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 Cytosolic Nucleoprotein of the Plant-Infecting Bunyavirus Tomato Spotted Wilt Recruits Endoplasmic Reticulum–Resident Proteins to Endoplasmic Reticulum Export Sites

In contrast with animal-infecting viruses, few known plant viruses contain a lipid envelope, and the processes leading to their membrane envelopment remain largely unknown. Plant viruses with lipid envelopes include viruses of the Bunyaviridae, which obtain their envelope from the Golgi complex. The envelopment process is predominantly dictated by two viral glycoproteins (Gn and Gc) and the viral nucleoprotein (N). During maturation of the plant-infecting bunyavirus Tomato spotted wilt, Gc localizes at endoplasmic reticulum (ER) membranes and becomes ER export competent only upon coexpression with Gn. In the presence of cytosolic N, Gc remains arrested in the ER but changes its distribution from reticular into punctate spots. Here, we show that these areas correspond to ER export sites (ERESs), distinct ER domains where glycoprotein cargo concentrates prior to coat protein II vesicle–mediated transport to the Golgi. Gc concentration at ERES is mediated by an interaction between its cytoplasmic tail (CT) and N. Interestingly, an ER-resident calnexin provided with Gc-CT was similarly recruited to ERES when coexpressed with N. Furthermore, disruption of actin filaments caused the appearance of a larger amount of smaller ERES loaded with N-Gc complexes, suggesting that glycoprotein cargo concentration acts as a trigger for de novo synthesis of ERES.     

Arabidopsis SEIPIN Proteins Modulate Triacylglycerol Accumulation and Influence Lipid Droplet Proliferation

The lipodystrophy protein SEIPIN is important for lipid droplet (LD) biogenesis in human and yeast cells. In contrast with the single SEIPIN genes in humans and yeast, there are three SEIPIN homologs in Arabidopsis thaliana, designated SEIPIN1, SEIPIN2, and SEIPIN3. Essentially nothing is known about the functions of SEIPIN homologs in plants. Here, a yeast (Saccharomyces cerevisiae) SEIPIN deletion mutant strain and a plant (Nicotiana benthamiana) transient expression system were used to test the ability of Arabidopsis SEIPINs to influence LD morphology. In both species, expression of SEIPIN1 promoted accumulation of large-sized lipid droplets, while expression of SEIPIN2 and especially SEIPIN3 promoted small LDs. Arabidopsis SEIPINs increased triacylglycerol levels and altered composition. In tobacco, endoplasmic reticulum (ER)-localized SEIPINs reorganized the normal, reticulated ER structure into discrete ER domains that colocalized with LDs. N-terminal deletions and swapping experiments of SEIPIN1 and 3 revealed that this region of SEIPIN determines LD size. Ectopic overexpression of SEIPIN1 in Arabidopsis resulted in increased numbers of large LDs in leaves, as well as in seeds, and increased seed oil content by up to 10% over wild-type seeds. By contrast, RNAi suppression of SEIPIN1 resulted in smaller seeds and, as a consequence, a reduction in the amount of oil per seed compared with the wild type. Overall, our results indicate that Arabidopsis SEIPINs are part of a conserved LD biogenesis machinery in eukaryotes and that in plants these proteins may have evolved specialized roles in the storage of neutral lipids by differentially modulating the number and sizes of lipid droplets.     

Metabolic Interactions between the Lands Cycle and the Kennedy Pathway of Glycerolipid Synthesis in Arabidopsis Developing Seeds

It has been widely accepted that the primary function of the Lands cycle is to provide a route for acyl remodeling to modify fatty acid (FA) composition of phospholipids derived from the Kennedy pathway. Lysophosphatidylcholine acyltransferase (LPCAT) is an evolutionarily conserved key enzyme in the Lands cycle. In this study, we provide direct evidence that the Arabidopsis thaliana LPCATs, LPCAT1 and LPCAT2, participate in the Lands cycle in developing seeds. In spite of a substantially reduced initial rate of nascent FA incorporation into phosphatidylcholine (PC), the PC level in the double mutant lpcat1 lpcat2-2 remained unchanged. LPCAT deficiency triggered a compensatory response of de novo PC synthesis and a concomitant acceleration of PC turnover that were attributable at least in part to PC deacylation. Acyl-CoA profile analysis revealed complicated metabolic alterations rather than merely reduced acyl group shuffling from PC in the mutant. Shifts in FA stereo-specific distribution in triacylglycerol of the mutant seed suggested a preferential retention of saturated acyl chains at the stereospecific numbering (sn)-1 position from PC and likely a channeling of lysophosphatidic acid, derived from PC, into the Kennedy pathway. Our study thus illustrates an intricate relationship between the Lands cycle and the Kennedy pathway.     

