Participation and Properties of Lipoxygenase and Hydroperoxide Lyase in Volatile C6-Aldehyde Formation from C18-Unsaturated. Fatty Acids in Isolated Tea Chloroplasts

Isolated tea chloroplasts utilized linoleic acid, linolenicacid and their 13-hydroperoxides as substrates for volatileC₆-aldehyde formation. Optimal pH values for oxygen uptake,hydroperoxide lyase and the overall reaction from C₁₈-fattyacids to C₆-aldehydes were 6.3, 7.0 and 6.3, respectively. Methyllinoleate, linoleyl alcohol and -linolenic acid were poor substratesfor the overall reaction, but linoleic and linolenic acids weregood substrates. The 13-hydroperoxides of the above fatty acidsand alcohol also showed substrate specificity similar to thatof fatty acids. Oxygen uptakes (relative V_max) with methyl linoleate,linoleyl alcohol, linolenic acid, -linolenic acid and arachidonicacid were comparable to or higher than that with linoleic acid.In winter leaves, the activity for C₆-aldehyde formation fromC₁₈-fatty acids was raduced to almost zero. This was due tothe reduction in oxygenation. The findings presented here provideevidence for the involvement of lipoxygenase and hydroperoxidelyase in C₆-aldehyde formation in isolated chloroplasts. (Received July 11, 1981; Accepted November 5, 1981)     

Developmental change in c(6)-aldehyde formation by soybean leaves

Damage to plant leaves by wounding or freezing induces the production of large amounts of C₆-compounds. However, the control of formation of these compounds in leaves is not yet clear. In the current study, C₆-aldehyde formation by freeze-injured soybean leaves of different ages (based on the leaf positions on the plant) at stage R1 of plant development was investigated. The results demonstrate that C₆-aldehyde formation by the soybean (Glycine max L.) leaves changes as leaves develop. Younger leaves produce high levels of C₆-aldehydes, mainly composed of hexanal. Subsequently, as the leaves develop, the level of C₆-aldehyde formation decreases markedly, followed by an increase with a large shift from hexanal to hexenals. Lipoxygenase and lipolytic acyl hydrolase activity was reduced, and, in contrast, hydroperoxide lyase activity increased. There was little difference in lipoxygenase substrate specificity for linoleic acid and linolenic acid, but hydroperoxide lyase preferentially utilized 13-hydroperoxy-9,11,15-octadecatrienoic acid. In the in vivo lipoxygenase substrate pool, the linoleic acid level declined and the relative level of linolenic acid increased. The change in ratios of linolenic acid to linoleic acid showed a similar trend during soybean leaf development to that of hexenals to hexanal.     

Natural Inhibitor for Volatile C6-Aldehyde Formation from C18-Unsaturated Fatty Acids

The homogenate of tea seed cotyledons contained an inhibitor for C₆-aldehyde formation from linoleic acid and linolenic acid by isolated tea chloroplasts. Seed homogenates of other plants, such as soybean, kidney bean, cucumber, Japanese radish and rice, also contained the inhibitor for C₆-aldehyde formation. The inhibitor from tea seed and cucumber seed inhibited C₆-aldehyde formation by the homogenate of cucumber hypocotyl. Hydroperoxides of linoleic acid detected were reduced when the tea seed inhibitor was added to the reaction mixture, but the enzyme activities of lipoxygenase and hydroperoxide lyase were not inhibited. This means that the inhibitor is a decomposer of fatty acid hydroperoxides as an intermediate of C₆-aldehyde formation. The tea seed inhibitor was formed during the seed ripening and it was stable during the seed germination. These findings obtained here suggest that the inhibitor is widely present in plant seeds and inhibits C₆-aldehyde formation by a variety of plant tissues.     

Volatile C6-Aldehyde Formation via Hydroperoxides from C18-Unsaturated Fatty Acids in Etiolated Alfalfa and Cucumber Seedlings

1. Etiolated seedlings of alfalfa and cucumber evolved n-hexanal from linoleic acid and cis-3-hexenal and trans-2-hexenal from linolenic acid when they were homogenized.

