AKR2A interacts with KCS1 to improve VLCFAs contents and chilling tolerance of Arabidopsis thaliana

Arabidopsis thaliana AKR2A plays an important role in plant responses to cold stress. However, its exact function in plant resistance to cold stress remains unclear. In the present study, we found that the contents of very long‐chain fatty acids (VLCFAs) in akr2a mutants were decreased, and the expression level of KCS1 was also reduced. Overexpression of KCS1 in the akr2a mutants could enhance VLCFAs contents and chilling tolerance. Yeast‐2‐hybrid and bimolecular fluorescence complementation (BIFC) results showed that the transmembrane motif of KCS1 interacts with the PEST motif of AKR2A both in vitro and in vivo. Overexpression of KCS1 in akr2a mutants rescued akr2a mutant phenotypes, including chilling sensitivity and a decrease of VLCFAs contents. Moreover, the transgenic plants co‐overexpressing AKR2A and KCS1 exhibited a greater chilling tolerance than the plants overexpressing AKR2A or KCS1 alone, as well as the wild‐type. AKR2A knockdown and kcs1 knockout mutants showed the worst performance under chilling conditions. These results indicate that AKR2A is involved in chilling tolerance via an interaction with KCS1 to affect VLCFA biosynthesis in Arabidopsis.     

Misexpression of FATTY ACID ELONGATION1 in the Arabidopsis Epidermis Induces Cell Death and Suggests a Critical Role for Phospholipase A2 in This Process

Very-long-chain fatty acids (VLCFAs) are important functional components of various lipid classes, including cuticular lipids in the higher plant epidermis and lipid-derived second messengers. Here, we report the characterization of transgenic Arabidopsis thaliana plants that epidermally express FATTY ACID ELONGATION1 (FAE1), the seed-specific β-ketoacyl-CoA synthase (KCS) catalyzing the first rate-limiting step in VLCFA biosynthesis. Misexpression of FAE1 changes the VLCFAs in different classes of lipids but surprisingly does not complement the KCS fiddlehead mutant. FAE1 misexpression plants are similar to the wild type but display an essentially glabrous phenotype, owing to the selective death of trichome cells. This cell death is accompanied by membrane damage, generation of reactive oxygen species, and callose deposition. We found that nuclei of arrested trichome cells in FAE1 misexpression plants cell-autonomously accumulate high levels of DNA damage, including double-strand breaks characteristic of lipoapoptosis. A chemical genetic screen revealed that inhibitors of KCS and phospholipase A2 (PLA2), but not inhibitors of de novo ceramide biosynthesis, rescue trichome cells from death. These results support the functional role of acyl chain length of fatty acids and PLA2 as determinants for programmed cell death, likely involving the exchange of VLCFAs between phospholipids and the acyl-CoA pool.     

Natural Variation in Seed Very Long Chain Fatty Acid Content Is Controlled by a New Isoform of KCS18 in Arabidopsis thaliana

Oil from oleaginous seeds is mainly composed of triacylglycerols. Very long chain fatty acids (VLCFAs) are major constituents of triacylglycerols in many seed oils and represent valuable feedstock for industrial purposes. To identify genetic factors governing natural variability in VLCFA biosynthesis, a quantitative trait loci (QTL) analysis using a recombinant inbred line population derived from a cross between accessions Bay-0 and Shahdara was performed in Arabidopsis thaliana. Two fatty acid chain length ratio (CLR) QTL were identified, with one major locus, CLR.2, accounting for 77% of the observed phenotypic variation. A fine mapping and candidate gene approach showed that a key enzyme of the fatty acid elongation pathway, the β-ketoacyl-CoA synthase 18 (KCS18), was responsible for the CLR.2 QTL detected between Bay-0 and Shahdara. Association genetics and heterologous expression in yeast cells identified a single point mutation associated with an alteration of KCS18 activity, uncovering the molecular bases for the modulation of VLCFA content in these two natural populations of Arabidopsis. Identification of this kcs18 mutant with altered activity opens new perspectives for the modulation of oil composition in crop plants.     

