Tobacco and Parsley 4-Coumarate:Coenzyme A Ligase Genes Are Temporally and Spatially Regulated in a Cell Type-Specific Manner during Tobacco Flower Development

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Abstract
The enzyme 4-coumarate:coenzyme A ligase (4CL) plays a key role in phenylpropanoid metabolism by supplying the precursors (coenzyme A esters of cinnamic acid derivatives) used for the biosynthesis of diverse natural products, many of which play functional roles in floral organs. In this study, we used in situ hybridization and histochemical localization of [beta]-glucuronidase (GUS) activity to define in detail the temporal and spatial patterns of 4CL-1 expression during tobacco flower development. Sectioned flowers from tobacco plants transgenic for a complete copy of the parsley (Petroselinum crispum) 4CL-1 gene were hybridized to probes that distinguished between 4CL-1 transcripts and endogenous tobacco 4CL transcripts. Both probes hybridized with similar cell type-specific patterns to carpels, anthers, petals, and sepals, and the sites of hybridization varied during flower development. The sites of hybridization generally coincided temporally and spatially with sites of 4CL-GUS expression, suggesting that most of the expression patterns are regulated by 4CL-1 promoter sequences, but lack of correlation between sites of 4CL mRNA accumulation and GUS activity in portions of the petal suggest that downstream sequences may mediate some aspects of developmentally regulated 4CL-1 expression. These results indicate that the introduced 4CL-1 gene correctly responds to endogenous tobacco developmental signals and demonstrate complex temporal and spatial patterns of expression during floral organ differentiation.