Structure,chromosomal location and expression of a rice gene encoding the microsome ω-3 fatty acid desaturase

The -3 fatty acid desaturases are membrane-bound enzymes catalyzing the conversion of linoleic acid to linolenic acid in lipids, and are located both in the microsome and plastid envelopes as two different isoforms. A cDNA encoding the microsome -3 fatty acid desaturase (OsFAD3) and the corresponding genomic clone were isolated from rice (Oryza sativa L.). The OsFAD3 gene was composed of 8 exons and 7 introns. A microsatellite was present in the second exon of the OsFAD3 gene, showing polymorphism between Indica and Japonica rice varieties. The mapping of this microsatellite showed that the OsFAD3 gene was located on chromosome 11. Expression of the OsFAD3 cDNA in tobacco hairy root tissues and subsequent analysis of fatty acid compositions demonstrated the activity of the microsome -3 fatty acid desaturase. The OsFAD3 mRNA was abundant in root tissues, but was hardly detectable in leaves. In root tissues, a high level of the OsFAD3 mRNA was observed at 15 °C and 20 °C, with its level decreasing markedly at temperatures below 10 °C. The accumulation of the OsFAD3 mRNA in leaf tissues remained at quite low levels, both at normal growth temperatures and at chilling temperatures. Similar temperature responses of the OsFAD3 gene were observed both in chilling- tolerant and in chilling-intolerant rice cultivars.     

A tobacco microsomal ω-3 fatty acid desaturase gene increases the linolenic acid content in transgenic sweet potato (Ipomoea batatas)

A tobacco microsomal P-3 fatty acid desaturase gene (NtFAD3) under the control of the CaMV 35S promoter or an improved CaMV 35S promoter (El2Q) was introduced into sweet potato. Transformed sweet potato plants were obtained from embryogenic calli following Agrobacterium tumefaciens-mediated transformation. The transgenic plants grew normally to form storage roots and showed properties similar to those of the non-transgenic plants. The fatty acid composition in the transgenic line with a NtFAD3 gene driven by the CaMV 35S promoter was similar to that in the non-transformant. However, in the transgenic line that had a NtFAD3 gene driven by the El2Q promoter, linoleic acid (18:2) and linolenic acid (18:3) contents were 47.7 mol% and 24.8 mol%, respectively, which were significantly different from the 53.6 mol% and 11.3 mol%, respectively, in the non-transformant. The NtFAD3 gene driven by the El2Q promoter was expressed more strongly than that driven by the CaMV 35S promoter, thereby increasing the linolenic acid content in the transgenic sweet potato plants.     

Hormonal regulation of tissue-specific ectopic expression of an Arabidopsis endoplasmic reticulum-type ω-3 fatty acid desaturase (FAD3) gene

A common feature of the membrane lipids of higher plants is a large content of polyunsaturated fatty acids, which typically consist of dienoic and trienoic fatty acids. Two types of omega-3 fatty acid desaturase. which are present in the plastids and in the endoplasmic reticulum (ER), respectively, are responsible for the conversion of dienoic to trienoic fatty acids. To establish a system for investigating the tissue-specific, and hor-mone-regulated expression of the ER-type desaturase gene (FAD3), transgenic plants of Arabidopsis thaliana (L.) Heynh. containing the firefly luciferase gene (LUC) fused to the FAD3 promoter (FAD3::LUC) were constructed. At different times during plant development, FAD3::LUC was actively expressed at two major sites, the vegetative shoot meristem and the floral organs. Transgenic plants with LUC fused to the promoter of FAD7 (FAD7::LUC) which encodes plastid-type desaturase, were also constructed. FAD3::LUC and FAD7::LUC were expressed in the same organs during reproductive growth, but not during vegetative growth. In plants exposed to both auxin and cytokinin, FAD3::LUC expression was ectopically induced in the root tissues. However, this induction by auxin and cytokinin was inhibited when abscisic acid was also present. FAD3::LUC expression could be induced in the roots by auxin and cytokinin if the hormones were applied during vegetative growth, but not if they were applied during germination or reproductive growth. Analysis of the fatty acid composition in the roots of Arabidopsis fad mutant and wild-type plants confirmed that the response of FAD3::LUC expression to various hormones reflected the response of endogenous FAD3 gene expression. These results suggest that the expression of ER-type desaturase is regulated through synergistic and antagonistic hormonal interactions, and that such hormonal regulation and the tissue specificity of the expression of this gene are further modified in accordance with the growth phase in plant development.     

