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 wild-type 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 μg/dm² versus 76.4 μg/dm², respectively). However, the thickness of the cutin layer on the abaxial surface of lacs2 leaves was only 22.3 ± 1.7 nm compared with 33.0 ± 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 ω-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 β-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.

Summary: A 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.

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 thatlacs9-1 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.