Arabidopsis ECERIFERUM2 Is a Component of the Fatty Acid Elongation Machinery Required for Fatty Acid Extension to Exceptional Lengths

Primary aerial surfaces of land plants are coated by a lipidic cuticle, which forms a barrier against transpirational water loss and protects the plant from diverse stresses. Four enzymes of a fatty acid elongase complex are required for the synthesis of very-long-chain fatty acid (VLCFA) precursors of cuticular waxes. Fatty acid elongase substrate specificity is determined by a condensing enzyme that catalyzes the first reaction carried out by the complex. In Arabidopsis (Arabidopsis thaliana), characterized condensing enzymes involved in wax synthesis can only elongate VLCFAs up to 28 carbons (C28) in length, despite the predominance of C29 to C31 monomers in Arabidopsis stem wax. This suggests additional proteins are required for elongation beyond C28. The wax-deficient mutant eceriferum2 (cer2) lacks waxes longer than C28, implying that CER2, a putative BAHD acyltransferase, is required for C28 elongation. Here, we characterize the cer2 mutant and demonstrate that green fluorescent protein-tagged CER2 localizes to the endoplasmic reticulum, the site of VLCFA biosynthesis. We use site-directed mutagenesis to show that the classification of CER2 as a BAHD acyltransferase based on sequence homology does not fit with CER2 catalytic activity. Finally, we provide evidence for the function of CER2 in C28 elongation by an assay in yeast (Saccharomyces cerevisiae).Land plants have a lipidic cuticle that seals the outer surface of all of their primary aerial organs. Structurally, the cuticle consists of two components, cutin and cuticular waxes. Together these form a hydrophobic barrier that plays a critical role in plant survival by restricting nonstomatal water loss (Riederer and Schreiber, 2001). Cuticles also protect the plant from biotic and abiotic stresses, profoundly affect plant-insect interactions (Müller, 2006), prevent epidermal fusions (Sieber et al., 2000), and are involved in drought stress signaling (Wang et al., 2011).Cutin is a polymer of mainly midchain- and ω-hydroxy and -epoxy 16 carbon (C16) and C18 fatty acids, which are cross-linked in ester bonds directly or through a glycerol backbone (Pollard et al., 2008). Cuticular waxes are aliphatic monomers that are deposited within the cutin matrix as intracuticular wax, and on top of it as epicuticular wax film and crystals. Wax is a heterogeneous mixture of very-long-chain fatty acids (VLCFAs) and their alkane, aldehyde, alcohol, ketone, and ester derivatives, which typically range from C24 to C32 in length (Samuels et al., 2008). The composition of cuticular wax varies greatly among species and tissues, often providing physical and chemical properties to the plant surface that are advantageous in specific environments.Genetic analyses have revealed that a fatty acid elongase (FAE) complex is responsible for the synthesis of VLCFA wax precursors (Millar et al., 1999; Fiebig et al., 2000; Kunst and Samuels, 2009). FAE complexes are heterotetramers of independently transcribed, monofunctional proteins localized to the endoplasmic reticulum (ER). Together, they catalyze a series of four reactions to elongate long-chain acyl-CoAs or very-long-chain acyl-CoAs by sequential addition of two carbon units. The condensing enzyme, or β-ketoacyl-CoA synthase (KCS), catalyzes the first reaction in this sequence and is both rate limiting and specific for the chain length of acyl-CoA synthesized (Millar and Kunst, 1997). Two very dissimilar families of KCSs have been identified in Arabidopsis (Arabidopsis thaliana): a FAE1-type family homologous to the first such KCS enzyme discovered in association with seed oil biosynthesis (Kunst et al., 1992; James et al., 1995; Lassner et al., 1996), and an ELONGATION DEFECTIVE (ELO)-like family homologous to the yeast (Saccharomyces cerevisiae) ELO family responsible for sphingolipid synthesis (Dunn et al., 2004). To date, no function has been ascribed to Arabidopsis ELOs. Of the 21 FAE1-type KCS enzymes in Arabidopsis (Joubès et al., 2008), 11 have been shown by microarray analysis to be up-regulated in the stem epidermis (Suh et al., 2005). Only one of these, ECERIFERUM6 (CER6/KCS6/CUT1; Millar et al., 1999; Fiebig et al., 2000; Joubès et al., 2008), has a dominant role in the elongation of VLCFAs for cuticular wax synthesis, as CER6 suppression results in a dramatic reduction of all wax monomers longer than C24 (Millar et al., 1999). Heterologous expression of CER6 in yeast has demonstrated that the CER6 condensing enzyme can produce C28 VLCFAs (O. Rowland and L. Kunst, unpublished data). However, CER6 appears to be unable to produce VLCFAs longer than C28 in yeast; this presents a problem as the bulk of Arabidopsis stem wax is made up of C29 alkanes, secondary alcohols, and ketones derived from C30 VLCFAs. Mutant screens have not revealed any other KCS enzymes necessary for VLCFA elongation past C28 in Arabidopsis. Therefore, there may be other proteins unrelated to condensing enzymes that are required for acyl chain extension beyond C28 that remain unknown.The wax-deficient mutant cer2 shows a dramatic reduction in all stem waxes longer than C28 and increased accumulation of waxes C28 or shorter, suggesting that CER2 has a role in the final steps of VLCFA elongation. Surprisingly, the cer2 mutation has been mapped to At4g24510 (Negruk et al., 1996; Xia et al., 1996), a gene homologous to plant BAHD acyltransferases. However, the CER2 protein was reported to localize exclusively to the nucleus (Xia et al., 1997). This does not fit with CER2 annotation as a BAHD acyltransferase, as all characterized BAHD acyltransferases are soluble cytosolic enzymes (D’Auria, 2006).The objective of this work was to more precisely evaluate the role of CER2 in fatty acid elongation using a new CER2 allele, cer2-5 (Columbia-0 [Col-0] ecotype). We provide evidence that CER2 has a metabolic function specific to wax synthesis, and that the CER2 homolog CER2-LIKE1 has an analogous role in leaf wax synthesis. Despite the classification of CER2 as a BAHD acyltransferase based on sequence homology, we demonstrate that CER2 cannot share the catalytic mechanism that has been confirmed for other members of the BAHD family, and provide biochemical support for a function of CER2 in VLCFA elongation by an assay in yeast.     

