Long chain acyl CoA synthetase 4 catalyzes the first step in peroxisomal indole-3-butyric acid to IAA conversion

Indole-3-butyric acid (IBA) is an endogenous storage auxin important for maintaining appropriate indole-3-acetic acid (IAA) levels, thereby influencingprimary root elongation and lateral root development. IBA is metabolized into free IAA in peroxisomes in a multistep process similar to fatty acid β-oxidation. We identified LONG CHAIN ACYL-COA SYNTHETASE 4 (LACS4) in a screen for enhanced IBA resistance in primary root elongation in Arabidopsis thaliana. LACSs activate substrates by catalyzing the addition of CoA, the necessary first step for fatty acids to participate in β-oxidation or other metabolic pathways. Here, we describe the novel role of LACS4 in hormone metabolism and postulate that LACS4 catalyzes the addition of CoA onto IBA, the first step in its β-oxidation. lacs4 is resistant to the effects of IBA in primary root elongation and dark-grown hypocotyl elongation, and has reduced lateral root density. lacs6 also is resistant to IBA, although both lacs4 and lacs6 remain sensitive to IAA in primary root elongation, demonstrating that auxin responses are intact. LACS4 has in vitro enzymatic activity on IBA, but not IAA or IAA conjugates, and disruption of LACS4 activity reduces the amount of IBA-derived IAA in planta. We conclude that, in addition to activity on fatty acids, LACS4 and LACS6 also catalyze the addition of CoA onto IBA, the first step in IBA metabolism and a necessary step in generating IBA-derived IAA.

An enhancer mutant revealed an acyl-CoA synthetase that catalyzes CoA addition to indole-3-butryic acid, required for the β-oxidation steps necessary to generate indole-3-butryic acid-derived IAA.     

Identification of a peroxisomal acyl-activating enzyme involved in the biosynthesis of jasmonic acid in Arabidopsis

Jasmonic acid (JA) is a lipid-derived signal that regulates a wide variety of developmental and defense-related processes in higher plants. JA is synthesized from linolenic acid via an enzymatic pathway that initiates in the plastid and terminates in peroxisomes. The C18 JA precursor 12-oxo-phytodienoic acid (OPDA) is converted in the peroxisome to 3-oxo-2-(2'-[Z]-pentenyl)cyclopentane-1-octanoic acid (OPC-8:0), which subsequently undergoes three rounds of beta-oxidation to yield JA. Although most JA biosynthetic enzymes have been identified, several key steps in the pathway remain to be elucidated. To address this knowledge gap, we employed co-expression analysis to identify genes that are coordinately regulated with known JA biosynthetic components in Arabidopsis. Among the candidate genes uncovered by this approach was a 4-coumarate-CoA ligase-like member of the acyl-activating enzyme (AAE) gene family, which we have named OPC-8:0 CoA Ligase1 (OPCL1). In response to wounding, opcl1 null mutants exhibited reduced levels of JA and hyperaccumulation of OPC-8:0. Recombinant OPCL1 was active against both OPDA and OPC-8:0, as well as medium-to-long straight-chain fatty acids. Subcellular localization studies with green fluorescent protein-tagged OPCL1 showed that the protein is targeted to peroxisomes. These findings establish a physiological role for OPCL1 in the activation of JA biosynthetic precursors in leaf peroxisomes, and further indicate that OPC-8:0 is a physiological substrate for the activation step. The results also demonstrate the utility of co-expression analysis for identification of factors that contribute to jasmonate homeostasis.     

Characterization of triacsin C inhibition of short-, medium-, and long-chain fatty acid: CoA ligases of human liver

