Functional analysis of alpha-DOX2, an active alpha-dioxygenase critical for normal development in tomato plants

Abstract

Plant alpha-dioxygenases initiate the synthesis of oxylipins by catalyzing the incorporation of molecular oxygen at the alpha-methylene carbon atom of fatty acids. Previously, alpha-DOX1 has been shown to display alpha-dioxygenase activity and to be implicated in plant defense. In this study, we investigated the function of a second alpha-dioxygenase isoform, alpha-DOX2, in tomato (Solanum lycopersicum) and Arabidopsis (Arabidopsis thaliana). Recombinant Slalpha-DOX2 and Atalpha-DOX2 proteins catalyzed the conversion of a wide range of fatty acids into 2(R)-hydroperoxy derivatives. Expression of Slalpha-DOX2 and Atalpha-DOX2 was found in seedlings and increased during senescence induced by detachment of leaves. In contrast, microbial infection, earlier known to increase the expression of alpha-DOX1, did not alter the expression of Slalpha-DOX2 or Atalpha-DOX2. The tomato mutant divaricata, characterized by early dwarfing and anthocyanin accumulation, carries a mutation at the Slalpha-DOX2 locus and was chosen for functional studies of alpha-DOX2. Transcriptional changes in such mutants showed the up-regulation of genes playing roles in lipid and phenylpropanoid metabolism, the latter being in consonance with the anthocyanin accumulation. Transgenic expression of Atalpha-DOX2 and Slalpha-DOX2 in divaricata partially complemented the compromised phenotype in mature plants and fully complemented it in seedlings, thus indicating the functional exchangeability between alpha-DOX2 from tomato and Arabidopsis. However, deletion of Atalpha-DOX2 in Arabidopsis plants did not provoke any visible phenotypic alteration indicating that the relative importance of alpha-DOX2 in plant physiology is species specific.

Figure 1.

Determination of α-dioxygenase activity of Slα-DOX2. A, GC-MS identification of products formed by incubation of palmitic acid with Slα-DOX2-containing insect cells. Top: Peaks due to pentadecanal (O-methyloxime syn/anti isomers), palmitic acid (methyl ester; corresponding to substrate remaining not converted), and 2-hydroxypalmitic acid (methyl ester/trimethylsilyl ether derivative) were observed. The reaction products observed arouse by decarboxylation or reduction of 2-hydroperoxypalmitic acid, the primary α-DOX product. Bottom: Mass spectrum of 2-hydroxypalmitic acid (methyl ester/trimethylsilyl ether derivative). B, Steric analysis of 2-hydroxylinolenic acid as its (−)-menthoxycarbonyl/methyl ester derivative. Top: 2-Hydroxylinolenic acid prepared from an incubation of linolenic acid with Slα-DOX2. Bottom: Synthetic 2(R,S)-hydroxylinolenic acid elution order 2(S) followed by 2(R). C, Fatty acid substrate specificity of oxygenation by Slα-DOX2 (mean ± se of n = 3–4 measurements). Enzymatic oxygenation rates were determined at 23°C after addition of approximately 100 μg total protein to 1.5 mL 0.1 m Tris, pH 7.4, containing 100 μm fatty acid substrate and 100 μm tert-butylhydroperoxide.

Figure 2.

Determination of α-dioxygenase activity of Atα-DOX2. A, MS identification of 2-hydroxypalmitic acid formed by incubation of palmitic acid with a homogenate of Atα-DOX2-expressing insect cells. The methyl ester/trimethylsilyl ether derivative was used. B, Steric analysis of 2-hydroxypalmitic acid as its (−)-menthoxycarbonyl/methyl ester derivative. Top: 2-Hydroxypalmitic acid prepared from an incubation of palmitic acid with Atα-DOX2. Bottom: Synthetic 2(R,S)-hydroxypalmitic acid elution order 2(S) followed by 2(R). C, Mass-spectral ions (m/z) recorded on Atα-DOX2-derived 2-hydroxy fatty acids (methyl ester/trimethylsilyl ether derivatives) and fatty aldehydes (O-methyloxime derivatives). D, Fatty acid substrate specificity of oxygenation by Atα-DOX2 (mean ± se of n = 3–4 measurements).

Figure 3.

Vegetative growth and fruit development in wild-type and div tomato plants. A, Genomic structure of Slα-DOX2 gene (GenBank accession no. FN428743), indicating the div mutation. B, Seedlings, young leaves, and top view of wild-type plants. C, Seedlings, young leaves, and top view of div plants. D, Lateral view of adult wild-type tomato plant (5 weeks old). E, Lateral view of adult div tomato plant (5 weeks old). F, Cross section of ripe fruit and senescent leaf of wild-type tomato plants. G, Cross section of ripe fruit and senescent leaf of div mutant. H, Gene expression levels of the three tomato α-dioxygenase genes Slα-DOX2, Slα-DOX1.1, and Slα-DOX1.2 in roots, hypocotyls, epicotyls, cotyledons, and leaves of seedlings of tomato wild-type or div plants. I, Gene expression levels of Slα-DOX2 during a 1-week period after detachment of young leaves of wild-type and div plants.

Figure 4.

Gene expression analysis in the div mutant compared to wild-type tomato plants. Differentially expressed transcripts obtained from microarray analyses were examined by RT-PCR. Fold change and statistical value FDR are indicated for each probe from Affymetrix GeneChip Tomato Genome Array. Tomato GAPDH was used to normalize transcript levels in each sample. Gene-specific primer sets used for the evaluation of RNA are shown in Supplemental Table S2.

Figure 5.

Expression of Atα-DOX2. A, Histochemical localization of GUS gene expression in transgenic plants containing an Atα-DOX2∷GUS chimeric construct. Bright-field micrographs reveal in blue the presence of GUS enzyme activity in seedlings and mature tissues of transgenic plants. B, RNA was extracted from different plant organs of healthy untreated plants. Blots were hybridized with riboprobes derived from an Atα-DOX2 cDNA. C, Atα-DOX2 expression during detachment of mature Arabidopsis leaves. Histochemical localization of GUS gene expression in adult leaves of Atα-DOX2∷GUS transgenic plants during a 1-week period after detachment. RNA blots were hybridized with riboprobes derived from an Atα-DOX2 cDNA. Loading control was analyzed by ethidium bromide staining followed by hybridization against an 18S rRNA radioactive probe.

Figure 6.

Complementation of tomato div mutant plants with Slα-DOX2 or Atα-DOX2 rescues the phenotypic defects of div plants. A, Young leaves and top view of complemented div 35S∷Slα-DOX2 plants. B, Young leaves and top view of complemented div 35S∷Atα-DOX2 plants. C, Lateral view of adult complemented div 35S∷Slα-DOX2 plants (5 weeks old). D, Lateral view of adult complemented div 35S∷Atα-DOX2 plants (5 weeks old). E, Adult leaf from the second node below the first inflorescence of a tomato wild-type plant (5 weeks old). F, Adult leaf from the second node below the first inflorescence of tomato div plant (5 weeks old). G, Adult leaf from the second node below the first inflorescence of tomato div 35S∷Slα-DOX2 plant (5 weeks old). H, Adult leaf from the second node below the first inflorescence of a tomato div 35S∷Atα-DOX2 plant (5 weeks old). See Figure 3 for phenotype of wild-type tomato and div seedlings and 5-week-old adult plants.