Increasing α-linolenic acid content in rice bran by embryo-specific expression of ω3/Δ15-desaturase gene

Rice bran, the byproduct of brown rice, with a million tons produced annually, is the major storage organ for lipids. Increasing the α-linolenic acid (ALA) content in rice bran by biotechnology strategies is beneficial for both human health and the oil industries. We introduced two ω3/Δ15 fatty acid desaturase genes cloned from rice and soybean into rice under the control of an embryo-specific promoter, REG. The ALA content (dry weight) in embryos and bran was increased up to 6.09 and 5.86 mg/g, respectively, in transgenic lines, which was 25.4- and 27.9-fold higher than the 0.24 and 0.21 mg/g in the nontransformant control. ALA accounted for 46.7 and 44.3 % of the total fatty acids in embryos and bran, respectively, of transgenic plants, which was comparable to that in linseed and perilla seeds. The trait of high ALA content was stably inherited. The enhanced ALA content was preferentially located at the sn-2 position in triacylglycerols, which are digestible and absorbable for humans. ALA-enriched rice bran may help alleviate human health problems caused by ALA deficiency, with the production of healthy bran oil for humans and feed for animals.     

Identification and evaluation of ω-3 fatty acid desaturase genes for hyperfortifying α-linolenic acid in transgenic rice seed

α-Linolenic acid (ALA) deficiency and a skewed of ω6:ω3 fatty acid ratio in the diet are a major explanation for the prevalence of cardiovascular diseases and inflammatory/autoimmune diseases. There is a need to enhance the ALA content and to reduce the ratio of linoleic acid (LA) to ALA. Six ω-3 (Δ-15) fatty acid desaturase (FAD) genes were cloned from rice and soybean. The subcellular localizations of the proteins were identified. The FAD genes were introduced into rice under the control of an endosperm-specific promoter, GluC, or a Ubi-1 promoter to evaluate their potential in increasing the ALA content in seeds. The ALA contents in the seeds of endoplasmic reticulum (ER)-localized GmFAD3-1 and OsFAD3 overexpression lines increased from 0.36 mg g?1 to 8.57 mg g?1 and 10.06 mg g?1, respectively, which was 23.8- and 27.9-fold higher than that of non-transformants. The trait of high ALA content was stably inheritable over three generations. Homologous OsFAD3 is more active than GmFAD3-1 in catalysing LA conversion to ALA in rice seeds. Overexpression of ER-localized GmFAD3-2/3 and chloroplast-localized OsFAD7/8 had less effect on increasing the ALA content in rice seeds. The GluC promoter is advantageous compared with Ubi-1 in this experimental system. The enhanced ALA was preferentially located at the sn-2 position in triacylglycerols. A meal-size portion of high ALA rice would meet >80% of the daily adult ALA requirement. The ALA-rich rice could be expected to ameliorate much of the global dietary ALA deficiency.     

Improve freezing tolerance in transgenic poplar by overexpressing a ω-3 fatty acid desaturase gene

Polyunsaturated fatty acids are a major component of glycerolipids in plant cellular membranes, and play important role in the maintenance of its fluidity. In this study, we isolated a novel ω-3 fatty acid desaturase gene (PtFAD3) from Populus tomentosa Carr. and transformed it into hybrid poplar (Populus alba × P. glandulossa) K84 lines by overexpression and RNAi silencing methods. Quantitative PCR analysis revealed varied expression levels of PtFAD3 among the transgenic lines. Two overexpressing lines (OE lines) and two gene down-regulated RNAi lines (DR lines) were chosen for further analysis. The level of the major trienoic fatty acid species, linolenic acid, increased by an average of 10% in the two OE lines and decreased by an average of 5% in the two DR lines. When young poplar cuttings with three leaves were exposed to a freezing temperature (?4°C) for 3 h without cold acclimation, survival rates of the two independent OE lines (70 and 77%) were far superior to the wild type (36.7%) and the two DR lines (10% for both). Damage caused by freezing was alleviated significantly in the two OE lines compared to wild-type controls. Phenotypic evaluation indicated that the variable responses to freezing were attributable to genotypic variation. Therefore, in the case of poplar, a significant increase in freezing tolerance was achieved on a small scale following the introduction of PtFAD3. This strategy can be used for freezing tolerance breeding in hybrid poplar and other economically important forest trees.     

