Biosynthesis and secretion of plant cuticular wax

The cuticle covers the aerial portions of land plants. It consists of amorphous intracuticular wax embedded in cutin polymer, and epicuticular wax crystalloids that coat the outer plant surface and impart a whitish appearance. Cuticular wax is mainly composed of long-chain aliphatic compounds derived from very long chain fatty acids. Wax biosynthesis begins with fatty acid synthesis in the plastid. Here we focus on fatty acid elongation (FAE) to very long chains (C24-C34), and the subsequent processing of these elongated products into alkanes, secondary alcohols, ketones, primary alcohols and wax esters. The identity of the gene products involved in these processes is starting to emerge. Other areas of this field remain enigmatic. For example, it is not known how the hydrophobic wax components are moved intracellularly, how they are exported out of the cell, or translocated through the hydrophilic cell wall. Two hypotheses are presented for intracellular wax transport: direct transfer of lipids from the endoplasmic reticulum to the plasma membrane, and Golgi mediated exocytosis. The potential roles of ABC transporters and non-specific lipid transfer proteins in wax export are also discussed. Biochemical-genetic and genomic approaches in Arabidopsis thaliana promise to be particularly useful in identifying and characterizing gene products involved in wax biosynthesis, secretion and function. The current review will, therefore, focus on Arabidopsis as a model for studying these processes.     

The plastid outer membrane localized LPTD1 is important for glycerolipid remodelling under phosphate starvation

Glycerolipid synthesis in plants is coordinated between plastids and the endoplasmic reticulum (ER). A central step within the glycerolipid synthesis is the transport of phosphatidic acid from ER to chloroplasts. The chloroplast outer envelope protein TGD4 belongs to the LptD family conserved in bacteria and plants and selectively binds and may transport phosphatidic acid. We describe a second LptD‐family protein in A. thaliana (atLPTD1; At2g44640) characterized by a barrel domain with an amino‐acid signature typical for cyanobacterial LptDs. It forms a cation selective channel in vitro with a diameter of about 9 Å. atLPTD1 levels are induced under phosphate starvation. Plants expressing an RNAi construct against atLPTD1 show a growth phenotype under normal conditions. Expressing the RNAi against atLPTD1 in the tgd4–1 background renders the plants more sensitive to light stress or phosphate limitation than the individual mutants. Moreover, lipid analysis revealed that digalactosyldiacylglycerol and sulfoquinovosyldiacylglycerol levels remain constant in the RNAi mutants under phosphate starvation, while these two lipids are enhanced in wild‐type. Based on our results, we propose a function of atLPTD1 in the transport of lipids from ER to chloroplast under phosphate starvation, which is combinatory with the function of TGD4.     

Lipid-dependent surface transport of the proton pumping ATPase: a model to study plasma membrane biogenesis in yeast

The proton pumping H+-ATPase, Pma1, is one of the most abundant integral membrane proteins of the yeast plasma membrane. Pma1 activity controls the intracellular pH and maintains the electrochemical gradient across the plasma membrane, two essential cellular functions. The maintenance of the proton gradient, on the other hand, also requires a specialized lipid composition of this membrane. The plasma membrane of eukaryotic cells is typically rich in sphingolipids and sterols. These two lipids condense to form less fluid membrane microdomains or lipid rafts. The yeast sphingolipid is peculiar in that it invariably contains a saturated very long-chain fatty acid with 26 carbon atoms. During cell growth and plasma membrane expansion, both C26-containing sphingolipids and Pma1 are first synthesized in the endoplasmatic reticulum from where they are transported by the secretory pathway to the cell surface. Remarkably, shortening the C26 fatty acid to a C22 fatty acid by mutations in the fatty acid elongation complex impairs raft association of newly synthesized Pma1 and induces rapid degradation of the ATPase by rerouting the enzyme from the plasma membrane to the vacuole, the fungal equivalent of the lysosome. Here, we review the role of lipids in mediating raft association and stable surface transport of the newly synthesized ATPase, and discuss a model, in which the newly synthesized ATPase assembles into a membrane environment that is enriched in C26-containing lipids already in the endoplasmatic reticulum. The resulting protein-lipid complex is then transported and sorted as an entity to the plasma membrane. Failure to successfully assemble this lipid-protein complex results in mistargeting of the protein to the vacuole.     