Acyl Editing and Headgroup Exchange Are the Major Mechanisms That Direct Polyunsaturated Fatty Acid Flux into Triacylglycerols

Triacylglycerols (TAG) in seeds of Arabidopsis (Arabidopsis thaliana) and many plant species contain large amounts of polyunsaturated fatty acids (PUFA). These PUFA are synthesized on the membrane lipid phosphatidylcholine (PC). However, the exact mechanisms of how fatty acids enter PC and how they are removed from PC after being modified to participate in the TAG assembly are unclear, nor are the identities of the key enzymes/genes that control these fluxes known. By reverse genetics and metabolic labeling experiments, we demonstrate that two genes encoding the lysophosphatidylcholine acyltransferases LPCAT1 and LPCAT2 in Arabidopsis control the previously identified “acyl-editing” process, the main entry of fatty acids into PC. The lpcat1/lpcat2 mutant showed increased contents of very-long-chain fatty acids and decreased PUFA in TAG and the accumulation of small amounts of lysophosphatidylcholine in developing seeds revealed by [¹⁴C]acetate-labeling experiments. We also showed that mutations in LPCATs and the PC diacylglycerol cholinephosphotransferase in the reduced oleate desaturation1 (rod1)/lpcat1/lpcat2 mutant resulted in a drastic reduction of PUFA content in seed TAG, accumulating only one-third of the wild-type level. These results indicate that PC acyl editing and phosphocholine headgroup exchange between PC and diacylglycerols control the majority of acyl fluxes through PC to provide PUFA for TAG synthesis.Plant oils are an important natural resource to meet the increasing demands of food, feed, biofuel, and industrial applications (Lu et al., 2011; Snapp and Lu, 2012). The fatty acid composition in the triacylglycerols (TAG), especially the contents of polyunsaturated fatty acids (PUFA) or other specialized structures, such as hydroxy, epoxy, or conjugated groups, determines the properties and thus the uses of plant oils (Dyer and Mullen, 2008; Dyer et al., 2008; Pinzi et al., 2009; Riediger et al., 2009). To effectively modify seed oils tailored for different uses, it is necessary to understand the fundamental aspects of how plant fatty acids are synthesized and accumulated in seed oils.In developing oilseeds, fatty acids are synthesized in plastids and are exported into the cytosol mainly as oleic acid, 18:1 (carbon number:double bonds), and a small amount of palmitic acid (16:0) and stearic acid (18:0; Ohlrogge and Browse, 1995). Further modification of 18:1 occurs on the endoplasmic reticulum in two major pathways (Fig. 1): (1) the 18:1-CoA may be elongated into 20:1- to 22:1-CoA esters by a fatty acid elongase, FAE1 (Kunst et al., 1992); (2) the dominant flux of 18:1 in many oilseeds is to enter the membrane lipid phosphatidylcholine (PC; Shanklin and Cahoon, 1998; Bates and Browse, 2012), where they can be desaturated by the endoplasmic reticulum-localized fatty acid desaturases including the oleate desaturase, FAD2, and the linoleate desaturase, FAD3, to produce the polyunsaturated linoleic acid (18:2) and α-linolenic acid (18:3; Browse et al., 1993; Okuley et al., 1994). The PUFA may be removed from PC to enter the acyl-CoA pool, or PUFA-rich diacylglycerol (DAG) may be derived from PC by removal of the phosphocholine headgroup (Bates and Browse, 2012). The PUFA-rich TAG are then produced from de novo-synthesized DAG or PC-derived DAG (Bates and Browse, 2012) and PUFA-CoA by the acyl-CoA:diacylglycerol acyltransferases (DGAT; Hobbs et al., 1999; Zou et al., 1999). Alternatively, PUFA may be directly transferred from PC onto DAG to form TAG by an acyl-CoA-independent phospholipid:diacylglycerol acyltransferase (PDAT; Dahlqvist et al., 2000). Recent results demonstrated that DGAT and PDAT are responsible for the majority of TAG synthesized in Arabidopsis (Arabidopsis thaliana) seeds (Zhang et al., 2009).Open in a separate windowFigure 1.Reactions involved in the flux of fatty acids into TAG. De novo glycerolipid synthesis is shown in white arrows, acyl transfer reactions are indicated by dashed lines, and the movement of the lipid glycerol backbone through the pathway is shown in solid lines. Major reactions (in thick lines) controlling the flux of fatty acid from PC into TAG are as follows: LPC acylation reaction of acyl editing by LPCAT (A); PC deacylation reaction of acyl editing by the reverse action of LPCAT or phospholipase A (B); and the interconversion of DAG and PC by PDCT (C). Substrates are in boldface, enzymatic reactions are in italics. FAD, Fatty acid desaturase; FAS, fatty acid synthase; GPAT, acyl-CoA:G3P acyltransferase; LPA, lysophosphatidic acid; LPAT, acyl-CoA:LPA