Detection of an enol intermediate in the hydroperoxide lyase chain cleavage reaction

AbstractGuava (Psidium guajava) hydroperoxide lyase (HPL) preparations were incubated with [1-14C](9Z,11E,13S,15Z)-13-hydroperoxy-9,11,15-octadecatrienoic acid for 1 min at 0°C, followed by rapid extraction/trimethylsilylation. Analysis of the trimethylsilylated products by gas chromatography–mass spectrometry and radio-high-performance liquid chromatography revealed a single predominant 14C-labelled compound, identified by its 1H-nuclear magnetic resonance, ultraviolet and mass spectra as the trimethylsilyl ether/ester of (9Z,11E)-12-hydroxy-9,11-dodecadienoic acid. Longer time incubations afford smaller yield of this enol due to its partial tautomerization into (9Z)-12-oxo-9-dodecenoic acid. The data obtained demonstrate that formation of (9Z)-12-oxo-9-dodecenoic acid in the HPL reaction is preceded by unstable enol oxylipin, and further suggest that hemiacetals are the true products of HPL catalysis

Oxylipins in moss development and defense

Oxylipins are oxygenated fatty acids that participate in plant development and defense against pathogen infection, insects and wounding. Initial oxygenation of substrate fatty acids is mainly catalyzed by lipoxygenases and alpha-dioxygenases but can also take place non-enzymatically by autoxidation or singlet oxygen-dependent reactions. The resulting hydroperoxides are further metabolized by secondary enzymes to produce a large variety of compounds, including the hormone jasmonic acid and short-chain green leaf volatiles. In flowering plants, which lack arachidonic acid, oxylipins are produced mainly from oxidation of polyunsaturated C18 fatty acids, notably linolenic and linoleic acids. Algae and mosses in addition possess polyunsaturated C20 fatty acids including arachidonic and eicosapentaenoic acids, which can also be oxidized by lipoxygenases and transformed into bioactive compounds. Mosses are phylogenetically placed between unicellular green algae and flowering plants, allowing evolutionary studies of the different oxylipin pathways. During the last years the moss Physcomitrella patens has become an attractive model plant for understanding oxylipin biosynthesis and diversity. In addition to the advantageous evolutionary position, functional studies of the different oxylipin-forming enzymes can be performed in this moss by targeted gene disruption or single point mutations by means of homologous recombination. Biochemical characterization of several oxylipin-producing enzymes and oxylipin profiling in P. patens reveal the presence of a wider range of oxylipins compared to flowering plants, including C18 as well as C20-derived oxylipins. Surprisingly, one of the most active oxylipins in plants, jasmonic acid, is not synthesized in this moss. In this review we present an overview of oxylipins produced in mosses and discuss the current knowledge related to the involvement of oxylipin-producing enzymes and their products in moss development and defense

Fatty acyl-CoA reductases of birds

<p>Abstract</p> <p>Background</p> <p>Birds clean and lubricate their feathers with waxes that are produced in the uropygial gland, a holocrine gland located on their back above the tail. The type and the composition of the secreted wax esters are dependent on the bird species, for instance the wax ester secretion of goose contains branched-chain fatty acids and unbranched fatty alcohols, whereas that of barn owl contains fatty acids and alcohols both of which are branched. Alcohol-forming fatty acyl-CoA reductases (FAR) catalyze the reduction of activated acyl groups to fatty alcohols that can be esterified with acyl-CoA thioesters forming wax esters.</p> <p>Results</p> <p>cDNA sequences encoding fatty acyl-CoA reductases were cloned from the uropygial glands of barn owl (<it>Tyto alba</it>), domestic chicken (<it>Gallus gallus domesticus</it>) and domestic goose (<it>Anser anser domesticus</it>). Heterologous expression in <it>Saccharomyces cerevisiae </it>showed that they encode membrane associated enzymes which catalyze a NADPH dependent reduction of acyl-CoA thioesters to fatty alcohols. By feeding studies of transgenic yeast cultures and <it>in vitro </it>enzyme assays with membrane fractions of transgenic yeast cells two groups of isozymes with different properties were identified, termed FAR1 and FAR2. The FAR1 group mainly synthesized 1-hexadecanol and accepted substrates in the range between 14 and 18 carbon atoms, whereas the FAR2 group preferred stearoyl-CoA and accepted substrates between 16 and 20 carbon atoms. Expression studies with tissues of domestic chicken indicated that FAR transcripts were not restricted to the uropygial gland.</p> <p>Conclusion</p> <p>The data of our study suggest that the identified and characterized avian FAR isozymes, FAR1 and FAR2, can be involved in wax ester biosynthesis and in other pathways like ether lipid synthesis.</p