2. The activities for n-hexanal formation from linoleic acid, lipoxygenase and hydro-peroxide lyase were maximum in dry seeds and 1~2 day-old etiolated seedlings of alfalfa, and in 6~7 day-old etiolated seedlings of cucumber.

3. n-Hexanal was produced from linoleic acid and 13-hydroperoxylinoleic acid by the crude extracts of etiolated alfalfa and cucumber seedlings. cis-3-Hexenal and trans-2-hexenal were produced from linolenic acid and 13-hydroperoxylinolenic acid by the crude extracts of etiolated alfalfa and cucumber seedlings. But these extracts, particulariy cucumber one, showed a high isomerizing activity from cis-3-hexenal to trans-2-hexenal.

4. When the C₈-aldehydes were produced from linoleic acid and linolenic acid by the crude extracts, formation of hydroperoxides of these C₁₈-fatty acids was observed.

5. When 9-hydroperoxylinoleic acid was used as a substrate, trans-2-nonenal was produced by the cucumber homogenate but not by the alfalfa homogenate.

6. As the enzymes concerned with C₆-aldehyde formation, lipoxygenase was partially purified from alfalfa and cucumber seedlings and hydroperoxide lyase, from cucumber seedlings. Lipoxygenase was found in a soluble fraction, but hydroperoxide lyase was in a membrane bound form. Alfalfa lipoxygenase catalyzed formation of 9- and 13-hydroperoxylinoleic acid (35: 65) from linoleic acid and cucumber one, mainly 13-hydroperoxylinoleic acid formation. Alfalfa hydroperoxide lyase catalyzed n-hexanal formation from 13-hydroperoxylinoleic acid, but cucumber one catalyzed formation of n-hexanal and trans-2-nonenal from 13- and 9-hydroperoxylinoleic acid, respectively.

7. From the above results, the biosynthetic pathway for C₆-aldehyde formation in etiolated alfalfa and cucumber seedlings is established that C₆-aldehydes (n-hexanal, cis-3-hexenal and trans-2-hexenal) are produced from linoleic acid and linolenic acid via their 13-hydroperoxides by lipoxygenase and hydroperoxide lyase.     

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.     

Distribution of lipoxygenase and hydroperoxide lyase in the leaves of various plant species

The enzyme activity responsible for volatile C₆-aldehyde formation was accompanied by lipoxygenase and hydroperoxide lyase in the green leaves of 28 plant species tested, but the level of each enzyme's activity varied. Lipoxygenase activity rather than hydroperoxide lyase activity appears to affect the overall C₆-aldehyde formation. There was a positive correlation (r = 0.712) between hydroperoxide lyase activity and the chlorophyll content of the green leaves; no correlation was found between lipoxygenase activity and chlorophyll content.     

Biosynthesis of C9-aldehydes in the moss Physcomitrella patens

After wounding, the moss Physcomitrella patens emits fatty acid derived volatiles like octenal, octenols and (2E)-nonenal. Flowering plants produce nonenal from C18-fatty acids via lipoxygenase and hydroperoxide lyase reactions, but the moss exploits the C20 precursor arachidonic acid for the formation of these oxylipins. We describe the isolation of the first cDNA (PpHPL) encoding a hydroperoxide lyase from a lower eukaryotic organism. The physiological pathway allocation and characterization of a downstream enal-isomerase gives a new picture for the formation of fatty acid derived volatiles from lower plants. Expression of a fusion protein with a yellow fluorescent protein in moss protoplasts showed that PpHPL was found in clusters in membranes of plastids. PpHPL can be classified as an unspecific hydroperoxide lyase having a substrate preference for 9-hydroperoxides of C18-fatty acids but also the predominant substrate 12-hydroperoxy arachidonic acid is accepted. Feeding experiments using arachidonic acid show an increase in the 12-hydroperoxide being metabolized to C8-aldehydes/alcohols and (3Z)-nonenal, which is rapidly isomerized to (2E)-nonenal. PpHPL knock out lines failed to emit (2E)-nonenal while formation of C8-volatiles was not affected indicating that in contrast to flowering plants, PpHPL is only involved in formation of a specific subset of volatiles.     