acyltransferase; PA, phosphatidic acid; PLC, phospholipase C; PLD, phospholipase D.The above TAG synthesis model highlights the importance of acyl fluxes through PC for PUFA enrichment in plant oils. However, the exact mechanisms of how fatty acids enter PC and how they are removed from PC after being modified to participate in the TAG assembly are unclear, nor are the identities of the enzymes/genes that control these fluxes known. The traditional view is that 18:1 enters PC through de novo glycerolipid synthesis (Fig. 1; Kennedy, 1961): the sequential acylation of glycerol-3-phosphate (G3P) at the sn-1 and sn-2 positions produces phosphatidic acid; subsequent removal of the phosphate group at the sn-3 position of phosphatidic acid by phosphatidic acid phosphatases (PAPs) produces de novo DAG; finally, PC is formed from DAG by a cytidine-5′-diphosphocholine:diacylglycerol cholinephosphotransferase (CPT; Slack et al., 1983; Goode and Dewey, 1999). However, metabolic labeling experiments in many different plant tissues by us and others (Williams et al., 2000; Bates et al., 2007, 2009; Bates and Browse, 2012; Tjellström et al., 2012) have demonstrated that the majority of newly synthesized fatty acids (e.g. 18:1) enter PC by a process termed “acyl editing” rather than by proceeding through de novo PC synthesis. Acyl editing is a deacylation-reacylation cycle of PC that exchanges the fatty acids on PC with fatty acids in the acyl-CoA pool (Fig. 1, A and B). Through acyl editing, newly synthesized 18:1 can be incorporated into PC for desaturation and PUFA can be released from PC to the acyl-CoA pool to be utilized for glycerolipid synthesis.Additionally, there is accumulating evidence that many plants utilize PC-derived DAG to synthesize TAG laden with PUFA (Bates and Browse, 2012). PC-derived DAG may be synthesized through the reverse reaction of the CPT (Slack et al., 1983, 1985) or by the phospholipases C and D (followed by PAP). However, our recent discovery indicates that the main PC-to-DAG conversion is catalyzed by a phosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT) through the phosphocholine headgroup exchange between PC and DAG (Fig. 1C; Lu et al., 2009; Hu et al., 2012). The PDCT is encoded by the REDUCED OLEATE DESATURATION1 (ROD1) gene (At3g15820) in Arabidopsis, which is responsible for about 40% of the flux of PUFA from PC through DAG into TAG synthesis (Lu et al., 2009). Acyl editing and PC-DAG interconversion through PDCT may work together to generate PUFA-rich TAG in oilseed plants (Bates and Browse, 2012).The enzymes/genes involved in the incorporation of 18:1 into PC through acyl editing are not known. However, stereochemical localization of newly synthesized fatty acid incorporation into PC predominantly at the sn-2 position (Bates et al., 2007, 2009; Tjellström et al., 2012) strongly suggest that the acyl editing cycle proceeds through the acylation of lysophosphatidylcholine (LPC) by acyl-CoA:lysophosphatidylcholine acyltransferases (LPCATs [Enzyme Commission 2.3.1.23]; Fig. 1A). High LPCAT activity has been detected in many different oilseed plants that accumulate large amounts of PUFA in TAG (Stymne and Stobart, 1987; Bates and Browse, 2012), suggesting the potential ubiquitous involvement of LPCAT in the generation of PUFA-rich TAG. Several possible pathways for the removal of acyl groups from PC to generate the lysophosphatidylcholine within the acyl editing cycle have been proposed. The acyl groups may be released from PC to enter the acyl-CoA pool via the reverse reactions of LPCATs (Stymne and Stobart, 1984) or by reactions of phospholipase A (Chen et al., 2011) followed by the acyl-CoA synthetases (Shockey et al., 2002). The main focus of this study was to identify the genes and enzymes involved in the incorporation of fatty acids into PC through acyl editing in Arabidopsis and to quantify the contribution of acyl editing and PDCT-based PC-DAG interconversion to controlling the flux of PUFA from PC into TAG. Herein, we demonstrate that mutants of two Arabidopsis genes encoding LPCATs (At1g12640 [LPCAT1] and At1g63050 [LPCAT2]) have reduced TAG PUFA content. Analysis of the acyl-editing cycle through metabolic labeling of developing seeds with [¹⁴C]acetate indicate that the lpcat1/lpcat2 double mutant was devoid of acyl editing-based incorporation of newly synthesized fatty acids into PC, indicating that these two genes are responsible for the acylation of LPC during acyl editing. Additionally, the triple mutant rod1/lpcat1/lpcat2 indicated that PDCT-based PC-DAG interconversion and acyl editing together provide two-thirds of the flux of PUFA from PC to TAG in Arabidopsis seeds.     