Short-, medium-, and long-chain fatty acid:CoA ligases from human liver were tested for their sensitivity to inhibition by triacsin C. The short-chain fatty acid:CoA ligase was inhibited less than 10% by concentrations of triacsin C as high as 80 microM. The two mitochondrial xenobiotic/medium-chain fatty acid:CoA ligases (XM-ligases), HXM-A and HXM-B, were partially inhibited by triacsin C, and the inhibitions were characterized by low affinity for triacsin C (K(I) values > 100 microM). These inhibitions were found to be the result of triacsin C competing with medium-chain fatty acid for binding at the active site. The microsomal and mitochondrial forms of long-chain fatty acid:CoA ligase (also termed long-chain fatty acyl-CoA synthetase, or long-chain acyl-CoA synthetase LACS) were potently inhibited by triacsin C, and the inhibition had identical characteristics for both LACS forms. Dixon plots of this inhibition were biphasic. There is a high-affinity site with a K(I) of 0.1 microM that accounts for a maximum of 70% of the inhibition. There is also a low affinity site with a K(I) of 6 microM that accounts for a maximum of 30% inhibition. Kinetic analysis revealed that the high-affinity inhibition of the mitochondrial and microsomal LACS forms is the result of triacsin C binding at the palmitate substrate site.The high-affinity triacsin C inhibition of both the mitochondrial and microsomal LACS forms was found to require a high concentration of free Mg(2+), with the EC(50) for inhibition being 3 mM free Mg(2+). The low affinity triacsin C inhibition was also enhanced by Mg(2+). The data suggests that Mg(2+) promotes triacsin C inhibition of LACS by enhancing binding at the palmitate binding site. In contrast, the partial inhibition of the XM-ligases by triacsin C, which showed only a low-affinity component, did not require Mg(2+).     

Arabidopsis contains nine long-chain acyl-coenzyme a synthetase genes that participate in fatty acid and glycerolipid metabolism

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 beta-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.     

Two Activities of Long-Chain Acyl-Coenzyme A Synthetase Are Involved in Lipid Trafficking between the Endoplasmic Reticulum and the Plastid in Arabidopsis

