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• W1997593467 abstract "Malondialdehyde (MDA) is a small, ubiquitous, and potentially toxic aldehyde that is produced in vivo by lipid oxidation and that is able to affect gene expression. Tocopherol deficiency in the vitamin E2 mutant vte2-1 of Arabidopsis thaliana leads to massive lipid oxidation and MDA accumulation shortly after germination. MDA accumulation correlates with a strong visual phenotype (growth reduction, cotyledon bleaching) and aberrant GST1 (glutathione S-transferase 1) expression. We suppressed MDA accumulation in the vte2-1 background by genetically removing tri-unsaturated fatty acids. The resulting quadruple mutant, fad3-2 fad7-2 fad8 vte2-1, did not display the visual phenotype or the aberrant GST1 expression observed in vte2-1. Moreover, cotyledon bleaching in vte2-1 was chemically phenocopied by treatment of wild-type plants with MDA. These data suggest that products of tri-unsaturated fatty acid oxidation underlie the vte2-1 seedling phenotype, including cellular toxicity and gene regulation properties. Generation of the quadruple mutant facilitated the development of an in situ fluorescence assay based on the formation of adducts of MDA with 2-thiobarbituric acid at 37 °C. Specificity was verified by measuring pentafluorophenylhydrazine derivatives of MDA and by liquid chromatography analysis of MDA-2-thiobarbituric acid adducts. Potentially applicable to other organisms, this method allowed the localization of MDA pools throughout the body of Arabidopsis and revealed an undiscovered pool of the compound unlikely to be derived from trienoic fatty acids in the vicinity of the root tip quiescent center. Malondialdehyde (MDA) is a small, ubiquitous, and potentially toxic aldehyde that is produced in vivo by lipid oxidation and that is able to affect gene expression. Tocopherol deficiency in the vitamin E2 mutant vte2-1 of Arabidopsis thaliana leads to massive lipid oxidation and MDA accumulation shortly after germination. MDA accumulation correlates with a strong visual phenotype (growth reduction, cotyledon bleaching) and aberrant GST1 (glutathione S-transferase 1) expression. We suppressed MDA accumulation in the vte2-1 background by genetically removing tri-unsaturated fatty acids. The resulting quadruple mutant, fad3-2 fad7-2 fad8 vte2-1, did not display the visual phenotype or the aberrant GST1 expression observed in vte2-1. Moreover, cotyledon bleaching in vte2-1 was chemically phenocopied by treatment of wild-type plants with MDA. These data suggest that products of tri-unsaturated fatty acid oxidation underlie the vte2-1 seedling phenotype, including cellular toxicity and gene regulation properties. Generation of the quadruple mutant facilitated the development of an in situ fluorescence assay based on the formation of adducts of MDA with 2-thiobarbituric acid at 37 °C. Specificity was verified by measuring pentafluorophenylhydrazine derivatives of MDA and by liquid chromatography analysis of MDA-2-thiobarbituric acid adducts. Potentially applicable to other organisms, this method allowed the localization of MDA pools throughout the body of Arabidopsis and revealed an undiscovered pool of the compound unlikely to be derived from trienoic fatty acids in the vicinity of the root tip quiescent center. The in vivo oxidation of polyunsaturated fatty acids and the diverse compounds generated by this process in healthy and diseased organisms are of increasing interest. Higher plants such as Arabidopsis thaliana offer particularly good systems in which to study many aspects of the biology of this type of molecule because of the availability of mutants in fatty acid synthesis and metabolism. Indeed, the complex cellular