Developing a platform for production of the oxylipin KODA 9-hydroxy-10-oxo-octadecadienoic acid in plants

Abstract KODA (9-hydroxy-10-oxo-12(Z),15(Z)-octadecadienoic acid) is a plant oxylipin involved in recovery from stress. As an agrichemical, KODA helps maintain crop production under various environmental stresses. In plants, KODA is synthesized from α-linolenic acids via 9-lipoxygenase (9-LOX) and allene oxide synthase (AOS), although the amount is usually low except in the free-floating aquatic plant Lemna paucicostata. To improve KODA biosynthetic yield in other plants such as Nicotiana benthamiana and Arabidopsis thaliana, we developed a system to overproduce KODA in vivo via ectopic expression of L. paucicostata 9-LOX and AOS. The transient expression in N. benthamiana showed that the expression of these two genes is sufficient to produce KODA in leaves. However, stable expression of 9-LOX and AOS (with consequent KODA production) in Arabidopsis plants succeeded only when the two proteins were localized in plastids or the endoplasmic reticulum/lipid droplets. Although only small amounts of KODA could be detected in leaf extracts of transgenic Nicotiana or Arabidopsis plants, subsequent incubation of the extracts increased KODA abundance over time. Therefore, KODA production in transgenic plants stably expressing 9-LOX and AOS requires specific subcellular localization of these two enzymes and incubation of leaf crude extracts, which liberates α-linolenic acid via breakdown of endogenous lipids.

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The lipoxygenase pathway in tulip (Tulipa gesneriana): detection of the ketol route

Biochemical Journal ◽

10.1042/bj3520501 ◽

2000 ◽

Vol 352 (2) ◽

pp. 501-509 ◽

Cited By ~ 17

Author(s):

Alexander N. GRECHKIN ◽

Lucia S. MUKHTAROVA ◽

Mats HAMBERG

Keyword(s):

Octadecadienoic Acid ◽

Optical Purity ◽

Cyclase Activity ◽

Allene Oxide ◽

Allene Oxide Synthase ◽

Lipoxygenase Pathway ◽

Tulipa Gesneriana ◽

Tulip Bulbs ◽

Oxide Synthase

The in vitro metabolism of [1-14C]linoleate, [1-14C]linolenate and their 9(S)-hydroperoxides was studied in cell-free preparations from tulip (Tulipa gesneriana) bulbs, leaves and flowers. Linoleate and its 9-hydroperoxide were converted by bulb and leaf preparations into three ketols: (12Z)-9-hydroxy-10-oxo-12-octadecadienoic acid (α-ketol), (11E)-10-oxo-13-hydroxy-11-octadecadienoic acid (γ-ketol) and a novel compound, (12Z)-10-oxo-11-hydroxy-12-octadecadienoic acid (10,11-ketol), in the approximate molar proportions of 10:3:1. The corresponding 15,16-dehydro α- and γ-ketols were the main metabolites of [1-14C]linolenate and its 9-hydroperoxide. Thus bulbs and leaves possessed 9-lipoxygenase and allene oxide synthase activities. Incubations with flower preparations gave α-ketol hydro(pero)xides as predominant metabolites. Bulb and leaf preparations possessed a novel enzyme activity, γ-ketol reductase, which reduces γ-ketol to 10-oxo-13-hydroxyoctadecanoic acid (dihydro-γ-ketol) in the presence of NADH. Exogenous linolenate 13(S)-hydroperoxide was converted mostly into chiral (9S,13S)-12-oxo-10-phytodienoate (99.5% optical purity) by bulb preparations, while [1-14C]linolenate was a precursor for ketols only. Thus tulip bulbs possess abundant allene oxide cyclase activity, the substrate for which is linolenate 13(S)-hydroperoxide, even though 13(S)-lipoxygenase products were not detectable in the bulbs. The majority of the cyclase activity was found in the microsomes (105g pellet). Cyclase activity was not found in the other tissues examined, but only in the bulbs. The ketol route of the lipoxygenase pathway, mediated by 9-lipoxygenase and allene oxide synthase activities, has not been detected previously in the vegetative organs of any plant species.

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Lipoxygenase pathway in tulip: biosynthesis of ketols

Biochemical Society Transactions ◽

10.1042/bst0280851 ◽

2000 ◽

Vol 28 (6) ◽

pp. 851-853 ◽

Cited By ~ 2

Author(s):

A. N. Grechkin ◽

L. S. Mukhtarova ◽

M. Hamberg

Keyword(s):

Octadecadienoic Acid ◽

Allene Oxide ◽

The Novel ◽

Allene Oxide Synthase ◽

Allene Oxide Cyclase ◽

Lipoxygenase Pathway ◽

Tulipa Gesneriana ◽

Oxide Synthase

The metabolism in vitro of [1-14C]linoleate, [1-14C]linolenate and their 9(S)-hydroperoxides in tulip (Tulipa gesneriana) was found to be under the control of 9-lipoxygenase and allene oxide synthase, and directed towards α-ketol, γ-ketol and the novel compound (12Z)-10-oxo-11-hydroxy- 12-octadecadienoic acid (10,11-ketol). Potent activity of allene oxide cyclase (in bulbs) and a new enzyme, γ-ketol reductase (in bulbs and leaves), was detected. Metabolism in flowers is directed predominantly towards α-ketol hydroperoxide.