In plants, fatty acids are synthesized within the plastid and need to be distributed to the different sites of lipid biosynthesis within the cell. Free fatty acids released from the plastid need to be converted to their corresponding coenzyme A thioesters to become metabolically available. This activation is mediated by long-chain acyl-coenzyme A synthetases (LACSs), which are encoded by a family of nine genes in Arabidopsis (Arabidopsis thaliana). So far, it has remained unclear which of the individual LACS activities are involved in making plastid-derived fatty acids available to cytoplasmic glycerolipid biosynthesis. Because of its unique localization at the outer envelope of plastids, LACS9 was regarded as a candidate for linking plastidial fatty export and cytoplasmic use. However, data presented in this study show that LACS9 is involved in fatty acid import into the plastid. The analyses of mutant lines revealed strongly overlapping functions of LACS4 and LACS9 in lipid trafficking from the endoplasmic reticulum to the plastid. In vivo labeling experiments with lacs4 lacs9 double mutants suggest strongly reduced synthesis of endoplasmic reticulum-derived lipid precursors, which are required for the biosynthesis of glycolipids in the plastids. In conjunction with this defect, double-mutant plants accumulate significant amounts of linoleic acid in leaf tissue.Two discrete but intimately connected pathways are involved in plant glycerolipid biosynthesis (Roughan et al., 1980). Both pathways follow exactly the same scheme of synthesis within the plastid and at the endoplasmic reticulum (ER) to assemble phosphatidic acid (PA) by two consecutive acylation reactions of glycerol-3-phosphate. Essential substrates for both pathways are fatty acids that are synthesized exclusively in plastids. De novo synthesized fatty acids can feed directly into the so-called prokaryotic lipid synthesis pathway localized within the plastid to produce phosphatidylglycerol (PG), the so-called C16:3 plants (e.g., Arabidopsis [Arabidopsis thaliana]), and also, other thylakoid lipids, like sulfoquinovosyldiacylglycerol, monogalactosyldiacylglycerol (MGDG), and digalactosyldiacylglycerol (DGDG; Heinz and Roughan, 1983). In addition, plastid-derived fatty acids are also substrates for eukaryotic lipid biosynthesis at the ER to produce important membrane lipid precursors, like PA and diacylglycerol (DAG). The main products of the lipid biosynthesis pathway in the ER are, however, phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylinositol. Recent studies revealed an additional mechanism to incorporate plastid-derived fatty acids at the ER by acyl editing of PC (Bates et al., 2007). In the proposed model (also designated as the Lands cycle; Lands, 1958), PC is continuously converted to lyso-PC, which becomes reacylated by newly exported fatty acids to generate PC again. However, irrespective of the route taken to attach the fatty acids to the glycerol backbone, the interconnection between plastidial and cytoplasmic lipid metabolism is, in most plant species, further complicated by the fact that the eukaryotic pathway is not only generating lipids for all extraplastidial compartments but also, synthesizing lipid precursors, which are delivered back to the plastid to become thylakoid lipids. Consequently, plastidial membrane lipids represent a mixture of molecules partially synthesized within the plastid and partially assembled at the ER. The contribution of ER and plastidial lipid synthesis to the overall mixture of thylakoid lipids differs strongly between different plant species, but in Arabidopsis, both sites of synthesis are responsible for approximately equal amounts of chloroplast lipids (Browse et al., 1986). Subtle biochemical differences reveal the site of synthesis of a specific lipid molecule. Because of different substrate specificities of the acyltransferases located at the ER and in the plastid, the resulting lipid molecules can be distinguished based on the fatty acids attached to the sn-2 position of the glycerol backbone. Whereas the lysophosphatidyl acyltransferase at the ER is highly specific for 18-carbon fatty acids, its plastidial homolog incorporates exclusively 16-carbon fatty acids into the sn-2 position.Another important difference between plastidial and cytoplasmic lipid metabolism is defined by the nature of the fatty acid substrate. In both cases, fatty acid thioesters are used; however, within the plastid, the fatty acids are provided as acyl-acyl carrier proteins (acyl-ACPs), whereas in the cytoplasm, acyl-CoAs are the established substrates. Acyl-ACP produced by plastidial fatty acid synthase can be used directly by enzymes of the plastidial lipid biosynthesis pathway, but fatty acids need to be exported and converted to acyl-CoA by long-chain acyl-CoA synthetases (LACS) to become substrate for the pathway operating at the ER. The precise mechanism of the fatty acid transport through the plastidial membrane is still unknown; however, the findings of acyl-ACP thioesterase activity in the stroma of plastids (Ohlrogge et al., 1978, 1979) and LACS activity at the outer envelope (Andrews and Keegstra, 1983; Block et al., 1983) suggested both enzymes to be involved in the export of fatty acids from plastids. This model was challenged by the identification of LACS9 as the major plastidial LACS isoform in Arabidopsis and the finding that its inactivation did not result in any substantial changes in lipid composition (Schnurr et al., 2002). Because LACS activity is encoded in Arabidopsis by a small gene family comprising nine genes (Shockey et al., 2002), there must be other LACS isoforms involved in providing acyl-CoA substrate to cytoplasmic lipid metabolism. Surprisingly, none of the lacs mutant lines analyzed so far, including single mutants of all members of the enzyme family, showed pronounced effects on glycerolipid metabolism. The data seem to suggest a network of overlapping LACS activities concealing the effects of individual members of the enzyme family. It may also indicate that mutual interactions between the different LACS enzymes are still poorly understood. To elucidate such interactions and identify those LACS activities contributing to glycerolipid metabolism, we established a comprehensive mutant collection comprising all possible double-mutant lines based on nine members of the LACS gene family. The individual mutants of this collection were screened for visual phenotypes potentially associated with modifications in lipid biosynthesis.Here, we show overlapping functions of LACS4 and LACS9 in Arabidopsis. The combined inactivation of both proteins results in severe morphological phenotypes of the adult plant that are tightly linked to changes in the fatty acid metabolism. The results suggest that both LACS activities are involved in fatty acid channeling and lipid processing. But instead of contributing to fatty acid export from the plastid, both proteins were found to be involved in the process of retrograde lipid flux from the ER to the plastid.     

The acyl-CoA synthetase encoded by LACS2 is essential for normal cuticle development in Arabidopsis

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 microg/dm(2) versus 76.4 microg/dm(2), 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 omega-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.     

