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.     

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.     

Photoinhibition in mutants of Arabidopsis deficient in thylakoid unsaturation

Thylakoid lipid composition in higher plants is characterized by a high level of fatty acid unsaturation. We have screened four mutants of Arabidopsis that have reduced levels of fatty acid unsaturation. Three of the mutant lines tested, fad5, fad6, and the fad3-2 fad7-2 fad8 triple mutant, were more susceptible to photoinhibition than wild-type Arabidopsis, whereas one mutant, fab1, was indistinguishable from wild type. The fad3-2 fad7-2 fad8 triple mutant, which contains no trienoic fatty acids in its thylakoid membranes, was most susceptible to photoinhibition. Detailed investigation of photoinhibition in the triple mutant revealed that the rate of photoinactivation of PSII was the same in wild-type and mutant plants. However, the recovery of photoinactivated PSII was slower in fad3-2 fad7-2 fad8, relative to wild type, at all temperatures below 27 degrees C. These results indicate that trienoic fatty acids of thylakoid membrane lipids are required for low-temperature recovery from photoinhibition in Arabidopsis.     

Arabidopsis mutants reveal that short- and long-term thermotolerance have different requirements for trienoic fatty acids

The photosynthetic thylakoid has the highest level of lipid unsaturation of any membrane. In Arabidopsis thaliana plants grown at 22°C, approximately 70% of the thylakoid fatty acids are trienoic - they have three double bonds. In Arabidopsis, and other species, the levels of trienoic fatty acids decline substantially at higher temperatures. Several genetic studies indicate that reduced unsaturation improves photosynthetic function and plant survival at high temperatures. Here, these studies are extended using the Arabidopsis triple mutant, fad3-2 fad7-2 fad8 that contains no detectable trienoic fatty acids. In the short-term, fluorescence analyses and electron-transport assays indicated that photosynthetic functions in this mutant are more thermotolerant than the wild type. However, long-term photosynthesis, growth, and survival of plants were all compromised in the triple mutant at high temperature. The fad3-2 fad7-2 fad8 mutant is deficient in jasmonate synthesis and this hormone has been shown to mediate some aspects of thermotolerance; however, additional experiments demonstrated that a lack of jasmonate was not a major factor in the death of triple-mutant plants at high temperature. The results indicate that long-term thermotolerance requires a basal level of trienoic fatty acids. Thus, the success of genetic and molecular approaches to increase thermotolerance by reducing membrane unsaturation will be limited by countervailing effects that compromise essential plant functions at elevated temperatures.     

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.     

Identification of a gene that complements an Arabidopsis mutant deficient in chloroplast omega 6 desaturase activity.

Membrane lipids of the fad6 (formerly fadC) mutant of Arabidopsis, which is deficient in chloroplast omega 6 desaturase activity, have increased levels of monounsaturated fatty acids and are deficient in trienoic fatty acids. A putative fad6 cDNA clone was isolated by probing a cDNA library with a degenerate oligonucleotide based on a conserved region within known omega 3 desaturase genes. Expression of the cDNA in transgenic plants of a fad6 mutant restored normal levels of all fatty acids. When used as a hybridization probe, the cDNA identified a restriction fragment-length polymorphism that co-segregated with the fad6 mutation. Thus, on the basis of a genetic complementation test and genetic map position, the fad6 gene is encoded by the cDNA. The cDNA encoded a 418-amino acid polypeptide of 47,727 D that displayed a high degree of sequence similarity to a delta 12 desaturase from the cyanobacterium Synechocystis. The fad6 gene exhibited less sequence homology to any known higher plant desaturase, including an endoplasmic reticulum-localized omega 6 desaturase corresponding to the Arabidopsis fad2 gene.     

Tocopherols modulate extraplastidic polyunsaturated fatty acid metabolism in Arabidopsis at low temperature

Tocopherols (vitamin E) are synthesized in plastids and have long been assumed to have essential functions restricted to these organelles. We previously reported that the vitamin e-deficient2 (vte2) mutant of Arabidopsis thaliana is defective in transfer cell wall development and photoassimilate transport at low temperature (LT). Here, we demonstrate that LT-treated vte2 has a distinct composition of polyunsaturated fatty acids (PUFAs): lower levels of linolenic acid (18:3) and higher levels of linoleic acid (18:2) compared with the wild type. Enhanced 18:3 oxidation was not involved, as indicated by the limited differences in oxidized lipid species between LT-treated vte2 and the wild type and by a lack of impact on the LT-induced vte2 phenotype in a vte2 fad3 fad7 fad8 quadruple mutant deficient in 18:3. PUFA changes in LT-treated vte2 occur primarily in phospholipids due to reduced conversion of dienoic to trienoic fatty acids in the endoplasmic reticulum (ER) pathway. Introduction of the ER fatty acid desaturase mutation, fad2, and to a lesser extent the plastidic fad6 mutation into the vte2 background suppressed the LT-induced vte2 phenotypes, including abnormal transfer cell wall development. These results provide biochemical and genetic evidence that plastid-synthesized tocopherols modulate ER PUFA metabolism early in the LT adaptation response of Arabidopsis.     