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Detection of the First Epoxyalcohol Synthase/Allene Oxide Synthase (CYP74 Clan) in the Lancelet (Branchiostoma belcheri, Chordata)

International Journal of Molecular Sciences ◽

10.3390/ijms22094737 ◽

2021 ◽

Vol 22 (9) ◽

pp. 4737

Author(s):

Yana Y. Toporkova ◽

Elena O. Smirnova ◽

Natalia V. Lantsova ◽

Lucia S. Mukhtarova ◽

Alexander N. Grechkin

Keyword(s):

Fatty Acids ◽

Fatty Acid ◽

Oxidative Metabolism ◽

Octadecadienoic Acid ◽

Cytochromes P450 ◽

Allene Oxide ◽

Allene Oxide Synthase ◽

Branchiostoma Belcheri ◽

Oxide Synthase ◽

Fatty Acid Hydroperoxides

The CYP74 clan cytochromes (P450) are key enzymes of oxidative metabolism of polyunsaturated fatty acids in plants, some Proteobacteria, brown and green algae, and Metazoa. The CYP74 enzymes, including the allene oxide synthases (AOSs), hydroperoxide lyases, divinyl ether synthases, and epoxyalcohol synthases (EASs) transform the fatty acid hydroperoxides to bioactive oxylipins. A novel CYP74 clan enzyme CYP440A18 of the Asian (Belcher’s) lancelet (Branchiostoma belcheri, Chordata) was biochemically characterized in the present work. The recombinant CYP440A18 enzyme was active towards all substrates used: linoleate and α-linolenate 9- and 13-hydroperoxides, as well as with eicosatetraenoate and eicosapentaenoate 15-hydroperoxides. The enzyme specifically converted α-linolenate 13-hydroperoxide (13-HPOT) to the oxiranyl carbinol (9Z,11R,12R,13S,15Z)-11-hydroxy-12,13-epoxy-9,15-octadecadienoic acid (EAS product), α-ketol, 12-oxo-13-hydroxy-9,15-octadecadienoic acid (AOS product), and cis-12-oxo-10,15-phytodienoic acid (AOS product) at a ratio of around 35:5:1. Other hydroperoxides were converted by this enzyme to the analogous products. In contrast to other substrates, the 13-HPOT and 15-HPEPE yielded higher proportions of α-ketols, as well as the small amounts of cyclopentenones, cis-12-oxo-10,15-phytodienoic acid and its higher homologue, dihomo-cis-12-oxo-3,6,10,15-phytotetraenoic acid, respectively. Thus, the CYP440A18 enzyme exhibited dual EAS/AOS activity. The obtained results allowed us to ascribe a name “B. belcheri EAS/AOS” (BbEAS/AOS) to this enzyme. BbEAS/AOS is a first CYP74 clan enzyme of Chordata species possessing AOS activity.

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Specific Adduction of Plant Lipid Transfer Protein by an Allene Oxide Generated by 9-Lipoxygenase and Allene Oxide Synthase

Journal of Biological Chemistry ◽

10.1074/jbc.m608580200 ◽

2006 ◽

Vol 281 (51) ◽

pp. 38981-38988 ◽

Cited By ~ 31

Author(s):

Bénédicte Bakan ◽

Mats Hamberg ◽

Ludivine Perrocheau ◽

Daniel Maume ◽

Hélène Rogniaux ◽

...

Keyword(s):

Lipid Transfer Protein ◽

Allene Oxide ◽

Allene Oxide Synthase ◽

Lipid Transfer ◽

Plant Lipid ◽

Transfer Protein ◽

Oxide Synthase

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Functional Characterization of An Allene Oxide Synthase Involved in Biosynthesis of Jasmonic Acid and Its Influence on Metabolite Profiles and Ethylene Formation in Tea (Camellia sinensis) Flowers

International Journal of Molecular Sciences ◽

10.3390/ijms19082440 ◽

2018 ◽

Vol 19 (8) ◽

pp. 2440 ◽

Cited By ~ 5

Author(s):

Qiyuan Peng ◽

Ying Zhou ◽

Yinyin Liao ◽

Lanting Zeng ◽

Xinlan Xu ◽

...