C6-aldehyde formation by fatty acid hydroperoxide lyase in the brown alga Laminaria angustata

Some marine algae can form volatile aldehydes such as n-hexanal, hexenals, and nonenals. In higher plants it is well established that these short-chain aldehydes are formed from C18 fatty acids via actions of lipoxygenase and fatty acid hydroperoxide lyase, however, the biosynthetic pathway in marine algae has not been fully established yet. A brown alga, Laminaria angustata, forms relatively higher amounts of C6- and C9-aldehydes. When linoleic acid was added to a homogenate prepared from the fronds of this algae, formation of n-hexanal was observed. When glutathione peroxidase was added to the reaction mixture concomitant with glutathione, the formation of n-hexanal from linoleic acid was inhibited, and oxygenated fatty acids accumulated. By chemical analyses one of the major oxygenated fatty acids was shown to be (S)-13-hydroxy-(Z, E)-9, 11-octadecadienoic acid. Therefore, it is assumed that n-hexanal is formed from linoleic acid via a sequential action of lipoxygenase and fatty acid hydroperoxide lyase (HPL), by an almost similar pathway as the counterpart found in higher plants HPL partially purified from the fronds has a rather strict substrate specificity, and only 13-hydroperoxide of linoleic acid, and 15-hydroperoxide of arachidonic acid are the essentially suitable substrates for the enzyme. By surveying various species of marine algae including Phaeophyta, Rhodophyta and Chlorophyta it was shown that almost all the marine algae have HPL activity. Thus, a wide distribution of the enzyme is expected.     

Altered rates of protein transport in Arabidopsis mutants deficient in chloroplast membrane unsaturation

Protein transfer across membranes is mediated by protein machinery embedded in the membrane. The complement of different lipid classes within a membrane is known to influence the efficiency of some protein translocation processes, but very little is known about whether the fatty acid composition of the membrane bilayer also affects protein transport. We investigated this issue using three mutants of Arabidopsis, fad6, fad5, and fad3 fad7 fad8, that have reduced levels of fatty acid unsaturation in their thylakoid membranes. Interestingly, the effect of reduced unsaturation was different for three distinct pathways of protein transport. In thylakoids from all three mutants, transport of the OE17 protein on the DeltapH/Tat pathway was reduced by up to 50% relative to wild-type controls, when assays were run at 10, 20 or 30 degrees C. By contrast, transport of the OE33 protein on the Sec pathway was substantially increased in all the mutants at the three temperatures. Transport of the CF(O)II protein (ATPg) on the 'spontaneous' pathway was largely unaffected by reduced unsaturation of the thylakoid membranes. Experiments with intact chloroplasts from wild-type Arabidopsis and the three mutants confirmed these changes in thylakoid transport reactions and also demonstrated an increased rate of protein import across the chloroplast envelope in each of the mutants. This increased capacity of chloroplast protein import may partially compensate for the reduced capacity of thylakoid transport by the DeltapH/Tat pathway. The fad5, fad6 and fad3 fad7 fad8 mutants used in this study grow normally at 15-20 degrees C, but exhibit reduced photosynthesis and growth, relative to wild-type controls, at low temperatures (4 degrees C). The results reported here indicate that protein transport and chloroplast biogenesis may well contribute to these low-temperature phenotypes.     

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.     

Differential distribution of the lipoxygenase pathway enzymes within potato chloroplasts