Arabidopsis LTPG Is a Glycosylphosphatidylinositol-Anchored Lipid Transfer Protein Required for Export of Lipids to the Plant Surface

Plant epidermal cells dedicate more than half of their lipid metabolism to the synthesis of cuticular lipids, which seal and protect the plant shoot. The cuticle is made up of a cutin polymer and waxes, diverse hydrophobic compounds including very-long-chain fatty acids and their derivatives. How such hydrophobic compounds are exported to the cuticle, especially through the hydrophilic plant cell wall, is not known. By performing a reverse genetic screen, we have identified LTPG, a glycosylphosphatidylinositol-anchored lipid transfer protein that is highly expressed in the epidermis during cuticle biosynthesis in Arabidopsis thaliana inflorescence stems. Mutant plant lines with decreased LTPG expression had reduced wax load on the stem surface, showing that LTPG is involved either directly or indirectly in cuticular lipid deposition. In vitro 2-p-toluidinonaphthalene-6-sulfonate assays showed that recombinant LTPG has the capacity to bind to this lipid probe. LTPG was primarily localized to the plasma membrane on all faces of stem epidermal cells in the growing regions of inflorescence stems where wax is actively secreted. These data suggest that LTPG may function as a component of the cuticular lipid export machinery.     

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.     

Membrane composition and physiological activity of plastids from an oenothera plastome mutator-induced chloroplast mutant

Plastids were isolated from a plastome mutator-induced mutant (pm7) of Oenothera hookeri and were analyzed for various physiological and biochemical attributes. No photosynthetic electron transport activity was detected in the mutant plastids. This is consistent with previous ultrastructural analysis showing the absence of thylakoid membranes in the pm7 plastids and with the observation of aberrant processing and accumulation of chloroplast proteins in the mutant. In comparison to wild type, the mutant tissue lacks chlorophyll, and has significant differences in levels of four fatty acids. The analyses did not reveal any differences in carotenoid levels nor in the synthesis of several chloroplast lipids. The consequences of the altered composition of the chloroplast membrane are discussed in terms of their relation to the aberrant protein processing of the pm7 plastids. The pigment, fatty acid, and lipid measurements were also performed on two distinct nuclear genotypes (A/A and A/C) which differ in their compatibility with the plastid genome (type I) contained in these lines. In these cases, only chlorophyll concentrations differed significantly.     

Effects of Iron Deficiency on Chloroplast Lipids

The lipids of plastids from three different nodes of Hubbardsquash plants were investigated. A comparison was made of theplastid lipids of plants grown on a complete nutrien with theplastid lipids of plants grown on an iron-deficient nutrientsolution. The iron-deficient leaves were not chlorotic at the time ofharvest. The separated lipids were quantitated by determiningphosphorus, sugar, and fatty acid content. Plastids from olderleaves contained more lipid phosphorus per lipid sugar thanplastids from younger leaves. The plastids treated with an iron-deficientnutrient seemed to have relatively less glycerylphosphoryl glycerol-lipidsthan did the complete plastids. However, few major differencesin the fatty acid composition existed between the iron-deficientand complete grown plastids. It was concluded that the rateof turnover of the plastid lipids may depend upon the individualspecies and the environment to which the plants are subjected.Further changes in the plastid lipids of the iron-dificientplants, as opposed to complete nutrient treated, occurred priorto the appearance of marked visible symptoms of chlorosis.     

Dictyostelium discoideum Dgat2 Can Substitute for the Essential Function of Dgat1 in Triglyceride Production but Not in Ether Lipid Synthesis

Triacylglycerol (TAG), the common energy storage molecule, is formed from diacylglycerol and a coenzyme A-activated fatty acid by the action of an acyl coenzyme A:diacylglycerol acyltransferase (DGAT). In order to conduct this step, most organisms rely on more than one enzyme. The two main candidates in Dictyostelium discoideum are Dgat1 and Dgat2. We show, by creating single and double knockout mutants, that the endoplasmic reticulum (ER)-localized Dgat1 enzyme provides the predominant activity, whereas the lipid droplet constituent Dgat2 contributes less activity. This situation may be opposite from what is seen in mammalian cells. Dictyostelium Dgat2 is specialized for the synthesis of TAG, as is the mammalian enzyme. In contrast, mammalian DGAT1 is more promiscuous regarding its substrates, producing diacylglycerol, retinyl esters, and waxes in addition to TAG. The Dictyostelium Dgat1, however, produces TAG, wax esters, and, most interestingly, also neutral ether lipids, which represent a significant constituent of lipid droplets. Ether lipids had also been found in mammalian lipid droplets, but the role of DGAT1 in their synthesis was unknown. The ability to form TAG through either Dgat1 or Dgat2 activity is essential for Dictyostelium to grow on bacteria, its natural food substrate.     