Arabidopsis CER8 encodes LONG-CHAIN ACYL-COA SYNTHETASE 1 (LACS1) that has overlapping functions with LACS2 in plant wax and cutin synthesis

Plant cuticle is an extracellular lipid-based matrix of cutin and waxes, which covers aerial organs and protects them from many forms of environmental stress. We report here the characterization of CER8 / LACS1 , one of nine Arabidopsis long-chain acyl-CoA synthetases thought to activate acyl chains. Mutations in LACS1 reduced the amount of wax in all chemical classes on the stem and leaf, except in the very long-chain fatty acid (VLCFA) class wherein acids longer than 24 carbons (C₂₄) were elevated more than 155%. The C₁₆ cutin monomers on lacs1 were reduced by 37% and 22%, whereas the C₁₈ monomers were increased by 28% and 20% on stem and leaf, respectively. Amounts of wax and cutin on a lacs1-1 lacs2-3 double mutant were much lower than on either parent, and lacs1-1 lacs2-3 had much higher cuticular permeability than either parent. These additive effects indicate that LACS1 and LACS2 have overlapping functions in both wax and cutin synthesis. We demonstrated that LACS1 has synthetase activity for VLCFAs C₂₀–C₃₀, with highest activity for C₃₀ acids. LACS1 thus appears to function as a very long-chain acyl-CoA synthetase in wax metabolism. Since C₁₆ but not C₁₈ cutin monomers are reduced in lacs1 , and C₁₆ acids are the next most preferred acid (behind C₃₀) by LACS1 in our assays, LACS1 also appears to be important for the incorporation of C₁₆ monomers into cutin polyester. As such, LACS1 defines a functionally novel acyl-CoA synthetase that preferentially modifies both VLCFAs for wax synthesis and long-chain (C₁₆) fatty acids for cutin synthesis.     

Synthesis of Long-Chain Acyl-CoA in Chloroplast Envelope Membranes

The chloroplast envelope is the site of a very active long-chain acylcoenzyme A (CoA) synthetase. Furthermore, we have recently shown that an acyl CoA thioesterase is also associated with envelope membrane (Joyard J, PK Stumpf 1980 Plant Physiol 65: 1039-1043). To clarify the interacting roles of both the acyl-CoA thioesterase and the acyl-CoA synthetase, the formation of acyl-CoA in envelope membranes was examined with different techniques which permitted the measurement of the actual rates of acyl-CoA formation. Using [¹⁴C]ATP or [¹⁴C]oleic acid as labeled substrates, it can be shown that the envelope acyl-CoA synthetase required both Mg²⁺ and dithiothreitol. Triton X-100 slightly stimulated the activity. The specificity of the acyl-CoA synthetase was determined either with [¹⁴C]ATP or with [³H]CoA as substrates. The results obtained in both cases were similar, that is, as substrates, the unsaturated fatty acids were more effective than saturated fatty acids, the velocity of the reaction increased from lauric acid to palmitic acid, and the maximum velocity was obtained with unsaturated C₁₈ fatty acids.     

Fatty Acid Elongation Is Independent of Acyl-Coenzyme A Synthetase Activities in Leek and Brassica napus

In both animal and plant acyl elongation systems, it has been proposed that fatty acids are first activated to acyl-coenzyme A (CoA) before their elongation, and that the ATP dependence of fatty acid elongation is evidence of acyl-CoA synthetase involvement. However, because CoA is not supplied in standard fatty acid elongation assays, it is not clear if CoA-dependent acyl-CoA synthetase activity can provide levels of acyl-CoAs necessary to support typical rates of fatty acid elongation. Therefore, we examined the role of acyl-CoA synthetase in providing the primer for acyl elongation in leek (Allium porrum L.) epidermal microsomes and Brassica napus L. cv Reston oil bodies. As presented here, fatty acid elongation was independent of CoA and proceeded at maximum rates with CoA-free preparations of malonyl-CoA. We also showed that stearic acid ([1-¹⁴C]18:0)-CoA was synthesized from [1-¹⁴C]18:0 in the presence of CoA-free malonyl-CoA or acetyl-CoA, and that [1-¹⁴C]18:0-CoA synthesis under these conditions was ATP dependent. Furthermore, the appearance of [1-¹⁴C]18:0 in the acyl-CoA fraction was simultaneous with its appearance in phosphatidylcholine. These data, together with the s of a previous study (A. Hlousek-Radojcic, H. Imai, J.G. Jaworski [1995] Plant J 8: 803–809) showing that exogenous [¹⁴C]acyl-CoAs are diluted by a relatively large endogenous pool before they are elongated, strongly indicated that acyl-CoA synthetase did not play a direct role in fatty acid elongation, and that phosphatidylcholine or another glycerolipid was a more likely source of elongation primers than acyl-CoAs.     