Identification of avian wax synthases

<p>Abstract</p> <p>Background</p> <p>Bird species show a high degree of variation in the composition of their preen gland waxes. For instance, <it>galliform </it>birds like chicken contain fatty acid esters of 2,3-alkanediols, while <it>Anseriformes </it>like goose or <it>Strigiformes </it>like barn owl contain wax monoesters in their preen gland secretions. The final biosynthetic step is catalyzed by wax synthases (WS) which have been identified in pro- and eukaryotic organisms.</p> <p>Results</p> <p>Sequence similarities enabled us to identify six cDNAs encoding putative wax synthesizing proteins in chicken and two from barn owl and goose. Expression studies in yeast under <it>in vivo </it>and <it>in vitro </it>conditions showed that three proteins from chicken performed WS activity while a sequence from chicken, goose and barn owl encoded a bifunctional enzyme catalyzing both wax ester and triacylglycerol synthesis. Mono- and bifunctional WS were found to differ in their substrate specificities especially with regard to branched-chain alcohols and acyl-CoA thioesters. According to the expression patterns of their transcripts and the properties of the enzymes, avian WS proteins might not be confined to preen glands.</p> <p>Conclusions</p> <p>We provide direct evidence that avian preen glands possess both monofunctional and bifunctional WS proteins which have different expression patterns and WS activities with different substrate specificities.</p

The Physcomitrella patens unique alpha-dioxygenase participates in both developmental processes and defense responses

[Background] Plant α-dioxygenases catalyze the incorporation of molecular oxygen into polyunsaturated fatty acids leading to the formation of oxylipins. In flowering plants, two main groups of α-DOXs have been described. While the α-DOX1 isoforms are mainly involved in defense responses against microbial infection and herbivores, the α-DOX2 isoforms are mostly related to development. To gain insight into the roles played by these enzymes during land plant evolution, we performed biochemical, genetic and molecular analyses to examine the function of the single copy moss Physcomitrella patens α-DOX (Ppα-DOX) in development and defense against pathogens.[Results] Recombinant Ppα-DOX protein catalyzed the conversion of fatty acids into 2-hydroperoxy derivatives with a substrate preference for α-linolenic, linoleic and palmitic acids. Ppα-DOX is expressed during development in tips of young protonemal filaments with maximum expression levels in mitotically active undifferentiated apical cells. In leafy gametophores, Ppα-DOX is expressed in auxin producing tissues, including rhizoid and axillary hairs. Ppα-DOX transcript levels and Ppα-DOX activity increased in moss tissues infected with Botrytis cinerea or treated with Pectobacterium carotovorum elicitors. In B. cinerea infected leaves, Ppα-DOX-GUS proteins accumulated in cells surrounding infected cells, suggesting a protective mechanism. Targeted disruption of Ppα-DOX did not cause a visible developmental alteration and did not compromise the defense response. However, overexpressing Ppα-DOX, or incubating wild-type tissues with Ppα-DOX-derived oxylipins, principally the aldehyde heptadecatrienal, resulted in smaller moss colonies with less protonemal tissues, due to a reduction of caulonemal filament growth and a reduction of chloronemal cell size compared with normal tissues. In addition, Ppα-DOX overexpression and treatments with Ppα-DOX-derived oxylipins reduced cellular damage caused by elicitors of P. carotovorum.[Conclusions] Our study shows that the unique α-DOX of the primitive land plant P. patens, although apparently not crucial, participates both in development and in the defense response against pathogens, suggesting that α-DOXs from flowering plants could have originated by duplication and successive functional diversification after the divergence from bryophytes.This work was supported by Agencia Nacional de Investigación e Innovación (ANII) [grants FCE2007_376, FCE2011_6095, fellowships BE_POS_2009_726 (A. Castro) and BE_POS_2010_2533 (L. Machado)], UdelaR Uruguay/CSIC Spain (Joint project), the Swedish Research Council, and Programa de Desarrollo de las Ciencias Básicas (PEDECIBA) Uruguay. The Ppα-DOX cDNA was obtained from the RIKEN Biological Research Center, Tsukuba, Japan

