Functional analysis of the DAT gene promoter using transient Catharanthus roseus and stable Nicotiana tabacum transformation systems

• Published in: Plant cell reports Vol. 30; no. 7; pp. 1173 - 1182
• Main Authors: Makhzoum, Abdullah, Petit-Paly, Geneviève, St. Pierre, Benoit, Bernards, Mark A.
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer-Verlag 01.07.2011; Springer; Springer Nature B.V
• Subjects: Acetyltransferases - genetics; Acetyltransferases - metabolism; Base Sequence; Biological and medical sciences; Biomedical and Life Sciences; Biotechnology; Catharanthus - genetics; Catharanthus - metabolism; Catharanthus roseus; Cell Biology; Cloning, Molecular; Electroporation; Enzymatic activity; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation, Plant; Genes, Plant; Genes, Reporter; Genetic Vectors; Life Sciences; Molecular Sequence Data; Nicotiana - genetics; Nicotiana - metabolism; Nicotiana tabacum; Open Reading Frames; Original Paper; Plant Biochemistry; Plant Leaves - genetics; Plant Leaves - metabolism; Plant Roots - enzymology; Plant Roots - genetics; Plant Sciences; Plant tissues; Plants, Genetically Modified - genetics; Plants, Genetically Modified - metabolism; Promoter Regions, Genetic; Roots; Seedlings - genetics; Seedlings - metabolism; Stomata; Transcription Initiation Site; Transfection; Transformation, Genetic; Vindoline; Acetyl-CoA:deacetylvindoline-4; Specific cell type; Indole alkaloids; Promoter analysis; acetyltransferase; Acyltransferases; Vegetals; Enzyme; Transcription promoter; Transferases; Indole; Functional analysis; Catharanthus roseus; Nicotiana tabacum; Transients; Apocynaceae; Alkaloid; Dicotyledones; Angiospermae; Acetyl-CoA:deacetylvindoline-4-O-acetyltransferase; Spermatophyta; Solanaceae; Catharanthus
• ISSN: 0721-7714; 1432-203X
• DOI: 10.1007/s00299-011-1025-y

The Catharanthus roseus DAT gene encodes the enzyme acetyl-CoA:deacetylvindoline-4- O- acetyltransferase involved in the last step of the indole alkaloid pathway leading to vindoline. This gene is characterized by specific cell type expression in idioblasts and laticifers. To understand the specific transcriptional regulation mechanism(s) of DAT , several DAT promoter GUS constructs were cloned into pCAMBIA1305.1. Agroinfiltration of different explant types of C. roseus resulted in organ-specific accumulation of GUS, albeit at various levels. Heterologous accumulation of GUS in transgenic tobacco revealed both general and non-specific expression with the exception of a stomata-specific expression when 2.3 kb of the DAT promoter was coupled with a portion of the DAT ORF. These results suggest that in addition to the 2.3 kb upstream of the DAT transcriptional start site, additional cis -acting elements may be responsible for the specific spatial expression of DAT in vivo. Furthermore, hairy roots transformed with DAT promoter GUS constructs demonstrated GUS expression in root tissues (visualized through GUS enzyme activity), even though DAT is repressed in non-transformed roots.