organization of plants offers many possibilities to explore the physiological effects, genesis, and distribution of nonenzymatic lipid oxidation products at the whole-organism level. The latter aspect, the whole-body distribution of lipid oxidation products at the cell and tissue level, is largely unexplored in complex organisms like plants.The interest in nonenzymatic products of lipid oxidation stems in part from the fact that they provide markers in pathogenesis (1Muckenschnabel I. Goodman B.A. Williamson B. Lyon G.D. Deighton N. J. Exp. Bot. 2002; 53: 207-214Crossref PubMed Scopus (85) Google Scholar, 2Loeffler C. Berger S. Guy A. Durand T. Bringmann G. Dreyer M. von Rad U. Durner J. Mueller M.J. Plant Physiol. 2005; 137: 328-340Crossref PubMed Scopus (108) Google Scholar) and that they may also have powerful biological activities in the control of stress response gene expression (2Loeffler C. Berger S. Guy A. Durand T. Bringmann G. Dreyer M. von Rad U. Durner J. Mueller M.J. Plant Physiol. 2005; 137: 328-340Crossref PubMed Scopus (108) Google Scholar, 3Farmer E.E. Davoine C. Curr. Opin. Plant Biol. 2007; 10: 380-386Crossref PubMed Scopus (219) Google Scholar, 4Mueller M.J. Curr. Opin. Plant Biol. 2004; 7: 441-448Crossref PubMed Scopus (159) Google Scholar, 5Vollenweider S. Weber H. Stolz S. Chetelat A. Farmer E.E. Plant J. 2000; 24: 467-476Crossref PubMed Google Scholar). Additionally, the susceptibility of fatty acids to oxidation is reported to increase in some mutants with decreased thermotolerance (6Larkindale J. Hall J.D. Knight M.R. Vierling E. Plant Physiol. 2005; 138: 882-897Crossref PubMed Scopus (584) Google Scholar). In most cases, lipid oxidation has been studied in aerial tissues, where organelles such as chloroplasts and also mitochondria are particularly rich in the trienoic fatty acid α-linolenic acid (7Harwood, J. L. (1982) in The Biochemistry of Plants: A Comprehensive Treatise (Stumpf, P. K., and Conn, E. E., eds) Vol. 4, pp. 1-55, Academic Press, New YorkGoogle Scholar, 8Falcone D.L. Ogas J.P. Somerville C.R. BMC Plant Biol. 2004; 4: 17Crossref PubMed Scopus (223) Google Scholar), a fatty acid that is highly susceptible to nonenzymatic oxidation (9Frankel, E. N. (2005) The Oily Press Lipid Library: Lipid Oxidation, 2nd Ed., Vol. 18, PJ Barnes & Associates, Bridgwater, UKGoogle Scholar).One of the α-linolenic acid oxidation products that has been studied in some detail in plants in terms of its origin and biological activities is malondialdehyde (MDA) 3The abbreviations used are: MDA, malondialdehyde; TBA, 2-thiobarbituric acid; HPLC, high performance liquid chromatography. 3The abbreviations used are: MDA, malondialdehyde; TBA, 2-thiobarbituric acid; HPLC, high performance liquid chromatography. (3Farmer E.E. Davoine C. Curr. Opin. Plant Biol. 2007; 10: 380-386Crossref PubMed Scopus (219) Google Scholar, 10Weber H. Chetelat A. Reymond P. Farmer E.E. Plant J. 2004; 37: 877-888Crossref PubMed Scopus (207) Google Scholar). MDA is a three-carbon aldehyde that is a major product of the oxidation of many fatty acids with three or more double bonds and is one of the most ubiquitous small molecules produced by lipid oxidation in many organisms (9Frankel, E. N. (2005) The Oily Press Lipid Library: Lipid Oxidation, 2nd Ed., Vol. 18, PJ Barnes & Associates, Bridgwater, UKGoogle Scholar, 11Esterbauer H. Schaur R.J. Zollner H. Free Radic. Biol. Med. 1991; 11: 81-128Crossref PubMed Scopus (5846) Google Scholar). In the mature expanded leaves of Arabidopsis, MDA is known to be produced in vivo from at least two genetically separable sources. The principal source, accounting for ∼75% of MDA in expanded leaves, is trienoic fatty acids, whereas the origin(s) of the second pool of MDA