The 5′ UTR intron-mediated enhancement of constitutive splicing of the tobacco microsome ω-3 fatty acid desaturase gene

Several plant genes have their first intron in the 5′ untranslated region (5′ UTR), and such 5′ UTR introns often show several biological functions, including the intron-mediated enhancement of protein expression through an increase of mRNA level (IME), intron-dependent spatial expression, and intron-mediated enhancement of translation. Here, we show another function of the 5′ UTR intron, i.e., the 5′ UTR intron-mediated enhancement of constitutive splicing. The NtFAD3 gene, which encodes a tobacco microsome ω-3 fatty acid desaturase, has a 552 nucleotide-long 5′ UTR intron (intron 1), and the other seven introns are located in the coding sequence. The splicing of the 5′ half region of the NtFAD3 was studied through an in vivo splicing assay using Arabidopsis leaf explants. The low splicing efficiency of intron 2 was much improved when the assay construct harbored intron 1. Deletion of intron 1 and the replacement of intron 1 to the NtFAD3 intron 8 decreased the splicing efficiency of intron 2. The splicing enhancers were redundant and dispersed in the 5′ splice site-proximal, 284-nucleotides region of intron 1. In addition, the interaction among the cis-elements, i.e., the splicing enhancers in the intron 1 and exon 2, were necessary for the efficient splicing of intron 2. The 5′ UTR intron-mediated constitutive splicing was partially inhibited when an SR-like protein, SR45, was deficient. These results indicated a novel function of the 5′ UTR intron, namely an enhancement of the constitutive splicing.     

Enhanced cold tolerance in transgenic tobacco expressing a chloroplast ω-3 fatty acid desaturase gene under the control of a cold-inducible promoter

A new cold-inducible genetic construct was cloned using a chloroplast-specific omega-3-fatty acid desaturase gene (FAD7) under the control of a cold-inducible promoter (cor15a) from Arabidopsis thaliana. RT-PCR confirmed a marked increase in FAD7 expression, in young Nicotiana tabacum (cv. Havana) plants harboring cor15a–FAD7, after a short-term exposure to cold. When young, cold-induced tobacco seedlings were exposed to low-temperature (0.5, 2 or 3.5°C) for up to 44 days, survival within independent cor15a–FAD7 transgenic lines (40.2–96%) was far superior to the wild type (6.7–10.2%). In addition, the major trienoic fatty acid species remained stable in cold-induced cor15a–FAD7 N. tabacum plants under prolonged cold storage while the levels of hexadecatrienoic acid (16:3) and octadecatrienoic acid (18:3) declined in wild type plants under the same conditions (79 and 20.7% respectively). Electron microscopy showed that chloroplast membrane ultrastructure in cor15a–FAD7 transgenic plants was unaffected by prolonged exposure to cold temperatures. In contrast, wild type plants experienced a loss of granal stacking and disorganization of the thylakoid membrane under the same conditions. Changes in membrane integrity coincided with a precipitous decline in leaf chlorophyll concentration and low survival rates in wild type plants. Cold-induced double transgenic N. alata (cv. Domino Mix) plants, harboring both the cor15a–FAD7 cold-tolerance gene and a cor15a–IPT dark-tolerance gene, exhibited dramatically higher survival rates (89–90%) than wild type plants (2%) under prolonged cold storage under dark conditions (2°C for 50 days).     

Functional complementation of a periila ω3 fatty acid desaturase under the seed-specific SeFAD2 promoter

Functional characterization of the fatty acid desaturase genes and seed-specific promoters is prerequisite for altering the unsaturated fatty acid content of oilseeds by genetic manipulation. The ω-6 fatty acid desaturase (FAD2) and ω-3 fatty acid desaturase (FAD3) catalyze extra-plastidial desaturation of oleic acid to linoleic acid and linoleic acid to linolenic acid, respectively. These are major constituents in seed storage oils. Here, we report the complementation of a perilla linoleic acid desaturase (PrFAD3) cDNA under the seed-specific sesame FAD2 (SeFAD2) promoter in the Arabidopsis fad3 mutant. PrFAD3 is functionally active and the SeFAD2 promoter is applicable for modifying fatty acid composition in developing seeds. Transient expression of the GUS gene under that promoter in the developing seeds and leaves of sesame, soybean, and corn via microprojectile bombardment indicated that the SeFAD2 promoter likely will be useful for altering the seed phenotypes of dicot and monocot crops.     

