Identification of the Arabidopsis palmitoyl-monogalactosyldiacylglycerol delta7-desaturase gene FAD5, and effects of plastidial retargeting of Arabidopsis desaturases on the fad5 mutant phenotype

Hexadeca 7,10,13-trienoic acid (16:3Delta(7,10,13)) is one of the most abundant fatty acids in Arabidopsis (Arabidopsis thaliana) and a functional component of thylakoid membranes, where it is found as an sn-2 ester of monogalactosyldiacylglycerol. The Arabidopsis fad5 mutant lacks activity of the plastidial palmitoyl-monogalactosyldiacylglycerol Delta7-desaturase FAD5, and is characterized biochemically by the absence of 16:3Delta(7,10,13) and physiologically by reduced chlorophyll content and a reduced recovery rate after photoinhibition. While the fad5 mutation has been mapped, the FAD5 gene was not unambiguously identified, and a formal functional characterization by complementation of fad5 mutant phenotypes has not been reported. Two candidate genes (At3g15850 and At3g15870) predicted to encode plastid-targeted desaturases at the fad5 chromosomal locus were cloned from fad5 plants and sequenced. A nonsense mutation changing codon TGG (Trp-98) into TGA (stop) was identified in At3g15850 (ADS3), whereas the fad5 At3g15870 allele was identical to wild type (after correction of a sequencing error in the published wild-type genomic At3g15870 sequence). Expression of a genomic clone or cDNA for wild-type At3g15850 conferred on fad5 plants the ability to synthesize 16:3Delta(7,10,13) and restored leaf chlorophyll content. Arabidopsis carrying a T-DNA insertion in At3g15870 had wild-type levels of both 16:3Delta(7,10,13) and chlorophyll. Together, these data formally prove that At3g15850 is FAD5. Interestingly, the fad5 phenotype was partially complemented when extraplastidial Delta9-desaturases of the Arabidopsis desaturase (ADS) family were expressed as fusions with a plastidial transit peptide. Tight correlation between leaf 16:3Delta(7,10,13) levels and chlorophyll content suggests a role for plastidial fatty acid desaturases in thylakoid formation.     

Cloning of a temperature-regulated gene encoding a chloroplast omega-3 desaturase from Arabidopsis thaliana.

Previous genetic evidence suggested that the fad8 and fad7 genes of Arabidopsis thaliana encode chloroplast membrane-associated omega-3 desaturases. A putative fad8 cDNA was isolated by heterologous hybridization using a gene encoding an endoplasmic reticulum-localized omega-3 desaturase (fad3) as a probe. The cDNA encodes a protein of 435 amino acid residues with a molecular mass of 50,134 D. Constitutive expression of the cDNA in transgenic plants of a fad7 mutant resulted in genetic complementation of the mutation, indicating that the fad7 and fad8 gene products are functionally equivalent. Expression of the fad8 cDNA in transgenic plants often resulted in the co-suppression of both the endogenous fad7 and fad8 genes in spite of the fact that these two genes share only about 75% nucleotide identity. In contrast to all other known plant desaturases, including fad7, the steady-state level of fad8 mRNA is strongly increased in plants grown at low temperature. This suggests that the role of fad8 is to provide increased omega-3 desaturase activity in plants that are exposed to low growth temperature. The fad8-1 mutation created a premature stop codon 149 amino acids from the amino-terminal end of the fad8 open reading frame, suggesting that this mutation results in a complete loss of fad8 activity.     

A Mutation at the fad8 Locus of Arabidopsis Identifies a Second Chloroplast [omega]-3 Desaturase

Two independently isolated mutations at the fad7 locus in Arabidopsis produced plants with a temperature-conditional phenotype. Leaves of fad7 mutants grown at 28[deg]C contained less than 30% of wild-type levels of trienoic fatty acids (16:3 plus 18:3) compared with more than 70% of wild-type levels for plants grown at 15[deg]C. Screening of an M2 population derived from the fad7-1 line led to the identification of a line, SH1, in which the proportion of trienoic acids was much less than in fad7 plants. The segregation pattern of F2 progeny from a cross between SH1 and wild type indicated that the additional fatty acid mutation in SH1 is at a new locus, designated fad8. In a genetic background that was wild type at the FAD7 locus, the fad8 mutation had no detectable effect on overall leaf fatty acid composition irrespective of the temperature at which plants were grown. However, fatty acid analyses of individual leaf lipids revealed small decreases in the levels of 18:3 in two chloroplast lipids. In fad8 plants grown at 22[deg]C, phospha-tidylglycerol contained 22.5% 18:3 compared with 33.5% in wild-type Arabidopsis. For sulfoquinovosyldiacylglycerol, the values were 31.4 and 44.5%, respectively. Together with information from studies of the cloned FAD8 gene (S. Gibson, V. Arondel, K. Iba, C. Somerville [1994] Plant Physiol 106: 1615-1621), these results indicate that the FAD8 locus encodes a chloroplast-localized 16:2/18:2 desaturase that has a substrate specificity similar to the FAD7 gene product but that is induced by low temperature.     