LEAFY COTYLEDON1 is a key regulator of fatty acid biosynthesis in Arabidopsis

In plants, fatty acids are de novo synthesized predominantly in plastids from acetyl-coenzyme A. Although fatty acid biosynthesis has been biochemically well studied, little is known about the regulatory mechanisms of the pathway. Here, we show that overexpression of the Arabidopsis (Arabidopsis thaliana) LEAFY COTYLEDON1 (LEC1) gene causes globally increased expression of fatty acid biosynthetic genes, which are involved in key reactions of condensation, chain elongation, and desaturation of fatty acid biosynthesis. In the plastidial fatty acid synthetic pathway, over 58% of known enzyme-coding genes are up-regulated in LEC1-overexpressing transgenic plants, including those encoding three subunits of acetyl-coenzyme A carboxylase, a key enzyme controlling the fatty acid biosynthesis flux. Moreover, genes involved in glycolysis and lipid accumulation are also up-regulated. Consistent with these results, levels of major fatty acid species and lipids were substantially increased in the transgenic plants. Genetic analysis indicates that the LEC1 function is partially dependent on ABSCISIC ACID INSENSITIVE3, FUSCA3, and WRINKLED1 in the regulation of fatty acid biosynthesis. Moreover, a similar phenotype was observed in transgenic Arabidopsis plants overexpressing two LEC1-like genes of Brassica napus. These results suggest that LEC1 and LEC1-like genes act as key regulators to coordinate the expression of fatty acid biosynthetic genes, thereby representing promising targets for genetic improvement of oil production plants.     

Isolation and characterization of eceriferum (cer) mutants induced by T-DNA insertions in Arabidopsis thaliana

Thirteen Arabidopsis thaliana mutants with deviating epicuticular wax layers (i.e., cer mutants) were isolated by screening 13 000 transformed lines produced by the seed transformation method. After crossing the 13 mutants to some of the previously known cer mutant lines, 12 of our mutants mapped to 6 of the 21 known complementation groups (cer1 through cer4 as well as cer6 and cer10), while the other mutant corresponded to a previously unknown locus, cer21. Mutant phenotypes of 6 of the 13 mutant lines were caused by T-DNA insertions within cer genes. We also analyzed the chemical composition of the epicuticular wax layers of the cer mutants isolated in this study relative to that of Arabidopsis wild-type plants. Our results suggest that the five genes we tagged regulate different steps in wax biosynthesis, i.e., the decarbonylation of fatty aldehydes to alkanes, the elongation of hexacosanoic acid to octacosanoic acid, the reduction of fatty aldehydes to primary alcohols and the production of free aldehydes, while an insertion in the fifth gene causes an alteration in the chain length distribution of the different classes of wax compounds.     

Genomic and biochemical analysis of lipid biosynthesis in the unicellular rhodophyte Cyanidioschyzon merolae: lack of a plastidic desaturation pathway results in the coupled pathway of galactolipid synthesis

