CoA Ligase Gene Research Articles

Structural comparison, modes of expression, and putative cis-acting elements of the two 4-coumarate: CoA ligase genes in potato

4-Coumarate:CoA ligase (4CL), a key enzyme of phenylpropanoid metabolism in plants, is encoded in potato (Solanum tuberosum L.) by two structurally similar genes (St4cl-1, St4cl-2). Computer-based sequence analyses revealed similarities at the amino acid sequence level with other enzymes dependent on ATP for activation of aromatic carboxylic acids, e.g. some bacterial peptide synthetases. All these enzymes have a common seven amino acid sequence motif containing one cysteine residue. Using an assay on the basis of the polymerase chain reaction, we show that the mRNAs from both 4CL genes accumulate to equal levels in suspension-cultured cells and whole plant tissues, independent of various kinds of activating stimulus applied and of the overall transcriptional activity of the genes. The apparent lack of differential expression, together with the fact that both 4CL genes and proteins are nearly identical in structure, make it unlikely that 4CL isoforms in potato have specific roles in metabolic channeling. Constitutive in vivo footprints in the TATA-box proximal region of the St4cl-1 promoter define putative cis-acting elements which may be involved in the responses of the 4CL genes to various endogenous and exogenous stimuli.

Nucleotide sequence of rice 4-coumarate:CoA ligase gene, 4-CL.1

Nucleotide sequence of rice 4-coumarate:CoA ligase gene, 4-CL.1

A phenylalanine ammonia-lyase gene from parsley: structure, regulation and identification of elicitor and light responsive cis-acting elements.

We demonstrate that phenylalanine ammonia-lyase (PAL) in parsley (Petroselinum crispum) is encoded by a small family of at least four genes. The levels of mRNA from three identified PAL genes increase considerably upon treatment of cultured parsley cells with UV light or fungal elicitor and upon wounding of parsley leaves or roots. In cultured cells these changes were shown to involve transcriptional activation. We present the first primary structure of a plant PAL gene (parsley PAL-1) and the deduced amino acid sequence of the enzyme. Inducible in vivo footprints in the PAL-1 promoter define two nucleotide sequences, within the motifs CTCCAACAAACCCCTTC and ATTCTCACCTACCA, involved in the responses to both UV irradiation and elicitor application. These motifs are conserved at similar positions in several elicitor or light-responsive genes from different species. In two cases they are found within short regions known to confer elicitor or UV-light inducibility. The conserved motifs in the parsley 4-coumarate:CoA ligase gene, which is coordinately regulated with PAL, also display UV-light inducible in vivo footprints. Taken together, our findings suggest a general role of these putative cis-acting elements in the responses of plants to such stresses.

Chromosomal localization of parsley 4-coumarate: CoA ligase genes by in situ hybridization with a complementary DNA

A near full-length cDNA (1.9 kb) was used as probe forin situ hybridization to assign one of the two highly homologous 4-coumarate: CoA ligase genes in parsley (Petroselinum crispum) to the short arm of a submetacentric chromosome. The results suggest, but do not definitely prove, that the second gene is located on a metacentric chromosome and is thus unlinked from the other.

Primary structures and catalytic properties of isoenzymes encoded by the two 4-coumarate: CoA ligase genes in parsley.

We have determined the primary structures of two 4-coumarate: CoA ligase (4CL) isoenzymes in parsley (Petroselinum crispum) by sequencing near full-length cDNAs corresponding to the two 4CL genes, Pc4CL-1 and Pc4CL-2, present in this plant. Comparison of the cDNA and genomic nucleotide sequences showed that each 4CL gene is organized in five exons separated by introns of varying lengths. The positions of introns are the same in both genes and 97-99% of the corresponding nucleotide sequences are identical. The two isoenzymes, which are nearly identical in their primary structures, were separated by ion-exchange chromatography, and were found to be indistinguishable with regard to substrate specificity. Assignment to Pc4CL-1 and Pc4CL-2 was achieved by comparison with catalytically active 4CL proteins, isolated from Escherichia coli cells which had been transformed with plasmids harboring the corresponding cDNAs.

Structure and elicitor or u.v.-light-stimulated expression of two 4-coumarate:CoA ligase genes in parsley

We have isolated genomic clones encoding 4-coumarate:CoA ligase (4CL), a key enzyme of general phenylpropanoid metabolism, and have analysed the structure and regulation of the genes contained on these clones. Restriction enzyme and sequence analysis indicated that two distinct 4CL genes, Pc4CL-1 and Pc4CL-2, are represented on the clones and that additional 4CL genes are not present in parsley. Two lines of evidence suggest that each gene is transcriptionally activated by both elicitor and u.v. irradiation: cDNA clones corresponding to each gene were found in cDNA libraries made with RNA from both elicitor-treated and u.v-irradiated cells, and run-off transcripts homologous to a Pc4CL-2-specific intron probe were induced by both treatments. This induction was about half of the induction measured using probes homologous to both genes. The transcription initiation sites of both genes were determined. Comparison of the nucleotide sequences of the two genes 5' to these sites showed that they are highly homologous for several hundred base pairs and that they contain features potentially involved in regulation by elicitor and u.v. irradiation.