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.     

Omega-3 fatty acid desaturase (FAD3, FAD7, FAD8) gene expression and linolenic acid content in cowpea leaves submitted to drought and after rehydration

Membrane polyunsaturated fatty acids (PUFA) and particularly linolenic acid (18:3, LA) are known to be implicated in plant tolerance to low temperature. Their role in resistance to drought is much less investigated. In this work, three full-length cDNAs corresponding to omega-3 fatty acid desaturases: fad3 (endoplasmic reticulum), fad7 and fad8 (chloroplastic) were isolated from Vigna unguiculata leaves. Two cowpea cultivars, one drought-tolerant, EPACE-1, and one drought-susceptible, 1183, were compared in terms of fad isoform gene expression and leaf LA contents in plants submitted to water stress followed by rehydration. In EPACE-1, LA content in the main leaf polar lipids increased in response to mild water deficit. Severe water deficits induced a decrease in MGDG LA content while those of PC and DGDG continued to increase. Variations in FAD gene expression, matched those in LA contents. In 1183, LA contents decreased in all lipid classes in response to water stress, as did FAD3 and FAD8 gene expression levels. Rehydration after a moderate water stress induced stimulation mostly in FAD3 gene expression in both cvs. LA contents were equivalent to control levels in EPACE-1. In 1183, they were back to control levels in PC shortly after rehydration but remained low in galactolipids. These results suggested that omega-3 FAD activities were involved in the increase in leaf membrane unsaturation, in the drought tolerant plants whereas the sensitive plants lost PUFAs in response to the treatment. The significance of this discrepancy between the two cvs. in terms of adaptation to drought is discussed.     

Sequence Analysis of a Plastidial Omega-3 Desaturase Gene from Brassica juncea

A genomic clone (λg₂) encoding a plastidial omega-3 desaturase has been isolated from a genomic library of Brassica juncea cv Pusa Bold and sequenced after subcloning. A continuous stretch of 3866 by of the clone sequenced showed homology with omega-3 desaturase from other plant sources but maximum homology was with that of a plastidial omega-3 fad 7 gene. This sequence designated as Bjgfad7 includes 1381 by 5’ untranslated region, 273 by 3’ untranslated region along with an open reading frame of 1290 by interrupted by 7 introns. The 5’ untranslated region contains putative cis elements responsible for seed specificity, light responsive and stress-inducible expression besides minimal promoter elements.     

CTD phosphatases in the attenuation of wound-induced transcription of jasmonic acid biosynthetic genes in Arabidopsis

Trienoic fatty acids (TAs), the major constituents in plant membrane lipids, play essential roles in stress signalling as precursors of the phytohormone jasmonic acid (JA). Arabidopsis FAD7 encodes a plastidial ω-3 fatty acid desaturase, which catalyses the production of TAs. In coordination with other JA-biosynthetic genes, expression of FAD7 is induced locally by wounding. This provides a feedforward mechanism for the rapid and sustainable accumulation of JA. To identify molecular components involved in this mechanism, a transgenic Arabidopsis line carrying the FAD7 promoter ( pFAD7 ) fused to the firefly luciferase gene ( LUC ) was constructed. Reciprocal crossing experiments revealed that the induction of FAD7 expression depends largely on JA biosynthesis and the SCF^COI1-mediated signalling mechanism, whereas JA alone is insufficient for its maximal induction. Full induction required synergistic interactions between JA-dependent and -independent wound signalling mechanisms. A genetic screen for aberrant pFAD7::LUC expression yielded a recessive mutant showing enhanced wound-induced LUC bioluminescence. The mutation was associated with the cpl1 locus encoding an RNA polymerase II C-terminal domain (CTD) phosphatase, and conferred wound hyper-responsiveness on the promoters of several JA-biosynthetic genes. The picture of signalling mechanisms underlying the wound-regulated FAD7 expression, and potential roles of CPL proteins as attenuators of wound-induced JA biosynthesis, are discussed.     

