Trienoic Fatty Acids Research Articles

2-Cys peroxiredoxins contribute to thylakoid lipid unsaturation by affecting ω-3 fatty acid desaturase 8.

Fatty acid unsaturation levels affect chloroplast function and plant acclimation to environmental cues. However, the regulatory mechanism(s) controlling fatty acid unsaturation in thylakoid lipids is poorly understood. Here, we have investigated the connection between chloroplast redox homeostasis and lipid metabolism by focusing on 2-Cys peroxiredoxins (Prxs), which play a central role in balancing the redox state within the organelle. The chloroplast redox network relies on NADPH-dependent thioredoxin reductase C (NTRC), which controls the redox balance of 2-Cys Prxs to maintain the reductive activity of redox-regulated enzymes. Our results show that Arabidopsis (Arabidopsis thaliana) mutants deficient in 2-Cys Prxs contain decreased levels of trienoic fatty acids, mainly in chloroplast lipids, indicating that these enzymes contribute to thylakoid membrane lipids unsaturation. This function of 2-Cys Prxs is independent of NTRC, the main reductant of these enzymes, hence 2-Cys Prxs operates beyond the classic chloroplast regulatory redox system. Moreover, the effect of 2-Cys Prxs on lipid metabolism is primarily exerted through the prokaryotic pathway of glycerolipid biosynthesis and fatty acid desaturase 8 (FAD8). While 2-Cys Prxs and FAD8 interact in leaf membranes as components of a large protein complex, the levels of FAD8 were markedly decreased when FAD8 is overexpressed in 2-Cys Prxs-deficient mutant backgrounds. These findings reveal a function for 2-Cys Prxs, possibly acting as a scaffold protein, affecting the unsaturation degree of chloroplast membranes.

Phytohormones regulate the non-redundant response of ω-3 fatty acid desaturases to low temperatures in Chorispora bungeana

To explore the contributions of ω-3 fatty acid desaturases (FADs) to cold stress response in a special cryophyte, Chorispora bungeana, two plastidial ω-3 desaturase genes (CbFAD7, CbFAD8) were cloned and verified in an Arabidopsis fad7fad8 mutant, before being compared with the microsomal ω-3 desaturase gene (CbFAD3). Though these genes were expressed in all tested tissues of C. bungeana, CbFAD7 and CbFAD8 have the highest expression in leaves, while CbFAD3 was mostly expressed in suspension-cultured cells. Low temperatures resulted in significant increases in trienoic fatty acids (TAs), corresponding to the cooperation of CbFAD3 and CbFAD8 in cultured cells, and the coordination of CbFAD7 and CbFAD8 in leaves. Furthermore, the cold induction of CbFAD8 in the two systems were increased with decreasing temperature and independently contributed to TAs accumulation at subfreezing temperature. A series of experiments revealed that jasmonie acid and brassinosteroids participated in the cold-responsive expression of ω-3 CbFAD genes in both C. bungeana cells and leaves, while the phytohormone regulation in leaves was complex with the participation of abscisic acid and gibberellin. These results point to the hormone-regulated non-redundant contributions of ω-3 CbFADs to maintain appropriate level of TAs under low temperatures, which help C. bungeana survive in cold environments.

Different Cis-Regulatory Elements Control the Tissue-Specific Contribution of Plastid ω-3 Desaturases to Wounding and Hormone Responses