The acyl lipids making up the plastid membranes in plants and algae are highly enriched in polyunsaturated fatty acids and are synthesized by two distinct pathways, known as the prokaryotic and eukaryotic pathways, which are located within the plastids and the endoplasmic reticulum, respectively. Here we report the results of biochemical as well as genomic analyses of lipids and fatty acids in the unicellular rhodophyte Cyanidioschyzon merolae. All of the glycerolipids usually found in photosynthetic algae were found, such as mono- and digalactosyl diacylglycerol, sulfolipid, phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol. However, the fatty acid composition was extremely simple. Only palmitic, stearic, oleic, and linoleic acids were found as major acids. In addition, 3-trans-hexadecanoic acid was found as a very minor component in phosphatidylglycerol. Unlike the case for most other photosynthetic eukaryotes, polyenoic fatty acids having three or more double bonds were not detected. These results suggest that polyunsaturated fatty acids are not necessary for photosynthesis in eukaryotes. Genomic analysis suggested that C. merolae lacks acyl lipid desaturases of cyanobacterial origin as well as stearoyl acyl carrier protein desaturase, both of which are major desaturases in plants and green algae. The results of labeling experiments with radioactive acetate showed that the desaturation leading to linoleic acid synthesis occurs on phosphatidylcholine located outside the plastids. Monogalactosyl diacylglycerol is therefore synthesized by the coupled pathway, using plastid-derived palmitic acid and endoplasmic reticulum-derived linoleic acid. These results highlight essential differences in lipid biosynthetic pathways between the red algae and the green lineage, which includes plants and green algae.     

Golgi- and Trans-Golgi Network-Mediated Vesicle Trafficking Is Required for Wax Secretion from Epidermal Cells

Lipid secretion from epidermal cells to the plant surface is essential to create the protective plant cuticle. Cuticular waxes are unusual secretory products, consisting of a variety of highly hydrophobic compounds including saturated very-long-chain alkanes, ketones, and alcohols. These compounds are synthesized in the endoplasmic reticulum (ER) but must be trafficked to the plasma membrane for export by ATP-binding cassette transporters. To test the hypothesis that wax components are trafficked via the endomembrane system and packaged in Golgi-derived secretory vesicles, Arabidopsis (Arabidopsis thaliana) stem wax secretion was assayed in a series of vesicle-trafficking mutants, including gnom like1-1 (gnl1-1), transport particle protein subunit120-4, and echidna (ech). Wax secretion was dependent upon GNL1 and ECH. Independent of secretion phenotypes, mutants with altered ER morphology also had decreased wax biosynthesis phenotypes, implying that the biosynthetic capacity of the ER is closely related to its structure. These results provide genetic evidence that wax export requires GNL1- and ECH-dependent endomembrane vesicle trafficking to deliver cargo to plasma membrane-localized ATP-binding cassette transporters.The aerial, nonwoody tissues of all land plants are covered by a waxy cuticle that protects the plant against nonstomatal water loss. The cuticle also provides the first barrier between the plant and its environment and mediates important biotic and abiotic interactions. The cuticle has two main components: cutin and waxes. Cutin is a tough, cross-linked polyester matrix primarily composed of C16 and C18 oxygenated fatty acids and glycerol (Pollard et al., 2008). Wax is a heterogenous mixture, primarily composed of very-long-chain (VLC) fatty acid derivatives (predominantly 29-carbon alkane in Arabidopsis [Arabidopsis thaliana] stems).As a result of biochemical approaches, forward genetic screens yielding the eceriferum (cer) mutants (Koornneef et al., 1989), and reverse genetics approaches (Greer et al., 2007), almost all of the enzymes in the wax biosynthesis pathway have been identified. The enzymes that elongate C16 or C18 fatty acids to VLC (greater than 20C) fatty acids are localized in the endoplasmic reticulum (ER; for review, see Haslam and Kunst, 2013). Primary alcohols are synthesized by the fatty acyl reductases (Rowland et al., 2006), while alkanes are generated via an undefined mechanism involving CER1, CER3, and an unidentified cytochrome b₅ (Bernard et al., 2012). These alkanes may be modified by the midchain alkane hydroxylase cytochrome P450 (MAH1) to generate secondary alcohols and ketones (Greer et al., 2007). All of these wax synthesis enzymes have also been localized to the ER (Greer et al., 2007; Bernard et al., 2012).In contrast to wax synthesis, comparatively little is known about how waxes are trafficked within the cell from their site of synthesis at the ER to the plasma membrane. ATP-binding cassette (ABC) transporters of the G subfamily are required for wax export, and when either half-transporter is disrupted, waxes accumulate in the ER (McFarlane et al., 2010). Two extracellular glycosylphosphatidylinositol-anchored lipid transfer proteins (LTPs) are further required for wax accumulation on the plant surface (DeBono et al., 2009; Kim et al., 2012). Although these components of the molecular machinery of wax transport at the plasma membrane have been identified, the intracellular mechanisms by which waxes are transported to the plasma membrane remain undefined.Several mechanisms have been hypothesized for the transport of waxes from the ER to the plasma membrane (for review, see Samuels et al., 2008). Waxes could be incorporated into vesicles at the ER, travel to and through the Golgi apparatus and the trans-Golgi network (TGN), and then move to the plasma membrane via vesicle secretion. These vesicles could carry wax components within their membranes, as computational modeling of wax components in lipid bilayers indicates that VLC alkanes partition entirely into the hydrophobic phase of the bilayer (Coll et al., 2007). Alternatively, lipoproteins may bind to lipid molecules in order to solubilize them so that they can be transported as cargo in the vesicle lumen, by analogy to mammalian systems where lipoproteins are secreted from hepatocytes into the circulatory system by exocytosis via post-Golgi vesicles (for review, see Mansbach and Siddiqi, 2010). However, no analogous lipid-binding apoproteins or transport vesicles have been found in plants. It is also possible that LTPs in membrane contact sites between the ER and the plasma membrane could transfer cuticular lipids directly from the ER to the plasma membrane. However, although these membrane contact sites have been observed in plant cells (Samuels and McFarlane, 2012), no structural or functional components of membrane contact sites are known.Early studies of VLC fatty acid trafficking used pulse-chase labeling to show that treatment with monensin, a post-Golgi trafficking inhibitor, results in decreased VLC fatty acid trafficking to the plasma membrane and a corresponding increase in these lipids in the Golgi apparatus (Bertho et al., 1991), suggesting a Golgi-dependent mechanism of VLC lipid trafficking to the plasma membrane. However, the “Golgi” fraction in this study contained significant elongase activity, which has subsequently been localized to the ER, making interpretation of these data difficult. While a variety of inhibitors are available that disrupt different stages in the secretory pathway (Zhang et al., 1993; Robinson et al., 2008), inhibitor studies of wax trafficking have proven ineffective, since the wax-producing epidermal cells do not effectively take up solutions carrying these inhibitors. This illustrates the difficulties of studying the transport of highly hydrophobic cargo, such as wax, within the single cell layer of epidermis.The objective of this study was to determine the intracellular trafficking mechanisms underlying cuticular wax transport from the ER to the plasma membrane. Arabidopsis mutants, which have been successfully applied in wax biosynthesis studies, were used to investigate wax secretion. Well-characterized mutants with defects in vesicle traffic and protein secretion were chosen to test the hypothesis that wax components are trafficked via endomembrane vesicles. These mutant analyses indicate that wax movement from the ER to the plasma membrane requires vesicle traffic at both the ER-Golgi interface and the TGN. Independent of secretion phenotypes, strong decreases in wax synthesis were observed in mutants with altered ER morphology, which implies that ER structure influences its biosynthetic capacity for wax production.     