Fatty Acid Desaturation during Chilling Acclimation Is One of the Factors Involved in Conferring Low-Temperature Tolerance to Young Tobacco Leaves

The FAD7 gene, a gene for a chloroplast [omega]-3 fatty acid desaturase, is responsible for the trienoic fatty acid (TA) formation in leaf tissues. The TA content of the leaf tissue of the 25[deg]C-grown transgenic tobacco (Nicotiana tabacum cv SR1) plants, in which the FAD7 gene from Arabidopsis thaliana was overexpressed, increased uniformly by about 10%. Fatty acid unsaturation in all major leaf polar lipid species increased in the 25[deg]C-grown FAD7 transformants but was approximately the same between the control plants and the FAD7 transformants when grown at 15[deg]C. Therefore, the overexpression of the exogenous FAD7 gene leads to the same consequence in the tobacco plants as the low-temperature-induced TA production that may be catalyzed by an endogenous, temperature-regulated chloroplast [omega]-3 fatty acid desaturase. In the 25[deg]C-grown control plants, the chilling treatment caused symptoms of leaf chlorosis and suppression of leaf growth. The 25[deg]C-grown FAD7 transgenic plants conferred alleviation of these chilling-induced symptoms. A reductions of the chilling injury similar to that of the FAD7 transformants was also observed in the 15[deg]C-preincubated control plants. These results indicate that the increased TA production during chilling acclimation is one of the prerequisites for the normal leaf development at low, nonfreezing temperatures.     

Cloning of higher plant omega-3 fatty acid desaturases.

Arabidopsis thaliana T-DNA transformants were screened for mutations affecting seed fatty acid composition. A mutant line was found with reduced levels of linolenic acid (18:3) due to a T-DNA insertion. Genomic DNA flanking the T-DNA insertion was used to obtain an Arabidopsis cDNA that encodes a polypeptide identified as a microsomal omega-3 fatty acid desaturase by its complementation of the mutation. Analysis of lipid content in transgenic tissues demonstrated that this enzyme is limiting for 18:3 production in Arabidopsis seeds and carrot hairy roots. This cDNA was used to isolate a related Arabidopsis cDNA, whose mRNA is accumulated to a much higher level in leaf tissue relative to root tissue. This related cDNA encodes a protein that is a homolog of the microsomal desaturase but has an N-terminal extension deduced to be a transit peptide, and its gene maps to a position consistent with that of the Arabidopsis fad D locus, which controls plastid omega-3 desaturation. These Arabidopsis cDNAs were used as hybridization probes to isolate cDNAs encoding homologous proteins from developing seeds of soybean and rapeseed. The high degree of sequence similarity between these sequences suggests that the omega-3 desaturases use a common enzyme mechanism.     

Arabidopsis fatty acid desaturase FAD2 is required for salt tolerance during seed germination and early seedling growth

Fatty acid desaturases play important role in plant responses to abiotic stresses. However, their exact function in plant resistance to salt stress is unknown. In this work, we provide the evidence that FAD2, an endoplasmic reticulum localized ω-6 desaturase, is required for salt tolerance in Arabidopsis. Using vacuolar and plasma membrane vesicles prepared from the leaves of wild-type (Col-0) and the loss-of-function Arabidopsis mutant, fad2, which lacks the functional FAD2, we examined the fatty acid composition and Na+-dependent H+ movements of the isolated vesicles. We observed that, when compared to Col-0, the level of vacuolar and plasma membrane polyunsaturation was lower, and the Na+/H+ exchange activity was reduced in vacuolar and plasma membrane vesicles isolated from fad2 mutant. Consistent with the reduced Na+/H+ exchange activity, fad2 accumulated more Na+ in the cytoplasm of root cells, and was more sensitive to salt stress during seed germination and early seedling growth, as indicated by CoroNa-Green staining, net Na+ efflux and salt tolerance analyses. Our results suggest that FAD2 mediated high-level vacuolar and plasma membrane fatty acid desaturation is essential for the proper function of membrane attached Na+/H+ exchangers, and thereby to maintain a low cytosolic Na+ concentration for salt tolerance during seed germination and early seedling growth in Arabidopsis.     