The Effect of Tobacco Plant Transformation with a Gene for Acyl-Lipid Δ9-Desaturase from <Emphasis Type="Italic">Synechococcus vulcanus</Emphasis> on Plant Chilling Tolerance

Tobacco plants with the introduced desC gene for acyl-lipid Δ9-desaturase from the thermophilic cyanobacterium Synechococcus vulcanus were cultivated on agar-solidified Murashige and Skoog nutrient medium supplemented with ferulic acid and antibiotics at 22°C and a 16-h photoperiod. Control plants were transformed with an empty pGA482 vector. The analysis of fatty acids (FAs) showed that, in transgenic plants, the level of 16:0 and 18:0 FAs decreased substantially, whereas the levels of di- and trienoic FAs increased. Transformed plants were more cold-tolerant. The tolerance to chilling was evaluated from electrolyte leakage from tissues damaged by cold treatments and from the accumulation of a product of lipid peroxidation, malondialdehyde. It was concluded that acyl-lipid Δ9-desaturase was actively expressed in transgenic tobacco plants and converted stearic acid into oleic acid, thus producing a substrate for further synthesis of di- and trienoic FAs. An increased proportion of polyunsaturated FAs in membrane lipids resulted in improved tobacco plant tolerance to chilling.     

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.     

The Critical Requirement for Linolenic Acid Is Pollen Development,Not Photosynthesis,in an Arabidopsis Mutant

The very high proportions of trienoic fatty acids found in chloroplast membranes of all higher plants suggest that these lipid structures might be essential for photosynthesis. We report here on the production of Arabidopsis triple mutants that contain negligible levels of trienoic fatty acids. Photosynthesis at 22[deg]C was barely affected, and vegetative growth of the mutants was identical with that of the wild type, demonstrating that any requirement for trienoic acyl groups in membrane structure and function is relatively subtle. Although vegetative growth and development were unaffected, the triple mutants are male sterlle and produce no seed under normal conditions. Comparisons of pollen development in wild-type and triple mutant flowers established that pollen grains in the mutant developed to the tricellular stage. Exogenous applications of [alpha]-llnolenate or jasmonate restored fertility. Taken together, the results demonstrate that the critical role of trienoic acids in the life cycle of plants is as the precursor of oxylipin, a signaling compound that regulates final maturation processes and the release of pollen.     

Trienoic fatty acids are required to maintain chloroplast function at low temperatures

The chloroplast membranes of all higher plants contain very high proportions of trienoic fatty acids. To investigate how these lipid structures are important in photosynthesis, we have generated a triple mutant line of Arabidopsis that contains negligible levels of trienoic fatty acids. For mutant plants grown at 22 degrees C, photosynthetic fluorescence parameters were indistinguishable from wild type at 25 degrees C. Lowering the measurement temperature led to a small decrease in photosynthetic quantum yield, Phi(II), in the mutant relative to wild-type controls. These and other results indicate that low temperature has only a small effect on photosynthesis in the short term. However, long-term growth of plants at 4 degrees C resulted in decreases in fluorescence parameters, chlorophyll content, and thylakoid membrane content in triple-mutant plants relative to wild type. Comparisons among different mutant lines indicated that these detrimental effects of growth at 4 degrees C are strongly correlated with trienoic fatty acid content with levels of 16:3 + 18:3, approximately one-third of wild type being sufficient to sustain normal photosynthetic function. In total, our results indicate that trienoic fatty acids are important to ensure the correct biogenesis and maintenance of chloroplasts during growth of plants at low temperatures.     

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.     

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.     