Trienoic fatty acids are essential constituents of biomembranes and precursors of jasmonates involved in plant defense responses. Two ω-3 desaturases, AtFAD7 and AtFAD8, synthetize trienoic fatty acids in the plastid. Promoter:GUS and mutagenesis analysis was used to identify cis-elements controlling AtFAD7 and AtFAD8 basal expression and their response to hormones or wounding. AtFAD7 promoter GUS activity was much higher than that of AtFAD8 in leaves, with specific AtFAD7 expression in the flower stamen and pistil and root meristem and vasculature. This specific tissue and organ expression of AtFAD7 was controlled by different cis-elements. Thus, promoter deletion and mutagenesis analysis indicated that WRKY proteins might be essential for basal expression of AtFAD7 in leaves. Two MYB target sequences present in the AtFAD7 promoter might be responsible for its expression in the flower stamen and stigma of the pistil and in the root meristem, and for the AtFAD7 wound-specific response. Two MYB target sequences detected in the distal region of the AtFAD8 gene promoter seemed to negatively control AtFAD8 expression, particularly in true leaves and flowers, suggesting that MYB transcription factors act as repressors of AtFAD8 gene basal expression, modulating the different relative abundance of both plastid ω-3 desaturases at the transcriptional level. Our data showed that the two ABA repression sequences detected in the AtFAD7 promoter were functional, suggesting an ABA-dependent mechanism involved in the different regulation of both ω-3 plastid desaturases. These results reveal the implication of different signaling pathways for the concerted regulation of trienoic fatty acid content in Arabidopsis.

In vivo anticancer effects of Momordica charantia seed fat on hepatocellular carcinoma in a rat model

BackgroundMomordica charantia or bitter melon is a well-known vegetable with a number of therapeutic actions in Ayurvedic medicine. Alpha-eleostearic acid, a conjugated trienoic fatty acid present in bitter melon is proven to have anticancer properties. Crude seed oil from local bitter melon varieties could be an effective and economical anticancer therapy. Objective(s)The study was conducted to evaluate the anticancer effect of the crude oil from the seeds of Matale green variety of bitter melon on a hepatocellular carcinoma-induced rat model. Materials and methodsHepatocellular carcinoma (HCC) was experimentally induced in Wistar rats. Crude seed oil of Matale green bitter melon (MGBM) was supplemented to one treatment group in concurrence with carcinoma induction and to another treatment group after the development of carcinoma. After 168 days, gross morphological, histopathological, biochemical, hematological and gene-expression analysis of treated and control groups were performed. ResultsOral supplementation of MGBM seed oil showed a statistically significant reduction (p < 0.05) in the average number, diameter and area of hepatic dysplastic nodules and a reduction in the size of histopathological neoplastic lesions in both treatment groups compared to the non-treated control group. The expression of tumor suppressor gene p53 and anti-apoptotic gene Bcl-2 were significantly increased while the expression of apoptotic gene caspase 3 was significantly reduced in the treatment group when MGBM supplementation was in concurrence with carcinogenesis (p < 0.05). ConclusionCrude seed oil from the MGBM has anticancer effects against experimentally induced HCC in Wistar rats, specially when supplemented in concurrence with carcinoma induction.

Role of Fatty Acid ω3 Acyl-Lipid Desaturases in Low-Temperature Hardening of Solanum tuberosum L.

The role of fatty acid ω3 acyl-lipid desaturases in low-temperature hardening (7 days at 3°C) of potato plants (Solanum tuberosum L., cv. Yubilei Zhukova) was studied. It was found that transcriptions of the three genes of ω3 acyl-lipid desaturases present in the potato genome significantly differed in their response to low temperature. The content of the FAD3 gene transcripts dramatically fell in the first day of cooling and was not restored until the end of the hardening period. The FAD7 gene was constitutively expressed through almost the entire adaptation period. The level of the FAD8 gene transcripts sharply increased in the first day of the hardening. The total fraction of trienoic fatty acids, which are synthesized by ω3 acyl-lipid desaturases, rose by 3% during the hardening, which is a relatively large value for potato. The hardened plants, in comparison with the unhardened ones, manifested a higher net photosynthesis/dark respiration ratio, higher (2.5-fold) concentration of soluble sugars, and stronger resistance to negative (–2 or –3°C) temperatures. It is suggested that the augmentation of the trienoic FA fraction, which are mainly localized in the thylakoid membranes of the potato chloroplasts, sustains the photosynthesis under low temperatures. Therefore, the maintenance of photosynthetic activity during the low-temperature acclimation supplies the plant with photoassimilates that are the principal sources of energy and metabolites necessary for the establishment of potato plant resistance to hypothermia.