Focus on Ethylene: Ethylene Biosynthesis Is Promoted by Very-Long-Chain Fatty Acids during Lysigenous Aerenchyma Formation in Rice Roots

In rice (Oryza sativa) roots, lysigenous aerenchyma, which is created by programmed cell death and lysis of cortical cells, is constitutively formed under aerobic conditions, and its formation is further induced under oxygen-deficient conditions. Ethylene is involved in the induction of aerenchyma formation. reduced culm number1 (rcn1) is a rice mutant in which the gene encoding the ATP-binding cassette transporter RCN1/OsABCG5 is defective. Here, we report that the induction of aerenchyma formation was reduced in roots of rcn1 grown in stagnant deoxygenated nutrient solution (i.e. under stagnant conditions, which mimic oxygen-deficient conditions in waterlogged soils). 1-Aminocyclopropane-1-carboxylic acid synthase (ACS) is a key enzyme in ethylene biosynthesis. Stagnant conditions hardly induced the expression of ACS1 in rcn1 roots, resulting in low ethylene production in the roots. Accumulation of saturated very-long-chain fatty acids (VLCFAs) of 24, 26, and 28 carbons was reduced in rcn1 roots. Exogenously supplied VLCFA (26 carbons) increased the expression level of ACS1 and induced aerenchyma formation in rcn1 roots. Moreover, in rice lines in which the gene encoding a fatty acid elongase, CUT1-LIKE (CUT1L; a homolog of the gene encoding Arabidopsis CUT1, which is required for cuticular wax production), was silenced, both ACS1 expression and aerenchyma formation were reduced. Interestingly, the expression of ACS1, CUT1L, and RCN1/OsABCG5 was induced predominantly in the outer part of roots under stagnant conditions. These results suggest that, in rice under oxygen-deficient conditions, VLCFAs increase ethylene production by promoting 1-aminocyclopropane-1-carboxylic acid biosynthesis in the outer part of roots, which, in turn, induces aerenchyma formation in the root cortex.Aerenchyma formation is a morphological adaptation of plants to complete submergence and waterlogging of the soil, and facilitates internal gas diffusion (Armstrong, 1979; Jackson and Armstrong, 1999; Colmer, 2003; Voesenek et al., 2006; Bailey-Serres and Voesenek, 2008; Licausi and Perata, 2009; Sauter, 2013; Voesenek and Bailey-Serres, 2015). To adapt to waterlogging in soil, rice (Oryza sativa) develops lysigenous aerenchyma in shoots (Matsukura et al., 2000; Colmer and Pedersen, 2008; Steffens et al., 2011) and roots (Jackson et al., 1985b; Justin and Armstrong, 1991; Kawai et al., 1998), which is formed by programmed cell death and subsequent lysis of some cortical cells (Jackson and Armstrong, 1999; Evans, 2004; Yamauchi et al., 2013). In rice roots, lysigenous aerenchyma is constitutively formed under aerobic conditions (Jackson et al., 1985b), and its formation is further induced under oxygen-deficient conditions (Colmer et al., 2006; Shiono et al., 2011). The former and latter are designated constitutive and inducible lysigenous aerenchyma formation, respectively (Colmer and Voesenek, 2009). The gaseous plant hormone ethylene regulates adaptive growth responses of plants to submergence (Voesenek and Blom, 1989; Voesenek et al., 1993; Visser et al., 1996a,b; Lorbiecke and Sauter, 1999; Hattori et al., 2009; Steffens and Sauter, 2009; van Veen et al., 2013). Ethylene also induces lysigenous aerenchyma formation in roots of some gramineous plants (Drew et al., 2000; Shiono et al., 2008). The treatment of roots with ethylene or its precursor (1-aminocyclopropane-1-carboxylic acid [ACC]) stimulates aerenchyma formation in rice (Justin and Armstrong, 1991; Colmer et al., 2006; Yukiyoshi and Karahara, 2014), maize (Zea mays; Drew et al., 1981; Jackson et al., 1985a; Takahashi et al., 2015), and wheat (Triticum aestivum; Yamauchi et al., 2014a,b). Moreover, treatment of roots with inhibitors of ethylene action or ethylene biosynthesis effectively blocks aerenchyma formation under hypoxic conditions in maize (Drew et al., 1981; Konings, 1982; Jackson et al., 1985a; Rajhi et al., 2011).Ethylene biosynthesis is accomplished by two main successive enzymatic reactions: conversion of S-adenosyl-Met to ACC by 1-aminocyclopropane-1-carboxylic acid synthase (ACS), and conversion of ACC to ethylene by 1-aminocyclopropane-1-carboxylic acid oxidase (ACO; Yang and Hoffman, 1984). The activities of both enzymes are enhanced during aerenchyma formation