The biosynthesis of cutin and suberin as an alternative source of enzymes for the production of bio-based chemicals and materials

Oxygenated fatty acids such as ricinoleic acid and vernolic acid can serve in the industry as synthons for the synthesis of a wide range of chemicals and polymers traditionally produced by chemical conversion of petroleum derivatives. Oxygenated fatty acids can also be useful to synthesize specialty chemicals such as cosmetics and aromas. There is thus a strong interest in producing these fatty acids in seed oils (triacylglycerols) of crop species. In the last 15 years or so, much effort has been devoted to isolate key genes encoding proteins involved in the synthesis of oxygenated fatty acids and to express them in the seeds of the model plant Arabidopsis thaliana or crop species. An often overlooked but rich source of enzymes catalyzing the synthesis of oxygenated fatty acids and their esterification to glycerol is the biosynthetic pathways of the plant lipid polyesters cutin and suberin. These protective polymers found in specific tissues of all higher plants are composed of a wide variety of oxygenated fatty acids, many of which have not been reported in seed oils (e.g. saturated ω-hydroxy fatty acids and α,ω-diacids). The purpose of this mini-review is to give an overview of the recent advances in the biosynthesis of cutin and suberin and discuss their potential utility in producing specific oxygenated fatty acids for specialty chemicals. Special emphasis is given to the role played by specific acyltransferases and P450 fatty acid oxidases. The use of plant surfaces as possible sinks for the accumulation of high value-added lipids is also highlighted.     

Regulation of salicylic acid, jasmonic acid and fatty acids in cucumber (Cucumis sativus L.) by spermidine promotes plant growth against salt stress

Phytohormones and fatty acids play a significant role in developmental stages of plant growth and defense against biotic and abiotic stresses. The purpose of this study was to determine the spermidine (Spd)-induced phytohormones and fatty acids changes involve the acclimation of cucumber plants against salt stress. Plants exposed to salt stress had significant reduction in their growth. Exogenously applied Spd increased the shoot length and protein content in salt-stressed plants. The accumulation of total phenol and malondialdehyde was higher in salt-affected plants than in their controls and these detrimental effects were mitigated by Spd treatment. Moreover, salt stress caused a significant increase in salicylic acid (SA) and jasmonic acid (JA); while Spd treatment ameliorated these salt stress effects by reducing SA and JA content. The marked accumulation of total free fatty acid was observed in salt-stressed plants, while the application of Spd to salt-stressed plants reduced the total free fatty acid content. In addition, Spd inhibited the stearic acid, linoleic acid and linolenic acid in salt-stressed plants. The results of current study suggest that exogenous application of Spd-induced phytohormones and fatty acids changes would be a reason for increasing the acclimation of cucumber plants under salt stress condition.     

A mutation in Arabidopsis cytochrome b5 reductase identified by high-throughput screening differentially affects hydroxylation and desaturation

As a model for analyzing the production of novel fatty acids in oilseeds, we used the genetic and molecular techniques available for Arabidopsis to characterize modifying mutations affecting the accumulation of hydroxy fatty acids in the seeds of Arabidopsis plants that express a transgene for the castor bean fatty acid hydroxylase, FAH12. We developed a high-throughput analytical system and used it to identify three complementation classes of mutations with reduced hydroxy fatty acid accumulation from among Arabidopsis M3 seed samples derived from chemical mutagenesis. We identified one of the mutations by positional cloning as a single base pair change in a gene encoding NADH:cytochrome b5 reductase (CBR1, At5g17770). When expressed in yeast, the mutant form of the enzyme was less active and less stable than the wild-type enzyme. Characterization of homozygous mutant lines with and without the FAH12 transgene (FAH12 cbr1-1 and cbr1-1, respectively) indicated that the only detectable consequence of the cbr1-1 mutation was on desaturase and hydroxylase reactions in the developing seed. The leaf and root fatty compositions, as well as the growth, development and seed production of mutant plants were indistinguishable from wild type. Interestingly, while the cbr1-1 mutation reduced the accumulation of hydroxy fatty acids in seeds by 85%, the effects on 18:1 and 18:2 desaturation reactions were much less (<25% and <60%, respectively). These results suggest that there is competition in developing seeds among the several reactions that utilize reduced cytochrome b5.     