Engineering Trienoic Fatty Acids into Cottonseed Oil Improves Low-Temperature Seed Germination, Plant Photosynthesis and Cotton Fiber Quality.

Alpha-linolenic acid (ALA, 18:3Δ9,12,15) and γ-linolenic acid \ (GLA, 18:3Δ6,9,12) are important trienoic fatty acids, which are beneficial for human health in their own right, or as precursors for the biosynthesis of long-chain polyunsaturated fatty acids. ALA and GLA in seed oil are synthesized from linoleic acid (LA, 18:2Δ9,12) by the microsomal ω-3 fatty acid desaturase (FAD3) and Δ6 desaturase (D6D), respectively. Cotton (Gossypium hirsutum L.) seed oil composition was modified by transforming with an FAD3 gene from Brassica napus and a D6D gene from Echium plantagineum, resulting in approximately 30% ALA and 20% GLA, respectively. The total oil content in transgenic seeds remained unaltered relative to parental seeds. Despite the use of a seed-specific promoter for transgene expression, low levels of GLA and increased levels of ALA were found in non-seed cotton tissues. At low temperature, the germinating cottonseeds containing the linolenic acid isomers elongated faster than the untransformed controls. ALA-producing lines also showed higher photosynthetic rates at cooler temperature and better fiber quality compared to both untransformed controls and GLA-producing lines. The oxidative stability of the novel cottonseed oils was assessed, providing guidance for potential food, pharmaceutical and industrial applications of these oils.

Novel Non-methylene-Interrupted Dienoic and Trienoic Fatty Acids with a Terminal Double Bond in Ovaries of the Limpet Cellana toreuma.

In this study, to obtain further information on the structural diversity of non-methylene-interrupted (NMI) fatty acids (FA) in the gonads of the limpet Cellana toreuma, previously unreported minor NMI FA, fractionated by argentation thin-layer chromatography (TLC), in the ovaries were characterized by gas chromatography-mass spectrometry (GC-MS) analysis of their 3-pyridylcarbinol derivatives. As a result, five novel NMI FA were identified as 5,14-pentadecadienoic acid (15: 2Δ5,14), 5,9,18-nonadecatrienoic acid (19:3Δ5,9,18), 5,9,20-heneicosatrienoic acid (21:3Δ5,9,20), 7,13,20-heneicosatrienoic acid (21:3Δ7,13,20), and 5,9,22-tricosatrienoic acid (23:3Δ5,9,22). Possible biosynthetic pathways for these FA are also discussed in relation to structural variations of NMI FA regioisomers. This study clearly reinforces the findings of our previous works with structurally diverse NMI FA with a terminal double bond.

Tissue Distribution and Specific Contribution of Arabidopsis FAD7 and FAD8 Plastid Desaturases to the JA- and ABA-Mediated Cold Stress or Defense Responses.

To overcome the difficulties to analyze membrane desaturases at the protein level, transgenic Arabidopsis plants expressing the plastidial AtFAD7 and AtFAD8 ω-3 desaturases fused to green fluorescent protein, under the control of their endogenous promoters, were generated and their tissue relative abundance was studied. Gene expression, glucuronidase promoter activity, immunoblot and confocal microscopy analyses indicated that AtFAD7 is the major ω-3 desaturase in leaves when compared to AtFAD8. This higher abundance of AtFAD7 was consistent with its higher promoter activity and could be related with its specificity for the abundant leaf galactolipids. AtFAD7 was also present in roots but at much lower level than leaves. AtFAD8 expression and protein abundance in leaves was consistent with its lower promoter activity, suggesting that transcriptional control modulates the abundance of both desaturases in leaves. AtFAD7 protein levels increased in response to wounding but not to jasmonate (JA), and decreased upon abscisic acid (ABA) treatment. Conversely, AtFAD8 protein levels increased upon cold or JA exposure and decreased at high temperatures, but did not respond to ABA or wounding. These results indicated specific and non-redundant roles for the plastidial ω-3 desaturases in defense, temperature stress or phytohormone mediated responses and a tight coordination of their activities between biotic and abiotic stress signaling pathways. Our data suggested that transcriptional regulation was crucial for this coordination. Finally, bimolecular fluorescence complementation analysis showed that both AtFAD7 and AtFAD8 interact with the AtFAD6 ω-6 desaturase in vivo, suggesting that quaternary complexes are involved in trienoic fatty acid production within the plastid membranes.

