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

Abstract

Main conclusion

The grapevine VvβVPE promoter is specifically expressed in the seed. The − 1306~− 1045 bp core region restricts expression in other tissues and organs.

Abstract

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~− 1045 bp region. All deletion promoters presenting various quantified GUS activities indicate that the region − 1704~− 1306 bp inhibits, and the region − 705~− 861 bp 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~− 1045 bp) is the key one responsible for seed-specific expression.

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Abbreviations

GUS:

β-Glucuronidase

MU:

4-Methyl umbelliferone

VPE:

Vacuolar processing enzyme

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (Grant No. 31171926), the earmarked fund for the China Agriculture Research System (CARS-30-y-7) and Key Technologies R & D Program of Xi’ an city (201806114 YF02NC10-4).

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Correspondence to Chaohong Zhang.

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Gong, P., Wei, R., Li, Y. et al. Molecular cloning and functional characterization of a seed-specific VvβVPE gene promoter from Vitis vinifera. Planta 250, 657–665 (2019). https://doi.org/10.1007/s00425-019-03197-0

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Keywords

  • GUS activity
  • Ovule development
  • Promoter deletion analysis
  • Seedless grapes
  • Seed-specific expression
  • β-type vacuolar processing enzyme (VPE)