under hypoxic conditions in maize root (He et al., 1996). Since the ACC content in roots of maize is increased by oxygen deficiency and is strongly correlated with ethylene production (Atwell et al., 1988), ACC biosynthesis is essential for ethylene production during aerenchyma formation in roots. In fact, exogenously supplied ACC induced ethylene production in roots of maize (Drew et al., 1979; Konings, 1982; Atwell et al., 1988) and wheat (Yamauchi et al., 2014b), even under aerobic conditions. Ethylene production in plants is inversely related to oxygen concentration (Yang and Hoffman, 1984). Under anoxic conditions, the oxidation of ACC to ethylene by ACO, which requires oxygen, is almost completely repressed (Yip et al., 1988; Tonutti and Ramina, 1991). Indeed, anoxic conditions stimulate neither ethylene production nor aerenchyma formation in maize adventitious roots (Drew et al., 1979). Therefore, it is unlikely that the root tissues forming inducible aerenchyma are anoxic, and that the ACO-mediated step is repressed. Moreover, aerenchyma is constitutively formed in rice roots even under aerobic conditions (Jackson et al., 1985b), and thus, after the onset of waterlogging, oxygen can be immediately supplied to the apical regions of roots through the constitutively formed aerenchyma.Very-long-chain fatty acids (VLCFAs; ≥20 carbons) are major constituents of sphingolipids, cuticular waxes, and suberin in plants (Franke and Schreiber, 2007; Kunst and Samuels, 2009). In addition to their structural functions, VLCFAs directly or indirectly participate in several physiological processes (Zheng et al., 2005; Reina-Pinto et al., 2009; Roudier et al., 2010; Ito et al., 2011; Nobusawa et al., 2013; Tsuda et al., 2013), including the regulation of ethylene biosynthesis (Qin et al., 2007). During fiber cell elongation in cotton ovules, ethylene biosynthesis is enhanced by treatment with saturated VLCFAs, especially 24-carbon fatty acids, and is suppressed by an inhibitor of VLCFA biosynthesis (Qin et al., 2007). The first rate-limiting step in VLCFA biosynthesis is condensation of acyl-CoA with malonyl-CoA by β-ketoacyl-CoA synthase (KCS; Joubès et al., 2008). KCS enzymes are thought to determine the substrate and tissue specificities of fatty acid elongation (Joubès et al., 2008). The Arabidopsis (Arabidopsis thaliana) genome has 21 KCS genes (Joubès et al., 2008). In the Arabidopsis cut1 mutant, which has a defect in the gene encoding CUT1 that is required for cuticular wax production (i.e. one of the KCS genes), the expression of AtACO genes and growth of root cells were reduced when compared with the wild type (Qin et al., 2007). Furthermore, expression of the AtACO genes was rescued by exogenously supplied saturated VLCFAs (Qin et al., 2007). These observations imply that VLCFAs or their derivatives work as regulatory factors for gene expression during some physiological processes in plants.reduced culm number1 (rcn1) was first identified as a rice mutant with a low tillering rate in a paddy field (Takamure and Kinoshita, 1985; Yasuno et al., 2007). The rcn1 (rcn1-2) mutant has a single nucleotide substitution in the gene encoding a member of the ATP-binding cassette (ABC) transporter subfamily G, RCN1/OsABCG5, causing an Ala-684Pro substitution (Yasuno et al., 2009). The mutation results in several mutant phenotypes, although the substrates of RCN1/OsABCG5 have not been determined (Ureshi et al., 2012; Funabiki et al., 2013; Matsuda et al., 2014). We previously found that the rcn1 mutant has abnormal root morphology, such as shorter root length and brownish appearance of roots, under stagnant (deoxygenated) conditions (which mimics oxygen-deficient conditions in waterlogged soils). We also found that the rcn1 mutant accumulates less of the major suberin monomers originating from VLCFAs in the outer part of adventitious roots, and this results in a reduction of a functional apoplastic barrier in the root hypodermis (Shiono et al., 2014a).The objective of this study was to elucidate the molecular basis of inducible aerenchyma formation. To this end, we examined lysigenous aerenchyma formation and ACC, ethylene, and VLCFA accumulation and their biosyntheses in rcn1 roots. Based on the results of these studies, we propose that VLCFAs are involved in inducible aerenchyma formation through the enhancement of ethylene biosynthesis in rice roots.     