Evaluation of the ratio of ω6 : ω3 fatty acids and vitamin e levels in the diet on the reproductive performance of cockerels

Three hundred and twenty 30-week old White Leghorn cockerels were housed in individual cages and distributed in a completely randomized factorial design of 5?×?3, with five oil sources (sunflower, soybean, canola, linseed and fish/soybean) and three levels of antioxidant (30, 200 and 400?mg of vitamin E/kg). The aim of this study was to evaluate the effect of the ratio of ω6?:?ω3 fatty acids by the inclusion of different oil sources and of dietary supplementation with vitamin E on the reproductive performance of cockerels. The use of the fish/soybean combination determined the lowest total antioxidant status of the semen. However, the addition of vitamin E to the fish/soybean-oil-based diet resulted in a linear increase in semen volume, motility and sperm vigour in the 38th week and again in the 52nd week for motility and for sperm vigour and fertility rate in the periods from 50?–?53 and 41?–?53 weeks of age. The use of canola oil in the diet resulted in the highest fertility rate during 50?–?53 and 41?–?53 weeks of life. Animals receiving the soybean oil based diet showed the smallest fertility rate in the range from 50?–?53 weeks of age and concomitantly the highest level of cholesterol in the spermatozoa in the range from 47?–?51 weeks. An interaction between the vitamin E level and soybean oil was verified by the linear increase in motility and sperm vigour at 38 weeks of age. Later, the contrary was shown by a linear reduction in fertility in the periods from 44?–?46, 47?–?49 and 41?–?53 weeks of age. Cockerels that had been fed on the sunflower-oil-based diet showed the highest content of saturated fatty acids in the spermatozoa from 48?–?51 weeks. An interaction effect was observed between the vitamin E level and sunflower oil shown by a linear increase in the content of saturated fatty acids in the spermatozoa and a linear reduction in the C18?:?1ω9, C18?:?2ω6 and PUFA (C18?:?2ω6?+?C20?:?4ω6) contents in the spermatozoa at 48?–?51 weeks and in sperm volume at 52 weeks of age.     

Biotechnological production and applications of the ω-3 polyunsaturated fatty acid docosahexaenoic acid

Docosahexaenoic acid (DHA) is a polyunsaturated fatty acid composed of 22 carbon atoms and six double bonds. Because the first double bond, as counted from the methyl terminus, is at position three, DHA belongs to the so-called -3 group. In recent years, DHA has attracted much attention because of its beneficial effect on human health. At present, fish oil is the major source of DHA, but alternatively it may be produced by use of microorganisms. Marine microorganisms may contain large quantities of DHA and are considered a potential source of this important fatty acid. Some of these organisms can be grown heterotrophically on organic substrates without light. These processes can be well controlled and DHA with constant quality can be produced all year round. This paper reviews recent advances in the biotechnological production of DHA by marine microorganisms.     

Role of ω-3 fatty acid desaturases in the regulation of the level of trienoic fatty acids during leaf cell maturation