identified in leaves is currently unknown (10Weber H. Chetelat A. Reymond P. Farmer E.E. Plant J. 2004; 37: 877-888Crossref PubMed Scopus (207) Google Scholar). Like other products of nonenzymatic lipid oxidation in plants, the localization of MDA at the level of tissues and organs is unknown. This latter problem presents a challenge, and a factor limiting the analysis of molecules like MDA in complex multicellular organisms is that it can be difficult to detect individual molecular species without first extracting the compounds and thus destroying tissue structure. Currently, it is not possible to localize the sites of accumulation of discrete molecular products of lipid oxidation in intact plant tissues. We have attempted to solve this problem for MDA.To localize MDA within the body of plants, several genetic backgrounds with altered abilities to produce MDA would ideally be employed. Fortunately, Arabidopsis mutants exist with either decreased or increased ability to accumulate MDA. For example, the fatty acid desaturase triple mutant fad3-2 fad7-2 fad8 (12McConn M. Browse J. Plant Cell. 1996; 8: 403-416Crossref PubMed Google Scholar) has lower MDA levels in mature leaves compared with the wild type (10Weber H. Chetelat A. Reymond P. Farmer E.E. Plant J. 2004; 37: 877-888Crossref PubMed Scopus (207) Google Scholar). In contrast, the vitamin E2 mutant vte2-1 (13Sattler S.E. Gilliland L.U. Magallanes-Lundback M. Pollard M. DellaPenna D. Plant Cell. 2004; 16: 1419-1432Crossref PubMed Scopus (473) Google Scholar), a loss-of-function mutant in homogentisate phytyltransferase, lacks both tocopherols and redox-active tocopherol pathway intermediates and overaccumulates MDA (14Sattler S.E. Mène-Saffrané L. Farmer E.E. Krischke M. Mueller M.J. DellaPenna D. Plant Cell. 2006; 18: 3706-3720Crossref PubMed Scopus (156) Google Scholar). Although the nonenzymatic oxidation of di-and tri-unsaturated fatty acids is pronounced in the vte2-1 mutant, there is little or no activation of the synthesis of enzymatically derived jasmonates (which are derived from trienoic fatty acids). Furthermore, the transcripts for key jasmonate-responsive marker genes are not up-regulated in vte2-1 relative to the wild type. The vte2-1 mutant strongly overproduces MDA as well as stable phytoprostanes during early the post-germination phase (14Sattler S.E. Mène-Saffrané L. Farmer E.E. Krischke M. Mueller M.J. DellaPenna D. Plant Cell. 2006; 18: 3706-3720Crossref PubMed Scopus (156) Google Scholar), and the seedlings also show a strong visual phenotype upon germination. Typically, on emerging from the testa, the seedlings display defects in their cotyledons. These defects are asymmetric; they affect one cotyledon more strongly than the other, often leaving this cotyledon poorly developed and with a bleached appearance (13Sattler S.E. Gilliland L.U. Magallanes-Lundback M. Pollard M. DellaPenna D. Plant Cell. 2004; 16: 1419-1432Crossref PubMed Scopus (473) Google Scholar).We compared the phenotypes and MDA levels in seedlings of the vte2-1 mutant and the fad3-2 fad7-2 fad8 triple mutant (hereafter termed fad3 fad7 fad8) with those of a novel quadruple mutant (fad3-2 fad7-2 fad8 vte2-1, hereafter termed fad3 fad7 fad8 vte2) derived from crossing the two parents. The results showed that the oxidation of trienoic fatty acids is specifically responsible for the vte2-1 phenotype, including cotyledon defects and up-regulation of GST1 expression. Furthermore, liquid chromatography analysis indicated that the only small 2-thiobarbituric acid (TBA)-reactive substance produced by these plants is MDA. Supported by quantitative MDA measurements using pentafluorophenylhydrazine derivatives, this genetic system was then used as a basis for the development