and characterization of two YUCCA flavin monooxygenase genes from cultivated strawberry (Fragaria × ananassa Duch.).

UNLABELLED: In strawberry (Fragaria × ananassa Duch.), auxin has been recognized as the main signal molecule coordinating the growth and initiation of ripening of fruits. The molecular mechanism regulating auxin biosynthesis in strawberry remains unknown. This project reports two YUCCA flavin monooxygenase genes FaYUC1-2 isolated from cultivated strawberry. FaYUC1 and FaYUC2 are most homologous to AtYUC6 and AtYUC4, respectively. Significant expression of FaYUC1-2 is found in vegetative meristems and reproductive organs, with overlapping but distinct patterns. During fruit development, both transcripts of FaYUC1 and FaYUC2 in achenes reach a peak around large green fruit (G2) stage, but the sudden rise in FaYUC2 transcript level is much steeper and begins earlier than that in FaYUC1. FaYUC2 is also obviously expressed in the receptacles from green fruits, hinting another auxin source for receptacle development, other than achenes. FaYUC1 over-expression Arabidopsis exhibits typical auxin hyper-accumulation phenotype in many aspects, such as the narrow and downward curled leaves, strong apical dominance, short and hairy root. It is also severely sterile, due to the disruption of floral meristems initiation and floral organs development. Transgenic analysis indicates that strawberry YUC gene may hold conserved role in auxin biosynthesis like their homologs in other plants. Integrated with the spatiotemporal expression features, these results led us to propose that FaYUC1-2 may involve in many developmental processes including flower and fruit development in strawberry. KEY MESSAGE: This paper is the first report of isolation and characterization of strawberry auxin biosynthesis genes. And their conserved functions in auxin biosynthesis were confirmed after ectopic expression.

Analysis of expressed sequence tags from grapevine flower and fruit and development of simple sequence repeat markers.

A total of 6,230 EST sequences were produced from 7,561 clones in a cDNA library generated from grapevine (Vitis vinifera cv. 'Summer Black') flower and fruit tissues in this study. After cluster and assembly analysis of the datasets, 3,582 unigenes (GenBank accession numbers GW836604-GW840185) were established, among which 381 were new grapevine EST sequences. Out of the 381 new ESTs, 289 could be mapped on the 19 grapevine chromosomes. 913 unique ESTs with known or putative functions were assigned to 11 putative cellular roles. 540 potentially workable grapevine EST-SSRs were developed from 3,582 unigenes and about 42.6% of these unigenes were identified as true-to-type SSR loci and could amplify polymorphic bands from 22 individual plants of V. vinifera L, indicating that grapevine EST datasets are a valuable source for the development of functional simple sequence repeat (SSR) markers.

Computational identification of MicroRNAs in strawberry expressed sequence tags and validation of their precise sequences by miR-RACE.

MicroRNAs (miRNAs) are small, endogenously expressed, nonprotein-coding RNAs that regulate gene expression at the post-transcriptional level in both animals and plants through repressing translation or inducing mRNA degradation. A comprehensive strategy to identify new miRNA homologs by mining the repository of available strawberry expressed sequence tags (ESTs) was developed. By adopting a range of filtering criteria, we identified 11 potential miRNAs belonging to 5 miRNA families from 47 890 Fragaria vesca EST sequences. Using 2 specific 5' and 3' miRNA RACE PCR reactions and a sequence-directed cloning method, we accurately determined both end sequences of 5 candidate miRNAs. Meanwhile, qRT-PCR was used to detect the expression of these 5 miRNAs in different strawberry organs and tissues at several growing stages. These newly identified F. vesca miRNAs (fve-miRNAs) and their expression information can improve our understanding of possible roles of fve-miRNAs in regulating the growth and development of F. vesca.