Peroxisomal Acyl-CoA synthetase activity is essential for seedling development in Arabidopsis thaliana

In plants and other eukaryotes, long-chain acyl-CoAs are assumed to be imported into peroxisomes for beta-oxidation by an ATP binding cassette (ABC) transporter. However, two genes in Arabidopsis thaliana, LACS6 and LACS7, encode peroxisomal long-chain acyl-CoA synthetase (LACS) isozymes. To investigate the biochemical and biological roles of peroxisomal LACS, we identified T-DNA knockout mutants for both genes. The single-mutant lines, lacs6-1 and lacs7-1, were indistinguishable from the wild type in germination, growth, and reproductive development. By contrast, the lacs6-1 lacs7-1 double mutant was specifically defective in seed lipid mobilization and required exogenous sucrose for seedling establishment. This phenotype is similar to the A. thaliana pxa1 mutants deficient in the peroxisomal ABC transporter and other mutants deficient in beta-oxidation. Our results demonstrate that peroxisomal LACS activity and the PXA1 transporter are essential for early seedling growth. The peroxisomal LACS activity would be necessary if the PXA1 transporter delivered unesterified fatty acids into the peroxisomal matrix. Alternatively, PXA1 and LACS6/LACS7 may act in parallel pathways that are both required to ensure adequate delivery of acyl-CoA substrates for beta-oxidation and successful seedling establishment.     

Enzymatic and genetic characterization of firefly luciferase and Drosophila CG6178 as a fatty acyl-CoA synthetase

Recently we found that firefly luciferase is a bifunctional enzyme, catalyzing not only the luminescence reaction but also long-chain fatty acyl-CoA synthesis. Further, the gene product of CG6178 (CG6178), an ortholog of firefly luciferase in Drosophila melanogaster, was found to be a long-chain fatty acyl-CoA synthetase and dose not function as a luciferase. We investigated the substrate specificities of firefly luciferase and CG6178 as an acyl-CoA synthetase utilizing a series of carboxylic acids. The results indicate that these enzymes synthesize acyl-CoA efficiently from various saturated medium-chain fatty acids. Lauric acid is the most suitable substrate for these enzymes, and the product of lauroyl CoA was identified with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Phylogenetic analysis indicated that firefly luciferase and CG6178 genes belong to the group of plant 4-coumarate:CoA ligases, and not to the group of medium- and long-chain fatty acyl-CoA synthetases in mammals. These results suggest that insects have a novel type of fatty acyl-CoA synthetase.     

Insertional mutant analysis reveals that long-chain acyl-CoA synthetase 1 (LACS1), but not LACS8, functionally overlaps with LACS9 in Arabidopsis seed oil biosynthesis

Triacylglycerols (TAGs) are major storage materials that accumulate in developing seeds and serve as carbon and energy reserves for germination and growth of the seedling. One of the critical reactions in TAG biosynthesis is activation of fatty acyl chains to fatty acyl CoAs, catalyzed by long-chain acyl CoA synthetases (LACSs). Of the nine LACSs identified in Arabidopsis, only LACS9 is known to reside in the plastid, the site of de novo fatty acid synthesis, and is considered the major LACS isoform involved in plastidial fatty acid export for TAG formation. Because the lacs9 null mutant did not show any detectable phenotype, it was hypothesized that at least one additional LACS enzyme must be active in the plastid. Expression analyses to identify potential plastid-localized LACSs involved in TAG biosynthesis revealed that, in addition to LACS9, isoforms LACS1, LACS2, LACS4 and LACS8 are transcribed in the seed. LACS8 showed the highest expression level in the embryo and a high sequence similarity with LACS9, and was therefore characterized further and shown to be associated with the ER, not the plastid. Furthermore, disruption of LACS8 in the lacs8 mutant and lacs8 lacs9 double mutant, and over-expression of LACS8, did not affect the seed fatty acid content. In contrast, 11 and 12% decreases in fatty acid content were detected in lacs1 lacs9 and lacs1 lacs8 lacs9 seeds, respectively, indicating that LACS1 and LACS9 have overlapping functions in TAG biosynthesis. This result is surprising because, unlike LACS9, LACS1 is localized in the ER and has been shown to be involved in cuticular lipid synthesis.     