Role of Aminoalcoholphosphotransferases 1 and 2 in Phospholipid Homeostasis in Arabidopsis

Aminoalcoholphosphotransferase (AAPT) catalyzes the synthesis of phosphatidylcholine (PC) and phosphotidylethanolamine (PE), which are the most prevalent membrane phospholipids in all eukaryotic cells. Here, we show that suppression of AAPTs results in extensive membrane phospholipid remodeling in Arabidopsis thaliana. Double knockout (KO) mutants that are hemizygous for either aapt1 or aapt2 display impaired pollen and seed development, leading to embryotic lethality of the double KO plants, whereas aapt1 or aapt2 single KO plants show no overt phenotypic alterations. The growth rate and seed yield of AAPT RNA interference (RNAi) plants are greatly reduced. Lipid profiling shows decreased total galactolipid and phospholipid content in aapt1-containing mutants, including aapt1, aapt1/aapt1 aapt2/AAPT2, aapt1/AAPT1 aapt2/aapt2, and AAPT RNAi plants. The level of PC in leaves was unchanged, whereas that of PE was reduced in all AAPT-deficient plants, except aapt2 KO. However, the acyl species of PC was altered, with increased levels of C34 species and decreased C36 species. Conversely, the levels of PE and phosphatidylinositol were decreased in C34 species. In seeds, all AAPT-deficient plants, including aapt2 KO, displayed a decrease in PE. The data show that AAPT1 and AAPT2 are essential to plant vegetative growth and reproduction and have overlapping functions but that AAPT1 contributes more than AAPT2 to PC production in vegetative tissues. The opposite changes in molecular species between PC and PE and unchanged PC level indicate the existence of additional pathways that maintain homeostatic levels of PC, which are crucial for the survival and proper development of plants.     

ROOT HAIR DEFECTIVE3 Family of Dynamin-Like GTPases Mediates Homotypic Endoplasmic Reticulum Fusion and Is Essential for Arabidopsis Development

In all eukaryotic cells, the endoplasmic reticulum (ER) forms a tubular network whose generation requires the fusion of ER membranes. In Arabidopsis (Arabidopsis thaliana), the membrane-bound GTPase ROOT HAIR DEFECTIVE3 (RHD3) is a potential candidate to mediate ER fusion. In addition, Arabidopsis has two tissue-specific isoforms of RHD3, namely RHD3-like (RL) proteins, and their function is not clear. Here, we show that a null allele of RHD3, rhd3-8, causes growth defects and shortened root hairs. A point mutant, rhd3-1, exhibits a more severe growth phenotype than the null mutant, likely because it exerts a dominant-negative effect on the RL proteins. Genetic analysis reveals that the double deletion of RHD3 and RL1 is lethal and that the rhd3 rl2 plants produce no viable pollen, suggesting that the RL proteins are redundant to RHD3. RHD3 family proteins can replace Sey1p, the homolog of RHD3 in yeast (Saccharomyces cerevisiae), in the maintenance of ER morphology, and they are able to fuse membranes both in vivo and in vitro. Our results suggest that RHD3 proteins mediate ER fusion and are essential for plant development and that the formation of the tubular ER network is of general physiological significance.In all eukaryotic cells, the endoplasmic reticulum (ER) comprises a continuous membrane system of sheets and tubules (Baumann and Walz, 2001; Shibata et al., 2006). ER tubules frequently connect through homotypic membrane fusion to form a reticular network (Lee and Chen, 1988; Prinz et al., 2000; Du et al., 2004). ER fusion in metazoans is mediated by the atlastins (ATLs), a class of dynamin-like, membrane-bound GTPases (Hu et al., 2009; Orso et al., 2009). ATL possesses a cytoplasmic N-terminal GTPase domain, followed by a helical domain, two closely spaced transmembrane domains, and a C-terminal cytosolic tail. ATL proteins localize mostly to ER tubules and they interact with the tubule-shaping proteins, reticulons and DP1 (Hu et al., 2009). A role for the ATLs in ER fusion is suggested by the fact that depletion of ATLs leads to long, nonbranched ER tubules in cultured cells (Hu et al., 2009) and to ER fragmentation in Drosophila melanogaster (Orso et al., 2009), possibly due to insufficient fusion between the tubules. Nonbranched ER tubules are also observed upon the expression of dominant-negative ATL mutants (Hu et al., 2009). In addition, antibodies to ATL inhibit ER network formation in Xenopus laevis egg extracts (Hu et al., 2009). Moreover, proteoliposomes containing purified D. melanogaster ATL undergo GTP-dependent fusion in vitro (Orso et al., 2009; Bian et al., 2011). The physiological significance of ER fusion is supported by the observation that mutations in human ATL1, the dominant isoform in the brain, cause hereditary spastic paraplegia (Zhao et al., 2001), a neurodegenerative disease characterized by axon shortening in corticospinal motor neurons and progressive spasticity and weakness of the lower limbs (Salinas et al., 2008).Many organisms lack ATL homologs. In yeast (Saccharomyces cerevisiae), another dynamin-like GTPase, Sey1p, has been found to share the same signature motifs and membrane topology as ATL (Hu et al., 2009). Recent work suggests that Sey1p mediates ER membrane fusion both in vivo and in vitro (Anwar et al., 2012). Cells lacking Sey1p grow normally (Hu et al., 2009), but additional mutation of an ER SNARE Ufe1p, which probably represents an alternative ER fusion mechanism in yeast, causes severe growth defects (Anwar et al., 2012). In Arabidopsis (Arabidopsis thaliana), the potential functional ortholog of ATL appears to be ROOT HAIR DEFECTIVE3 (RHD3; Hu et al., 2009), which was initially discovered by a genetic screen of root hair-defective mutants (Schiefelbein and Somerville, 1990). It is sequence related to Sey1p over the entire length (Wang et al., 1997; Brands and Ho, 2002). Mutations of RHD3 cause short and wavy root hairs (Schiefelbein and Somerville, 1990; Wang et al., 1997; Stefano et al., 2012) and defects in cell expansion (Wang et al., 2002).Despite the sequence homology between Sey1p and RHD3, it was reported that Sey1p could not replace RHD3 in plants and vice versa (Chen et al., 2011). Therefore, it is not clear whether RHD3 can mediate ER fusion. Another complication in plants is that the Arabidopsis RHD3 family also contains two RHD3-like (RL) proteins (Hu et al., 2003): RL1 is expressed only in pollen, whereas RL2 is expressed ubiquitously, but both are present at very low levels. Deletion of either RL protein causes no detectable defects in root hair development or overall growth (Chen et al., 2011). Whether RL proteins support the role of RHD3 in a tissue-specific manner remains to be investigated.Here, we have analyzed the function of RHD3 and RL proteins in Arabidopsis. We show that RHD3 and the two RL proteins play redundant roles but function during different stages of Arabidopsis development. In addition, we show that RHD3 proteins can functionally replace Sey1p in yeast and mediate ER membrane fusion.     