Etioplast Development in Dark-grown Leaves of Zea mays L

The ultrastructure of etioplasts and the acyl lipid and the fatty acid composition of sequential 2-centimeter sections cut from the base (youngest) to the top (oldest) of nonilluminated 5-day-old etiolated leaves of Zea mays L., and the acyl lipid and fatty acid composition of the etioplasts isolated from them have been investigated. There is a 2.5-fold increase in the size of the plastids from the base to the tip of the leaf, and an increase both in the size of the prolamellar body and in the length of lamellae attached to it. The etioplasts in the bundle sheath and mesophyll cells of the older, but not the younger leaf tissue, are morphologically distinct. The monogalactosyl and digalactosyldiglycerides, phosphatidylcholine, phosphatidylglycerol, and phosphatidylinositol were the only detectable acyl lipids in the isolated etioplast fractions. Together with phosphatidylethanolamine these were also the major acyl lipids in the whole leaf sections. With increasing age of the leaf tissue, increases occurred in two of the major plastid lipids, monogalactosyldiglyceride and phosphatidylglycerol, while the levels of essentially nonplastid lipids remained constant or declined slightly. The monogalactosyldiglyceride to digalactosyldiglyceride ratio increased from 0.4 to 1.1 in the tissue sections of increasing age and from 0.7 to 1.2 in the etioplasts isolated from them. Similarly, the galactolipid to phospholipid ratio increased from 0.8 to 1.4 in the tissue and from 0.5 to 4.5 in the isolated plastids. In the latter, the proportions of phosphatidylglycerol (as a per cent of total phospholipid) increased from 20 to 41% with increasing age of plastids.