Two long-chain acyl-CoA synthetases from Arabidopsis thaliana involved in peroxisomal fatty acid beta-oxidation

Post-germinative growth of oilseeds is dependent on the breakdown of the stored lipid reserves. Long-chain acyl-CoA synthetase activities (LACS) are critically involved in this process by activating the released free fatty acids and thus feeding the beta-oxidation cycle in glyoxysomes. Here we report on the identification of two LACS genes, AtLACS6 and AtLACS7 from Arabidopsis thaliana coding for peroxisomal LACS proteins. The subcellular localization was verified by co-expression studies of spectral variants of the green fluorescent protein (GFP). While AtLACS6 is targeted by a type 2 (PTS2) peroxisomal targeting sequence, for AtLACS7 a functional PTS1 as well as a PTS2 could be demonstrated. Possible explanations for this potentially redundant targeting information will be discussed. Expression studies of both genes revealed a strong induction 1 day after germination resembling the expression pattern of other genes involved in beta-oxidation. Analysis of the substrate specificities of the two LACS proteins demonstrated enzymatic activity for both enzymes with the whole spectrum of fatty acids found in stored lipid reserves. These results suggest that both LACS proteins might have overlapping functions and are able to initiate beta-oxidation in plant peroxisomes.     

Sunflower (Helianthus annuus) long‐chain acyl‐coenzyme A synthetases expressed at high levels in developing seeds

Long chain fatty acid synthetases (LACSs) activate the fatty acid chains produced by plastidial de novo biosynthesis to generate acyl‐CoA derivatives, important intermediates in lipid metabolism. Oilseeds, like sunflower, accumulate high levels of triacylglycerols (TAGs) in their seeds to nourish the embryo during germination. This requires that sunflower seed endosperm supports very active glycerolipid synthesis during development. Sunflower seed plastids produce large amounts of fatty acids, which must be activated through the action of LACSs, in order to be incorporated into TAGs. We cloned two different LACS genes from developing sunflower endosperm, HaLACS1 and HaLACS2, which displayed sequence homology with Arabidopsis LACS9 and LACS8 genes, respectively. These genes were expressed at high levels in developing seeds and exhibited distinct subcellular distributions. We generated constructs in which these proteins were fused to green fluorescent protein and performed transient expression experiments in tobacco cells. The HaLACS1 protein associated with the external envelope of tobacco chloroplasts, whereas HaLACS2 was strongly bound to the endoplasmic reticulum. Finally, both proteins were overexpressed in Escherichia coli and recovered as active enzymes in the bacterial membranes. Both enzymes displayed similar substrate specificities, with a very high preference for oleic acid and weaker activity toward stearic acid. On the basis of our findings, we discuss the role of these enzymes in sunflower oil synthesis.     

Combined activity of LACS1 and LACS4 is required for proper pollen coat formation in Arabidopsis

Very long chain lipids are important components of the plant cuticle that establishes the boundary surface of aerial organs. In addition, these lipids were detected in the extracellular pollen coat (tryphine), where they play a crucial role in appropriate pollen‐stigma communication. As such they are involved in the early interaction of pollen with the stigma. A substantial reduction in tryphine lipids was shown to compromise pollen germination and, consequently, resulted in male sterility. We investigated the role of two long‐chain acyl‐CoA synthetases (LACSs) in Arabidopsis with respect to their contribution to the production of tryphine lipids. LACS was shown to provide CoA‐activated very long chain fatty acids (VLCFA‐CoAs) to the pathways of wax biosynthesis. The allocation of sufficient quantities of VLCFA‐CoA precursors should therefore be relevant to the generation of tryphine lipids. Here, we report on the identification of lacs1 lacs4 double knock‐out mutant lines that were conditionally sterile and showed significant reductions in pollen coat lipids. Whereas the contributions of both LACS proteins to surface wax levels were roughly additive, their co‐operation in tryphine lipid biosynthesis was clearly more complex. The inactivation of LACS4 resulted in increased levels of tryphine lipids accompanied by morphological anomalies of the pollen grains. The additional inactivation of LACS1 neutralized the morphological defects, decreased the tryphine lipids far below wild‐type levels and resulted in conditionally sterile pollen.     