Tung Tree (Vernicia fordii, Hemsl.) Genome and Transcriptome Sequencing Reveals Co-Ordinate Up-Regulation of Fatty Acid β-Oxidation and Triacylglycerol Biosynthesis Pathways During Eleostearic Acid Accumulation in Seeds.

The tung tree (Vernicia fordii) is one of only a few plant species that produces high oil-yielding seeds rich in α-eleostearic acid (α-ESA, 18:3Δ9cis, 11trans, 13trans), a conjugated trienoic fatty acid with valuable industrial and medical properties. Previous attempts have been made to engineer tung oil biosynthesis in transgenic oilseed crops, but these efforts have met with limited success. Here we present a high-quality genome assembly and developing seed transcriptomic data set for this species. Whole-genome shotgun sequencing generated 176 Gb of genome sequence data used to create a final assembled sequence 1,176,320 kb in size, with a scaffold N50 size of >474 kb, and containing approximately 47,000 protein-coding genes. Genomic and transcriptomic data revealed full-length candidate genes for most of the known and suspected reactions that are necessary for fatty acid desaturation/conjugation, acyl editing and triacylglycerol biosynthesis. Seed transcriptomic analyses also revealed features unique to tung tree, including unusual transcriptional profiles of fatty acid biosynthetic genes, and co-ordinated (and seemingly paradoxical) simultaneous up-regulation of both fatty acid β-oxidation and triacylglycerol biosynthesis in mid-development seeds. The precise temporal control of the expression patterns for these two pathways may account for α-ESA enrichment in tung seeds, while controlling the levels of potentially toxic by-products. Deeper understanding of these processes may open doors to the design of engineered oilseeds containing high levels of α-ESA.

Membrane Lipid Composition upon Normal and Patterned Wood Formation in Betula Pendula Roth

A comparative study of two forms of Betula pendula Roth differing in wood structure (B. pendula var. pendula with common straight-grained wood and B. pendula var. carelica with abnormal patterned wood) has demonstrated that the level of phospholipids reflects the content of parenchyma cells in the wood and can be regarded as an indicator of the abnormality degree in the tissue structure. The content of glycolipids testifies to the dynamics of the plastid apparatus condition and correlates with the activity of the storage function in the wood parenchyma cells. It is shown that two directions in the differentiation of cambial derivatives, namely by the pathway of cell death (vessels and fibrous elements) and the preservation of a live protoplast (parenchyma cells), correlate with different fatty acid compositions of membrane lipids. In the first case, the share of saturated fatty acids in the composition of phospholipids and glycolipids increases, indicating a deterioration of the functional characteristics of cell membranes. In the latter case, the share of unsaturated, mainly dienoic fatty acids, increases, which reflects a rise in the activity of cell membranes. During periods with the highest rate of metabolism, the content of polyunsaturated, mainly trienoic fatty acids increase in membrane lipids.

The pht4;1-3 mutant line contains a loss of function allele in the Fatty Acid Desaturase 7 gene caused by a remnant inactivated selection marker-a cautionary tale.

A striking and unexpected biochemical phenotype was found in an insertion mutant line in the model plant Arabidopsis thaliana. One of two investigated insertion mutant lines in the gene encoding the phosphate transporter PHT4;1 demonstrated a prominent loss of trienoic fatty acids, whereas the other insertion line was indistinguishable from wild type in this aspect. We demonstrate that the loss of trienoic fatty acids was due to a remnant inactive negative selection marker gene in this particular transposon tagged line, pht4;1-3. This constitutes a cautionary tale that warns of the importance to confirm the loss of this type of selection markers and the importance of verifying the relationship between a phenotype and genotype by more than one independent mutant line or alternatively genetic complementation.

