The phenylpropanoid enzyme 4-coumarate:coenzyme A ligase (4CL) plays a key role in linking general phenylpropanoid metabolism to end-product specific biosynthetic pathways. During vascular system and floral organ differentiation, the parsley 4CL-1 gene is expressed in a restricted set of tissues and cell types where 4CL activity is required to supply precursors for the synthesis of diverse phenylpropanoid-derived products such as lignin and flavonoids. In order to localize cis-acting elements which specify complex patterns of 4CL-1 expression, we analyzed the expression of internally deleted promoter fragment-GUS fusions in tobacco plants and parsley protoplasts. Elements located between -244 and -78 were required for most aspects of developmentally regulated expression. Within this region, three separate promotor domains containing partially redundant cis-elements directed vascular-specific expression when combined with a TATA-proximal domain. A negative cis-acting element which represses phloem expression was revealed in one of the domains and appears to be responsible for restricting vascular expression to the xylem. Distinct but overlapping promoter domain combinations were required for expression in floral organs, suggesting that different combinations of cis-acting elements may direct expression in different organs. Gel retardation assays were used to demonstrate the formation of DNA-protein complexes between factors present in nuclear extracts of parsley tissue culture cells and various tobacco organs and a 4CL-1 promoter fragment. Competition experiments showed that complex formation required the presence of a 42 bp promoter domain shown to be critical for 4CL-1 expression in vascular and floral tissues. The results are discussed in light of the coordinate expression of 4CL and other phenylpropanoid genes.
The differential response of cultured parsley cells to u.v. irradiation and elicitor treatment is a paradigm for analysis of specific plant defense responses. We demonstrate that freshly isolated parsley protoplasts, in the absence of detectable cell wall, maintain fully the ability to be activated by these important environmental factors. Stimulated protoplasts synthesize typical qualitative patterns and amounts of potentially protective flavonoid glycosides and coumarin phytoalexins following either u.v. irradiation or treatment with fungal elicitor, respectively. Induced accumulation of mRNAs and enzymes of the phenylpropanoid biosynthetic pathways is nearly identical in protoplasts and cells. Stimulation of protoplasts with elicitor requires only a short period of contact, which is not sufficient for cell wall regeneration. Importantly, there is no activation of these pathways during protoplast preparation. These results establish that parsley protoplasts respond appropriately to two physically distinct stimuli and might serve as an especially suitable system for the analysis of signal transduction and gene activation.
The 4CL-1 gene is one of two highly homologous parsley genes encoding 4-coumarate:coenzyme A ligase, a key enzyme of general phenylpropanoid metabolism. Expression of these genes is essential for the biosynthesis of both defense-related and developmentally required phenylpropanoid derivatives. We examined the developmental regulation of the 4CL-1 promoter by analyzing the expression of 4CL-1-8-glucuronidase fusions in transgenic tobacco plants. A 597-base pair 4CL-1 promoter fragment specified histochemically detectable expression in a complex array of vegetative and floral tissues and cell types. The activity of a series of 5' deleted promoter fragments was analyzed in parsley protoplasts and transgenic tobacco plants. Deletions past -210 base pairs led to a drastic decline in 8-glucuronidase activity in protoplasts and loss of tissue-specific expression in transgenic tobacco. These results were put into the context of potential protein-DNA interactions by in vivo footprint analysis of the 4CL-1 promoter in parsley cells. Loss of promoter activity in parsley protoplasts and transgenic tobacco was correlated with the deletion or disruption of the dista1 portion of a large (100-base pair) footprinted region within the first 200 base pairs of the 4CL-1 promoter.
The 4CL-1 gene is one of two highly homologous parsley genes encoding 4-coumarate:coenzyme A ligase, a key enzyme of general phenylpropanoid metabolism. Expression of these genes is essential for the biosynthesis of both defense-related and developmentally required phenylpropanoid derivatives. We examined the developmental regulation of the 4CL-1 promoter by analyzing the expression of 4CL-1-beta-glucuronidase fusions in transgenic tobacco plants. A 597-base pair 4CL-1 promoter fragment specified histochemically detectable expression in a complex array of vegetative and floral tissues and cell types. The activity of a series of 5' deleted promoter fragments was analyzed in parsley protoplasts and transgenic tobacco plants. Deletions past -210 base pairs led to a drastic decline in beta-glucuronidase activity in protoplasts and loss of tissue-specific expression in transgenic tobacco. These results were put into the context of potential protein-DNA interactions by in vivo footprint analysis of the 4CL-1 promoter in parsley cells. Loss of promoter activity in parsley protoplasts and transgenic tobacco was correlated with the deletion or disruption of the distal portion of a large (100-base pair) footprinted region within the first 200 base pairs of the 4CL-1 promoter.
Cultured parsley cells (Petroselinum crispum) treated with an elicitor from the soybean pathogen, Phytophthora megasperma f. sp. glycinea, were used as source for the separation, extensive purification and characterization of S-adenosyl-ʟ-methionine: xanthotoxol and S-adenosyl-ʟ-m ethionine: bergaptol O-methyltransferases. The products, xanthotoxin and bcrgapten, were among the most abundant coumarin derivatives accumulated in the culture fluid of elicitor-treated parsley cells. The latter enzyme also catalyzed the methylation of 5-hydroxyxanthotoxin to isopimpinellin, another major coumarin derivative in this system. The activities of both enzymes showed transient increases upon elicitor application prior to the accumulation of the products. No activity was detectable in untreated cells. Activity increases of the O-methyltransferases occurred a few hours later than those of phenylalanine ammonia-lyase and 4-coumarate:CoA ligase.
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