Up‐regulation of GhTT2‐3A in cotton fibres during secondary wall thickening results in brown fibres with improved quality

Brown cotton fibres are the most widely used naturally coloured raw materials for the eco‐friendly textile industry. Previous studies have indicated that brown fibre pigments belong to proanthocyanidins (PAs) or their derivatives, and fibre coloration is negatively associated with cotton productivity and fibre quality. To date, the molecular basis controlling the biosynthesis and accumulation of brown pigments in cotton fibres is largely unknown. In this study, based on expressional and transgenic analyses of cotton homologs of ArabidopsisPA regulator TRANSPARENT TESTA 2 (TT2) and fine‐mapping of the cotton dark‐brown fibre gene (Lc1), we show that a TT2 homolog, GhTT2‐3A, controls PA biosynthesis and brown pigmentation in cotton fibres. We observed that GhTT2‐3A activated GhbHLH130D, a homolog of ArabidopsisTT8, which in turn synergistically acted with GhTT2‐3A to activate downstream PA structural genes and PA synthesis and accumulation in cotton fibres. Furthermore, the up‐regulation of GhTT2‐3A in fibres at the secondary wall‐thickening stage resulted in brown mature fibres, and fibre quality and lint percentage were comparable to that of the white‐fibre control. The findings of this study reveal the regulatory mechanism controlling brown pigmentation in cotton fibres and demonstrate a promising biotechnological strategy to break the negative linkage between coloration and fibre quality and/or productivity.     

Combinatorial Effects of Fatty Acid Elongase Enzymes on Nervonic Acid Production in Camelina sativa

Very long chain fatty acids (VLCFAs) with chain lengths of 20 carbons and longer provide feedstocks for various applications; therefore, improvement of VLCFA contents in seeds has become an important goal for oilseed enhancement. VLCFA biosynthesis is controlled by a multi-enzyme protein complex referred to as fatty acid elongase, which is composed of β-ketoacyl-CoA synthase (KCS), β-ketoacyl-CoA reductase (KCR), β-hydroxyacyl-CoA dehydratase (HCD) and enoyl reductase (ECR). KCS has been identified as the rate-limiting enzyme, but little is known about the involvement of other three enzymes in VLCFA production. Here, the combinatorial effects of fatty acid elongase enzymes on VLCFA production were assessed by evaluating the changes in nervonic acid content. A KCS gene from Lunaria annua (LaKCS) and the other three elongase genes from Arabidopsis thaliana were used for the assessment. Five seed-specific expressing constructs, including LaKCS alone, LaKCS with AtKCR, LaKCS with AtHCD, LaKCS with AtECR, and LaKCS with AtKCR and AtHCD, were transformed into Camelina sativa. The nervonic acid content in seed oil increased from null in wild type camelina to 6-12% in LaKCS-expressing lines. However, compared with that from the LaKCS-expressing lines, nervonic acid content in mature seeds from the co-expressing lines with one or two extra elongase genes did not show further increases. Nervonic acid content from LaKCS, AtKCR and AtHCD co-expressing line was significantly higher than that in LaKCS-expressing line during early seed development stage, while the ultimate nervonic acid content was not significantly altered. The results from this study thus provide useful information for future engineering of oilseed crops for higher VLCFA production.     

Very-long-chain fatty acid biosynthesis is controlled through the expression and specificity of the condensing enzyme

