The enzyme 4-coumarate:coenzyme A ligase (4CL) is important in providing activated thioester substrates for phenylpropanoid natural product biosynthesis. We tested different hybrid poplar (Populus trichocarpa ؋ Populus deltoides) tissues for the presence of 4CL isoforms by fast-protein liquid chromatography and detected a minimum of three 4CL isoforms. These isoforms shared similar hydroxycinnamic acid substrate-utilization profiles and were all inactive against sinapic acid, but instability of the native forms precluded extensive further analysis. 4CL cDNA clones were isolated and grouped into two major classes, the predicted amino acid sequences of which were 86% identical. Genomic Southern blots showed that the cDNA classes represent two poplar 4CL genes, and northern blots provided evidence for their differential expression. Recombinant enzymes corresponding to the two genes were expressed using a baculovirus system. The two recombinant proteins had substrate utilization profiles similar to each other and to the native poplar 4CL isoforms (4-coumaric acid > ferulic acid > caffeic acid; there was no conversion of sinapic acid), except that both had relatively high activity toward cinnamic acid. These results are discussed with respect to the role of 4CL in the partitioning of carbon in phenylpropanoid metabolism.The enzyme 4CL (EC 6.2.1.12) catalyzes the formation of CoA thioesters of hydroxycinnamic acids by a two-step reaction mechanism that involves the hydrolysis of ATP (Gross and Zenk, 1974). These thioesters serve as substrates for a number of important reactions within plant phenylpropanoid metabolism. Depending on the species and tissue, formation of hydroxycinnamate esters and amides often involves CoA derivatives of 4-coumaric, caffeic, and/or ferulic acid, whereas flavonoid and stilbene biosynthesis requires cinnamoyl and/or 4-coumaroyl CoA esters as the substrates (Hahlbrock and Scheel, 1989;Dixon and Paiva, 1995;Holton and Cornish, 1995). The biosynthesis of salicylic acid from cinnamic acid may occur via oxidation of a cinnamoyl-CoA intermediate (Ryals et al., 1996), and within virtually all higher plants, generation of lignin monomers is presumed to require the conversion of 4-coumaric acid, ferulic acid, and sinapic acid to the corresponding CoA esters in preparation for side-chain reduction .In keeping with this metabolic demand for different hydroxycinnamoyl CoA esters, 4CL preparations from plants are normally found to be able to use a number of hydroxycinnamic acid derivatives as substrates (Gross and Zenk, 1974; Hahlbrock, 1975, 1977;Grand et al., 1983;Lozoya et al., 1988;Voo et al., 1995;Lee and Douglas, 1996). It has also been proposed, however, that different isoforms of 4CL might possess different patterns of substrate preference, and characterization of 4CL isoforms from some plants supports this (Knobloch and Hahlbrock, 1975;Ranjeva et al., 1976;Wallis and Rhodes, 1977;Grand et al., 1983). Such diversity could conceivably enable particular 4CL isoforms to efficiently supply an appro...
A meristem-specific gene coding for deoxyuridine triphosphatase (EC 3.6.1.23) (dUTPase) in tomato was isolated, and its developmental expression in vegetative and floral apices was monitored. An 18-kD polypeptide, P18, was isolated as a consequence of its accumulation in arrested floral meristems of anantha mutant plants. The corresponding cDNA isolated from an expression library exhibited a 40 to 60% similarity with the pseudoprotease sequences of poxviruses, genes that have been suggested to encode dUTPases. Enzymatic tests and conservation of peptide motifs common to bacterial and vira1 genes verified that the P18 cDNA clone indeed represents a eukaryotic dUTPase. lmmunogold localization and in situ hybridization experiments showed that polypeptides and transcripts of dUTPase are maintained at high levels in apical meristematic cells of vegetative and floral meristems. dUTPase gene activity is also high in the potentially meristematic cells of the provascular and vascular system. Its expression is lower in the immediate parenchymal derivatives of the apical meristematic cells, and this downregulation marks, perhaps, the transition from totipotency to the first differentiated state. INTRODUCTIONPrimary tissues of the plant shoot and root arise continuously from apical meristems. Apical meristems differentiate during embryogeny and are developmentally autonomous. They retain their meristematic activity throughout the life cycle while generating primary tissues and new meristematic centers that form lateral organs (Walbot, 1985;Goldberg, 1988;Sussex, 1989;Poethig, 1990). Unlike vegetative meristems, floral meristems are not embryonic in origin. Transmissible physiological signals in the vegetative apex initiate floral evocation, which results in transformation to a floral apex (Bernier, 1988). Floral meristems, moreover, are considered to be determinate because in most cases they form only inflorescences. Understanding meristems is, therefore, a prerequisite for the understanding of plant development (Sussex, 1989). An impressive body of descriptive and classical experimental studies on this subject is discussed comprehensively by Esau (1977), Cutter (1980), and Steeves and Sussex (1989).In an attempt to dissect the developmental processes in tomato meristems, we undertook the isolation of gene markers common to all plant meristems and others that are more specific to floral meristems. The repertoire of soluble and insoluble proteins of growing leaves and mature flowers was compared with that of anantha floral meristems. Floral meristems of anantha mutant plants are arrested at an early To whom correspondence should be addressed. preorganogenesis stage and then duplicated repeatedly (Helm, 1951;Paddock and Alexander, 1952), thus providing an excellent source of meristematic tissue (Lifschitz, 1988). Selected polypeptides that appeared more abundant in anantha meristems were purified, antibodies were prepared, and cDNA clones were isolated. The tomato deoxyuridine triphosphatase (dUTPase) gene reported here was ...
A meristem-specific gene coding for deoxyuridine triphosphatase (EC 3.6.1.23) (dUTPase) in tomato was isolated, and its developmental expression in vegetative and floral apices was monitored. An 18-kD polypeptide, P18, was isolated as a consequence of its accumulation in arrested floral meristems of anantha mutant plants. The corresponding cDNA isolated from an expression library exhibited a 40 to 60% similarity with the pseudoprotease sequences of poxviruses, genes that have been suggested to encode dUTPases. Enzymatic tests and conservation of peptide motifs common to bacterial and viral genes verified that the P18 cDNA clone indeed represents a eukaryotic dUTPase. Immunogold localization and in situ hybridization experiments showed that polypeptides and transcripts of dUTPase are maintained at high levels in apical meristematic cells of vegetative and floral meristems. dUTPase gene activity is also high in the potentially meristematic cells of the provascular and vascular system. Its expression is lower in the immediate parenchymal derivatives of the apical meristematic cells, and this downregulation marks, perhaps, the transition from totipotency to the first differentiated state.
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