Identification of genes encoding enzymes catalyzing the early steps of carrot polyacetylene biosynthesis

Lucas Busta, Won Cheol Yim, Evan William Labrant, Peng Wang, Lindsey Grimes, Kiah Malyszka, John C. Cushman, Patricia Santos, Dylan K. Kosma, Edgar B. Cahoon

Research output: Contribution to journalArticlepeer-review

5 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)1507-1521
Number of pages15
JournalPlant physiology
Volume178
Issue number4
DOIs
StatePublished - Dec 1 2018
Externally publishedYes

Bibliographical note

Funding Information:
1This research was supported by a National Science Foundation grant (Plant Genome IOS-13-39385 to E.B.C.) and a National Science Foundation of China grant (no. 31300260 to P.W.). 2Author for contact: ecahoon2@unl.edu. 3Senior author. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Edgar B. Cahoon (ecahoon2@unl.edu). L.B., W.C.Y., P.S., D.K.K., and E.B.C. designed the research plans; L.B., W.C.Y., E.W.L., L.G., K.M., P.S., and P.W. performed the experiments; L.B. and W.C.Y. analyzed the data and prepared figures; L.B. wrote the article with contributions from all other authors; J.C.C. provided research oversight to W.C.Y.; E.B.C. supervised the research. [OPEN]Articles can be viewed without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.18.01195

Funding Information:
1This research was supported by a National Science Foundation grant (Plant Genome IOS-13-39385 to E.B.C.) and a National Science Foundation of China grant (no. 31300260 to P.W.).We wish to acknowledge Lu Gan for advice on gene cloning and vector construction, Evan Updike for assistance with transforming Arabidopsis plants and selecting transgenic seeds, Samantha Link for careful monitoring of carrot plants, Zach Wahrenburg for helpful discussions of polyacetylene purification, Reinhard Jetter for providing comments on an early version of the manuscript, Brett Tylerfor providing the Phytopthora cell walls, and the RTSF Genomics Core at Michigan State University for sequencing services.

Publisher Copyright:
© 2018 American Society of Plant Biologists. All rights reserved.

Copyright:
Copyright 2019 Elsevier B.V., All rights reserved.

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