Linolenic acid was the major fatty acid in the total lipid of each of the etioplast fractions, but it was only the major fatty acid in the total lipid of the oldest leaf tissue. Its proportion in both total lipid extracts and individual lipids increased with age. The trans Δ³ hexadecenoic acid was absent from all lipids. The protochlorophyllide content of the tissue increased with age. The results are discussed in relation to the use of illuminated etiolated leaves for studying chloroplast development.

     

Analysis of Lipid Export in Hydrocarbonoclastic Bacteria of the Genus Alcanivorax: Identification of Lipid Export-Negative Mutants of Alcanivorax borkumensis SK2 and Alcanivorax jadensis T9

Triacylglycerols (TAGs), wax esters (WEs), and polyhydroxyalkanoates (PHAs) are the major hydrophobic compounds synthesized in bacteria and deposited as cytoplasmic inclusion bodies when cells are cultivated under imbalanced growth conditions. The intracellular occurrence of these compounds causes high costs for downstream processing. Alcanivorax species are able to produce extracellular lipids when the cells are cultivated on hexadecane or pyruvate as the sole carbon source. In this study, we developed a screening procedure to isolate lipid export-negative transposon-induced mutants of bacteria of the genus Alcanivorax for identification of genes required for lipid export by employing the dyes Nile red and Solvent Blue 38. Three transposon-induced mutants of A. jadensis and seven of A. borkumensis impaired in lipid secretion were isolated. All isolated mutants were still capable of synthesizing and accumulating these lipids intracellularly and exhibited no growth defect. In the A. jadensis mutants, the transposon insertions were mapped in genes annotated as encoding a putative DNA repair system specific for alkylated DNA (Aj17), a magnesium transporter (Aj7), and a transposase (Aj5). In the A. borkumensis mutants, the insertions were mapped in genes encoding different proteins involved in various transport processes, like genes encoding (i) a heavy metal resistance (CZCA2) in mutant ABO_6/39, (ii) a multidrug efflux (MATE efflux) protein in mutant ABO_25/21, (iii) an alginate lyase (AlgL) in mutants ABO_10/30 and ABO_19/48, (iv) a sodium-dicarboxylate symporter family protein (GltP) in mutant ABO_27/29, (v) an alginate transporter (AlgE) in mutant ABO_26/1, or (vi) a two-component system protein in mutant ABO_27/56. Site-directed MATE, algE, and algL gene disruption mutants, which were constructed in addition, were also unable to export neutral lipids and confirmed the phenotype of the transposon-induced mutants. The putative localization of the different gene products and their possible roles in lipid excretion are discussed. Beside this, the composition of the intra- and extracellular lipids in the wild types and mutants were analyzed in detail.Almost all prokaryotes synthesize lipophilic storage substances as an integral part of their metabolism under limited nitrogen or phosphorus conditions if there is an excess of a suitable carbon source at the same time. The accumulated storage lipids serve as energy and carbon sources during starvation periods, and they are mobilized again under conditions of carbon and energy deficiency. The majority of the members of many genera synthesize hydrophobic polymers, such as poly(3-hydroxybutyrate) (PHB) or other types of polyhydroxyalkanoates (PHAs), whereas the accumulation of triacylglycerols (TAGs; trioxoesters of glycerol and long-chain fatty acids [FAs]) or wax esters (WEs; oxoesters of primary long-chain fatty acids and primary long-chain fatty alcohols) occurs in fewer prokaryotes (66). TAG accumulation has been reported for species of the genera Streptomyces, Mycobacterium, Nocardia, Rhodococcus (4, 6, 65), and recently also Alcanivorax and other hydrocarbonoclastic marine bacteria (32). Accumulation of WEs has been frequently reported for species of the genus Acinetobacter (66) but also for marine bacteria, such as Marinobacter (50) and Alcanivorax (11, 32).In general, the accumulation of at least one type of these compounds occurs intracellularly under imbalanced growth conditions in almost all prokaryotes. The localization of neutral lipids in marine organisms is not restricted to the cell cytoplasm, as extracellular lipid deposition has been shown in studies with Alcaligenes sp. PHY9 and Pseudomonas nautica (24). The production of extracellular wax esters by Alcanivorax jadensis T9 growing on hexadecane was described a few years ago (11). Species of the genus Alcanivorax belong to an unusual group of marine hydrocarbon-degrading bacteria, which have been recognized and described over the past few years and were shown to play an important role in the biological removal of petroleum hydrocarbons from contaminated sites (69). Species of the genus Alcanivorax are, like some species of the genera Neptunomonas (27) and Marinobacter (23), marine hydrocarbon-degrading bacteria. Moreover, Alcanivorax and related bacteria constitute the group of obligate hydrocarbonoclastic marine bacteria (OHCB), which exhibit a narrow range of utilizable carbon sources (obligate hydrocarbon utilization), with only a few species being able to metabolize substrates other than hydrocarbons (69). Alcanivorax borkumensis SK2 became a model strain of OHCB, and its importance and pivotal role in hydrocarbon biodegradation have recently been emphasized (33). The predominance of A. borkumensis in early stages of petroleum degradation has also been reported in microcosm studies as well as for a field-scale experiment (26).From a biotechnological point of view, the production of extracellular lipids is important. Secretion of lipophilic products into the culture medium rather than its intracellular accumulation can significantly reduce the costs of product recovery. Another advantage is that the production of WEs and TAGs would not be directly limited by cell density or cell volume. Until now, the mechanism responsible for the export of lipids in bacteria of the genus Alcanivorax or other bacteria had not been known. In this study, we report on a screening procedure to select mutants defective in lipid export for identification of the gene(s) involved in the export mechanism. After transposon-induced mutagenesis we found different mutants which were not able to export TAGs (mutants of A. borkumensis) when the cells were cultivated in the presence of pyruvate as the sole carbon source. Mutants of A. jadensis defective in export of WEs and/or wax diesters (DE) were also identified. The possible influences of the gene products on the export mechanism in Alcanivorax species were analyzed and are discussed.     