HAPLESS13, the Arabidopsis μ1 Adaptin,Is Essential for Protein Sorting at the trans-Golgi Network/Early Endosome

In plant cells, secretory and endocytic routes intersect at the trans-Golgi network (TGN)/early endosome (EE), where cargos are further sorted correctly and in a timely manner. Cargo sorting is essential for plant survival and therefore necessitates complex molecular machinery. Adaptor proteins (APs) play key roles in this process by recruiting coat proteins and selecting cargos for different vesicle carriers. The µ1 subunit of AP-1 in Arabidopsis (Arabidopsis thaliana) was recently identified at the TGN/EE and shown to be essential for cytokinesis. However, little was known about other cellular activities affected by mutations in AP-1 or the developmental consequences of such mutations. We report here that HAPLESS13 (HAP13), the Arabidopsis µ1 adaptin, is essential for protein sorting at the TGN/EE. Functional loss of HAP13 displayed pleiotropic developmental defects, some of which were suggestive of disrupted auxin signaling. Consistent with this, the asymmetric localization of PIN-FORMED2 (PIN2), an auxin transporter, was compromised in the mutant. In addition, cell morphogenesis was disrupted. We further demonstrate that HAP13 is critical for brefeldin A-sensitive but wortmannin-insensitive post-Golgi trafficking. Our results show that HAP13 is a key link in the sophisticated trafficking network in plant cells.Plant cells contain sophisticated endomembrane compartments, including the endoplasmic reticulum, the Golgi, the trans-Golgi network (TGN)/early endosome (EE), the prevacuolar compartments/multivesicular bodies (PVC/MVB), various types of vesicles, and the plasma membrane (PM; Ebine and Ueda, 2009; Richter et al., 2009). Intracellular protein sorting between the various locations in the endomembrane system occurs in both secretory and endocytic routes (Richter et al., 2009; De Marcos Lousa et al., 2012). Vesicles in the secretory route start at the endoplasmic reticulum, passing through the Golgi before reaching the TGN/EE, while vesicles in the endocytic route start from the PM before reaching the TGN/EE (Dhonukshe et al., 2007; Viotti et al., 2010). The TGN/EE in Arabidopsis (Arabidopsis thaliana) is an independent and highly dynamic organelle transiently associated with the Golgi (Dettmer et al., 2006; Lam et al., 2007; Viotti et al., 2010), distinct from the animal TGN. Once reaching the TGN/EE, proteins delivered by their vesicle carriers are subject to further sorting, being incorporated either into vesicles that pass through the PVC/MVB before reaching the vacuole for degradation or into vesicles that enter the secretory pathway for delivery to the PM (Ebine and Ueda, 2009; Richter et al., 2009). Therefore, the TGN/EE is a critical sorting compartment that lies at the intersection of the secretory and endocytic routes.Fine-tuned control of intracellular protein sorting at the TGN/EE is essential for plant development (Geldner et al., 2003; Dhonukshe et al., 2007, 2008; Richter et al., 2007; Kitakura et al., 2011; Wang et al., 2013). An auxin gradient is crucial for pattern formation in plants, whose dynamic maintenance requires the polar localization of auxin efflux carrier PINs through endocytic recycling (Geldner et al., 2003; Blilou et al., 2005; Paciorek et al., 2005; Abas et al., 2006; Jaillais et al., 2006; Dhonukshe et al., 2007; Kleine-Vehn et al., 2008). Receptor-like kinases (RLKs) have also been recognized as major cargos undergoing endocytic trafficking, which are either recycled back to the PM or sent for vacuolar degradation (Geldner and Robatzek, 2008; Irani and Russinova, 2009). RLKs are involved in most if not all developmental processes of plants (De Smet et al., 2009).Intracellular protein sorting relies on sorting signals within cargo proteins and on the molecular machinery that recognizes sorting signals (Boehm and Bonifacino, 2001; Robinson, 2004; Dhonukshe et al., 2007). Adaptor proteins (AP) play a key role (Boehm and Bonifacino, 2001; Robinson, 2004) in the recognition of sorting signals. APs are heterotetrameric protein complexes composed of two large subunits (β and γ/α/δ/ε), a small subunit (σ), and a medium subunit (µ) that is crucial for cargo selection (Boehm and Bonifacino, 2001). APs associate with the cytoplasmic side of secretory and endocytic vesicles, recruiting coat proteins and recognizing sorting signals within cargo proteins for their incorporation into vesicle carriers (Boehm and Bonifacino, 2001). Five APs have been identified so far, classified by their components, subcellular localization, and function (Boehm and Bonifacino, 2001; Robinson, 2004; Hirst et al., 2011). Of the five APs, AP-1 associates with the TGN or recycling endosomes (RE) in yeast and mammals (Huang et al., 2001; Robinson, 2004), mediating the sorting of cargo proteins to compartments of the endosomal-lysosomal system or to the basolateral PM of polarized epithelial cells (Gonzalez and Rodriguez-Boulan, 2009). Knockouts of AP-1 components in multicellular organisms resulted in embryonic lethality (Boehm and Bonifacino, 2001; Robinson, 2004).We show here that the recently identified Arabidopsis µ1 adaptin AP1M2 (Park et al., 2013; Teh et al., 2013) is a key component in the cellular machinery mediating intracellular protein sorting at the TGN/EE. AP1M2 was previously named HAPLESS13 (HAP13), whose mutant allele hap13 showed male gametophytic lethality (Johnson et al., 2004). In recent quests for AP-1 in plants, HAP13/AP1M2 was confirmed as the Arabidopsis µ1 adaptin based on its interaction with other components of the AP-1 complex as well as its localization at the TGN (Park et al., 2013; Teh et al., 2013). A novel mutant allele of HAP13/AP1M2, ap1m2-1, was found to be defective in the intracellular distribution of KNOLLE, leading to defective cytokinesis (Park et al., 2013; Teh et al., 2013). However, it was not clear whether HAP13/AP1M2 mediated other cellular activities and their developmental consequences. Using the same mutant allele, we found that functional loss of HAP13 (hap13-1/ap1m2-1) resulted in a full spectrum of growth defects, suggestive of compromised auxin signaling and of defective RLK signaling. Cell morphogenesis was also disturbed in hap13-1. Importantly, hap13-1 was insensitive to brefeldin A (BFA) washout, indicative of defects in guanine nucleotide exchange factors for ADP-ribosylation factor (ArfGEF)-mediated post-Golgi trafficking. Furthermore, HAP13/AP1M2 showed evolutionarily conserved function during vacuolar fusion, providing additional support to its identity as a µ1 adaptin. These results demonstrate the importance of the Arabidopsis µ1 adaptin for intracellular protein sorting centered on the TGN/EE.     