A mutant of Arabidopsis deficient in c(18:3) and c(16:3) leaf lipids

Leaf tissue of a mutant of Arabidopsis thaliana contains reduced levels of both 16:3 and 18:3 fatty acids and has correspondingly increased levels of the 16:2 and 18:2 precursors due to a single recessive nuclear mutation. The kinetics of in vivo labeling of lipids with [¹⁴C]acetate and quantitative analysis of the fatty acid compositions of individual lipids suggests that reduced activity of a glycerolipid n-3 desaturase is responsible for the altered lipid composition of the mutant. The effects of the mutation are most pronounced when plants are grown at temperatures above 26°C but are relatively minor below 18°C, suggesting a temperature-sensitive enzyme. Since the desaturation of both 16- and 18-carbon fatty acids is altered, it appears that the affected enzyme lacks specificity with respect to acyl group chain length and that it is located in the chloroplast where 16:3-monogalactosyldiglyceride is synthesized. Because the degree of unsaturation of all the major glycerolipids was similarly affected by the mutation, it is inferred that either the affected desaturase does not exhibit head group specificity or there is substantial transfer of trienoic acyl groups between different lipid classes. Both chloroplast and extrachloroplast lipids are equally affected by the mutation. Thus, either the desaturase is located both outside and inside the chloroplast, or 18:3 formed inside the chloroplast is reexported to other cellular sites.     

A suppressor of fab1 challenges hypotheses on the role of thylakoid unsaturation in photosynthetic function

Leaf membrane lipids of the Arabidopsis (Arabidopsis thaliana) fatty acid biosynthesis 1 (fab1) mutant contain a 35% to 40% increase in the predominant saturated fatty acid 16:0, relative to wild type. This increase in membrane saturation is associated with loss of photosynthetic function and death of mutant plants at low temperatures. We have initiated a suppressor screen for mutations that allow survival of fab1 plants at 2 degrees C. Five suppressor mutants identified in this screen all rescued the collapse of photosynthetic function observed in fab1 plants. While fab1 plants died after 5 to 7 weeks at 2 degrees C, the suppressors remained viable after 16 weeks in the cold, as judged by their ability to resume growth following a return to 22 degrees C and to subsequently produce viable seed. Three of the suppressors had changes in leaf fatty acid composition when compared to fab1, indicating that one mechanism of suppression may involve compensating changes in thylakoid lipid composition. Surprisingly, the suppressor phenotype in one line, S31, was associated with a further substantial increase in lipid saturation. The overall leaf fatty acid composition of S31 plants contained 31% 16:0 compared with 23% in fab1 and 17% in wild type. Biochemical and genetic analysis showed that S31 plants contain a new allele of fatty acid desaturation 5 (fad5), fad5-2, and are therefore partially deficient in activity of the chloroplast 16:0 Delta7 desaturase. A double mutant produced by crossing fab1 to the original fad5-1 allele also remained alive at 2 degrees C, indicating that the fad5-2 mutation is the suppressor in the S31 (fab1 fad5-2) line. Based on the biophysical characteristics of saturated and unsaturated fatty acids, the increased 16:0 in fab1 fad5-2 plants would be expected to exacerbate, rather than ameliorate, low-temperature damage. We propose instead that a change in shape of the major thylakoid lipid, monogalactosyldiacylglycerol, mediated by the fad5-2 mutation, may compensate for changes in lipid structure resulting from the original fab1 mutation. Our identification of mutants that suppress the low-temperature phenotype of fab1 provides new tools to understand the relationship between thylakoid lipid structure and photosynthetic function.     

Enhancement of low-temperature tolerance in transgenic tomato plants overexpressing Lefad7 through regulation of trienoic fatty acids

We studied how tomato (Lycopersicon esculentum Mill.) chloroplast omega-3 fatty acid desaturase gene (Lefad7) overexpression enhanced low-temperature (LT) tolerance in transgenic tomato plants. In these plants, the content of linolenic acid (18:3) markedly increased and, correspondingly, the content of linoleic acid (18:2) decreased. Similar changes were found after 6 h under LT (4°C) treatment. Under LT stress, wild type (WT) tomato plants showed a much greater increase in relative electrolyte leakage and malondialdehyde (MDA) contents compared with transgenic plants. Transgenic plants exhibited higher activities of antioxidative enzymes and a lower content of reactive oxygen species (ROS). Transgenic plants maintained a relatively higher level of the net photosynthetic rate (P _N) and chlorophyll (Chl) content than WT plants under LT stress. Taken together, we suggested that overexpression of Lefad7 enhanced LT tolerance by changing the composition of membrane lipids in tomato plants, with the increased content of trienoic fatty acids and reduced content of dienoic fatty acids that led to series of physiological alterations.