A defect in glyoxysomal fatty acid beta-oxidation reduces jasmonic acid accumulation in Arabidopsis.

The final steps of jasmonic acid (JA) biosynthesis are thought to involve peroxisomal beta-oxidation, but this has not been directly demonstrated. The last and key step in fatty acid beta-oxidation is catalyzed by 3-ketoacyl-CoA thiolase (KAT) (EC 2.3.1.16). A mutant of Arabidopsis thaliana ecotype Landsberg erecta, which lacks a functional KAT protein and is defective in glyoxysomal fatty acid beta-oxidation has been reported. In this study, the mutant was found to accumulate reduced level of JA in both its wounded cotyledons and leaves, while only the cotyledons accumulate 3-oxo-2-(pent-2'-enyl)-cyclopentane-1-octanoic acid (OPC-8:0). This indicates that a defect in one of the thiolase isoenzymes impairs beta-oxidation of OPC-8:0 to JA. The mutant had sufficient thiolase activity for the synthesis of JA in the unwounded but not in the wounded tissues. Activities of the enzymes in the JA pathway that catalyze the steps, which precede beta-oxidation were not altered by the mutation in a thiolase protein. Thus, reduced levels of JA in the wounded tissues of the mutant were attributed to the defect in a thiolase protein.     

Cold responses of Arabidopsis mutants impaired in freezing tolerance

Mutants of Arabidopsis thaliana L. (Heynh), characterized asdeficient in their freezing tolerance after cold acclimation,were surveyed for some of the normal responses to cold exposure.In foliar tissue, the coldinducibility of three proteins, thelevels of sucrose and glucose, the fatty acyl composition oflipids, and the accumulation of anthocyanin was examined. Fourmutations (sfr3, sfr4, sfr6, and sfr7) reduced or eliminatedthe accumulation of anthocyanin during cold acclimation. Onemutation (sfr4) prevented the normally cold-induced elevationof sucrose and glucose levels; both sfr4 and another mutation(sfr7) affected fatty acid composition after (and only after)cold acclimation. On the other hand mutations sfr1, sfr2 andsfr5 did not differ significantly from the wild type in anyof the parameters tested, suggesting that they have other, perhapshighly specific, effects on lowtemperature responses. Key words: Arabidopsis thaliana, cold acclimation, freezing tolerance, mutation     

Fatty acid export from the chloroplast. Molecular characterization of a major plastidial acyl-coenzyme A synthetase from Arabidopsis

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.     

Effect of sweet cherry genes PaLACS2 and PaATT1 on cuticle deposition,composition and permeability in Arabidopsis

The cuticular membrane (CM) of sweet cherry (Prunus avium L.) fruit is severely strained during development. Strain results from a cessation of CM deposition during early development and is possibly caused by a downregulation of genes involved in CM synthesis. The objectives of our study were to investigate the effects of ectopic expression of two sweet cherry genes, PaLACS2 (a putative long-chain acyl-CoA synthetase) and PaATT1 (a putative cytochrome P450 monooxygenase), in Arabidopsis thaliana (L.). Effects on the expression of endogenous LACS2, ATT1 and LACS1 genes, wax and cutin composition, and cuticle permeability were investigated in 13 transgenic lines. Of these, six lines are selected for presentation based on the magnitude of the response. The amount of cutin increased in the PaLACS2 overexpression line C-L-29 and in the complemented lacs2-1 knockout mutant line l-L-14, but overexpression had no effect on cutin composition or wax. Wax deposition decreased in the complemented knockout lines l-L-14 and l-L-21. Overexpressing PaATT1 in A. thaliana line C-A-6 had no significant effect on cutin and wax deposition. In the complemented knockout lines a-A-7 and a-A-12, cutin deposition increased, whereas wax deposition was unaffected. The permeability of the cuticle for water and toluidine blue decreased in the PaLACS2 and PaATT1 complemented knockout lines. The results suggest that (1) PaLACS2 and PaATT1 expressed in A. thaliana are involved in cutin biosynthesis, and (2) their functions are consistent with those of a typical long-chain acyl-CoA synthetase (PaLACS2) and of a cytochrome P450 monooxygenase (PaATT1).