Impact of unusual fatty acid synthesis on futile cycling through beta-oxidation and on gene expression in transgenic plants

Arabidopsis expressing the castor bean (Ricinus communis) oleate 12-hydroxylase or the Crepis palaestina linoleate 12-epoxygenase in developing seeds typically accumulate low levels of ricinoleic acid and vernolic acid, respectively. We have examined the presence of a futile cycle of fatty acid degradation in developing seeds using the synthesis of polyhydroxyalkanoate (PHA) from the intermediates of the peroxisomal beta-oxidation cycle. Both the quantity and monomer composition of the PHA synthesized in transgenic plants expressing the 12-epoxygenase and 12-hydroxylase in developing seeds revealed the presence of a futile cycle of degradation of the corresponding unusual fatty acids, indicating a limitation in their stable integration into lipids. The expression profile of nearly 200 genes involved in fatty acid biosynthesis and degradation has been analyzed through microarray. No significant changes in gene expression have been detected as a consequence of the activity of the 12-epoxygenase or the 12-hydroxylase in developing siliques. Similar results have also been obtained for transgenic plants expressing the Cuphea lanceolata caproyl-acyl carrier protein thioesterase and accumulating high amounts of caproic acid. Only in developing siliques of the tag1 mutant, deficient in the accumulation of triacylglycerols and shown to have a substantial futile cycling of fatty acids toward beta-oxidation, have some changes in gene expression been detected, notably the induction of the isocitrate lyase gene. These results indicate that analysis of peroxisomal PHA is a better indicator of the flux of fatty acid through beta-oxidation than the expression profile of genes involved in lipid metabolism.     

植物脂肪酸脱饱和酶特性及转基因研究进展

脂肪酸代谢是有机体的基本代谢之一。植物体内首先合成的是饱和脂肪酸,然后在脂肪酸脱饱和酶作用下形成不饱和脂肪酸。目前已经从很多植物中克隆到了脂肪酸合成相关的酶,并对其功能进行了鉴定。详细介绍了近年来应用基因工程技术对植物油中不饱和脂肪酸含量和组分进行改造所取得的进展,并对其在植物抗性育种中的应用进行了展望。     

Cellular fatty acid uptake: the contribution of metabolism

PURPOSE OF REVIEW: The aim of this review is to highlight the importance of fatty acid metabolism as a major determinant in fatty acid uptake. In particular, we emphasize how the activation, intracellular transport and downstream metabolism of fatty acids influence their uptake into cells. RECENT FINDINGS: Studies examining fatty acid entry into cells have focused primarily on the roles of plasma membrane proteins or the question of passive diffusion. Recent studies, however, strongly suggest that a driving force governing fatty acid uptake is the metabolic demand for fatty acids. Both gain and loss-of-function experiments indicate that fatty acid uptake can be modulated by activation at both the plasma membrane and internal sites, by intracellular fatty acid binding proteins, and by enzymes in synthetic or degradative metabolic pathways. Although the mechanism is not known, it appears that converting fatty acids to acyl-CoAs and downstream metabolic intermediates increases cellular fatty acid uptake, probably by limiting efflux. SUMMARY: Altered fatty acid metabolism and the accumulation of triacylglycerol and lipid metabolites has been strongly associated with insulin resistance and diabetes, but we do not fully understand how the entry of fatty acids into cells is regulated. Future studies of cellular fatty acid uptake should consider the influence of fatty acid metabolism and the possible interactions between fatty acid metabolism or metabolites and fatty acid transport proteins.     

Metabolic engineering of fatty acid biosynthetic pathway in sesame (<Emphasis Type="Italic">Sesamum indicum</Emphasis> L.): assembling tools to develop nutritionally desirable sesame seed oil

Vegetable oils are an essential component of human diet, in terms of their health beneficial roles. Despite their importance, the fatty acid profile of most commonly used edible oil seed crop plants are imbalanced; this skewed ratio of fatty acids in the diet has been shown to be a major reason for the occurrence of cardiovascular and autoimmune diseases. Until recently, it was not possible to exert significant control over the fatty acid composition of vegetable oils derived from different plants. However, the advent of metabolic engineering, knowledge of the genetic networks and regulatory hierarchies in plants have offered novel opportunities to tailor-made the composition of vegetable oils for their optimization in regard to food functionality and dietary requirements. Sesame (Sesamum indicum L.) is one of the ancient oilseed crop in Indian subcontinent but its seed oil is devoid of balanced proportion of ω-6:ω-3 fatty acids. A recent study by our group has shed new lights on metabolic engineering strategies for the purpose of nutritional improvement of sesame seed oil to divert the carbon flux from the production of linoleic acid (C18:2) to α-linolenic acid (C18:3). Apart from that, this review evaluates current understanding of regulation of fatty acid biosynthetic pathways in sesame and attempts to identify the major options of metabolic engineering to produce superior sesame seed oil.     