MAIGO5 Functions in Protein Export from Golgi-Associated Endoplasmic Reticulum Exit Sites in Arabidopsis

Plant cells face unique challenges to efficiently export cargo from the endoplasmic reticulum (ER) to mobile Golgi stacks. Coat protein complex II (COPII) components, which include two heterodimers of Secretory23/24 (Sec23/24) and Sec13/31, facilitate selective cargo export from the ER; however, little is known about the mechanisms that regulate their recruitment to the ER membrane, especially in plants. Here, we report a protein transport mutant of Arabidopsis thaliana, named maigo5 (mag5), which abnormally accumulates precursor forms of storage proteins in seeds. mag5-1 has a deletion in the putative ortholog of the Saccharomyces cerevisiae and Homo sapiens Sec16, which encodes a critical component of ER exit sites (ERESs). mag mutants developed abnormal structures (MAG bodies) within the ER and exhibited compromised ER export. A functional MAG5/SEC16A–green fluorescent protein fusion localized at Golgi-associated cup-shaped ERESs and cycled on and off these sites at a slower rate than the COPII coat. MAG5/SEC16A interacted with SEC13 and SEC31; however, in the absence of MAG5/SEC16A, recruitment of the COPII coat to ERESs was accelerated. Our results identify a key component of ER export in plants by demonstrating that MAG5/SEC16A is required for protein export at ERESs that are associated with mobile Golgi stacks, where it regulates COPII coat turnover.     

Phosphatidic Acid Regulates Microtubule Organization by Interacting with MAP65-1 in Response to Salt Stress in Arabidopsis

Membrane lipids play fundamental structural and regulatory roles in cell metabolism and signaling. Here, we report that phosphatidic acid (PA), a product of phospholipase D (PLD), regulates MAP65-1, a microtubule-associated protein, in response to salt stress. Knockout of the PLDα1 gene resulted in greater NaCl-induced disorganization of microtubules, which could not be recovered during or after removal of the stress. Salt affected the association of MAP65-1 with microtubules, leading to microtubule disorganization in pldα1cells, which was alleviated by exogenous PA. PA bound to MAP65-1, increasing its activity in enhancing microtubule polymerization and bundling. Overexpression of MAP65-1 improved salt tolerance of Arabidopsis thaliana cells. Mutations of eight amino acids in MAP65-1 led to the loss of its binding to PA, microtubule-bundling activity, and promotion of salt tolerance. The pldα1 map65-1 double mutant showed greater sensitivity to salt stress than did either single mutant. These results suggest that PLDα1-derived PA binds to MAP65-1, thus mediating microtubule stabilization and salt tolerance. The identification of MAP65-1 as a target of PA reveals a functional connection between membrane lipids and the cytoskeleton in environmental stress signaling.     

Coexpressing Escherichia coli Cyclopropane Synthase with Sterculia foetida Lysophosphatidic Acid Acyltransferase Enhances Cyclopropane Fatty Acid Accumulation