The lipoxygenase pathway is responsible for the production of oxylipins, which are important compounds for plant defence responses. Jasmonic acid, the final product of the allene oxide synthase/allene oxide cyclase branch of the pathway, regulates wound-induced gene expression. In contrast, C6 aliphatic aldehydes produced via an alternative branch catalysed by hydroperoxide lyase, are themselves toxic to pests and pathogens. Current evidence on the subcellular localization of the lipoxygenase pathway is conflicting, and the regulation of metabolic channelling between the two branches of the pathway is largely unknown. It is shown here that while a 13-lipoxygenase (LOX H3), allene oxide synthase and allene oxide cyclase proteins accumulate upon wounding in potato, a second 13-lipoxygenase (LOX H1) and hydroperoxide lyase are present at constant levels in both non-wounded and wounded tissues. Wound-induced accumulation of the jasmonic acid biosynthetic enzymes may thus commit the lipoxygenase pathway to jasmonic acid production in damaged plants. It is shown that all enzymes of the lipoxygenase pathway differentially localize within chloroplasts, and are largely found associated to thylakoid membranes. This differential localization is consistently observed using confocal microscopy of GFP-tagged proteins, chloroplast fractionation, and western blotting, and immunodetection by electron microscopy. While LOX H1 and LOX H3 are localized both in stroma and thylakoids, both allene oxide synthase and hydroperoxide lyase protein localize almost exclusively to thylakoids and are strongly bound to membranes. Allene oxide cyclase is weakly associated with the thylakoid membrane and is also detected in the stroma. Moreover, allene oxide synthase and hydroperoxide lyase are differentially distributed in thylakoids, with hydroperoxide lyase localized almost exclusively to the stromal part, thus closely resembling the localization pattern of LOX H1. It is suggested that, in addition to their differential expression pattern, this segregation underlies the regulation of metabolic fluxes through the alternative branches of the lipoxygenase pathway.     

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.     

Lipoxygenase, Hydroperoxide Lyase and Volatile C6-Aldehyde Formation from C18-Fatty Acids during Development of Phaseolus vulgaris L.

Kidney bean plants (Phaseolus vulgaris) were found to have thecapability to produce C₆-aldehydes (hexanal and hexenals) fromlinoleic and linolenic acids. The various organs tested hadlipoxygenase and hydroperoxide lyase activities responsiblefor the C₆-aldehyde formation. Young leaves showed relativelyhigh activities for C₆-aldehyde formation. However, the activitiesof the leaves decreased gradually with leaf expansion. Seedlingsand seeds containing cotyledons showed low activities for C₆-aldehydeformation, because of the occurrence of an inhibitory factorin the cotyledons. The substrate specificity of the enzymeswas essentially the same among the various developmental stagesof leaves tested. (Received February 5, 1982; Accepted March 19, 1982)     

Genetic Enhancement of Cold Tolerance by Expression of a Gene for Chloroplast [omega]-3 Fatty Acid Desaturase in Transgenic Tobacco

The increased production of trienoic fatty acids, hexadecatrienoic (16:3) and linolenic (18:3) acids, is a response connected with cold acclimation of higher plants and is thought to protect plant cells against cold damage. Transgenic tobacco (Nicotiana tabacum cv SR1) plants that contain increased levels of 16:3 and 18:3 fatty acids, and correspondingly decreased levels of their precursors, hexadecadienoic and linoleic acids, were engineered by introduction of a chloroplast [omega]-3 fatty acid desaturase gene (the fad7 gene) isolated from Arabidopsis thaliana. When exposed to 1[deg]C for 7 d and then cultured at 25[deg]C, the suppression of leaf growth observed in the wild-type plants was significantly alleviated in the transgenic plants with the fad7 gene. The low-temperature- induced chlorosis was also much reduced in the plants transformed with the fad7 gene. These results indicate that increased levels of trienoic fatty acids in genetically engineered plants enhance cold tolerance.     

Sequence variation and genomic organization of fatty acid desaturase-2 (fad2) and fatty acid desaturase-6 (fad6) cDNAs in maize