Trienoic fatty acids, namely -linolenic acid and hexadecatrienoic acid, present in leaf lipids are produced by -3 fatty acid desaturases located in the endoplasmic reticulum and plastid membranes. The changes in the level of trienoic fatty acids during leaf maturation were investigated in wild-type plants of Arabidopsis thaliana (L.) Heynh. and in the fad7 mutant deficient in the activity of a plastid -3 desaturase. The levels of trienoic fatty acids increased in 26 °C- and 15 °C-grown wild-type plants with maturation of leaves. The increase in trienoic fatty acids was mainly due to galactolipids enriched in plastid membranes. In addition, the relative levels of trienoic fatty acids in major glycerolipids, including phospholipids enriched in the endoplasmic reticulum membranes, also increased with leaf maturation. By contrast, when the fad7 mutant was grown at 26 °C, the relative levels of trienoic fatty acids in individual lipids decreased with leaf maturation. The decreases in the levels of trienoic fatty acids, however, were alleviated when the fad7 mutant was grown at 15 °C. These results suggest that the plastid -3 desaturase plays a major role in increasing the levels of trienoic fatty acids with leaf maturation.Abbreviations 163 hexadecatrienoic acid - 183 -linolenic acid - DGD digalactosyldiacylglycerol - MGD monogalactosyldiacylglycerol - PC phosphatidylcholine - PE phosphatidylethanolamine - TA trienoic fatty acid - WT wild type - -3 refers to the position of the double bond from the methyl end of a fatty acid This research was supported in part by Grants-in-Aid for Scientific research (#07251214 and #06804050 to K.I.) from the Ministry of Education, Science and Culture, Japan, and by the research grant from Shorai Foundation.     

Isolation and expression of CsFAD7 and CsFAD8, two genes encoding ω-3 fatty acid desaturase from Camellia sinensis

The plastidial ω-3 fatty acid desaturase catalyses the production of trienoic fatty acids (TAs) in plant chloroplasts and plays an important role in plant responses to environmental stress. In this study, the full-length cDNAs encoding two plastidial ω-3 desaturases, designated CsFAD7 and CsFAD8 (GenBank Accession No. JX943516 and KC847167, respectively), were isolated from the tea plant (Camellia sinensis L.) using RT-PCR and RACE. Codon usage analysis revealed that U- and A-ended codons were preferentially used in these two genes. Sequence analysis showed that the deduced amino acid sequences of CsFAD7 and CsFAD8 had high homology to plastidial ω-3 desaturases from other plant species. Expression analysis by real-time PCR revealed that both genes are tissue-specific and expressed the highest levels in shoots. Meanwhile, CsFAD7 and CsFAD8 responded to various abiotic stresses and hormones, but in very different manners. Taken together, these results suggest that CsFAD7 and CsFAD8 are both responsive to abiotic stress signals; however, they may play very different roles during stress tolerance in tea plants.     

Effect of fatty acids of ω6 series on the biosynthesis of arachidonic acid in HTC cells

Summary The influence of the preincubation of HTC cells with fatty acids of 6 series and columbinic acid (St, 9c, 12c 18:3) on the biosynthesis of arachidonic acid was studied. The cells were incubated on a chemically defined medium with or without the addition of unlabeled linoleic, -linolenic, eicosatrienoic, arachidonic, docosatetraenoic, docosapentaenoic and columbinic acids. After 24 hr of preincubation in the presence of the aforementioned fatty acids, [1-¹⁴C]eicosa-8,11,14-trienoic acid was added to the culture medium as the only lipidic source. Twenty-four hours later the synthesis of arachidonic acid and the fatty acid composition of the cells were determined. At 20 MM concentration the 6 fatty acids studied except docosapentaenoic acid produced an increase on the biosynthesis of arachidonic acid compared to the cells incubated in the absence of unlabeled fatty acids in the medium. The fatty acids added to the culture medium were incorporated into the cells and modified their fatty acid composition. Columbinic acid, with a similar structure to linoleic acid, also produced a significant increase on the conversion of eicosatrienoic acid to arachidonic acid. These results would suggest that the effect of both, linoleic and columbinic acids, may be adscribed to their configuration and not necessarily to their transformation in higher homologs, since columbinic acid is unable to be desaturated.All authors are members of the Carrera del Investigador Cientifico of the Consejo Nacional de Investigaciones Cientifícas y Técnicas, Argentina.     

Comparison of the Δ~(12) fatty acid desaturase gene between high-oleic and normal-oleic peanut genotypes

Δ12 fatty acid desaturase gene has been targeted as a logical candidate controlling the high oleate trait in peanut seeds.By RT-PCR method,the full-length cDNAs of Δ12 fatty acid desaturase gene were isolated from peanut(Arachis hypogaea L.) genotypes with normal and high ratio of oleic to linoleic acid,which were designated AhFAD2B and AhFAD2B',respectively.Sequence alignment of their coding regions revealed that an extra A was inserted at the position +442 bp of AhFAD2B' sequence of high oleic acid genoty...