Effect of dietary flax seed and oil on milk yield, gross composition, and fatty acid profile in dairy cows: A meta-analysis and meta-regression

Several experiments were conducted over the past few years to evaluate the feeding value of flax seed and oil in dairy cow diets. The current meta-analysis and meta-regression was undertaken to assess the overall effect of different forms of flax, as a source of trienoic (cis-9,cis-12,cis-15 18:3) fatty acids (FA), on lactation performance and on transfer efficiency of its constituent n-3 FA from diet to milk fat. Comparisons were first conducted with nonsupplemented controls or with diets containing either saturated (mainly 16:0 or 18:0 or both), monoenoic (mainly cis-9 18:1), or dienoic (mainly cis-9,cis-12 18:2) FA. Results indicate that supplementing flax seed and oil decreased dry matter intake, as well as actual and energy-corrected milk yield without affecting the efficiency of utilization of dietary dry matter or energy as compared with nonsupplemented iso-energetic controls. When compared with the other 3 types of dietary fat evaluated, flax rich in trienoic FA supported a yield of energy-corrected milk similar to supplements rich in saturated, monoenoic, or dienoic FA. Greater milk fat concentration and feed efficiency were observed with saturated supplements. However, milk fat concentration and yield were lower with dienoic FA than with flax supplements. Further analyses were conducted to compare the effect of different forms of flax oil, seed, or fractions of seed. Among the 6 categories evaluated, mechanically processed whole seed (rolled or ground) allowed the greatest yield of energy-corrected milk and the best feed efficiency when compared with free oil, intact or extruded whole seed, protected flax, and flax hulls. Feeding protected flax and flax hulls allowed the greatest milk fat concentration of cis-9,cis-12,cis-15 18:3. Moreover, the greatest transfer efficiencies of this fatty acid from diet to milk were recorded with the same 2 treatments, plus the mechanically processed whole seed. These results make this last category the most suitable treatment, among the 6 flax forms evaluated, to combine optimum lactation performance and protection of flax constituent cis-9,cis-12,cis-15 18:3.

Arachidonic acid is important for efficient use of light by the microalga Lobosphaera incisa under chilling stress

The oleaginous microalga Lobosphaera incisa (Trebouxiophyceae, Chlorophyta) contains arachidonic acid (ARA, 20:4 n−6) in all membrane glycerolipids and in the storage lipid triacylglycerol. The optimal growth temperature of the wild-type (WT) strain is 25°C; chilling temperatures (≤15°C) slow its growth. This effect is more pronounced in the delta-5-desaturase ARA-deficient mutant P127, in which ARA is replaced with dihomo-γ-linolenic acid (DGLA, 20:3 n−6). In nutrient-replete cells grown at 25°C, the major chloroplast lipid monogalactosylglycerol (MGDG) was dominated by C18/C16 species in both strains. Yet ARA constituted over 10% of the total fatty acids in the WT MGDG as a component of C20/C18 and C20/C20 species, whereas DGLA was only a minor component of MGDG in P127. Both strains increased the percentage of 18:3 n−3 in membrane lipids under chilling temperatures. The temperature downshift led to a dramatic increase in triacylglycerol at the expense of chloroplast lipids. WT and P127 showed a similarly high photochemical quantum yield of photosystem II, whereas non-photochemical quenching (NPQ) and violaxanthin de-epoxidation were drastically higher in P127, especially at 15°C. Fluorescence anisotropy measurements indicated that ARA-containing MGDG might contribute to sustaining chloroplast membrane fluidity upon dropping to the chilling temperature. We hypothesize that conformational changes in chloroplast membranes and increased rigidity of the ARA-deficient MGDG of P127 at chilling temperatures are not compensated by trienoic fatty acids. This might ‘lock’ violaxanthin de-epoxidase in the activated state causing high constitutive NPQ and alleviate the risk of photodamage under chilling conditions in the mutant.