of a controlled in situ method for the detection of MDA-(TBA)2 adducts, which can be excited to fluoresce in a region where chlorophyll absorption and fluorescence are nearly minimal. The new in situ assay allowed the comparison of the vte2-1 phenotype with MDA distribution throughout the plant. Additionally, the assay allowed the localization of a pool of nontrienoic fatty acid-derived MDA in Arabidopsis root tips. The method should facilitate the mapping of MDA pools in the tissues of complex organisms.EXPERIMENTAL PROCEDURESPlant Growth Conditions—The seedlings were grown for 2 or 3 days on agar (1%, w/v) containing 0.5× Murashige and Skoog basal medium during a 12-h photoperiod (100 μmol of photons·m-2·s-1) at 20 °C.Construction and Selection of fad3 fad7 fad8 vte2 Quadruple Mutants—All mutants used are in the Columbia genetic background. The male sterile fad3 fad7 fad8 mutant was fertilized with vte2-1 pollen, and F1 plants were allowed to self-pollinate. The homozygous fad3 fad7 fad8 mutants were isolated from the F2 progeny (960 plants) by gas chromatography analysis for 18:3 (15Miquel M. Browse J. J. Biol. Chem. 1992; 267: 1502-1509Abstract Full Text PDF PubMed Google Scholar). Ten plants homozygous for the fad3 fad7 fad8 mutations were detected. Three vte2 heterozygotes were detected among these plants. Homozygous vte2 mutants were isolated from the following (F3) generation by cleaved amplified polymorphism sequence analysis. For VTE2/vte2 differentiation, a 632-bp-long fragment was amplified with forward primer 5′-TTTCACTGGCATCTTGGAGGTAATG-3′ and reverse primer 5′-AAGTGGCAACTGTTTGTAGTAGAAG-3′ in a PCR carried out at an annealing temperature of 51 °C. Restriction enzyme digestion of the vte2 allele with SacI endonuclease (Fermentas, Ontario, Canada) generated two fragments (477 and 154 bp), whereas VTE2 was not digested. Three quadruple mutant lines were obtained. Two of these lines (733 and 782) were propagated. Seed production from the quadruple mutants was rescued by spraying with methyl jasmonate (30 μm, sonicated in water containing 0.01% (v/v) Tween 20) at 2-day intervals. At the F5 generation, sufficient seeds were obtained for experiments.RNA Gel Blotting—Total RNA was extracted from 2-day-old seedlings grown on wet filter papers at 20 °C during a 12-h photoperiod (100 μmol of photons·m-2·s-1). RNA was extracted as described (5Vollenweider S. Weber H. Stolz S. Chetelat A. Farmer E.E. Plant J. 2000; 24: 467-476Crossref PubMed Google Scholar), separated by electrophoresis, and electroblotted onto nitrocellulose membranes (Amersham Biosciences). The membranes were probed with a cDNA corresponding to the GST1 gene At1g02930 (5Vollenweider S. Weber H. Stolz S. Chetelat A. Farmer E.E. Plant J. 2000; 24: 467-476Crossref PubMed Google Scholar).MDA Quantification—Seedlings (2 days old, 200 mg fresh weight) were harvested frozen in liquid nitrogen and ground to a fine powder. MDA levels in different mutants of Arabidopsis were quantified by a gas chromatography/mass spectrophotometry method as described (10Weber H. Chetelat A. Reymond P. Farmer E.E. Plant J. 2004; 37: 877-888Crossref PubMed Scopus (207) Google Scholar, 16Yeo H.C. Liu J. Helbock H.J. Ames B.N. Methods Enzymol. 