Arabidopsis 3-Ketoacyl-Coenzyme A Synthase9 Is Involved in the Synthesis of Tetracosanoic Acids as Precursors of Cuticular Waxes,Suberins, Sphingolipids,and Phospholipids

Very-long-chain fatty acids (VLCFAs) with chain lengths from 20 to 34 carbons are involved in diverse biological functions such as membrane constituents, a surface barrier, and seed storage compounds. The first step in VLCFA biosynthesis is the condensation of two carbons to an acyl-coenzyme A, which is catalyzed by 3-ketoacyl-coenzyme A synthase (KCS). In this study, amino acid sequence homology and the messenger RNA expression patterns of 21 Arabidopsis (Arabidopsis thaliana) KCSs were compared. The in planta role of the KCS9 gene, showing higher expression in stem epidermal peels than in stems, was further investigated. The KCS9 gene was ubiquitously expressed in various organs and tissues, including roots, leaves, and stems, including epidermis, silique walls, sepals, the upper portion of the styles, and seed coats, but not in developing embryos. The fluorescent signals of the KCS9::enhanced yellow fluorescent protein construct were merged with those of BrFAD2::monomeric red fluorescent protein, which is an endoplasmic reticulum marker in tobacco (Nicotiana benthamiana) epidermal cells. The kcs9 knockout mutants exhibited a significant reduction in C24 VLCFAs but an accumulation of C20 and C22 VLCFAs in the analysis of membrane and surface lipids. The mutant phenotypes were rescued by the expression of KCS9 under the control of the cauliflower mosaic virus 35S promoter. Taken together, these data demonstrate that KCS9 is involved in the elongation of C22 to C24 fatty acids, which are essential precursors for the biosynthesis of cuticular waxes, aliphatic suberins, and membrane lipids, including sphingolipids and phospholipids. Finally, possible roles of unidentified KCSs are discussed by combining genetic study results and gene expression data from multiple Arabidopsis KCSs.Very-long-chain fatty acids (VLCFAs) are fatty acids of 20 or more carbons in length and are essential precursors of functionally diverse lipids, cuticular waxes, aliphatic suberins, phospholipids, sphingolipids, and seed oils in the Brassicaceae. These lipids are involved in various functions, such as acting as protective barriers between plants and the environment, impermeable barriers to water and ions, energy-storage compounds in seeds, structural components of membranes, and lipid signaling, which is involved in the hypersensitive response (Pollard et al., 2008; Kunst and Samuels, 2009; Franke et al., 2012). VLCFAs are synthesized by the microsomal fatty acid elongase complex, which catalyzes the cyclic addition of a C2 moiety obtained from malonyl-CoA to C16 or C18 acyl-CoA. The fatty acid elongation process has been shown to proceed through a series of four reactions: condensation of the C2 carbon moiety to acyl-CoA by 3-ketoacyl coenzyme A synthase (KCS), reduction of KCS by 3-ketoacyl coenzyme A reductase (KCR), dehydration of 3-hydroxyacyl-CoA by 3-hydroxyacyl-CoA dehydratase (PAS2), and reduction of trans-2,3-enoyl-CoA by trans-2-enoyl-CoA reductase (ECR). Except for KCS isoforms with redundancy, disruption of KCR1, ECR/ECERIFERUM10 (CER10), or PAS2 exhibited severe morphological abnormalities and embryo lethality, suggesting that VLCFA homeostasis is essential for plant developmental processes (Zheng et al., 2005; Bach et al., 2008; Beaudoin et al., 2009).Cuticular waxes that cover plant aerial surfaces are known to be involved in limiting nonstomatal water loss and gaseous exchanges (Boyer et al., 1997; Riederer and Schreiber, 2001), repelling lipophilic pathogenic spores and dust (Barthlott and Neinhuis, 1997), and protecting plants from UV light (Reicosky and Hanover, 1978). VLCFAs that are synthesized in the epidermal cells are either directly used or further modified into aldehydes, alkanes, secondary alcohols, ketones, primary alcohols, and wax esters for the synthesis of cuticular waxes. Reverse genetic analysis and Arabidopsis (Arabidopsis thaliana) epidermal peel microarray analysis (Suh et al., 2005) has enabled the research community to identify the functions of many genes involved in cuticular wax biosynthesis (Kunst and Samuels, 2009): CER1 (Bourdenx et al., 2011; Bernard et al., 2012), WAX2/CER3 (Chen et al., 2003; Rowland et al., 2007; Bernard et al., 2012), and MAH1(Greer et al., 2007; Wen and Jetter, 2009) have been shown to be involved in the decarbonylation pathway to form aldehydes, alkanes, secondary alcohols, and ketones, and acyl-coenzyme A reductase (FAR; Aarts et al., 1997; Rowland et al., 2006) and WSD1 (Li et al., 2008) have been shown to be involved in the decarboxylation pathway for the synthesis of primary alcohols and wax esters. The export of wax precursors to the extracellular space is mediated by a heterodimer of the ATP-binding cassette transporters in the plasma membrane (Pighin et al., 2004; Bird et al., 2007; McFarlane et al., 2010). In addition, glycosylphosphatidylinositol-anchored LTP (LTPG1) and LTPG2 contribute either directly or indirectly to the export of cuticular wax (DeBono et al., 2009; Lee et al., 2009; Kim et al., 2012).VLCFAs that are synthesized