Developmental regulation and spatial expression of a plastidial fatty acid desaturase from Olea europaea

Trienoic fatty acids are major components of chloroplast membranes and are also precursors of the oxylipins, such as methyl jasmonate, that play important roles in signal transduction pathways relating to plant development and responses to stress. A cDNA encoding a plastidial ω-3 fatty acid desaturase responsible for trienoic formation has been isolated from a library made from ripening fruits of Olea europaea L. The predicted protein contains 436 amino acid residues including a consensus chloroplast specific transit peptide. Alignment with other desaturase sequences showed strong homology with the plastidial ω-3 desaturases fad7 and fad8. Since fad8 is only expressed at low temperatures and the olive fruit were developing at > 20 °C, it is concluded that the isolated cDNA is most likely to be derived from fad7. Northern hybridisation showed a transient expression of the putative fad7 gene at early stages of drupe (5–7 WAF) and mesocarp (16–19 WAF) development. In situ hybridisation showed particularly prominent expression in the palisade and vascular tissue of young leaves, the embryo sac and transmitting tissue of the carpel, and the tapetum, pollen grains and vascular tissue of anthers. The distinctive spatial, temporal and environmental regulation of the putative fad7 gene is consistent with major roles, not only in thylakoid membrane formation, but also in the provision of α-linolenate-derived signalling molecules that are particularly important in plant tissues involved in transportation and reproduction.     

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.     

Consequences of omega -6-oleate desaturase deficiency on lipid dynamics and functional properties of mitochondrial membranes of Arabidopsis thaliana

We probed the role of the polyunsaturated fatty acids on the dynamic and functional properties of mitochondrial membranes using the fad2 mutant of Arabidopsis thaliana, deficient in omega-6-oleate desaturase. In mitochondria of this mutant, the oleic acid content exceeded 70% of the total fatty acids, and the lipid/protein ratio was greatly enhanced. As a consequence, local microviscosity, probed by anthroyloxy fatty acid derivatives, was increased by 30%, whereas the lipid lateral diffusion, assayed using 1-pyrenedodecanoic acid, was approximately 4 times increased. Functional parameters such as oxygen consumption rate under phosphorylating and nonphosphorylating conditions and proton permeability of the inner mitochondrial membrane were significantly reduced in fad2 mitochondrial membranes, while the thermal dependence of the respiration was enhanced. Moreover, metabolic control analysis of the respiration clearly showed an enhancement of the control exerted by the membrane proton leaks. Our data suggest that the loss of omega-6-oleate desaturase activity in Arabidopsis cells induced an enhancement of both microviscosity and lipid/protein ratio of mitochondrial membranes, which in turn were responsible for the change in lateral mobility of lipids and for bioenergetic parameter modifications.     

Genetic variation of six desaturase genes in flax and their impact on fatty acid composition

Flax (Linum usitatissimum L.) is one of the richest plant sources of omega-3 fatty acids praised for their health benefits. In this study, the extent of the genetic variability of genes encoding stearoyl-ACP desaturase (SAD), and fatty acid desaturase 2 (FAD2) and 3 (FAD3) was determined by sequencing the six paralogous genes from 120 flax accessions representing a broad range of germplasm including some EMS mutant lines. A total of 6 alleles for sad1 and sad2, 21 for fad2a, 5 for fad2b, 15 for fad3a and 18 for fad3b were identified. Deduced amino acid sequences of the alleles predicted 4, 2, 3, 4, 6 and 7 isoforms, respectively. Allele frequencies varied greatly across genes. Fad3a, with 110 SNPs and 19 indels, and fad3b, with 50 SNPs and 5 indels, showed the highest levels of genetic variations. While most of the SNPs and all the indels were silent mutations, both genes carried nonsense SNP mutations resulting in premature stop codons, a feature not observed in sad and fad2 genes. Some alleles and isoforms discovered in induced mutant lines were absent in the natural germplasm. Correlation of these genotypic data with fatty acid composition data of 120 flax accessions phenotyped in six field experiments revealed statistically significant effects of some of the SAD and FAD isoforms on fatty acid composition, oil content and iodine value. The novel allelic variants and isoforms identified for the six desaturases will be a resource for the development of oilseed flax with unique and useful fatty acid profiles.     