1999; 300: 70-78Crossref PubMed Scopus (28) Google Scholar). The internal standard for quantification was D2-MDA generated from (2D2)-1,1,3,3-tetraethoxypropane (Dr. Ehrenstorfer GmbH, Augsburg, Germany) as described (10Weber H. Chetelat A. Reymond P. Farmer E.E. Plant J. 2004; 37: 877-888Crossref PubMed Scopus (207) Google Scholar).Fluorescence Detection of Aldehyde-TBA Adducts—A fluorescence filter with a 515 ± 10-nm excitation filter and a 555 ± 15-nm emission filter was constructed by Leica Microsystems (Glattbrugg, Switzerland) based on fluorescence values in Ref. 17Yagi K. Biochem. Med. 1976; 15: 212-216Crossref PubMed Scopus (2030) Google Scholar. Fluorescence was detected with a Leica MZ16 FA microscope and photographed with a Leica DC300F camera.TBA Treatments—Plant tissues were treated at 37 °C for 5 h with either 35 mm TBA (Sigma) or 35 mm trichloroacetic acid (Merck) as a control. Tissues were examined by microscopy within 1 h of the end of the 5-h staining period.HPLC Analysis—MDA-TBA adducts were separated based on Ref. 18Scott I.M. Clarke S.M. Wood J.E. Mur L.A. Plant Physiol. 2004; 135: 1040-1049Crossref PubMed Scopus (201) Google Scholar. All solutions and mobile phases were prepared in purified water (NANOpure, Skan AG, Basel-Allschwil, Switzerland). Wild-type (Col-0), vte2-1, and fad3 fad7 fad8 seeds were germinated in the dark in water for 3 days. These seedlings were then incubated in 35 mm TBA solution (0.02 g of seedlings (fresh weight)/ml) for 5 h at 37 °C. The seedlings were ground in liquid nitrogen and extracted in 5% (w/v) trichloroacetic acid in the presence of 0.1% (w/v) butylated hydroxytoluene (0.02 g of seedlings/ml). The suspensions were shaken for 5 min at room temperature and then centrifuged at 10,000 × g for 2 min. The supernatant was directly analyzed by HPLC. A standard MDA-TBA adduct was prepared from 5 mm 1,1,3,3-tetraethoxypropane (Sigma) incubated in 35 mm TBA for 5 h at 37 °C. The reaction was diluted in 5% (w/v) trichloroacetic acid and 0.1% (w/v) butylated hydroxytoluene for injection. HPLC was performed using a LaChrom Elite HPLC system (Merck-Hitachi, Dietikon, Switzerland): L-2100 pump, L-2200 autosampler, L-2300 column oven, L-2450 diode array detector, and L-2480 fluorescence detector. HPLC was controlled with the EZChrom Elite Program Version 3.1.7 (Merck-Hitachi). Separation was performed by injection of a 10-μl sample into a ChromCart column (Nucleosil 120-5 C18, 5 μm, 4 × 250 mm; Macherey-Nagel, Ohringen, Switzerland). The autosampler was maintained at 4 °C and the column at 26 °C. The mobile phase was 24% (v/v) acetonitrile (Merck) in 0.1% (v/v) trifluoroacetic acid (Fluka, Buchs, Switzerland) run isocratically for 8 min at a flow rate of 1 ml/min. Detector wavelengths were set to 515 nm (excitation) and 555 nm (emission). The MDA-(TBA)2 adduct eluted at 3 min. In separate experiments designed to look for additional fluorescent compounds eluted from plant materials treated with TBA, a gradient of acetonitrile (from 24 to 100% (v/v) in 0.1% trifluoroacetic acid) was introduced after the 8-min isocratic run (same flow rate). The solvent system was then changed to 0-100% (v/v) methanol in 0.1% (v/v) trifluoroacetic acid (same flow rate and fluorescence parameters). The MDA-(TBA)2 adduct eluted at 21.7 min.Liquid Chromatography/Mass Spectrometry Analysis—Ultra-HPLC/time-of-flight mass spectrometry analyses were performed on a Micromass LCT Premier time-of-flight mass spectrometer (Waters, Milford, MA) with an electrospray interface and coupled with an ACQUITY UPLC system (Waters). Electrospray ionization conditions were as follows: capillary voltage, 2500 V; cone voltage, 90 V; microchannel plate detector voltage, 2650 V; source temperature, 120 °C; desolvation temperature, 250 °C; cone gas flow, 10 liters/h; and desolvation gas flow, 550 liters/h. Detection was in the negative ion mode in the m/z range 100-1000, with a scan time of 0.25 s, and interscan delay of 0.01 s, and a centered mode. A solution of leucine/enkephalin (Sigma) was used for the lock mass. Separation was carried out on a Waters ACQUITY UPLC BEH C18 column (50 × 1.0-mm inner diameter, 1.7 μm). Gradient analysis was performed at a flow rate of 0.25 ml/min with solvent system A (0.1% formic