The differences between two tea varieties in their response to natural cold conditions

ABSTRACTIn this study, two tea varieties (Camellia sinensis cv. CsCr02 and CsCr03) were studied under natural cold conditions to investigate the differences of thylakoid membrane structure, fatty acid desaturase (FAD) genes, and fatty acid (FA) composition in response to low temperatures. Quantitative real-time polymerase chain reaction assay confirmed that the expression of CsFAD8 in leaves of the two tea varieties was much higher than that of CsFAD7. The expression of both genes in CsCr03 was higher than in CsCr02. Electron microscopy showed that the damage to the thylakoid membrane in CsCr03 was less severe than in CsCr02 during the decrease of natural temperature. Changes in thylakoid membrane integrity coincided with the gradual decline in chlorophyll concentration and chlorosis of the tea leaves. With the decrease of natural temperature, a decline in the trienoic FA species (16:3 and 18:3) and an increase in the dienoic species (18:2) were observed in the two varieties. These results indicate that CsCr03 has higher cold adaptability compared to CsCr02.

Correlation between total lipids, linolenic acid and membrane injury under PEG-induced dehydration in leaves of Vigna genotypes differing in drought resistance

Membrane lipids contain trienoic fatty acids which enhance fluidity, an essential feature for optimal membrane performance. However highly unsaturated molecules are prone to lipoperoxidation processes. In V. unguiculata, linolenic acid (C18:3) accounts for more than 90% of the total fatty acids of galactolipids. High C18:3 contents causes galactolipids molecules, especially monogalactosyl-diacylglycerol (MGDG), to become more susceptible to drought-induced peroxidative or enzymatic degradation. In this work 8 Vigna genotypes differing in drought resistance in the field were compared to search for an eventual correlation between genotypes ability to resist to water stress and some characteristics of their membrane lipids in well-watered plants. Plants were grown in a greenhouse with semi-controlled environmental conditions, and kept fully hydrated. Leaves were collected from one month old plants to perform electrolyte leakage test under PEG-induced dehydration and lipid analysis. Results showed that both a reduced amount of total lipids, which reflects a small amount of membranes, and a reduced amount of C18:3 in MGDG, which indicates a low membrane fluidity, were strongly correlated with a high membrane resistance to dehydration, and therefore seem to be useful screening tools for drought-tolerant genotypes. Taking into account what is known concerning drought resistance of the genotypes under study, it was also concluded that the degree of membrane resistance to osmotic stress could be extrapolated to drought resistance in the field.

Enhancement of α-linolenic acid content in transgenic tobacco seeds by targeting a plastidial ω-3 fatty acid desaturase (fad7) gene of Sesamum indicum to ER.

Expression of sesame plastidial FAD7 desaturase modified with the endoplasmic reticulum targeting and retention signals, enhances the α-linolenic acid accumulation in seeds of Nicotiana tabacum. In plants, plastidial ω-3 fatty acid desaturase-7 (FAD7) catalyzes the formation of C16 and C18 trienoic fatty acids using organellar glycerolipids and participate in the membrane lipid formation. The plastidial ω-3 desaturases (FAD7) share high sequence homology with the microsomal ω-3 desaturases (FAD3) at the amino acid level except the N-terminal organelle transit peptide. In the present study, the predicted N-terminal plastidial signal peptide of fad7 gene was replaced by the endoplasmic reticulum signal peptide and an endoplasmic reticulum retention signal was placed at the C-terminal. The expression of the modified sesame ω-3 desaturase increases the α-linolenic acid content in the range of 4.78-6.77 % in the seeds of transgenic tobacco plants with concomitant decrease in linoleic acid content. The results suggested the potential of the engineered plastidial ω-3 desaturase from sesame to influence the profile of α-linolenic acid in tobacco plant by shifting the carbon flux from linoleic acid, and thus it can be used in suitable genetic engineering strategy to increase the α-linolenic acid content in sesame and other vegetable oils.