The Arabidopsis FATTY ACID ELONGATION1 (FAE1) gene encodes a putative seed-specific condensing enzyme. It is the first of four enzyme activities that comprise the microsomal fatty acid elongase (FAE) involved in the biosynthesis of very-long-chain fatty acids (VLCFAs). FAE1 has been expressed in yeast and in tissues of Arabidopsis and tobacco, where significant quantities of VLCFAs are not found. The introduction of FAE1 alone in these systems is sufficient for the production of VLCFAs, for wherever FAE1 was expressed, VLCFAs accumulated. These results indicate that FAE1 is the rate-limiting enzyme for VLCFA biosynthesis in Arabidopsis seed, because introduction of extra copies of FAE1 resulted in higher levels of the VLCFAs. Furthermore, the condensing enzyme is the activity of the elongase that determines the acyl chain length of the VLCFAs produced. In contrast, it appears that the other three enzyme activities of the elongase are found ubiquitously throughout the plant, are not rate-limiting and play no role in the control of VLCFA synthesis. The ability of yeast containing FAE1 to synthesize VLCFAs suggests that the expression and the acyl chain length specificity of the condensing enzyme, along with the apparent broad specificities of the other three FAE activities, may be a universal eukaryotic mechanism for regulating the amounts and acyl chain length of VLCFAs synthesized.     

Very-Long-Chain Fatty Acids Are Involved in Polar Auxin Transport and Developmental Patterning in Arabidopsis

Very-long-chain fatty acids (VLCFAs) are essential for many aspects of plant development and necessary for the synthesis of seed storage triacylglycerols, epicuticular waxes, and sphingolipids. Identification of the acetyl-CoA carboxylase PASTICCINO3 and the 3-hydroxy acyl-CoA dehydratase PASTICCINO2 revealed that VLCFAs are important for cell proliferation and tissue patterning. Here, we show that the immunophilin PASTICCINO1 (PAS1) is also required for VLCFA synthesis. Impairment of PAS1 function results in reduction of VLCFA levels that particularly affects the composition of sphingolipids, known to be important for cell polarity in animals. Moreover, PAS1 associates with several enzymes of the VLCFA elongase complex in the endoplasmic reticulum. The pas1 mutants are deficient in lateral root formation and are characterized by an abnormal patterning of the embryo apex, which leads to defective cotyledon organogenesis. Our data indicate that in both tissues, defective organogenesis is associated with the mistargeting of the auxin efflux carrier PIN FORMED1 in specific cells, resulting in local alteration of polar auxin distribution. Furthermore, we show that exogenous VLCFAs rescue lateral root organogenesis and polar auxin distribution, indicating their direct involvement in these processes. Based on these data, we propose that PAS1 acts as a molecular scaffold for the fatty acid elongase complex in the endoplasmic reticulum and that the resulting VLCFAs are required for polar auxin transport and tissue patterning during plant development.     

Increased content of very-long-chain fatty acids in the lipids of halophyte vegetative organs

Qualitative and quantitative compositions of esterified fatty acids (FAs) in the total lipids from the leaves, shoots, and roots of halophile plants, such as suaeda (Suaeda altissima), samphire (Salicornia europaea), and wormwood (Artemisia lerchiana), collected in their natural environments were estimated by GLC techniques. It was shown that the vegetative organs of these halophytes contained 24 FA species, and 16 of them were tentatively identified as the very-long-chain FAs (VLCFAs). There were four VLCFA groups, viz. C₂₀, C₂₁, C₂₂, and C₂₃, each including saturated, mono-, and diunsaturated components; C₂₄ and C₂₅ FAs were also present. The concentration of VLCFAs in the total FAs comprised 4–64%. In vegetative organs of higher plants not subjected to genetic transformation, such a high VLCFA content was found for the first time. Saturated and even-numbered components predominated among the VLCFAs, and the roots exceeded severalfold the above-ground organs in the total VLCFA content. Possible pathways of VLCFA biosynthesis in plants, VLCFA content in the vegetative tissues, and the physiological role of membrane lipid FA composition in the plant salt metabolism are discussed.     

