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, 178 (4), 1507-1521

Identification of Genes Encoding Enzymes Catalyzing the Early Steps of Carrot Polyacetylene Biosynthesis

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Identification of Genes Encoding Enzymes Catalyzing the Early Steps of Carrot Polyacetylene Biosynthesis

Lucas Busta et al. Plant Physiol.

Abstract

Polyacetylenic lipids accumulate in various Apiaceae species after pathogen attack, suggesting that these compounds are naturally occurring pesticides and potentially valuable resources for crop improvement. These compounds also promote human health and slow tumor growth. Even though polyacetylenic lipids were discovered decades ago, the biosynthetic pathway underlying their production is largely unknown. To begin filling this gap and ultimately enable polyacetylene engineering, we studied polyacetylenes and their biosynthesis in the major Apiaceae crop carrot (Daucus carota subsp. sativus). Using gas chromatography and mass spectrometry, we identified three known polyacetylenes and assigned provisional structures to two novel polyacetylenes. We also quantified these compounds in carrot leaf, petiole, root xylem, root phloem, and root periderm extracts. Falcarindiol and falcarinol predominated and accumulated primarily in the root periderm. Since the multiple double and triple carbon-carbon bonds that distinguish polyacetylenes from ubiquitous fatty acids are often introduced by Δ12 oleic acid desaturase (FAD2)-type enzymes, we mined the carrot genome for FAD2 genes. We identified a FAD2 family with an unprecedented 24 members and analyzed public, tissue-specific carrot RNA-Seq data to identify coexpressed members with root periderm-enhanced expression. Six candidate genes were heterologously expressed individually and in combination in yeast and Arabidopsis (Arabidopsis thaliana), resulting in the identification of one canonical FAD2 that converts oleic to linoleic acid, three divergent FAD2-like acetylenases that convert linoleic into crepenynic acid, and two bifunctional FAD2s with Δ12 and Δ14 desaturase activity that convert crepenynic into the further desaturated dehydrocrepenynic acid, a polyacetylene pathway intermediate. These genes can now be used as a basis for discovering other steps of falcarin-type polyacetylene biosynthesis, to modulate polyacetylene levels in plants, and to test the in planta function of these molecules.

Figures

Figure 1.
Figure 1.
Gas chromatographic-mass spectrometric identification of polyacetylenes from Daucus carota. A to D, Total ion chromatograms of putative polyacetylene-containing TLC fractions. Each peak is labeled with a number (1.1, 2.1, 3.1, 4.1, 4.2) that refers to its underlying compound. E to I, Composite mass spectra for each numbered compound (1.14.2), generated by overlaying the spectrum acquired for each compound at 70, 40, 20, and 10 eV. J to N, Structures of the numbered compounds and likely fragmentation mechanisms giving rise to their spectra in E to I. O to R, Spectrum, structure, and fragmentation of the product obtained from hydrogenation of compounds 2.1, 4.1 and 4.2, respectively. m/z, mass to charge ratio; Pd/C, palladium on carbon; LAH, lithium aluminum hydride; eV, electron volts.
Figure 2.
Figure 2.
Polyacetylene abundance and composition in aerial and subterranean tissues of Daucus carota. A, Absolute abundance of polyacetylenes in leaf, petiole, xylem, phloem, and epidermis given in micrograms per milligram of dry tissue. B, Relative abundance of polyacetylenes in leaf, petiole, xylem, phloem, and epidermis given as percent of total polyacetylenes. For both A and B, bar lengths and error bars represent the average and sd of six independent samples each from a different carrot plant.
Figure 3.
Figure 3.
Analysis of FAD2 amino acid sequences from carrot Daucus carota. Subsets of the multiple sequence alignment of carrot FAD2 sequences and previously characterized FAD2 enzymes from other plant species. Subsets were chosen based on the presence of highly conserved His residues (yellow highlights, “consensus” score on bottom) and residues associated with Δ12 desaturase versus divergent FAD2 activity (pink highlights, “correlation with divergent function” score on bottom). The maximum likelihood phylogenetic tree was generated using nucleotide sequences underlying the amino acids shown in alignment subsets. Sequence data used in this figure can be found in the Phytozome/GenBank databases under the accession numbers listed in Supplemental Table S3.
Figure 4.
Figure 4.
Expression of FAD2s from carrot. A, Expression of carrot FAD2 genes (plotted as z-scores) in carrot leaves, xylem, phloem, and whole-root samples, as well as in carrot suspension cultures before (CTRL1 and 2, i.e. two replicates) and after (ELI1 and 2, i.e. two replicates) treatment with an elicitor extracted from mycelia of Phytopthora megasperma. The dendrogram connecting the gene names on the y axis was generated from ward. D2 clustering analysis of the z-scores presented in Supplemental Figure S5, not the heat map presented in A, which shows a subset of the full heat map presented in Supplemental Figure S5. Blue tip points denote putative Δ12 FAD2 desaturase genes and green tips denote putative divergent FAD2s as determined by the clustering in Fig. 3. B, Coexpression between DCAR_013547 and other carrot FAD2s with a Pearson correlation coefficients q < 0.1. Sequence data used in this figure can be found in the Phytozome/GenBank databases under the accession numbers listed in Supplemental Table S3.
Figure 5.
Figure 5.
Functional testing of candidate carrot desaturase and acetylenase enzymes by heterologous expression. A, Pathway to falcarin-type polyacetylenes. B, GC traces (ion abundance versus time) of oleate, linoleate, crepenynate, and dehydrocrepenynate standards. C, Single ion trace (m/z 67) of the fatty acid methyl esters from induced yeast strain BY4741 containing the pYES vector. D, Single ion trace (m/z 67) of the fatty acid methyl esters from induced yeast strain BY4741 containing the pYES vector harboring DCAR_013547. E, EI mass spectrum of the linoleate standard. F, EI mass spectrum of the indicated peak in D. G, Single ion trance (m/z 79) of fatty acid methyl esters from Arabidopsis fad3fae1 seeds transformed with an empty the pBINGlyRed3 vector. H, Single ion trance (m/z 79) of the fatty acid methyl esters from Arabidopsis fad3fae1 seeds transformed with the pBINGlyRed3 vector harboring DCAR_013552. I, EI mass spectrum of the crepenynate standard. J, EI mass spectrum of the indicated peak in H. K, Single ion trance (m/z 91) of the fatty acid methyl esters from Arabidopsis fad3fae1 seeds transformed with the pBINGlyRed3 vector harboring both DCAR_013547 and DCAR_013552. L, EI mass spectrum of the dehydrocrepenynate standard. M, EI mass spectrum of the indicated peak in K. m/z, mass to charge ratio. Sequence data used in this figure can be found in the Phytozome/GenBank databases under the accession numbers listed in Supplemental Table S3.

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