Cyclopropane fatty acids (CPAs) are desirable as renewable chemical feedstocks for the production of paints, plastics, and lubricants. Toward our goal of creating a CPA-accumulating crop, we expressed nine higher plant cyclopropane synthase (CPS) enzymes in the seeds of fad2fae1 Arabidopsis (Arabidopsis thaliana) and observed accumulation of less than 1% CPA. Surprisingly, expression of the Escherichia coli CPS gene resulted in the accumulation of up to 9.1% CPA in the seed. Coexpression of a Sterculia foetida lysophosphatidic acid acyltransferase (SfLPAT) increases CPA accumulation up to 35% in individual T1 seeds. However, seeds with more than 9% CPA exhibit wrinkled seed morphology and reduced size and oil accumulation. Seeds with more than 11% CPA exhibit strongly decreased seed germination and establishment, and no seeds with CPA more than 15% germinated. That previous reports suggest that plant CPS prefers the stereospecific numbering (sn)-1 position whereas E. coli CPS acts on sn-2 of phospholipids prompted us to investigate the preferred positions of CPS on phosphatidylcholine (PC) and triacylglycerol. Unexpectedly, in planta, E. coli CPS acts primarily on the sn-1 position of PC; coexpression of SfLPAT results in the incorporation of CPA at the sn-2 position of lysophosphatidic acid. This enables a cycle that enriches CPA at both sn-1 and sn-2 positions of PC and results in increased accumulation of CPA. These data provide proof of principle that CPA can accumulate to high levels in transgenic seeds and sets the stage for the identification of factors that will facilitate the movement of CPA from PC into triacylglycerol to produce viable seeds with additional CPA accumulation.Modified fatty acids (mFAs; sometimes referred to as unusual fatty acids) obtained from plants play important roles in industrial applications as lubricants, protective coatings, plastics, inks, cosmetics, etc. The hundreds of potential industrial uses of mFAs have led to considerable interest in exploring their production in transgenic crop plants. mFAs are produced by a limited number of species, and the transfer of genes encoding mFA-producing enzymes from source plants to heterologous hosts has generally resulted in only modest accumulation, usually less than 20% of the desired mFA in transgenic seed (Napier, 2007) compared with levels found in the natural source. For example, ricinoleic acid accounts for more than 90% of the fatty acid of castor bean (Ricinus communis) seeds, and tung (Aleuites fordii) seeds accumulate more than 80% α-eleostearic acid (Thelen and Ohlrogge, 2002; Drexler et al., 2003). In order to elevate the content of mFAs in the engineered plants to that found in the native plant, it is necessary to (1) optimize the synthesis of mFA (Mekhedov et al., 2001), (2) minimize its degradation (Eccleston and Ohlrogge, 1998), and (3) optimize its incorporation into triacylglycerol (TAG; Bafor et al., 1990; Bates and Browse, 2011; van Erp et al., 2011).Cyclic fatty acids (CFAs) are desirable for numerous industrial applications. The strained bond angles of the carbocyclic ring contribute to their unique chemistry and physical properties, and hydrogenation of CFAs results in ring opening to produce methyl-branched fatty acids. Branched chain fatty acids are ideally suited for the oleochemical industry as feedstocks for the production of lubricants, plastics, paints, dyes, and coatings (Carlsson et al., 2011). Cyclopropane fatty acids (CPAs) have been found in certain gymnosperms, Malvales, Litchi spp., and other Sapindales species. They accumulate to as much as 40% in seeds of Litchi chinensis (Vickery, 1980; Gaydou et al., 1993). Sterculia foetida accumulates the desaturated CFA (i.e. cyclopropene fatty acid) to more than 60% of its seed oil (Bohannon and Kleiman, 1978; Pasha and Ahmad, 1992). The first step in its synthesis is the formation of the CPA by the cyclopropane synthase (CPS) enzyme, which transfers a methyl group to C9 of the oleoyl-phospholipid followed by cyclization to form the cyclopropane ring (Grogan and Cronan, 1997; Bao et al., 2002, 2003). None of the known natural sources of CPA are suitable for its commercial