Biochemical characterization of Sinorhizobium meliloti mutants reveals gene products involved in the biosynthesis of the unusual lipid A very long-chain fatty acid

Sinorhizobium meliloti forms a symbiosis with the legume alfalfa, whereby it differentiates into a nitrogen-fixing bacteroid. The lipid A species of S. meliloti are modified with very long-chain fatty acids (VLCFAs), which play a central role in bacteroid development. A six-gene cluster was hypothesized to be essential for the biosynthesis of VLCFA-modified lipid A. Previously, two cluster gene products, AcpXL and LpxXL, were found to be essential for S. meliloti lipid A VLCFA biosynthesis. In this paper, we show that the remaining four cluster genes are all involved in lipid A VLCFA biosynthesis. Therefore, we have identified novel gene products involved in the biosynthesis of these unusual lipid modifications. By physiological characterization of the cluster mutant strains, we demonstrate the importance of this gene cluster in the legume symbiosis and for growth in the absence of salt. Bacterial LPS species modified with VLCFAs are substantially less immunogenic than Escherichia coli LPS species, which lack VLCFAs. However, we show that the VLCFA modifications do not suppress the immunogenicity of S. meliloti LPS or affect the ability of S. meliloti to induce fluorescent plant defense molecules within the legume. Because VLCFA-modified lipids are produced by other rhizobia and mammalian pathogens, these findings will also be important in understanding the function and biosynthesis of these unusual fatty acids in diverse bacterial species.     

Inhibition of saturated very-long-chain fatty acid biosynthesis by mefluidide and perfluidone,selective inhibitors of 3-ketoacyl-CoA synthases

The trifluoromethanesulphonanilides mefluidide and perfluidone are used in agriculture as plant growth regulators and herbicides. Despite the fact that mefluidide and perfluidone have been investigated experimentally for decades, their mode of action is still unknown. In this study, we used a cascade approach of different methods to clarify the mode of action and target site of mefluidide and perfluidone. Physiological profiling using an array of biotests and metabolic profiling in treated plants of Lemna paucicostata suggested a common mode of action in very-long-chain fatty acid (VLCFA) synthesis similar to the known 3-ketoacyl-CoA synthase (KCS) inhibitor metazachlor. Detailed analysis of fatty acid composition in Lemna plants showed a decrease of saturated VLCFAs after treatment with mefluidide and perfluidone. To study compound effects on enzyme level, recombinant KCSs from Arabidopsis thaliana were expressed in Saccharomyces cerevisiae. Enzyme activities of seven KCS proteins from 17 tested were characterized by their fatty acid substrate and product spectrum. For the KCS CER6, the VLCFA product spectrum in vivo, which consists of tetracosanoic acid, hexacosanoic acid and octacosanoic acid, is reported here for the first time. Similar to metazachlor, mefluidide and perfluidone were able to inhibit KCS1, CER6 and CER60 enzyme activities in vivo. FAE1 and KCS2 were inhibited by mefluidide only slightly, whereas metazachlor and perfluidone were strong inhibitors of these enzymes with IC₅₀ values in μM range. This suggests that KCS enzymes in VLCFA synthesis are the primary herbicide target of mefluidide and perfluidone.     

A jojoba beta-Ketoacyl-CoA synthase cDNA complements the canola fatty acid elongation mutation in transgenic plants.

beta-Ketoacyl-coenzyme A (CoA) synthase (KCS) catalyzes the condensation of malonyl-CoA with long-chain acyl-CoA. This reaction is the initial step of the microsomal fatty acyl-CoA elongation pathway responsible for formation of very long chain fatty acids (VLCFAs, or fatty acids with chain lengths > 18 carbons). Manipulation of this pathway is significant for agriculture, because it is the basis of conversion of high erucic acid rapeseed into canola. High erucic acid rapeseed oil, used as an industrial feedstock, is rich in VLCFAs, whereas the edible oil extracted from canola is essentially devoid of VLCFAs. Here, we report the cloning of a cDNA from developing jojoba embryos involved in microsomal fatty acid elongation. The jojoba cDNA is homologous to the recently cloned Arabidopsis FATTY ACID ELONGATION1 (FAE1) gene that has been suggested to encode KCS. We characterize the jojoba enzyme and present biochemical data indicating that the jojoba cDNA does indeed encode KCS. Transformation of low erucic acid rapeseed with the jojoba cDNA restored KCS activity to developing embryos and altered the transgenic seed oil composition to contain high levels of VLCFAs. The data reveal the key role KCS plays in determining the chain lengths of fatty acids found in seed oils.     