Keyword(s):

Mass Spectrometry ◽

Jasmonic Acid ◽

Camellia Sinensis ◽

Allene Oxide ◽

Allene Oxide Synthase ◽

Tea Leaves ◽

Ethylene Formation ◽

Horticultural Crops ◽

Metabolite Profiles ◽

Oxide Synthase

Jasmonic acid (JA) is reportedly involved in the interaction between insects and the vegetative parts of horticultural crops; less attention has, however, been paid to its involvement in the interaction between insects and the floral parts of horticultural crops. Previously, we investigated the allene oxide synthase 2 (AOS2) gene that was found to be the only JA synthesis gene upregulated in tea (Camellia sinensis) flowers exposed to insect (Thrips hawaiiensis (Morgan)) attacks. In our present study, transient expression analysis in Nicotiana benthamiana plants confirmed that CsAOS2 functioned in JA synthesis and was located in the chloroplast membrane. In contrast to tea leaves, the metabolite profiles of tea flowers were not significantly affected by 10 h JA (2.5 mM) treatment as determined using ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry, and gas chromatography-mass spectrometry. Moreover, JA treatment did not significantly influence ethylene formation in tea flowers. These results suggest that JA in tea flowers may have different functions from JA in tea leaves and other flowers.

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Molecular cloning of an allene oxide synthase: a cytochrome P450 specialized for the metabolism of fatty acid hydroperoxides.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.90.18.8519 ◽

1993 ◽

Vol 90 (18) ◽

pp. 8519-8523 ◽

Cited By ~ 190

Author(s):

W. C. Song ◽

C. D. Funk ◽

A. R. Brash

Keyword(s):

Cytochrome P450 ◽

Fatty Acid ◽

Molecular Cloning ◽

Allene Oxide ◽

Allene Oxide Synthase ◽

Oxide Synthase ◽

Fatty Acid Hydroperoxides

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Jasmonates: what ALLENE OXIDE SYNTHASE does for plants

Journal of Experimental Botany ◽

10.1093/jxb/erz254 ◽

2019 ◽

Vol 70 (13) ◽

pp. 3373-3378 ◽

Cited By ~ 2

Author(s):

Edward E Farmer ◽

Alain Goossens

Keyword(s):

Allene Oxide ◽

Allene Oxide Synthase ◽

Oxide Synthase

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Allene oxide synthase and hydroperoxide lyase product accumulation in Artemisia species

Plant Science ◽

10.1016/j.plantsci.2005.03.004 ◽

2005 ◽

Vol 169 (1) ◽

pp. 139-146 ◽

Cited By ~ 2

Author(s):

Meshack Afitlhile ◽

Hirotada Fukushige ◽

Charles McCraken ◽

David Hildebrand

Keyword(s):

Hydroperoxide Lyase ◽

Allene Oxide ◽

Allene Oxide Synthase ◽

Artemisia Species ◽

Oxide Synthase ◽

Product Accumulation

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ALLENE OXIDE SYNTHASE and HYDROPEROXIDE LYASE, Two Non-Canonical Cytochrome P450s in Arabidopsis thaliana and Their Different Roles in Plant Defense

International Journal of Molecular Sciences ◽

10.3390/ijms20123064 ◽

2019 ◽

Vol 20 (12) ◽

pp. 3064 ◽

Cited By ~ 3

Author(s):

Sachin Rustgi ◽

Armin Springer ◽

ChulHee Kang ◽

Diter von Wettstein ◽

Christiane Reinbothe ◽

...

Keyword(s):

Arabidopsis Thaliana ◽

Plant Defense ◽

Metabolic Regulation ◽

Insect Pests ◽

Green Leaf Volatiles ◽

Hydroperoxide Lyase ◽

Allene Oxide ◽

Allene Oxide Synthase ◽

Cytochrome P450 Enzymes ◽

Oxide Synthase

The channeling of metabolites is an essential step of metabolic regulation in all living organisms. Multifunctional enzymes with defined domains for metabolite compartmentalization are rare, but in many cases, larger assemblies forming multimeric protein complexes operate in defined metabolic shunts. In Arabidopsis thaliana, a multimeric complex was discovered that contains a 13-lipoxygenase and allene oxide synthase (AOS) as well as allene oxide cyclase. All three plant enzymes are localized in chloroplasts, contributing to the biosynthesis of jasmonic acid (JA). JA and its derivatives act as ubiquitous plant defense regulators in responses to both biotic and abiotic stresses. AOS belongs to the superfamily of cytochrome P450 enzymes and is named CYP74A. Another CYP450 in chloroplasts, hydroperoxide lyase (HPL, CYP74B), competes with AOS for the common substrate. The products of the HPL reaction are green leaf volatiles that are involved in the deterrence of insect pests. Both enzymes represent non-canonical CYP450 family members, as they do not depend on O2 and NADPH-dependent CYP450 reductase activities. AOS and HPL activities are crucial for plants to respond to different biotic foes. In this mini-review, we aim to summarize how plants make use of the LOX2–AOS–AOC2 complex in chloroplasts to boost JA biosynthesis over volatile production and how this situation may change in plant communities during mass ingestion by insect pests.

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