title: Use of the VvMybA1 gene for non-destructive quantification of promoter activity via color histogram analysis in grapevine (Vitis vinifera) and tobacco.
abstract: We report the development of a convenient plant-based reporter system to analyze promoters and facilitate selection of genetically engineered plants. The VvMybA1 gene of grapevine (Vitis vinifera L.) regulates the last metabolic step of anthocyanin biosynthesis and its ectopic expression leads to anthocyanin production in otherwise non-pigmented cells. To develop an anthocyanin-based quantitative reporter system, the VvMybA1 gene was isolated from V. vinifera 'Merlot' and placed under control of three promoters to test its ability to distinguish different activity levels. Promoters included a double enhanced CaMV35S (d35S) promoter, a double enhanced CsVMV (dCsVMV) promoter or a bi-directional dual promoter (BDDP), resulting in transformation vectors DAT, CAT and DEAT, respectively. These vectors were introduced into grapevine and tobacco via Agrobacterium-mediated transformation for transient and stable expression analysis. A linear relationship between the mean red brightness (MRB) and optical density (OD) values with a 0.99 regression coefficient was identified in a dilution series of anthocyanin, thus allowing the use of histogram data for non-destructive and real-time assessment of transcriptional activity. Results of histogram-based analysis of color images from transformed grapevine somatic embryos (SE) and various tissues of transgenic tobacco showed a consistent six to sevenfold promoter activity increase of DEAT over DAT. This expression increase was verified by spectroscopic measurement of anthocyanin concentrations in sepal tissue of transgenic tobacco plants. These results were congruent with previously findings of promoter activity derived from GUS fluorometric assay, thus demonstrating for the first time that the VvMybA1 gene could offer a simple, versatile and reliable plant-based alternative for quantitative promoter analysis in plants.
authors.affiliation: Grape Biotechnology Core Laboratory, Mid-Florida Research and Education Center, University of Florida/IFAS, 2725 Binion Road, Apopka, FL 32703-8504, USA.
authors.forename: Zhijian T
authors.initials: ZT
authors.lastname: Li
authors.forename: Sadanand A
authors.initials: SA
authors.lastname: Dhekney
authors.forename: Dennis J
authors.initials: DJ
authors.lastname: Gray
mesh_terms: D060054:Agrobacterium / Q000235:genetics*; D000872:Anthocyanins / Q000096:biosynthesis / Q000235:genetics*; D005470:Fluorometry; D018506:Gene Expression Regulation, Plant; D017930:Genes, Reporter; D005822:Genetic Vectors; D030821:Plants, Genetically Modified / Q000235:genetics*; D011401:Promoter Regions, Genetic; D014026:Nicotiana / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics*; D027843:Vitis / Q000235:genetics*
chemical_list: D000872:Anthocyanins; D014157:Transcription Factors