Enhancing the accumulation of omega-3 long chain polyunsaturated fatty acids in transgenic Arabidopsis thaliana via iterative metabolic engineering and genetic crossing

The synthesis and accumulation of long chain polyunsaturated fatty acids such as eicosapentaenoic acid has previously been demonstrated in the seeds of transgenic plants. However, the obtained levels are relatively low, indicating the need for further studies and the better definition of the interplay between endogenous lipid synthesis and the non-native transgene-encoded activities. In this study we have systematically compared three different transgenic configurations of the biosynthetic pathway for eicosapentaenoic acid, using lipidomic profiling to identify metabolic bottlenecks. We have also used genetic crossing to stack up to ten transgenes in Arabidopsis. These studies indicate several potential approaches to optimize the accumulation of target fatty acids in transgenic plants. Our data show the unexpected channeling of heterologous C20 polyunsaturated fatty acids into minor phospholipid species, and also the apparent negative metabolic regulation of phospholipid-dependent ??6-desaturases. Collectively, this study confirms the benefits of iterative approaches to metabolic engineering of plant lipid synthesis.     

Polyhydroxyalkanoate synthesis in transgenic plants as a new tool to study carbon flow through beta-oxidation

Transgenic plants producing peroxisomal polyhydroxy- alkanoate (PHA) from intermediates of fatty acid degradation were used to study carbon flow through the beta-oxidation cycle. Growth of transgenic plants in media containing fatty acids conjugated to Tween detergents resulted in an increased accumulation of PHA and incorporation into the polyester of monomers derived from the beta-oxidation of these fatty acids. Tween-laurate was a stronger inducer of beta-oxidation, as measured by acyl-CoA oxidase activity, and a more potent modulator of PHA quantity and monomer composition than Tween-oleate. Plants co-expressing a peroxisomal PHA synthase with a capryl-acyl carrier protein thioesterase from Cuphea lanceolata produced eightfold more PHA compared to plants expressing only the PHA synthase. PHA produced in double transgenic plants contained mainly saturated monomers ranging from 6 to 10 carbons, indicating an enhanced flow of capric acid towards beta-oxidation. Together, these results support the hypothesis that plant cells have mechanisms which sense levels of free or esterified unusual fatty acids, resulting in changes in the activity of the beta-oxidation cycle as well as removal and degradation of these unusual fatty acids through beta-oxidation. Such enhanced flow of fatty acids through beta-oxidation can be utilized to modulate the amount and composition of PHA produced in transgenic plants. Furthermore, synthesis of PHAs in plants can be used as a new tool to study the quality and relative quantity of the carbon flow through beta-oxidation as well as to analyse the degradation pathway of unusual fatty acids.     

Biosynthesis of very-long-chain polyunsaturated fatty acids in transgenic oilseeds: constraints on their accumulation

Omega6- and omega3-polyunsaturated C20 fatty acids represent important components of the human diet. A more regular consumption and an accordingly sustainable source of these compounds are highly desirable. In contrast with the very high levels to which industrial fatty acids have to be enriched in plant oils for competitive use as chemical feedstocks, much lower percentages of very-long-chain polyunsaturated fatty acids (VLCPUFA) in edible plant oils would satisfy nutritional requirements. Seed-specific expression in transgenic tobacco (Nicotiana tabacum) and linseed (Linum usitatissimum) of cDNAs encoding fatty acyl-desaturases and elongases, absent from all agronomically important plants, resulted in the very high accumulation of Delta6-desaturated C18 fatty acids and up to 5% of C20 polyunsaturated fatty acids, including arachidonic and eicosapentaenoic acid. Detailed lipid analyses of developing seeds from transgenic plants were interpretated as indicating that, after desaturation on phosphatidylcholine, Delta6-desaturated products are immediately channeled to the triacylglycerols and effectively bypass the acyl-CoA pool. Thus, the lack of available Delta6-desaturated acyl-CoA substrates in the acyl-CoA pool limits the synthesis of elongated C20 fatty acids and disrupts the alternating sequence of lipid-linked desaturations and acyl-CoA dependent elongations. As well as the successful production of VLCPUFA in transgenic oilseeds and the identification of constraints on their accumulation, our results indicate alternative strategies to circumvent this bottleneck.     