acid and water) and solvent system B (0.1% formic acid and acetonitrile) producing a gradient of 5-98% solvent system B in 3.0 min. The injected volume was 2 μl.Chemical Phenotype of vte2-1—Wild-type (Col-0) and vte2-1 (13Sattler S.E. Gilliland L.U. Magallanes-Lundback M. Pollard M. DellaPenna D. Plant Cell. 2004; 16: 1419-1432Crossref PubMed Scopus (473) Google Scholar) seeds were imbibed in water at 20 °C during a 16-h photoperiod (100 μmol of photons·m-2·s-1) for 1 day. The seedlings were then incubated on wet filter papers for 2 days in the presence of MDA (0 or 1 μmol) in 1-liter glass jars during the same 16-h photoperiod and at the same temperature. MDA was generated from 1,1,3,3-tetraethoxypropane by addition to freshly prepared 1 m HCl (100 μl) at room temperature. The MDA solution was applied to cotton wicks. For the wild-type control (0 μm MDA), only HCl was applied. vte2-1 seedlings (1 day post-imbibition) were incubated under the same conditions but in the absence of MDA. After 3 days, seedlings were photographed under the Leica MZ16 FA microscope with the Leica DC300F camera using IM50 software (Leica Microsystems).RESULTSGenetic Suppression of the vte2-1 Seedling Phenotype—During germination, the tocopherol-deficient mutant vte2-1 exhibits a strong phenotype that includes a failure to fully expand one (or, more rarely, both) cotyledons. Additionally, the affected cotyledon is often devoid of chlorophyll (13Sattler S.E. Gilliland L.U. Magallanes-Lundback M. Pollard M. DellaPenna D. Plant Cell. 2004; 16: 1419-1432Crossref PubMed Scopus (473) Google Scholar). Moreover, this phenotype is correlated with the accumulation of potentially toxic compounds derived mainly from the nonenzymatic oxidation of trienoic fatty acids (10Weber H. Chetelat A. Reymond P. Farmer E.E. Plant J. 2004; 37: 877-888Crossref PubMed Scopus (207) Google Scholar, 13Sattler S.E. Gilliland L.U. Magallanes-Lundback M. Pollard M. DellaPenna D. Plant Cell. 2004; 16: 1419-1432Crossref PubMed Scopus (473) Google Scholar). To test whether the vte2-1 phenotype is specifically associated with trienoic fatty acids, two fad3 fad7 fad8 vte2 quadruple mutant lines were isolated from a cross between the trienoic fatty acid-deficient mutant fad3 fad7 fad8 (12McConn M. Browse J. Plant Cell. 1996; 8: 403-416Crossref PubMed Google Scholar) and vte2-1 (see “Experimental Procedures”). Three days after germination of the tocopherol-deficient vte2-1 seedlings, the plants were smaller than Col-0 and displayed a clear vte2-1 phenotype (Fig. 1) (13Sattler S.E. Gilliland L.U. Magallanes-Lundback M. Pollard M. DellaPenna D. Plant Cell. 2004; 16: 1419-1432Crossref PubMed Scopus (473) Google Scholar). However, the wild-type/fad3 fad7 fad8 triple mutant visual phenotype was restored in the two lines of the quadruple mutant (Fig. 1). The growth of seedlings of quadruple mutants (lines 733 and 782) appeared to be highly similar to that of the wild type and the fad3 fad7 fad8 triple mutant, and no bleaching was observed on the cotyledons of the fad3 fad7 fad8 vte2 quadruple mutants. Indeed, none of the fad3 fad7 fad8 vte2 quadruple mutant seedlings presented the cotyledon defects that typify the vte2-1 mutant at this stage of growth.MDA Levels and GST1 Gene Expression in Different Arabidopsis Mutants—The vte2-1 phenotype in seedlings is associated with a strong increase in nonenzymatic lipid peroxidation, resulting in the production of hydroperoxy and hydroxyl fatty acids, linear phytoprostanes, and MDA (13Sattler S.E. Gilliland L.U. Magallanes-Lundback M. Pollard M. DellaPenna D. Plant Cell. 2004; 16: 1419-1432Crossref PubMed Scopus (473) Google Scholar, 14Sattler S.E. Mène-Saffrané L. Farmer E.E. Krischke M. Mueller M.J. DellaPenna D. Plant