in the endodermis of primary roots, seed coats, and the chalaza-micropyle region of seeds are used as precursors for the synthesis of aliphatic suberins. The suberin layer is known to function as a barrier against uncontrolled water, gas, and ion loss and provides protection from environmental stresses and pathogens (Pollard et al., 2008; Franke et al., 2012). For aliphatic suberin biosynthesis, the ω-carbon of the VLCFAs is oxidized by the fatty acyl ω-hydroxylase (Xiao et al., 2004; Li et al., 2007; Höfer et al., 2008; Molina et al., 2008, 2009; Compagnon et al., 2009; Li-Beisson et al., 2009), and the ω-hydroxy VLCFAs are further oxidized into α,ω-dicarboxylic acids by the HOTHEAD-like oxidoreductase (Kurdyukov et al., 2006). α,ω-Dicarboxylic acids are acylated to glycerol-3-P via acyl-CoA:glycerol-3-P acyltransferase (Beisson et al., 2007; Li et al., 2007; Li-Beisson et al., 2009; Yang et al., 2010) or to ferulic acid. In addition, C18, C20, and C22 fatty acids are also reduced by FAR enzymes to primary fatty alcohols, which are a common component in root suberin (Vioque and Kolattukudy, 1997). Finally, the aliphatic suberin precursors are likely to be extensively polymerized and cross linked with the polysaccharides or lignins in the cell wall.In addition, VLCFAs are found in sphingolipids, including glycosyl inositolphosphoceramides, glycosylceramides, and ceramides and phospholipids, such as phosphatidylethanolamine (PE) and phosphatidyl-Ser (PS), which are present in the extraplastidial membrane (Pata et al., 2010; Yamaoka et al., 2011). For sphingolipid biosynthesis, VLCFA-CoAs and Ser are condensed to form 3-keto-sphinganine, which is subsequently reduced to produce sphinganine, a long chain base (LCB). LCBs are known to be further modified by 4-hydroxylation, 4-desaturation, and 8-desaturation (Lynch and Dunn, 2004; Chen et al., 2006, 2012; Pata et al., 2010). The additional VLCFAs are linked with 4-hydroxy LCBs via an amino group to form ceramides (Chen et al., 2008). The presence of VLCFA in sphingolipids may contribute to an increase of their hydrophobicity, membrane leaflet interdigitation, and the transition from a fluid to a gel phase, which is required for microdomain formation. In plants, PS is synthesized from CDP-diacylglycerol and Ser by PS synthase or through an exchange reaction between a phospholipid head group and Ser by a calcium-dependent base-exchange-type PS synthase (Vincent et al., 1999; Yamaoka et al., 2011). PE biosynthesis proceeds through decarboxylation via PS decarboxylase (Nerlich et al., 2007), the phosphoethanolamine transfer from CDP-ethanolamine to diacylglycerol (Kennedy pathway), and the exchange of the head group of PE with Ser via a base-exchange enzyme (Marshall and Kates, 1973). In particular, PS containing a relatively large amount of VLCFAs is enriched in endoplasmic reticulum (ER)-derived vesicles that may function in stabilizing small (70- to 80-nm-diameter) vesicles (Vincent et al., 2001).During the fatty acid elongation process, the first committed step is the condensation of C₂ units to acyl-CoA by KCS. Arabidopsis harbors a large family containing 21 KCS members (Joubès et al., 2008). Characterization of Arabidopsis KCS mutants with defects in VLCFA synthesis revealed in planta roles and substrate specificities (based on differences in carbon chain length and degree of unsaturation) of KCSs. For example, FAE1, a seed-specific condensing enzyme, was shown to catalyze C20 and C22 VLCFA biosynthesis for seed storage lipids (James et al., 1995). KCS6/CER6/CUT1 and KCS5/CER60 are involved in the elongation of fatty acyl-CoAs longer than C28 VLCFA for cuticular waxes in epidermis and pollen coat lipids (Millar et al., 1999; Fiebig et al., 2000; Hooker et al., 2002). KCS20 and KCS2/DAISY are functionally redundant in the two-carbon elongation to C22 VLCFA, which is required for cuticular wax and root suberin biosynthesis (Franke et al., 2009; Lee et al., 2009). When KCS1 and KCS9 were expressed in yeast (Saccharomyces cerevisiae), KCS1 showed broad substrate specificity for saturated and monounsaturated C16 to C24 acyl-CoAs and KCS9 utilized the C16 to C22 acyl-CoAs (Trenkamp et al., 2004; Blacklock and Jaworski, 2006; Paul et al., 2006). Recently, CER2 encoding putative BAHD acyltransferase was reported to be a fatty acid elongase that was involved in the elongation of C28 fatty acids for the synthesis of wax precursors (Haslam et al., 2012).In this study, the expression patterns and subcellular localization of KCS9 were examined, and an Arabidopsis kcs9 mutant was isolated to investigate the roles of KCS9 in planta. Diverse classes of lipids, including cuticular waxes, aliphatic suberins, and sphingolipids, as well as fatty acids in various organs were analyzed from the wild type, the kcs9 mutant, and complementation lines. The combined results of this study revealed that KCS9 is involved in the elongation of C22 to C24 fatty acids, which are essential precursors for the biosynthesis of cuticular waxes, aliphatic suberins, and membrane lipids, including sphingolipids. To the best of our knowledge, this is the first study where a KCS9 isoform involved in sphingolipid biosynthesis was identified.     

