Grapevine promoter directs gene expression in the nectaries of transgenic tobacco

Abstract

A major obstacle in the genetic manipulation of fruit ripening in grapevine (Vitis vinifera L.) remains the lack of regulatory elements (promoters) to regulate tissue‐ and ripening‐specific expression of the transgene in the grape berry. In this study, a cDNA previously shown to identify a fruit‐ and ripening‐specific proline‐rich protein from grapevine (mrip1) was used to isolate a fruit‐ and ripening‐specific promoter. Sequence analysis of the 5.5‐kb region directly upstream of the mrip1 transcription initiation site (TIS) revealed a 2.8‐kb AT‐rich region directly upstream of the mrip1 TIS and the presence of the coding and the 5′ untranscribed flanking region (UTR) of a proline‐rich protein further upstream. In silico analysis of the 2.8 kb directly upstream of the mrip1 TIS revealed several putative cis‐acting regulatory elements. These include a spectrum of hormone‐, light‐, phytochrome‐, sugar‐ and stress‐responsive elements. Based on the sugar‐responsive 2.2 kb upstream of the mrip1 TIS, a 2.2‐kb fragment of the mrip1 5′ UTR flanking region was selected for functional analysis of the promoter. Functionality of mrip1:sgfpS65T fusion gene was confirmed by transient expression in green pepper pericarp tissue, before introduction into tobacco by Agrobacterium‐mediated transformation. In transgenic tobacco, transcription of the mrip1:sgfpS65T fusion was developmentally regulated and specific to the ovary and nectary‐tissue specific of the developing flower. While low in immature flowers, green fluorescent protein (GFP) rapidly accumulated to the high level of expression visualized in the flower in full‐bloom, followed by a decrease in the final stages of ovary development. These observations suggest that the 2.2 kb mrip1 promoter is functional and that this promoter region harbours cis‐elements necessary for tissue‐ and developmental‐specific regulation of GFP expression. It furthermore suggests that the transcriptional activation of mrip1 is mediated by developmental signals present in both grapevine berries and tobacco flowers.