Seeds of the tung tree (Vernicia fordii) produce large quantities of triacylglycerols (TAGs) containing ;80% eleostearic acid, an unusual conjugated fatty acid. We present a comparative analysis of the genetic, functional, and cellular properties of tung type 1 and type 2 diacylglycerol acyltransferases (DGAT1 and DGAT2), two unrelated enzymes that catalyze the committed step in TAG biosynthesis. We show that both enzymes are encoded by single genes and that DGAT1 is expressed at similar levels in various organs, whereas DGAT2 is strongly induced in developing seeds at the onset of oil biosynthesis. Expression of DGAT1 and DGAT2 in yeast produced different types and proportions of TAGs containing eleostearic acid, with DGAT2 possessing an enhanced propensity for the synthesis of trieleostearin, the main component of tung oil. Both DGAT1 and DGAT2 are located in distinct, dynamic regions of the endoplasmic reticulum (ER), and surprisingly, these regions do not overlap. Furthermore, although both DGAT1 and DGAT2 contain a similar C-terminal pentapeptide ER retrieval motif, this motif alone is not sufficient for their localization to specific regions of the ER. These data suggest that DGAT1 and DGAT2 have nonredundant functions in plants and that the production of storage oils, including those containing unusual fatty acids, occurs in distinct ER subdomains.

To investigate the role of jasmonate in the defense of plants against fungal pathogens, we have studied a mutant of Arabidopsis, fad3-2 fad7-2 fad8, that cannot accumulate jasmonate. Mutant plants were extremely susceptible to root rot caused by the fungal root pathogen Pythium mastophorum (Drechs.), even though neighboring wild-type plants were largely unaffected by this fungus. Application of exogenous methyl jasmonate substantially protected mutant plants, reducing the incidence of disease to a level close to that of wild-type controls. A similar treatment with methyl jasmonate did not protect the jasmonate-insensitive mutant coi1 from infection, showing that protective action of applied jasmonate against P. mastophorum was mediated by the induction of plant defense mechanisms rather than by a direct antifungal action. Transcripts of three jasmonate-responsive defense genes are induced by Pythium challenge in the wildtype but not in the jasmonate-deficient mutant. Pythium species are ubiquitous in soil and root habitats world-wide, but most (including P. mastophorum) are considered to be minor pathogens. Our results indicate that jasmonate is essential for plant defense against Pythium and, because of the high exposure of plant roots to Pythium inoculum in soil, may well be fundamental to survival of plants in nature. Our results further indicate that the fad3-2 fad7-2 fad8 mutant is an appropriate genetic model for studying the role of this important signaling molecule in pathogen defense.

Long-chain acyl-CoA synthetase (LACS) activities are encoded by a family of at least nine genes in Arabidopsis (Arabidopsis thaliana). These enzymes have roles in lipid synthesis, fatty acid catabolism, and the transport of fatty acids between subcellular compartments. Here, we show that the LACS2 gene (At1g49430) is expressed in young, rapidly expanding tissues, and in leaves expression is limited to cells of the adaxial and abaxial epidermal layers, suggesting that the LACS2 enzyme may act in the synthesis of cutin or cuticular waxes. A lacs2 null mutant was isolated by reverse genetics. Leaves of mutant plants supported pollen germination and released chlorophyll faster than wild-type leaves when immersed in 80% ethanol, indicating a defect in the cuticular barrier. The composition of surface waxes extracted from lacs2 leaves was similar to the wild type, and the total wax load was higher than the wild type (111.4 mg/dm 2 versus 76.4 mg/dm 2 , respectively). However, the thickness of the cutin layer on the abaxial surface of lacs2 leaves was only 22.3 6 1.7 nm compared with 33.0 6 2.0 nm for the wild type. In vitro assays showed that 16-hydroxypalmitate was an excellent substrate for recombinant LACS2 enzyme. We conclude that the LACS2 isozyme catalyzes the synthesis of v-hydroxy fatty acyl-CoA intermediates in the pathway to cutin synthesis. The lacs2 phenotype, like the phenotypes of some other cutin mutants, is very pleiotropic, causing reduced leaf size and plant growth, reduced seed production, and lower rates of seedling germination and establishment. The LACS2 gene and the corresponding lacs2 mutant will help in future studies of the cutin synthesis pathway and in understanding the consequences of reduced cutin production on many aspects of plant biology.