production. Therefore, it would be desirable to create an oilseed crop plant that accumulates high levels of CPA by heterologously expressing CPS in seeds. However, to date, heterologous expression of plant cyclopropane synthase genes has led to only approximately 1.0% CPA in the transgenic seeds (Yu et al., 2011).Two pathways for the biosynthesis of TAG exist in plants (Bates and Browse, 2012; Fig. 1). The de novo biosynthesis from glycerol-3-phosphate and acyl-CoA occurs via the Kennedy pathway and includes three acyltransferases: glycerol-2-phosphate acyltransferase, acyl-CoA:lysophosphatidic acid acyltransferase (LPAT), and acyl-CoA:diacylglycerol acyltransferase (DGAT; Kennedy, 1961). Alternatively, acyl-CoAs can be redirected from phosphatidylcholine (PC) via the action of a phospholipase C, choline phosphotransferase, phosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT; Hu et al., 2012; Lu et al., 2009), or phospholipid:diacylglycerol acyltransferase (PDAT; Dahlqvist et al., 2000). An acyl group can be released from PC to generate lysophosphatidylcholine (LPC) by the back reaction of acyl-CoA:LPC acyltransferase (Stymne and Stobart, 1984; Wang et al., 2012) or a phospholipase A/acyl-CoA synthase (Chen et al., 2011).Open in a separate windowFigure 1.Schematic representation of the plant TAG biosynthesis network. Acyl editing can provide PC-modified fatty acids for de novo diacylglycerol/TAG synthesis. ACS, acyl-CoA synthase; CPT, CDP-choline:diacylglycerol choline phosphotransferase; G3P, glycerol-3-phosphate; GPAT, acyl-CoA:glycerol-3-phosphate acyltransferase; LPC acyltransferase, acyl-CoA:LPC acyltransferase; mFAS, modified fatty acid synthase (in this work, mFAS is CPS); PAP, phosphatidic acid phosphatase; PLA, phospholipase A; PLC, phospholipase C.LPAT is a pivotal enzyme controlling the metabolic flow of lysophosphatidic acid (LPA) into different phosphatidic acids (PAs) in diverse tissues. Membrane-associated LPAT activities, identified in bacteria, yeast, plant, and animal cells, catalyze the transfer of acyl groups from acyl-CoA to LPA to synthesize PA. In plants and other organisms, LPAT activities have been identified in the endoplasmic reticulum (Kim et al., 2005), plasma membrane (Bursten et al., 1991), and mitochondria (Zborowski and Wojtczak, 1969). In higher plants, endoplasmic reticulum-localized LPAT plays an essential role transferring fatty acid from CoA esters to the sn-2 position of LPA in the synthesis of PA, a key intermediate in the biosynthesis of membrane phospholipids and storage lipids in developing seeds (Maisonneuve et al., 2010). LPAT from developing seeds of flax (Linum usitatissimum), rape (Brassica napus), and castor bean preferentially incorporate oleoyl-CoA, weakly incorporate cyclopropane acyl-CoA, and were unable to incorporate methyl-branched acyl-CoA when presented with an equimolar mix of these potential substrates (Nlandu Mputu et al., 2009). Thus, LPAT activity from agronomic plants constitutes a potential bottleneck for the incorporation of branched chain acyl-CoA into PA. In this work, we investigate the utility of an LPAT from a cyclopropanoid-syntheizing plant, S. foetida, with respect to its ability to enhance CPA accumulation. In our efforts to enhance CPA accumulation in transgenic plants, we screened CPS genes from diverse sources and identified Escherichia coli CPS (EcCPS) as an effective enzyme for the production of CPA in plants. However, EcCPS is reported to prefer the sn-2 position of E. coli phospholipid (Hildebrand and Law, 1964), and the data presented here show that its expression primarily leads to the accumulation of CPA at the stereospecific numbering (sn)-1 position. Moreover, coexpression of S. foetida lysophosphatidic acid acyltransferase (SfLPAT) results in the incorporation of CPA at the sn-2 position of LPA. Thus, coexpression of EcCPS and SfLPAT enables a cycle that enriches the accumulation of CPA at both sn-1 and sn-2 positions of PC and increases the accumulation of CPA. This work underscores the utility of coexpressing an acyltransferase from mFA-accumulating species with mFA-synthesizing enzymes to help mitigate bottlenecks in mFA TAG synthesis.     