Production of conjugated dienoic and trienoic fatty acids by lactic acid bacteria and bifidobacteria

The production of conjugated linoleic (CLA) and linolenic (CLNA) acids by lactic acid bacteria (LAB) and bifidobacteria strains was assessed. The initial selection was based on optical density measurements by the UV method and GC analysis. The LAB strains showed a lower ability to synthesise CLA and CLNA. Yet, two Lactobacillus sakei strains were the best producers. Moreover, the production of CLA and CLNA by Lactobacillus mucosae is reported here for the first time. In contrast, bifidobacteria strains were more efficient to synthesise CLA and CLNA. Among these, the t9, t11-CLA was the main isomer, which is a potent anticarcinogenic compound. All tested strains produced the c9,t11,c15-CLNA isomer. High substrate concentrations of LA and LNA negatively affected the conversion rate of CLA and CLNA by Bifidobacterium animalis subsp. lactis INL2, even though the bacterial growth was not affected.

Non-redundant Contribution of the Plastidial FAD8 ω-3 Desaturase to Glycerolipid Unsaturation at Different Temperatures in Arabidopsis

Plastidial ω-3 desaturase FAD7 is a major contributor to trienoic fatty acid biosynthesis in the leaves of Arabidopsis plants. However, the precise contribution of the other plastidial ω-3 desaturase, FAD8, is poorly understood. Fatty acid and lipid analysis of several ω-3 desaturase mutants, including two insertion lines of AtFAD7 and AtFAD8, showed that FAD8 partially compensated the disruption of the AtFAD7 gene at 22°C, indicating that FAD8 was active at this growth temperature, contrasting to previous observations that circumscribed the FAD8 activity at low temperatures. Our data revealed that FAD8 had a higher selectivity for 18:2 acyl-lipid substrates and a higher preference for lipids other than galactolipids, particularly phosphatidylglycerol, at any of the temperatures studied. Differences in the mechanism controlling AtFAD7 and AtFAD8 gene expression at different temperatures were also detected. Confocal microscopy and biochemical analysis of FAD8-YFP over-expressing lines confirmed the chloroplast envelope localization of FAD8. Co-localization experiments suggested that FAD8 and FAD7 might be located in close vicinity in the envelope membrane. FAD8-YFP over-expressing lines showed a specific increase in 18:3 fatty acids at 22°C. Together, these results indicate that the function of both plastidial ω-3 desaturases is coordinated in a non-redundant manner.

Threonine 286 of fatty acid desaturase 7 is essential for ω-3 fatty acid desaturation in the green microalga Chlamydomonas reinhardtii.

Omega-3 fatty acid desaturases catalyze the conversion of dienoic fatty acids (C18:2 and C16:2) into trienoic fatty acids (C18:3 and C16:3), accounting for more than 50% of the total fatty acids in higher plants and the green microalga Chlamydomonas reinhardtii. Here, we describe a Thr residue located in the fourth transmembrane domain of fatty acid desaturase 7 (FAD7) that is essential for the biosynthesis of ω-3 fatty acids in C. reinhardtii. The ω-3 fatty acid deficiency in strain CC-620, which contains a putative missense mutation at Thr286 of CrFAD7, was recovered by the overexpression of CC-125 CrFAD7. A Ser substitution in position 286 was able to partially complement the phenotype of the ω-3 fatty acid deficiency, but other substitution variants, such as Tyr, His, Cys, and Gly, failed to do so. Prediction of the phosphorylation target site revealed that Thr286 may be phosphorylated. Analysis of the structural conformation of CC-620 CrFAD7 via topology prediction (and bends in the helix) shows that this missense mutation may collapse the catalytic structure of CrFAD7. Taken together, this study suggests that Thr286 is essential for the maintaining the catalytic structure of CrFAD7.

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.