Chloroacetamides affect the plasma membrane

In the present study membrane fatty acids were analyzed to find a link between the biosynthesis inhibition of very-long-chain fatty acids and the phytotoxic effects of herbicidal chloroacetamides. Accordingly, we have isolated membranes of cucumber seedlings (Cucumis sativus) by two-phase partitioning and analyzed their fatty acid content. Saturated VLCFAs ranging from C20 to C26 were found in high amounts (22%) in the plasma membrane fraction. Non-modified VLCFAs were predominantly present in phospholipids, while saturated 2-hydroxylated VLCFAs were identified in cerebrosides. Treatment of intact seedlings with chloroacetamides markedly reduced the VLCFA content in the plasma membrane. This result could be specified by fatty-acid labeling using [14C]malonate as a substrate for fatty acid elongation. De novo incorporation of VLCFAs into the plasma membrane and into microsomal membranes, respectively, was severely impaired by chloroacetamides with I50 values between 10 to 100 nM. These results confirm the previous finding that chloroacetamides inhibit VLCFA biosynthesis localized in the microsomes (B?ger et al., Pest Manage. Sci. 56, 497-508, 2000). The direct consequence of this inhibition is a strong decrease of VLCFAs required as constituents of the plasma membrane and the substitution by shorter acyl chains. Apparently, physical properties and function of the plasma membrane are affected eventually leading to death of the plant.     

The impact of reducing fatty acid desaturation on the composition and thermal stability of rapeseed oil

Oilseed rape (Brassica napus) is the third largest source of vegetable oil globally. In addition to food uses, there are industrial applications that exploit the ability of the species to accumulate the very‐long‐chain fatty acid (VLCFA) erucic acid in its seed oil, controlled by orthologues of FATTY ACID ELONGASE 1 (Bna.FAE1.A8 and Bna.FAE1.C3). The proportion of polyunsaturated fatty acids (PUFAs) in rapeseed oil is predicted to affect its thermal stability and is controlled by orthologues of FATTY ACID DESATURASE 2, particularly Bna.FAD2.C5. Our aim was to develop rapeseed lines combining high erucic and low PUFA characters and to assess the impact on thermal stability of the oil they produce. The new type of rapeseed oil (high erucic low polyunsaturate; HELP) contained a substantially greater proportion of erucic acid (54%) compared with high erucic rapeseed oil (46%). Although the total VLCFA content was greater in oil from HELP lines (64%) than from high erucic rapeseed (57%), analysis of triacylglycerol composition showed negligible incorporation of VLCFAs into the sn‐2 position. Rancimat analysis showed that the thermal stability of rapeseed oil was improved greatly as a consequence of reduction of PUFA content, from 3.8 and 4.2 h in conventional low erucic and high erucic rapeseed oils, respectively, to 11.3 and 16.4 h in high oleic low PUFA (HOLP) and HELP oils, respectively. Our results demonstrate that engineering of the lipid biosynthetic pathway of rapeseed, using traditional approaches, enables the production of renewable industrial oils with novel composition and properties.     

Very long chain fatty acid and lipid signaling in the response of plants to pathogens

Recent findings indicate that lipid signaling is essential for plant resistance to pathogens. Besides oxylipins and unsaturated fatty acids known to play important signaling functions during plant-pathogen interactions, the very long chain fatty acid (VLCFA) biosynthesis pathway has been recently associated to plant defense through different aspects. VLCFAs are indeed required for the biosynthesis of the plant cuticle and the generation of sphingolipids. Elucidation of the roles of these lipids in biotic stress responses is the result of the use of genetic approaches together with the identification of the genes/proteins involved in their biosynthesis. This review focuses on recent observations which revealed the complex function of the cuticle and cuticle-derived signals, and the key role of sphingolipids as bioactive molecules involved in signal transduction and cell death regulation during plant-pathogen interactions.Key words: very long chain fatty acids (VLCFAs), plant-pathogen interactions, lipid signaling, sphingolipids, epicuticular waxes, lipid rafts, cuticle, plant defense