NdhV Is a Subunit of NADPH Dehydrogenase Essential for Cyclic Electron Transport in Synechocystis sp. Strain PCC 6803

Two mutants sensitive to heat stress for growth and impaired in NADPH dehydrogenase (NDH-1)-dependent cyclic electron transport around photosystem I (NDH-CET) were isolated from the cyanobacterium Synechocystis sp. strain PCC 6803 transformed with a transposon-bearing library. Both mutants had a tag in the same sll0272 gene, encoding a protein highly homologous to NdhV identified in Arabidopsis (Arabidopsis thaliana). Deletion of the sll0272 gene (ndhV) did not influence the assembly of NDH-1 complexes and the activities of CO₂ uptake and respiration but reduced the activity of NDH-CET. NdhV interacted with NdhS, a ferredoxin-binding subunit of cyanobacterial NDH-1 complex. Deletion of NdhS completely abolished NdhV, but deletion of NdhV had no effect on the amount of NdhS. Reduction of NDH-CET activity was more significant in ΔndhS than in ΔndhV. We therefore propose that NdhV cooperates with NdhS to accept electrons from reduced ferredoxin.Cyanobacterial NADPH dehydrogenase (NDH-1) complexes are localized in the thylakoid membrane (Ohkawa et al., 2001, 2002; Zhang et al., 2004; Xu et al., 2008; Battchikova et al., 2011b) and participate in a variety of bioenergetic reactions, such as respiration, cyclic electron transport around photosystem I (NDH-CET), and CO₂ uptake (Ogawa, 1991; Mi et al., 1992; Ohkawa et al., 2000). Structurally, the cyanobacterial NDH-1 complexes closely resemble energy-converting complex I in eubacteria and the mitochondrial respiratory chain regardless of the absence of homologs of three subunits in cyanobacterial genomes that constitute the catalytically active core of complex I (Friedrich et al., 1995; Friedrich and Scheide, 2000; Arteni et al., 2006). Over the past decade, new subunits of NDH-1 complexes specific to oxygenic photosynthesis have been identified in several cyanobacterial strains. They are NdhM to NdhQ and NdhS (Prommeenate et al., 2004; Battchikova et al., 2005, 2011b; Nowaczyk et al., 2011; Wulfhorst et al., 2014; Zhang et al., 2014; Zhao et al., 2014b, 2015), in addition to NdhL first identified in the cyanobacterium Synechocystis sp. strain PCC 6803 (hereafter Synechocystis 6803) about 20 years ago (Ogawa, 1992). Among them, NdhS possesses a ferredoxin (Fd)-binding motif and was shown to bind Fd, which suggested that Fd is one of the electron donors to NDH-1 complexes (Mi et al., 1995; Battchikova et al., 2011b; Ma and Ogawa, 2015). Deletion of NdhS strongly reduced the activity of NDH-CET but had no effect on respiration and CO₂ uptake (Battchikova et al., 2011b; Ma and Ogawa, 2015). The NDH-CET plays an important role in coping with various environmental stresses regardless of its elusive mechanism. For example, this function can greatly alleviate heat-sensitive growth phenotypes (Wang et al., 2006a; Zhao et al., 2014a). Thus, heat treatment strategy can help in identifying the proteins essential to NDH-CET.Here, a new oxygenic photosynthesis-specific (OPS) subunit NdhV was identified in Synechocystis 6803 with the help of heat treatment strategy, and its deletion did not influence the assembly of NDH-1L and NDH-1MS complexes and the activities of CO₂ uptake and respiration but impaired the NDH-CET activity. We give evidence that NdhV interacts with NdhS and is another component of Fd-binding domain of cyanobacterial NDH-1 complex. A possible role of NdhV on the NDH-CET activity is discussed.     

Overexpression of Arabidopsis Ceramide Synthases Differentially Affects Growth,Sphingolipid Metabolism,Programmed Cell Death,and Mycotoxin Resistance

Ceramide synthases catalyze an N-acyltransferase reaction using fatty acyl-coenzyme A (CoA) and long-chain base (LCB) substrates to form the sphingolipid ceramide backbone and are targets for inhibition by the mycotoxin fumonisin B₁ (FB₁). Arabidopsis (Arabidopsis thaliana) contains three genes encoding ceramide synthases with distinct substrate specificities: LONGEVITY ASSURANCE GENE ONE HOMOLOG1 (LOH1; At3g25540)- and LOH3 (At1g19260)-encoded ceramide synthases use very-long-chain fatty acyl-CoA and trihydroxy LCB substrates, and LOH2 (At3g19260)-encoded ceramide synthase uses palmitoyl-CoA and dihydroxy LCB substrates. In this study, complementary DNAs for each gene were overexpressed to determine the role of individual isoforms in physiology and sphingolipid metabolism. Differences were observed in growth resulting from LOH1 and LOH3 overexpression compared with LOH2 overexpression. LOH1- and LOH3-overexpressing plants had enhanced biomass relative to wild-type plants, due in part to increased cell division, suggesting that enhanced synthesis of very-long-chain fatty acid/trihydroxy LCB ceramides promotes cell division and growth. Conversely, LOH2 overexpression resulted in dwarfing. LOH2 overexpression also resulted in the accumulation of sphingolipids with C16 fatty acid/dihydroxy LCB ceramides, constitutive induction of programmed cell death, and accumulation of salicylic acid, closely mimicking phenotypes observed previously in LCB C-4 hydroxylase mutants defective in trihydroxy LCB synthesis. In addition, LOH2- and LOH3-overexpressing plants acquired increased resistance to FB₁, whereas LOH1-overexpressing plants showed no increase in FB₁ resistance, compared with wild-type plants, indicating that LOH1 ceramide synthase is most strongly inhibited by FB₁. Overall, the findings described here demonstrate that overexpression of Arabidopsis ceramide synthases results in strongly divergent physiological and metabolic phenotypes, some of which have significance for improved plant performance.Ceramides are central intermediates in