Role of acetyl-coenzyme A synthetase in leaves of Arabidopsis thaliana

Acetyl-coenzyme A synthetase (ACS) is a plastidic enzyme that forms acetyl-coenzyme A (acetyl-CoA) from acetate and coenzyme A using the energy from ATP. Traditionally it has been thought to be the major source for the production of acetyl-CoA destined for fatty acid formation. Recent work suggested that the accumulation of lipids in developing Arabidopsis seeds was more closely correlated with the expression of pyruvate dehydrogenase complex than with the expression of ACS, suggesting that most of the carbon for fatty acid formation in the plastids of seeds comes from pyruvate rather than from acetate. To explore the role of this enzyme, Arabidopsis plants with altered amounts of ACS were generated by overexpressing its cDNA in either the sense or the antisense configuration. The resulting plants had in vitro enzyme activities that ranged from about 5% to over 400% of wild-type levels. The rate of [1-14C]acetate conversion into fatty acids was closely related to the in vitro ACS activity, showing that the amount of enzyme clearly limited the capacity of leaves to convert exogenous acetate to fatty acids. There was, however, no relationship between the ACS level and the capacity of the plants to incorporate 14CO2 into 14C-labeled fatty acids. These data strongly support the idea that, although plants can convert acetate into fatty acids, relatively little carbon moves through this pathway under normal conditions.     

Manipulation of galactolipid Fatty Acid composition with substituted pyridazinones

The fatty acid composition of the major lipids of the chloroplast membranes, the mono- and digalactosyl diglycerides, can be definably altered with various substituted pyridazinones. Galactolipid fatty acid composition of wheat (Triticum aestivum L.) can be altered so that there is a decrease in linolenic acid accompanied by an increase in linoleic acid without a shift in the relative proportion of saturated to unsaturated fatty acids; the fatty acid composition can be shifted toward a higher proportion of saturated fatty acids; or the fatty acid composition of the monogalactosyl diglycerides can be altered in preference to the digalactosyl diglycerides. Also, the light-mediated parallel accumulation of chlorophyll and linolenic acid can be separated with a substituted pyridazinone. The substituted pyridazinones may be useful tools in clarifying the role the galactolipids and their component fatty acids play in the structure and function of chloroplast membranes in higher plants.     

Increased flow of fatty acids toward beta-oxidation in developing seeds of Arabidopsis deficient in diacylglycerol acyltransferase activity or synthesizing medium-chain-length fatty acids

Synthesis of polyhydroxyalkanoates (PHAs) from intermediates of fatty acid beta-oxidation was used as a tool to study fatty acid degradation in developing seeds of Arabidopsis. Transgenic plants expressing a peroxisomal PHA synthase under the control of a napin promoter accumulated PHA in developing seeds to a final level of 0. 06 mg g(-1) dry weight. In plants co-expressing a plastidial acyl-acyl carrier protein thioesterase from Cuphea lanceolata and a peroxisomal PHA synthase, approximately 18-fold more PHA accumulated in developing seeds. The proportion of 3-hydroxydecanoic acid monomer in the PHA was strongly increased, indicating a large flow of capric acid toward beta-oxidation. Furthermore, expression of the peroxisomal PHA synthase in an Arabidopsis mutant deficient in the enzyme diacylglycerol acyltransferase resulted in a 10-fold increase in PHA accumulation in developing seeds. These data indicate that plants can respond to the inadequate incorporation of fatty acids into triacylglycerides by recycling the fatty acids via beta-oxidation and that a considerable flow toward beta-oxidation can occur even in a plant tissue primarily devoted to the accumulation of storage lipids.     

