Gene structure and molecular analysis of the laccase-like multicopper oxidase (LMCO) gene family in Arabidopsis thaliana.

Completed genome sequences have made it clear that multicopper oxidases related to laccase are widely distributed as multigene families in higher plants. Laccase-like multicopper oxidase (LMCO) sequences culled from GenBank and the Arabidopsis thaliana genome, as well as those from several newly cloned genes, were used to construct a gene phylogeny that clearly divided plant LMCOs into six distinct classes, at least three of which predate the evolutionary divergence of angiosperms and gymnosperms. Alignments of the predicted amino acid sequences highlighted regions of variable sequence flanked by the highly conserved copper-binding domains that characterize members of this enzyme family. All of the predicted proteins contained apparent signal sequences. The expression of 13 of the 17 LMCO genes in A. thaliana was assessed in different tissues at various stages of development using RT-PCR. A diversity of expression patterns was demonstrated with some genes being expressed in a constitutive fashion, while others were only expressed in specific tissues at a particular stage of development. Only a few of the LMCO genes were expressed in a pattern that could be considered consistent with a major role for these enzymes in lignin deposition. These results are discussed in the context of other potential physiological functions for plant LMCOs, such as iron metabolism and wound healing.

Regulation of plant arginase by wounding, jasmonate, and the phytotoxin coronatine.

In mammalian cells, induced expression of arginase in response to wound trauma and pathogen infection plays an important role in regulating the metabolism of L-arginine to either polyamines or nitric oxide (NO). In higher plants, which also utilize arginine for the production of polyamines and NO, the potential role of arginase as a control point for arginine homeostasis has not been investigated. Here, we report the characterization of two genes (LeARG1 and LeARG2) from Lycopersicon esculentum (tomato) that encode arginase. Phylogenic analysis showed that LeARG1 and -2, like all other plant arginases, are more similar to agmatinase than to arginases from vertebrates, fungi, and bacteria. Nevertheless, recombinant LeARG1 and -2 exhibited specificity for L-arginine over agmatine and related guanidino substrates. The plant enzymes, like mammalian arginases, were inhibited (K(i) approximately 14 microM) by the NO precursor N(G)-hydroxy-L-arginine. These results indicate that plant arginases define a distinct group of ureohydrolases that function as authentic L-arginases. LeARG1 and LeARG2 transcripts accumulated to their highest levels in reproductive tissues. In leaves, LeARG2 expression and arginase activity were induced in response to wounding and treatment with jasmonic acid (JA), a potent signal for plant defense responses. Wound- and JA-induced expression of LeARG2 was not observed in the tomato jasmonic acid-insensitive1 mutant, indicating that this response is strictly dependent on an intact JA signal transduction pathway. Infection of wild-type plants with a virulent strain of Pseudomonas syringae pv. tomato also up-regulated LeARG2 expression and arginase activity. This response was mediated by the bacterial phytotoxin coronatine, which exerts its virulence effects by co-opting the host JA signaling pathway. These results highlight striking similarities in the regulation of arginase in plants and animals and suggest that stress-induced arginase may perform similar roles in diverse biological systems.

The tomato homolog of CORONATINE-INSENSITIVE1 is required for the maternal control of seed maturation, jasmonate-signaled defense responses, and glandular trichome development.

Jasmonic acid (JA) is a fatty acid-derived signaling molecule that regulates a broad range of plant defense responses against herbivores and some microbial pathogens. Molecular genetic studies in Arabidopsis have established that JA also performs a critical role in anther and pollen development but is not essential for other developmental aspects of the plant's life cycle. Here, we describe the phenotypic and molecular characterization of a sterile mutant of tomato (jasmonic acid-insensitive1 [jai1]) that is defective in JA signaling. Although the mutant exhibited reduced pollen viability, sterility was caused by a defect in the maternal control of seed maturation, which was associated with the loss of accumulation of JA-regulated proteinase inhibitor proteins in reproductive tissues. jai1 plants exhibited several defense-related phenotypes, including the inability to express JA-responsive genes, severely compromised resistance to two-spotted spider mites, and abnormal development of glandular trichomes. We demonstrate that these defects are caused by the loss of function of the tomato homolog of CORONATINE-INSENSITIVE1 (COI1), an F-box protein that is required for JA-signaled processes in Arabidopsis. These findings indicate that the JA/COI1 signaling pathway regulates distinct developmental processes in different plants and suggest a role for JA in the promotion of glandular trichome-based defenses.

Identification of a jasmonate-regulated allene oxide synthase that metabolizes 9-hydroperoxides of linoleic and linolenic acids.

Allene oxide synthase (AOS) is a cytochrome P-450 (CYP74A) that catalyzes the first step in the conversion of 13-hydroperoxy linolenic acid to jasmonic acid and related signaling molecules in plants. Here, we report the molecular cloning and characterization of a novel AOS-encoding cDNA (LeAOS3) from Lycopersicon esculentum whose predicted amino acid sequence classifies it as a member of the CYP74C subfamily of enzymes that was hitherto not known to include AOSs. Recombinant LeAOS3 expressed in Escherichia coli showed spectral characteristics of a P-450. The enzyme transformed 9- and 13-hydroperoxides of linoleic and linolenic acid to alpha-ketol, gamma-ketol, and cyclopentenone compounds that arise from spontaneous hydrolysis of unstable allene oxides, indicating that the enzyme is an AOS. Kinetic assays demonstrated that LeAOS3 was approximately 10-fold more active against 9-hydroperoxides than the corresponding 13-isomers. LeAOS3 transcripts accumulated in roots, but were undetectable in aerial parts of mature plants. In contrast to wild-type plants, LeAOS3 expression was undetectable in roots of a tomato mutant that is defective in jasmonic acid signaling. These findings suggest that LeAOS3 plays a role in the metabolism of 9-lipoxygenase-derived hydroperoxides in roots, and that this branch of oxylipin biosynthesis is regulated by the jasmonate signaling cascade.