sphingolipid biosynthesis and mediators of programmed cell death (PCD) in plants (Dunn et al., 2004; Saucedo-García et al., 2011; Ternes et al., 2011a). Ceramides are synthesized by ceramide synthase (or sphingosine N-acyltransferase; EC 2.3.1.24), which catalyzes the formation of an amide linkage between a sphingoid long-chain base (LCB) and a fatty acid using LCB and fatty acyl-CoA substrates (Mullen et al., 2012). The LCB substrate can have two or three hydroxyl groups that are referred to as dihydroxy or trihydroxy LCBs, respectively (Chen et al., 2010). The fatty acyl-CoA substrates typically have chain lengths of C16 or C22 to C26 (Dunn et al., 2004). The latter are referred to as very-long-chain fatty acids (VLCFAs). The ceramide product of ceramide synthase is used primarily as a substrate for the synthesis of either of the two major glycosphingolipids found in plants: glucosylceramide (GlcCer) and glycosyl inositolphosphoceramide (GIPC; Chen et al., 2010). These glycosphingolipids are major structural components of the plasma membrane and other endomembranes of plant cells (Verhoek et al., 1983; Sperling et al., 2005). In this role, they contribute to membrane physical properties that are important for the ability of plant cells to adjust to environmental extremes and to Golgi-mediated protein trafficking of proteins, including cell wall metabolic enzymes and auxin transporters that underlie plant growth (Borner et al., 2005; Markham et al., 2011; Mortimer et al., 2013; Yang et al., 2013). Alternatively, ceramides can be converted to ceramide-1-phosphates by ceramide kinase activity (Liang et al., 2003). The interchange of ceramides between their free and phosphorylated forms has been linked to the regulation of PCD and PCD-associated resistance to pathogens via the hypersensitive response (HR; Liang et al., 2003; Bi et al., 2014; Simanshu et al., 2014).The Arabidopsis (Arabidopsis thaliana) genome contains three ceramide synthase genes denoted LONGEVITY ASSURANCE GENE ONE HOMOLOG1 (LOH1; At3g25540), LOH2 (At3g19260), and LOH3 (At1g13580; Markham et al., 2011; Ternes et al., 2011a). These studies suggest that LOH1 and LOH3 polypeptides are structurally related and catalyze primarily the amidation reaction of trihydroxy LCBs and CoA esters of VLCFAs. The LOH2 polypeptide is more distantly related to LOH1 and LOH3 and catalyzes primarily the condensation of dihydroxy LCBs and C16 fatty acyl-CoAs (Chen et al., 2008; Markham et al., 2011; Ternes et al., 2011a). The ceramide products of LOH1 and LOH3 are most prevalent in GIPC, whereas the ceramide products of LOH2 are more enriched in GlcCer (Markham and Jaworski, 2007; Chen et al., 2008; Ternes et al., 2011b). Similar to plants, the six ceramide synthase isoforms found in humans and mice have distinct specificities for their LCB and acyl-CoA substrates, and these specificities contribute to the formation of complex sphingolipids with differing structures and functions (Venkataraman et al., 2002; Riebeling et al., 2003; Mizutani et al., 2005, 2006; Laviad et al., 2008).In Arabidopsis, LOH1 and LOH3 are partially redundant, but the combined activities of the corresponding polypeptides are essential for plant cell viability, as null double mutants of these genes are lethal (Markham et al., 2011). In contrast, mutants of LOH2 are viable and display no apparent growth phenotype, which brings into question the role of LOH2 ceramide synthase in plant performance (Markham et al., 2011; Ternes et al., 2011a). Overall, these observations indicate that sphingolipids with LOH1-/LOH3-derived trihydroxy LCBs and VLCFA ceramides are essential, but LOH2-derived dihydroxy LCBs and C16 fatty acid ceramides are not required by plant cells. Related to this, LCB C-4 hydroxylase mutants that are deficient in trihydroxy LCBs accumulate elevated amounts of sphingolipids with dihydroxy LCB- and C16 fatty acid-containing ceramides via LOH2 activity (Chen et al., 2008). These mutants are severely impaired in growth and do not transition from vegetative to reproductive growth (Chen et al., 2008).Ceramide synthases are known targets for competitive inhibition by sphingosine analog mycotoxins, including fumonisin B₁ (FB₁) and AAL toxin, produced by pathogenic fungi such as various Fusarium spp. and Alternaria alternata f. sp. lycopersici (Abbas et al., 1994). Inhibition of ceramide synthase results in the accumulation of LCBs that are believed to trigger PCD and result in cytotoxicity (Abbas et al., 1994). In studies of LOH mutants, treatment of Arabidopsis seedlings with FB₁ resulted in not only increases in LCBs but also increases in C16 fatty acid-containing sphingolipids and decreases in VLCFA-containing sphingolipids (Markham et al., 2011; Ternes et al., 2011a). The interpretation of this observation was that FB₁ preferentially inhibits LOH1 and LOH3 ceramide synthases but inhibits LOH2 ceramide synthase to a lesser extent (Markham et al., 2011; Ternes et al., 2011a).Given the findings from Arabidopsis mutants that LOH1 and LOH3 ceramide synthases have distinct substrate specificities and sensitivity to FB₁ relative to LOH2, we hypothesized that the overexpression of each of these ceramide synthases would lead to the production of different sphingolipid compositions as well as different growth phenotypes. This report details experiments designed to test this hypothesis. Among the results presented is a large divergence in the effects of the overexpression of LOH1 and LOH3 versus LOH2 on the growth of Arabidopsis. LOH2 overexpression was also shown to result in sphingolipid compositional, growth, and physiological phenotypes that closely mimic those observed previously in LCB C-4 hydroxylase mutants (Chen et al., 2008).