Cell. 2006; 18: 3706-3720Crossref PubMed Scopus (156) Google Scholar). Moreover, the accumulation of these compounds in the vte2-1 mutant correlates with the up-regulation of 160 genes (14Sattler S.E. Mène-Saffrané L. Farmer E.E. Krischke M. Mueller M.J. DellaPenna D. Plant Cell. 2006; 18: 3706-3720Crossref PubMed Scopus (156) Google Scholar). To further test the correlation between the accumulation of trienoic fatty acid oxidation products and the modification of gene expression, MDA levels were quantified, and the transcript levels for GST1, an oxidative stress marker gene (19Alvarez M.E. Pennell R.I. Meijer P.J. Ishikawa A. Dixon R.A. Lamb C. Cell. 1998; 92: 773-784Abstract Full Text Full Text PDF PubMed Scopus (907) Google Scholar), were monitored in 2-day-old seedlings of the different mutants (Fig. 2). In the vte2-1 seedlings, the MDA concentration increased strongly to levels 14-fold higher than in Col-0 and 22-fold higher than in the fad3 fad7 fad8 triple mutant. In the fad3 fad7 fad8 vte2 quadruple mutants (lines 733 and 782), we observed a lower MDA level than in the wild type. This level was comparable with the MDA level in the fad3 fad7 fad8 triple mutant. The restoration to a nearly wild-type visual phenotype in the quadruple mutant correlated with a low MDA level. To further test the correlation between the accumulation of trienoic fatty acid oxidation products and gene expression observed in the vte2-1 mutant, the expression of the GST1 gene was analyzed. GST1 is up-regulated in the vte2-1 mutant (14Sattler S.E. Mène-Saffrané L. Farmer E.E. Krischke M. Mueller M.J. DellaPenna D. Plant Cell. 2006; 18: 3706-3720Crossref PubMed Scopus (156) Google Scholar) and also responds strongly to MDA (5Vollenweider S. Weber H. Stolz S. Chetelat A. Farmer E.E. Plant J. 2000; 24: 467-476Crossref PubMed Google Scholar, 10Weber H. Chetelat A. Reymond P. Farmer E.E. Plant J. 2004; 37: 877-888Crossref PubMed Scopus (207) Google Scholar). GST1 mRNA was strongly expressed in vte2-1 mutant seedlings (2 days), but not in the wild type (Fig. 2A). The elimination of trienoic fatty acids in the fad3 fad7 fad8 vte2 quadruple mutant, which correlated with suppression of MDA accumulation, led to a strong reduction of GST1 transcript levels compared with vte2-1 (Fig. 2A).FIGURE 2MDA levels and GST1 gene expression in the different Arabidopsis mutants. A, quantification of MDA levels in the different Arabidopsis mutants. Seedlings were 2 days old. F.W., fresh weight. B, GST1 gene expression tested for the wild type (Col-0) and fad3 fad7 fad8 triple, vte2-1, and fad3 fad7 fad8 vte2 quadruple (lines 733 and 782) mutants 2 days after germination (mean ± S.D., n = 5-8).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Chemical Phenotype of vte2-1—To further investigate the potential role of α-linolenic acid oxidation products in the vte2-1 phenotype, we attempted to chemically phenocopy the vte2-1 mutation. This was achieved by exposing germinated seeds of Col-0 to MDA. Wild-type seedlings exposed to MDA partially resembled vte2-1 seedlings and displayed one bleached cotyledon and shorter and narrower hypocotyls (Fig. 3C).FIGURE 3Chemical phenocopy of the vte2-1 phenotype. A, wild-type (WT) seedlings; B, vte2-1 seedlings; C, wild-type seedlings exposed to MDA. Seedlings were photographed at 3 days. The arrowheads indicate the bleached cotyledons in the vte2-1 mutant and in the wild-type plants treated with MDA.