Increased levels of oleic acid may enhance the nutritional and functional value of corn. Corn oil is primarily composed of palmitic, stearic, oleic, linoleic and linolenic fatty acids. Delta-12 desaturase in plants converts oleic acid (18:1) to linoleic acid (18:2) by inserting a double bond at the delta-12 position. Fatty acid desaturase-2 (fad2) encodes delta-12 desaturase that functions in the endoplasmic reticulum while fatty acid desaturase-6 (fad6) encodes delta-12 desaturase that functions in plastids. Complementary DNA (cDNA) clones from putative maize homologs for fad2 and fad6 were identified and the entire clones DNA sequenced. The maize fad2 cDNAs showed an amino-acid identity of 67-77% to fad2 of Glycine, Arabidopsis and Brassica species. Our cDNA sequence comparisons suggested that more than one fad2 gene is transcribed in maize embryos. Two different fad2 cDNAs from an embryo cDNA library map to separate chromosomal positions, providing evidence consistent with two different isoforms of fad2 expressed in the embryo. The fad2 cDNAs from multiple tissue sources clustered into three groups on a phenogram, and map to different positions on chromosomes 4, 5 and 10, which suggests at least three different isoforms of fad2 may be expressed in the maize plant. The two maize fad6 cDNAs share 81% amino-acid identity with the Arabidopsis fad6 and map to chromosome 1. Northern analysis revealed that fad2 is transcribed in embryos at 14, 21, 28 and 35 days after pollination, with the highest level observed at day 14. None of the fad2 or fad6 clones mapped to maize chromosome bins associated with QTLs for the ratio of oleic/linoleic acid, notably bin 6.04 which contains the linoleic1 locus and the largest reported QTL for the oleic/linoleic ratio. This suggests, but does not prove, that some of the QTLs for the oleic/linoleic acid ratio do not involve allelic variants of fad2 or fad6 but rather involve other genes that may influence flux through the enzymes encoded by fad2 or fad6.     

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.     

Polyunsaturated fatty acid metabolism in fish cells: differential metabolism of (n-3) and (n-6) series acids by cultured cells originating from a freshwater teleost fish and from a marine teleost fish

1. The incorporation and metabolism of (n-3) and (n-6) polyunsaturated fatty acids (PUFA) supplemented to growing cultures were studied in rainbow trout (RTG-2) and turbot (TF) cell lines. 2. A fatty acid concentration of 20 microM considerably altered the fatty acid composition of the cells without affecting lipid class composition or the appearance of cytoplasmic lipid droplets. 3. Both cell lines exhibited considerable delta 6 desaturase activities. 4. Whereas delta 5 desaturase activity was expressed in RTG-2 cells, delta 4 desaturase activity was absent and, conversely, delta 4 desaturase activity was expressed in TF cells, but there was an apparent deficiency in the C18 to C20 elongase multi-enzyme complex. 5. The delta 6 desaturase activity in both cell lines showed little preference between 18:2(n-6) and 18:3(n-3) but the delta 5 desaturase activity of RTG-2 cells and the delta 4 desaturase activity of TF cells showed a preference for (n-3)PUFA. 6. Two fish oil concentrates were assessed for their ability to generate fatty acid compositions in the cell lines more closely resembling those of intact fish tissues.     

Direct fungicidal activities of C6-aldehydes are important constituents for defense responses in Arabidopsis against Botrytis cinerea

C6-aldehydes, such as (Z)-3-hexenal, (E)-2-hexenal, and n-hexanal, are volatile compounds formed by hydroperoxide lyase (HPL) and found in most terrestrial plants. They are fungicidal and bactericidal compounds, and are also signaling compounds to induce defense responses in plants. Transgenic plants having overexpressed or suppressed HPL activity (SH or ASH, respectively) showed lower or higher susceptibility against a necrotrophic fungal pathogen, Botrytis cinerea. In this study, we examined whether the modulated susceptibility was accountable to the direct fungicidal activity or to the signaling potency of C6-aldehydes. When wild-type Arabidopsis leaves were inoculated with B. cinerea, HPL expression was upregulated, and concomitantly, the amounts of C6-aldehydes increased. Higher amounts of C6-aldehydes found in inoculated SH plants inhibited growth of B. cinerea in vitro, while lower amounts found in ASH plants caused no inhibitory effect on the fungi. Thus, it was suggested that direct fungicidal activity of C6-aldehydes accounted for the modulated susceptibility. With SH plants higher amounts of camalexin could be found, but with the ASH plants no difference from wild-type plants could be found. Surplus amounts of C6-aldehydes could induce formation of camalexin as signaling compounds; however, this was not the case with wild-type and ASH plants. Accordingly, it could be assumed that direct fungicidal activity of C6-aldehydes were prominently responsible to the defense against B. cinerea but their signaling roles could be little responsible if any.     

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.     

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.