Production of a Brassica napus Low-Molecular Mass Acyl-Coenzyme A-Binding Protein in Arabidopsis Alters the Acyl-Coenzyme A Pool and Acyl Composition of Oil in Seeds

Low-molecular mass (10 kD) cytosolic acyl-coenzyme A-binding protein (ACBP) has a substantial influence over fatty acid (FA) composition in oilseeds, possibly via an effect on the partitioning of acyl groups between elongation and desaturation pathways. Previously, we demonstrated that the expression of a Brassica napus ACBP (BnACBP) complementary DNA in the developing seeds of Arabidopsis (Arabidopsis thaliana) resulted in increased levels of polyunsaturated FAs at the expense of eicosenoic acid (20:1^cisΔ11) and saturated FAs in seed oil. In this study, we investigated whether alterations in the FA composition of seed oil at maturity were correlated with changes in the acyl-coenzyme A (CoA) pool in developing seeds of transgenic Arabidopsis expressing BnACBP. Our results indicated that both the acyl-CoA pool and seed oil of transgenic Arabidopsis lines expressing cytosolic BnACBP exhibited relative increases in linoleic acid (18:2^cisΔ9,12; 17.9%–44.4% and 7%–13.2%, respectively) and decreases in 20:1^cisΔ11 (38.7%–60.7% and 13.8%–16.3%, respectively). However, alterations in the FA composition of the acyl-CoA pool did not always correlate with those seen in the seed oil. In addition, we found that targeting of BnACBP to the endoplasmic reticulum resulted in FA compositional changes that were similar to those seen in lines expressing cytosolic BnACBP, with the most prominent exception being a relative reduction in α-linolenic acid (18:3^cisΔ9,12,15) in both the acyl-CoA pool and seed oil of the former (48.4%–48.9% and 5.3%–10.4%, respectively). Overall, these data support the role of ACBP in acyl trafficking in developing seeds and validate its use as a biotechnological tool for modifying the FA composition of seed oil.Cytosolic low-molecular mass (approximately 10 kD) acyl-coenzyme A-binding protein (ACBP) consists of a four-α-helix domain capable of binding acyl-CoAs with high affinity in a wide range of eukaryotic organisms (Faergeman et al., 2007). It is believed to serve a housekeeping function of maintaining free acyl-CoA concentrations at low nanomolar levels and, thus, prevents micelle formation and the partitioning of acyl-CoA into membranes (Knudsen et al., 1999). This protein is also considered to contribute to another facet of acyl-CoA pool maintenance via its role in the intracellular transport of acyl-CoAs in the aqueous environment of the cytosol (Rasmussen et al., 1994). Moreover, it has also been shown to exhibit more specialized functions in metabolic processes in which acyl-CoA is actively involved, depending on the tissue and physiological state (Guerrero et al., 2006; Xiao and Chye 2011; Yurchenko and Weselake, 2011).In the developing seeds of oleaginous plants, fatty acids (FAs) are synthesized de novo in plastids and are activated to acyl-CoAs upon their transfer to the cytosol, after which time they can undergo additional modifications (e.g. elongation and desaturation) on the membranes of the endoplasmic reticulum (ER; for review, see Rawsthorne, 2002). While FA elongation is performed on the acyl-CoA substrate, the introduction of the second and third double bonds requires the acyl group to be esterified to phosphatidylcholine (PC; Jaworski, 1987). The composition of the acyl-CoA pool, therefore, is highly dynamic and represents a net result of both de novo synthesis and acyl-editing processes (Bates et al., 2009).The acyl-CoA pool provides substrate for acyltransferases involved in the biosynthesis of triacylglycerol (TAG), which is a major component of seed oil (Weselake et al., 2009). More specifically, TAG synthesis typically occurs via a series of acyl-CoA-dependent acylations of a glycerol backbone derived from sn-glycerol-3-phosphate in a pathway known as the sn-glycerol-3-phosphate or Kennedy pathway (for review, see Snyder et al., 2009; Weselake et al., 2009), although acyl-CoA-independent reactions can also be involved in the production of TAG (Stobart et al., 1997; Banaś et al., 2000; Dahlqvist et al., 2000) and thus contribute to its final composition. Low-molecular mass ACBPs have been demonstrated to modulate the activities of Kennedy pathway acyltransferases in a manner dependent upon the ratio of ACBP to acyl-CoA, stimulating TAG biosynthesis under conditions of acyl-CoA excess and inhibiting acyltransferase activities when relative amounts of acyl-CoA are low compared with ACBP, thus regulating the size of the acyl-CoA pool (for review, see Yurchenko and Weselake, 2011).The acyl-CoA pool in seeds is also influenced through a distinct route involving lysophosphatidylcholine acyltransferase (LPCAT), which catalyzes the acyl-CoA-dependent acylation of lysophosphatidylcholine at the sn-2 position to form PC (Ichihara et al., 1995). Acyl groups esterified to PC become substrates for FA desaturation and other modifications (Miquel and Browse, 1992; Broun et al., 1998) and can then be returned back to the acyl-CoA pool or channeled into TAG through acyl-CoA-independent mechanisms (Stymne and Stobart, 1984; Weselake, 2005; Lager et al., 2013). The efficiency of this acyl group channeling to and from PC is an important determinant of the overall composition of FAs in the acyl-CoA pool and, subsequently, in seed oil.Previously, we demonstrated that the expression of the Brassica napus low-molecular mass ACBP (hereafter referred to as BnACBP) in the presence of Arabidopsis (Arabidopsis thaliana) LPCAT isoforms in an in vitro system enhanced the incorporation of oleic acid (18:1^cisΔ9; hereafter referred to as 18:1) into PC and the release of linoleic acid (18:2^cisΔ9,12; hereafter referred to as 18:2) from PC into acyl-CoA (Yurchenko et al., 2009). In line with these results, the expression of BnACBP complementary DNA (cDNA) in Arabidopsis developing seeds was also shown to result in elevated levels of the polyunsaturated fatty acids (PUFAs) 18:2 and α-linolenic acid (18:3^cisΔ9,12,15; hereafter referred to as 18:3) in seed oil, mainly at the expense of eicosenoic acid (20:1^cisΔ11; hereafter referred to as 20:1) and saturated fatty acids (SFAs; Yurchenko et al., 2009). Based on these findings, BnACBP was proposed to be involved in acyl exchange between acyl-CoA and PC pools, which may affect the rate of FA modifications and, ultimately, the FA composition of seed oil (Yurchenko et al., 2009).In this study, we endeavored to provide further evidence that low-molecular mass ACBP functions in acyl trafficking by investigating whether changes in the FA composition of TAG in Arabidopsis seeds expressing BnACBP were correlated with modifications in the composition of the acyl-CoA pool. In addition, since FA modifications such as elongation and desaturation as well as TAG synthesis occur on ER membranes, we also examined the effect of changing the subcellular localization of BnACBP (from the cytosol to the ER) on the acyl composition of TAG and the acyl-CoA pool in transgenic Arabidopsis. Consequently, we generated localized pools of acyl-CoAs that could be readily accessed by acyltransferases involved in seed oil biosynthesis. Taken together, our findings provide insight into the role of low-molecular mass ACBP in seed oil metabolism and suggest that ACBP (either in its native cytosolic form or as an ER-targeted fusion protein) may serve as a useful tool in biotechnological modifications of FA composition in oil crops.