Long-chain acyl-coenzyme A (CoA) synthetases (LACSs) activate free fatty acids to acyl-CoA thioesters and as such play critical roles in fatty acid metabolism. This important class of enzymes factors prominently in several fatty acid-derived metabolic pathways, including phospholipid, triacylglycerol, and jasmonate biosynthesis and fatty acid ␤-oxidation. In an effort to better understand the factors that control fatty acid metabolism in oilseeds, we have sought to identify and characterize genes that encode LACSs in Arabidopsis. Nine cDNAs were identified, cloned, and tested for their ability to complement a LACS-deficient strain of yeast (Saccharomyces cerevisiae). Seven of the nine successfully restored growth, whereas two cDNAs encoding putative peroxisomal isoforms did not. Lysates from yeast cells overexpressing each of the nine cDNAs were active in LACS enzyme assays using oleic acid as a substrate. The substrate specificities of the enzymes were determined after overexpression in LACS-deficient Escherichia coli. Most of the LACS enzymes displayed highest levels of activity with the fatty acids that make up the common structural and storage lipids in Arabidopsis tissues. Analysis of the tissue-specific expression profiles for these genes revealed one flower-specific isoform, whereas all others were expressed in various tissues throughout the plant. These nine cDNAs are thought to constitute the entire LACS family in Arabidopsis, and as such, will serve as powerful tools in the study of acyl-CoA metabolism in oilseeds.Long-chain acyl-CoA synthetase (LACS, EC 6.2.1.3) occupies a critical position in the biosynthetic pathways of nearly all fatty acid-derived molecules. LACS esterifies free fatty acids to acyl-CoAs, a key activation step that is necessary for the utilization of fatty acids by most lipid metabolic enzymes. LACS catalyzes the formation of acyl-CoA by a two-step mechanism (Groot et al., 1976). In the first step, the free fatty acid is converted to an acyl-AMP intermediate, called an adenylate, through the pyrophosphorolysis of ATP. The activated carbonyl carbon of the adenylate is then coupled to the thiol group of CoA, releasing AMP and the acyl-CoA final product (Groot et al., 1976).However, this reaction scheme is not exclusive to LACS. The formation of the enzyme-bound adenylate is a common mechanism for acyl activation shared by a wide variety of enzymes in organisms that span the biological spectrum, from algal polyketide synthetases (Bibb et al., 1994) to arthropod luciferases (Conti et al., 1996) to bacterial peptide antibiotic synthetases (Conti et al., 1997). This mechanistic similarity is reflected in the conservation of certain amino acid motifs between all enzymes of this group. One motif in particular (PROSITE PS00455) is very highly conserved and acts as the unifying feature of this large group of enzymes, called the AMPbinding protein (AMPBP) superfamily (Babbitt et al., 1992), to which LACS belongs.LACS is a particularly challenging and interesting target for molecular analy...

SummaryA central goal of green chemistry is to produce industrially useful fatty acids in oilseed crops.Although genes encoding suitable fatty acid-modifying enzymes are available from many wild species, progress has been limited because the expression of these genes in transgenic plants produces low yields of the desired products. For example, Ricinus communis fatty acid hydroxylase 12 (FAH12) produces a maximum of only 17% hydroxy fatty acids (HFAs) when expressed in Arabidopsis . cDNA clones encoding R. communis enzymes for additional steps in the seed oil biosynthetic pathway were identified. Expression of these cDNAs in FAH12 transgenic plants revealed that the R. communis type-2 acyl-coenzyme A:diacylglycerol acyltransferase (RcDGAT2) could increase HFAs from 17% to nearly 30%. Detailed comparisons of seed neutral lipids from the single-and double-transgenic lines indicated that RcDGAT2 substantially modified the triacylglycerol (TAG) pool, with significant increases in most of the major TAG species observed in native castor bean oil. These data suggest that RcDGAT2 prefers acyl-coenzyme A and diacylglycerol substrates containing HFAs, and biochemical analyses of RcDGAT2 expressed in yeast cells confirmed a strong preference for HFA-containing diacylglycerol substrates. Our results demonstrate that pathway engineering approaches can be used successfully to increase the yields of industrial feedstocks in plants, and that members of the DGAT2 gene family probably play a key role in this process.

