Molecular cloning and functional characterization of a seed-specific VvβVPE gene promoter from Vitis vinifera.

Abstract

The grapevine VvβVPE promoter is specifically expressed in the seed. The -1306~-1045 bp core region restricts expression in other tissues and organs. Vacuolar processing enzyme (VPE) is a cysteine proteinase regulating vacuolar protein maturation and executing programmed cell death (PCD) in plants. Vitis vinifera (Vv)βVPE is a β-type VPE showing seed-specific expression that processes seed proteins during ovule development. However, the regulation of the seed-specific gene expression is far from understood. In this study, we characterize VvβVPE promoter (pVvβVPE) from 12 seeded and seedless grape genotypes. 94.56% of the pVvβVPE coding sequence is consistent. Two βVPE promoters were constructed and transformed into Arabidopsis thaliana via β-glucuronidase (GUS) fused expression vectors, using cv. Pinot Noir and cv. Thompson as seed and seedless candidates. GUS staining in different tissues and organs revealed that VvβVPE expresses specifically in the embryo, including the cotyledon, hypocotyl and suspensor, but not in the leaf, stem, root or flowers of the seedling. Using promoter deletion analysis, we created four incomplete VvβVPE promoters and found each pVvβVPE deletion could drive GUS gene to express in seeds. Interestingly, seed specificity disappeared when the promoter missed the core - 1306~- 1045bp region. All deletion promoters presenting various quantified GUS activities indicate that the region - 1704~- 1306bp inhibits, and the region - 705~- 861bp promotes gene expression of VvβVPE. Our results demonstrate that pVvβVPE is a seed-specific promoter in both seeded and seedless grapes. Moreover, the core region of pVvβVPE (- 1306~- 1045bp) is the key one responsible for seed-specific expression.