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Development of an in Situ Test to Detect MDA in Plant Tissues—To facilitate the in situ microscopic detection of MDA, a custom fluorescence filter was constructed. The filter parameters were based on specific fluorescence values for the adduct between MDA and TBA (17Yagi K. Biochem. Med. 1976; 15: 212-216Crossref PubMed Scopus (2030) Google Scholar), with a 515 ± 10-nm excitation filter and a 555 ± 15-nm emission filter. The filter was first tested on germinating Col-0 and vte2-1 seedlings to see whether fluorescence could be observed in the seedlings. A stronger fluorescence in vte2-1 than in wild-type seedlings was expected because of the higher measurable levels of MDA in the former. Fig. 4 shows wild-type (Col-0) and vte2-1 seedlings (3 days post-germination) that had been incubated with 35 mm TBA for 5 h at 37 °C and then observed by microscopy. Under white light, a weak red coloration was observed in the root tips of both Col-0 and the vte2-1 mutant. In parallel, the fluorescence at 555 ± 15 nm was observed in each genotype. For wild-type seedlings, strong fluorescence was observed at the root tip, with weaker fluorescence in the cotyledons. On the other hand, in the vte2-1 mutant, the fluorescence was present throughout the entire seedling. This observation is consistent with the fact that the MDA level in the vte2-1 mutant is 14-fold higher than that in Col-0 3 days after germination (14Sattler S.E. Mène-Saffrané L. Farmer E.E. Krischke M. Mueller M.J. DellaPenna D. Plant Cell. 2006; 18: 3706-3720Crossref PubMed Scopus (156) Google Scholar).FIGURE 4Development of an assay for the in situ detection of MDA. Seedlings of Col-0 and vte2-1 (3 days post-germination) were incubated at 37 °C for 5 h in 35 mm TBA solution. A weak red coloration at the root tips corresponding to TBA adducts was observed under white light in the wild type and in the vte2-1 mutant (upper panels). Fluorescence (fluor.) was detected at 555 nm (lower panels). Scale bars = 1 mm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)At this point, it was not certain that the fluorescence observed emanated specifically from MDA-(TBA)2 adducts. To test for the specificity of fluorescence in the vte2-1 mutant, extracts of this plant previously incubated with TBA (for 5 h at 37 °C) were analyzed by HPLC. Using the same wavelengths as used for the fluorescence microscopy, the HPLC chromatograms (Fig. 5) revealed a single peak for the synthetic reference adduct. Only one fluorescent peak was detectable by HPLC analysis of wild-type and fad3 fad7 fad8 triple mutant seedlings, and a larger single peak was observed with vte2-1. These peaks migrated at the same retention time as the synthetic MDA-(TBA)2 adduct. More extensive HPLC analysis of the wild type using gradients of methanol or acetonitrile revealed only one peak corresponding to the MDA-(TBA)2 adduct from wild-type plants. Liquid chromatography/mass spectrometry was used to verify the identity of the MDA-(TBA)2 adduct produced in both the wild-type and fad3 fad7 fad8 triple mutant samples. The mass for the adduct obtained in the negative ion mode (M - H = 322.99) corresponded to that for the synthetic adduct (Fig. 6).FIGURE 5Liquid chromatography analysis of fluorescent MDA-TBA adducts. Seedlings of the wild type (A), vte2-1 (B), and fad3 fad7 fad8 (C) were incubated in 35 mm TBA at 37 °C for 5 h (0.02 g of seedlings (fresh weight)" @default.
• W1997593467 created "2016-06-24" @default.
• W1997593467 creator A5010231464 @default.
• W1997593467 creator A5027126344 @default.
• W1997593467 creator A5040376721 @default.
• W1997593467 creator A5044611624 @default.
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• W1997593467 date "2007-12-01" @default.
• W1997593467 modified "2023-10-18" @default.
• W1997593467 title "Genetic Removal of Tri-unsaturated Fatty Acids Suppresses Developmental and Molecular Phenotypes of an Arabidopsis Tocopherol-deficient Mutant" @default.
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