Producing unusual fatty acids (FAs) in crop plants has been a long-standing goal of green chemistry. However, expression of the enzymes that catalyze the primary synthesis of these unusual FAs in transgenic plants typically results in low levels of the desired FA. For example, seed-specific expression of castor (Ricinus communis) fatty acid hydroxylase (RcFAH) in Arabidopsis (Arabidopsis thaliana) resulted in only 17% hydroxy fatty acids (HFAs) in the seed oil. In order to increase HFA levels, we investigated castor phospholipid:diacylglycerol acyltransferase (PDAT). We cloned cDNAs encoding three putative PDAT enzymes from a castor seed cDNA library and coexpressed them with RcFAH12. One isoform, RcPDAT1A, increased HFA levels to 27%. Analysis of HFA-triacylglycerol molecular species and regiochemistry, along with analysis of the HFA content of phosphatidylcholine, indicates that RcPDAT1A functions as a PDAT in vivo. Expression of RcFAH12 alone leads to a significant decrease in FA content of seeds. Coexpression of RcPDAT1A and RcDGAT2 (for diacylglycerol acyltransferase 2) with RcFAH12 restored FA levels to nearly wild-type levels, and this was accompanied by a major increase in the mass of HFAs accumulating in the seeds. We show the usefulness of RcPDAT1A for engineering plants with high levels of HFAs and alleviating bottlenecks due to the production of unusual FAs in transgenic oilseeds.

Acyl-activating enzymes are a diverse group of proteins that catalyze the activation of many different carboxylic acids, primarily through the formation of a thioester bond. This group of enzymes is found in all living organisms and includes the acyl-coenzyme A synthetases, 4-coumarate:coenzyme A ligases, luciferases, and non-ribosomal peptide synthetases. The members of this superfamily share little overall sequence identity, but do contain a 12-amino acid motif common to all enzymes that activate their acid substrates using ATP via an enzyme-bound adenylate intermediate. Arabidopsis possesses an acyl-activating enzyme superfamily containing 63 different genes. In addition to the genes that had been characterized previously, 14 new cDNA clones were isolated as part of this work. The protein sequences were compared phylogenetically and grouped into seven distinct categories. At least four of these categories are plant specific. The tissue-specific expression profiles of some of the genes of unknown function were analyzed and shown to be complex, with a high degree of overlap. Most of the plant-specific genes represent uncharacterized aspects of carboxylic acid metabolism. One such group contains members whose enzymes activate short-and medium-chain fatty acids. Altogether, the results presented here describe the largest acyl-activating enzyme family present in any organism thus far studied at the genomic level and clearly indicate that carboxylic acid activation metabolism in plants is much more complex than previously thought.Carboxylic acid activation plays a vital role in numerous metabolic pathways in all living organisms. Activation of carboxylic acids provides the precursors for pathways that lead to the biosynthesis and/or breakdown of many types of important metabolites, including lipids, amino acids, sugars, and a variety of secondary metabolites. Given the chemical diversity of substrates requiring activation, several types of carboxylic acid activating enzymes have evolved to fulfill this role. Although some of these enzymes couple carboxyl groups to amines or alcohols, most acyl-activating enzymes are acid-thiol ligases (EC 6.2.1). Even within this smaller group, the similarity in the types of products formed is not strictly reflected in the routes by which these enzymes carry out their respective reactions: at least three different catalytic mechanisms are used to create the various thioester products (Groot et al., 1976; Stein et al., 1996;Sánchez et al., 2000). We are particularly interested in the acyl-activating enzymes (AAEs). This group of enzymes has previously been called the acyl adenylate-forming (Conti et al., 1996;Chang et al., 1997) or AMP-binding protein (Fulda et al., 1997) superfamily. In this report, we define AAEs as those enzymes that first activate their respective carboxylic acid substrates through the pyrophosphorylysis of ATP, forming an enzyme-bound acyl-AMP intermediate called an adenylate. The carbonyl carbon of the adenylate most commonly then undergoes nucleophilic attac...

Acyl-coenzyme A (CoA) synthetases (ACSs, EC 6.2.1.3) catalyze the formation of fatty acyl-CoAs from free fatty acid, ATP, and CoA. Essentially all de novo fatty acid synthesis occurs in the plastid. Fatty acids destined for membrane glycerolipid and triacylglycerol synthesis in the endoplasmic reticulum must be first activated to acyl-CoAs via an ACS. Within a family of nine ACS genes from Arabidopsis, we identified a chloroplast isoform, LACS9. LACS9 is highly expressed in developing seeds and young rosette leaves. Both in vitro chloroplast import assays and transient expression of a green fluorescent protein fusion indicated that the LACS9 protein is localized in the plastid envelope. A T-DNA knockout mutant (lacs9-1) was identified by reverse genetics and these mutant plants were indistinguishable from wild type in growth and appearance. Analysis of leaf lipids provided no evidence for compromised export of acyl groups from chloroplasts. However, direct assays demonstrated that lacs9-1 plants contained only 10% of the chloroplast long-chain ACS activity found for wild type. The residual long-chain ACS activity in mutant chloroplasts was comparable with calculated rates of fatty acid synthesis. Although another isozyme contributes to the activation of fatty acids during their export from the chloroplast, LACS9 is a major chloroplast ACS.