Identification,characterization and field testing of Brassica napus mutants producing high‐oleic oils

Producing healthy, high‐oleic oils and eliminating trans‐fatty acids from foods are two goals that can be addressed by reducing activity of the oleate desaturase, FAD2, in oilseeds. However, it is essential to understand the consequences of reducing FAD2 activity on the metabolism, cell biology and physiology of oilseed crop plants. Here, we translate knowledge from studies of fad2 mutants in Arabidopsis (Arabidopsis thaliana) to investigate the limits of non‐GMO approaches to maximize oleic acid in the seed oil of canola (Brassica napus), a species that expresses three active FAD2 isozymes. A series of hypomorphic and null mutations in the FAD2.A5 isoform were characterized in yeast (Saccharomyes cerevisiae). Then, four of these were combined with null mutations in the other two isozymes, FAD2.C5 and FAD2.C1. The resulting mutant lines contained 71–87% oleic acid in their seed oil, compared with 62% in wild‐type controls. All the mutant lines grew well in a greenhouse, but in field experiments we observed a clear demarcation in plant performance. Mutant lines containing less than 80% oleate in the seed oil were indistinguishable from wild‐type controls in growth parameters and seed oil content. By contrast, lines with more than 80% oleate in the seed oil had significantly lower seedling establishment and vigor, delayed flowering and reduced plant height at maturity. These lines also had 7–11% reductions in seed oil content. Our results extend understanding of the B. napusFAD2 isozymes and define the practical limit to increasing oil oleate content in this crop species.     

The impact of alteration of polyunsaturated fatty acid levels on C6-aldehyde formation of Arabidopsis thaliana leaves.

C6-aldehydes are synthesized via lipoxygenase/hydroperoxide lyase action on polyunsaturated fatty acid (PUFA) substrates in plant leaves. The source pools and subcellular location of the processes are unknown. A close relationship is found between the composition of PUFA and the composition of C6-aldehydes. In the current study, this relationship was tested using the Arabidopsis PUFA mutant lines act1, fad2, fad3, fad5, fad6, and fad7. The results indicate that C6-aldehyde formation is influenced by the alteration of C18 PUFA levels. Mutants act1 and fad5, which are deficient in C16 unsaturated fatty acids, had wild-type levels of C6-aldehyde production. Mutants deficient in the chloroplast hexadecenoic acid/oleic acid desaturase (fad6) or hexadecadienoic acid/linoleic acid desaturase (fad7) had altered C6-aldehyde formation in a pattern similar to the changes in the PUFA. Mutations that impair phosphatidylcholine desaturase activity, such as fad2 and fad3, however, resulted in increased E-2-hexenal formation. The enzymes involved in C6-aldehyde production were partially characterized, including measurement of pH optima. The differences in C6-aldehyde formation among the fatty acid mutants of Arabidopsis appeared not to result from alteration of lipoxygenase/hydroperoxide lyase pathway enzymes. Investigation of the fatty acid composition in leaf phospholipids, glycolipids, and neutral lipids and analysis of the fatty acid composition of chloroplast and extrachloroplast lipids indicate that chloroplasts and glycolipids of chloroplasts may be the source or major source of C6-aldehyde formation in Arabidopsis leaves.     

The sunflower plastidial ω3-fatty acid desaturase (HaFAD7) contains the signalling determinants required for targeting to, and retention in, the endoplasmic reticulum membrane in yeast but requires co-expressed ferredoxin for activity

Although plant plastidial ω3-desaturases are closely related to microsomal desaturases, heterologous expression in yeast of the Helianthus annuus FAD7 ω3-desaturase showed low activity in contrast to similar expression of microsomal FAD3 ω3-desaturases. However, the removal of the plastidial transit peptide and the incorporation of a KKNL motif to the C-terminus of HaFAD7 increased the activity by 10-fold compared to the native protein. N-terminal fusion of transmembrane-domains from either the yeast microsomal ELO3, (a type III signal anchor domain), or FAE1, an endoplasmic reticulum membrane anchoring domain, resulted in moderate increases in enzyme activity (5- and 7-fold, respectively), suggesting that the first, most hydrophobic transmembrane domain of HaFAD7 is sufficient to direct targeting to, and insertion into, the endoplasmic reticulum membrane. Furthermore, fusing a hemagglutinin (HA) epitope tag upstream of an endogenous C-terminal KEK motif resulted in a significant loss of activity compared to the un-tagged construct, indicating that the endogenous KEK C-terminal di-lysine motif is capable of directing in yeast the ER-retention of this normally plastidial-located protein. Western blotting analysis of constructs with internal HA epitope revealed that in whole cell extracts, with the exception of the one bound to C-terminal, it did not display a reduced level of protein accumulation. Whilst ferredoxin was shown to be required for HaFAD7 activity in yeast, it appears not necessary for protein stability and accumulation of this plastidial desaturase in the endoplasmic reticulum.