Plant epicuticular lipids: alteration by herbicidal carbamates

The effect of several carbamates and trichloroacetic acid on the biosynthesis of epicuticular lipids from leaves of pea (Pisum sativum) was tested by chemical and visual methods. The carbamates tested included S-(2,3-dichloroallyl) diisopropylthiocarbamate (diallate), N-(3-chlorophenyl) isopropylcarbamate (chloropropham), S-ethyl dipropylthiocarbamate, and 2-chloroallyl diethyldithiocarbamate. Diallate reduced epicuticular lipids by 50% when the plants were root-treated and by 80% when vapor-treated. These results were supported by scanning electron microscopy and carbon replica techniques with transmission electron microscopy. The ratio of wax lipid components in the diallate-treated plants remained unchanged, with the exception of the primary alcohols, which were reduced. Diallate appears to interfere with the biosynthesis of a precursor to the elongation-decarboxylation pathway of lipid synthesis. N-(3-Chlorophenyl)isopropylcarbamate had no significant effect on total amounts of extractable epicuticular lipids, nor did it alter the structure of the wax formation on the leaves. The scanning electron microscopy micrographs indicated that S-ethyl dipropylthiocarbamate significantly reduced wax formation on pea leaves. 2-Chloroallyl diethyldithiocarbamate altered the structure of the wax formations, but not the total amount of wax (scanning electron microscopy). Trichloroacetic acid had little effect on wax deposition compared to diallate or S-ethyl dipropylthiocarbamate (scanning electron microscopy). The implication of the effect of the carbamates on epicuticular lipids and penetration of subsequent topically applied chemicals is discussed.     

Multiple reaction monitoring mass spectrometry is a powerful tool to study glycerolipid composition in plants with different level of desaturase activity

In plants, two lipid desaturation pathways exist. A so-called prokaryotic pathway is active in plastids and responsible for unsaturation of 16 carbon fatty acids. An eukaryotic one, in the endoplasmic reticulum, acts on 18 carbon fatty acids. Desaturase activities are affected in stressed plants, and conversely, they have an impact on the capability of plants to adapt to stress. So knowing lipid unsaturation is important for physiological studies. Analysis of lipids by mass spectrometry, in the multiple reaction mode, gives access to the molecular species present in each membrane lipid class. We illustrate the powerfulness of this technique by applying it to phospholipids and galactolipids extracted from plants where the desaturation pathways are present at variable level.