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

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.

FAD2 Gene Radiation and Positive Selection Contributed to Polyacetylene Metabolism Evolution in Campanulids

“…1). Early intermediates of this pathway have been identified by radiochemical studies (Barley et al, 1988;Knispel et al, 2013), and the pathway has been examined in diverse campanulid lineages (Lee et al, 1998;Cahoon et al, 2003;Carlsson et al, 2004;Nam and Kappock, 2007;Busta et al, 2018). Linoleic acid is converted to crepenynic acid by installing the first acetylenic bond in the D 12 position (the 12 th carbon relative to the fatty acid head group; this notation is used consistently throughout).…”

“…The enzyme (D 12 -acetylenase) catalyzing the first committed step in the crepenynate pathway was characterized in the Asteraceae plant Crepis alpina and was found to be similar to Fatty Acid Desaturase 2 (FAD2; Lee et al, 1998). Additional D 12 -acetylenases have subsequently been isolated and characterized from other PA-producing plants in campanulids, such as parsley (Petroselinum crispum), English ivy (Hedera helix), and carrot (Daucus carota; Cahoon et al, 2003;Busta et al, 2018). All these enzymes were found to be encoded by FAD2 family members, which were traditionally known as D 12 -desaturases (Higashi and Murata, 1993;Okuley et al, 1994b), indicating that these acetylenases are FAD2s with diverged function.…”

The FATTY ACID DESATURASE2 Family in Tomato Contributes to Primary Metabolism and Stress Responses

“…While Δ12-desaturases influence levels of abiotic stress tolerance by modifying the physical properties of membranes, certain divergent FAD2 enzymes contribute to biotic stress responses by participating in the synthesis of defensive secondary metabolites. The antimicrobial polyacetylenes falcarindiol and falcarinol accumulate in the vasculature of tomato in response to fungal infection (De Wit and Kodde, 1981;Elgersma et al, 1984), and data from three other plant families (Apiaceae, Asteraceae, and Araliaceae) indicate that pathogen-inducible divergent FAD2-like acetylenases synthesize necessary precursors for these phytoalexins (Kirsch et al, 1997;Cahoon et al, 2003;Busta et al, 2018). In avocado (Persea americana), pathogen-inducible divergent FAD2 expression is also correlated with accumulation of an antimicrobial diene, (Z,Z)-1-acetoxy-2-hydroxy-4-oxoheneicosa-12,15-diene (Wang et al, 2004).…”

“…For example, soybean has seven FAD2 gene family members (Lakhssassi et al, 2017), cotton has nine (Feng et al, 2017), safflower has 11 (Cao et al, 2013), and parsley (Petroselinum crispum) and carrot (Daucus carota ssp. sativus) have 17 and 24, respectively, the highest number of family members documented so far, indicating that functional divergence of FAD2 is not limited to oil seed crops (Somssich et al, 1989;Busta et al, 2018).…”

“…Although the functions of the majority of FAD2 homologs are untested, analyses of plant species that produce unusual fatty acids or fatty acid derivatives have identified so-called divergent FAD2 proteins with novel enzymatic capabilities. FAD2 family members that encode hydroxylases (van de Loo et al, 1995;Broun et al, 1998a), epoxidases (Lee et al, 1998), acetylenases (Cahoon et al, 2001;Okada et al, 2013;Busta et al, 2018), or conjugases (Cahoon et al, 1999;Liu et al, 2001;Dyer et al, 2002;Reed et al, 2002;Cahoon and Kinney, 2004) have been identified in medicinal species from a diversity of taxa, including the Apiaceae, Asteraceae, Balsaminaceae, Brassicaceae, Cucurbitaceae, Euphorbiaceae, and Santalaceae. Despite the evidence for functional diversification of FAD2 in multiple plant families, the potential for such diversification in major crops has been largely unexplored.…”