Vacuolar processing enzyme (VvßVPE) from Vitis vinifera, processes seed proteins during ovule development, and accelerates seed germination in VvßVPE heterologously over-expressed Arabidopsis.

Vacuolar processing enzymes (VPEs), belonging to cysteine protease, are responsible for processing seed protein during maturation. Stenospermocarpic grapes occur self-abortion in fertilized embryos during the ovule development, which affects the formation of matured seed proteins. However, little is known about VPE functions in ovule self-defeating. Here, we investigated the role of one seed-type VPE gene, VvßVPE. Sequence analysis showed that all ORFs (Open reading frames) of VvßVPE from 19 seed/seedless genotypes are highly conserved. At the transcriptional level, VvßVPE was specifically expressed during ovule development, with distinct expression patterns: it increased gradually in seeded grapes; while weakly expressed in seedless grapes. Whereas, at the translational level, 3 forms of VvßVPE were expressed during ovule development in seeded grape: precursor ßVPE (pßVPE), intermediate ßVPE (ißVPE) and finally, active mature ßVPE (mßVPE). By contrast, in seedless grape, VvßVPE only exists as pßVPE at whole developmental stage of ovule. for confirming these expression patterns, 12 seeded/seedless genotypes were sampled and analyzed. Furthermore, VPE enzyme activity was increased in Arabidopsis overexpressing VvßVPE, leading to faster germination. Our study indicated that VvßVPE is essential for grapevine ovule maturation through various forms and is involved in seed germination.

The metacaspase gene family of Vitis vinifera L.: characterization and differential expression during ovule abortion in stenospermocarpic seedless grapes.

In both plants and animals, programmed cell death (PCD) is an indispensable process that removes redundant cells. In seedless grapes (Vitis vinifera), abnormal PCD in ovule cells and subsequent ovule abortion play key roles in stenospermocarpy. Metacaspase, a type of cysteine-dependent protease, plays an essential role in PCD. To reveal the characteristics of the metacaspase (MC) gene family and the relationship between metacaspases and the seedless trait, we identified the 6 V. vinifera metacaspases VvMC1-VvMC6, from the grape genome, using BLASTN against the 9 known Arabidopsis metacaspases. We also obtained full-length cDNAs by RT-PCR. Each of the 6 grape metacaspases contains small (p10-like) and a large (p20-like) conserved structural domains. Phylogenetic analysis of 6 grape and 9 Arabidopsis metacaspases showed that all metacaspases could be grouped into two classes: Type I and Type II. Each phylogenetic branch shares a similar exon/intron structure. Furthermore, the putative promoters of the grape metacaspases contained cis-elements that are involved in grape endosperm development. Moreover, expression analysis of metacaspases using real-time quantitative PCR demonstrated that VvMC1 and VvMC2 were able to be detected in any tissue, and VvMC3, VvMC4, VvMC5 and VvMC6 exhibited tissue-specific expression. Lastly, in cv. Thompson seedless grapes VvMC1, VvMC3, and VvMC4 were significantly up-regulated at the 35 DAF during ovule development, roughly same stage as endosperm abortion. In addition, the expression trend of VvMC2 and VvMC5 was similar between cv. Pinot Noir and cv. Thompson grape ovule development and that of VvMC6 was sustained in a relatively low level except the expression of cv. Pinot Noir significantly up-regulated in 25 DAF. Our data provided new insights into PCD by identifying the grape metacaspase gene family and provide a useful reference for further functional analysis of metacaspases in grape.