Cold-induced accumulation of ω-3 polyunsaturated fatty acid in a liverwort, Marchantia polymorpha L

The liverwort Marchantia polymorpha L. synthesizes various long-chain polyunsaturated fatty acids including arachidonic acid and eicosapentaenoic acid, neither of which is produced by higher plants. Here we report the effects of temperature on long-chain polyunsaturated fatty acid accumulation in the liverwort. The accumulation of ω-3 polyunsaturated fatty acids increased significantly as the growth temperature decreased. Specifically, the relative content of eicosapentaenoic acid to total fatty acids at 5 °C was approximately 3-fold higher than at 25 °C. On the other hand, the accumulation of ω-6 polyunsaturated fatty acids decreased at low temperatures. An analysis of gene expression indicated that the mRNA of the MpFAD3 gene for ER ω-3 desaturase increased significantly at 5 °C. These results indicate that in the liverwort the n-3 pathway was enhanced at low temperature, mainly via expression of the cold-induced ω-3 desaturase gene, leading to increased accumulation of eicosapentaenoic acid.     

Triacylglycerols in prokaryotic microorganisms

Triacylglycerols (TAG) are fatty acid triesters of glycerol; there are diverse types of TAG with different properties depending on their fatty acid composition. The occurrence of TAG as reserve compounds is widespread among eukaryotic organisms such as yeast, fungi, plants and animals, whereas occurrence of TAG in bacteria has only rarely been described. However, accumulation of TAG seems to be widespread among bacteria belonging to the actinomycetes group, such as species of Mycobacterium, Streptomyces, Rhodococcus and Nocardia. Fatty acids in acylglycerols in cells of Rhodococcus opacus PD630 accounted for up to 87% of the cellular dry weight. TAG biosynthesis, justifying an oleaginous status, seems to be restricted mainly to this group of bacteria, but occurs to a minor extent also in a few other bacteria. The compositions and structures of bacterial TAG vary considerably depending on the microorganism and on the carbon source, and unusual acyl moieties, such as phenyldecanoic acid and 4,8,12 trimethyl tridecanoic acid, are also included. The principal function of bacterial TAG seems to be as a reserve compound. Other functions that have been discussed include regulation of cellular membrane fluidity by keeping unusual fatty acids away from membrane phospholipids, or acting as a sink for reducing equivalents. In recent years, basic aspects of the physiology and biochemistry of bacterial TAG accumulation, and the molecular biology of the lipid inclusion bodies have been reported. TAG are used for nutritional, therapeutic and pharmaceutical purposes and serve as a source of oleochemicals.     

Industrial oils from transgenic plants

Unusual fatty acids that have useful industrial properties occur widely in the seed oils of many non-agronomic plant species. Researchers are attempting to use biotechnology to produce high levels of these fatty acids in the seeds of existing crop plants. cDNAs for a wide variety of unusual fatty acid biosynthetic enzymes have been identified, particularly through the use of expressed sequence tags. However, it has not yet been possible to use these cDNAs to produce large amounts of unusual fatty acids in seeds of transgenic plants. This difficulty points to the need for a greater understanding of fatty acid metabolism in oilseeds.     

Fatty-acid-induced release of manganes from chloroplasts

The effect of both endogenous and exogenous unsaturated free fatty acids on manganese release from chloroplasts of chill-resistant (spinach) and chill-sensitive (tomato, bean) plants was studied. The level of endogenous free fatty acids increased 2–3-fold during cold and dark storage of leaves of chill-sensitive plants and was accompanied by depletion of about 60% of total chloroplast manganese content. Similar effects were observed when accumulation of free fatty acids in chloroplasts was achieved by storage of growing tomato plants for a few days in the dark at room temperature. In contrast, the cold and dark treatment of leaves of chill-resistant plant (spinach) affected neither free fatty acid, manganese levels nor Hill-reaction activity in chloroplasts. Incubation of chloroplasts of both chill-sensitive and chill-resistant plants with bean leaf galactolipase resulted in an accumulation of free fatty acids and a release of approx. 60% of total manganese content. The same amount of total manganese content was released following 3 h incubation of chloroplasts with linolenic acid at fatty acid/chlorophyll ratio (w/w, 2:1–10:1). The efficiency of C₁₈ unsaturated fatty acids/linolenic, linoleic, oleic on manganese release from chloroplasts was established in decreasing order C_18:3 > C_18:2 > C_18:1. The results indicate that the inhibitory effect of both endogenous and exogenous fatty acids on Hill reaction depends on the release from chloroplasts of functionally active, loosely bound manganese. Thus, similarly to both Tris and hydroxylamine treatments of chloroplasts, the incubation of chloroplast preparations with unsaturated fatty acids may be a useful tool for manganese depletion of chloroplasts.