Ligand-receptor co-evolution shaped the jasmonate pathway in land plants

The phytohormone jasmonoyl-isoleucine (JA-Ile) regulates defense, growth and developmental responses in vascular plants. Bryophytes have conserved sequences for all JA-Ile signaling pathway components but lack JA-Ile. We show that, in spite of 450 million years of independent evolution, the JA-Ile receptor COI1 is functionally conserved between the bryophyte Marchantia polymorpha and the eudicot Arabidopsis thaliana but COI1 responds to different ligands in each species. We identified the ligand of Marchantia MpCOI1 as two isomeric forms of the JA-Ile precursor dinor-OPDA (dinor-cis-OPDA and dinor-iso-OPDA). We demonstrate that AtCOI1 functionally complements Mpcoi1 mutation and confers JA-Ile responsiveness and that a single-residue substitution in MpCOI1 is responsible for the evolutionary switch in ligand specificity. Our results identify the ancestral bioactive jasmonate and clarify its biosynthetic pathway, demonstrate the functional conservation of its signaling pathway, and show that JA-Ile and COI1 emergence in vascular plants required co-evolution of hormone biosynthetic complexity and receptor specificity

Dosage differences in 12-OXOPHYTODIENOATE REDUCTASE genes modulate wheat root growth

Wheat, an essential crop for global food security, is well adapted to a wide variety of soils. However, the gene networks shaping different root architectures remain poorly understood. We report here that dosage differences in a cluster of monocot-specific 12-OXOPHYTODIENOATE REDUCTASE genes from subfamily III (OPRIII) modulate key differences in wheat root architecture, which are associated with grain yield under water-limited conditions. Wheat plants with loss-of-function mutations in OPRIII show longer seminal roots, whereas increased OPRIII dosage or transgenic over-expression result in reduced seminal root growth, precocious development of lateral roots and increased jasmonic acid (JA and JA-Ile). Pharmacological inhibition of JA-biosynthesis abolishes root length differences, consistent with a JA-mediated mechanism. Transcriptome analyses of transgenic and wild-type lines show significant enriched JA-biosynthetic and reactive oxygen species (ROS) pathways, which parallel changes in ROS distribution. OPRIII genes provide a useful entry point to engineer root architecture in wheat and other cereals.Fil: Gabay, Gilad. University of California at Davis; Estados UnidosFil: Wang, Hanchao. University of California at Davis; Estados Unidos. University Of Haifa; IsraelFil: Zhang, Junli. University of California at Davis; Estados UnidosFil: Moriconi, Jorge Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Burguener, Germán Federico. University of California at Davis; Estados UnidosFil: Gualano, Leonardo David. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Howell, Tyson. University of California at Davis; Estados UnidosFil: Lukaszewski, Adam. University of California; Estados UnidosFil: Staskawicz, Brian. University of California; Estados UnidosFil: Cho, Myeong-Je. University of California; Estados UnidosFil: Tanaka, Jaclyn. University of California; Estados UnidosFil: Fahima, Tzion. University Of Haifa; IsraelFil: Ke, Haiyan. University of California; Estados UnidosFil: Dehesh, Katayoon. University of California; Estados UnidosFil: Zhang, Guo-Liang. Fudan University; ChinaFil: Gou, Jin Ying. Beijing Key Laboratory Of Crop Genetic Improvement; China. Fudan University; ChinaFil: Hamberg, Mats. Karolinska Huddinge Hospital. Karolinska Institutet; SueciaFil: Santa Maria, Guillermo Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Dubcovsky, Jorge. University of California at Davis; Estados Unidos. Howard Hughes Medical Institute; Estados Unido

Ligand-receptor co-evolution shaped the jasmonate pathway in land plants.

The phytohormone jasmonoyl-isoleucine (JA-Ile) regulates defense, growth and developmental responses in vascular plants. Bryophytes have conserved sequences for all JA-Ile signaling pathway components but lack JA-Ile. We show that, in spite of 450 million years of independent evolution, the JA-Ile receptor COI1 is functionally conserved between the bryophyte Marchantia polymorpha and the eudicot Arabidopsis thaliana but COI1 responds to different ligands in each species. We identified the ligand of Marchantia MpCOI1 as two isomeric forms of the JA-Ile precursor dinor-OPDA (dinor-cis-OPDA and dinor-iso-OPDA). We demonstrate that AtCOI1 functionally complements Mpcoi1 mutation and confers JA-Ile responsiveness and that a single-residue substitution in MpCOI1 is responsible for the evolutionary switch in ligand specificity. Our results identify the ancestral bioactive jasmonate and clarify its biosynthetic pathway, demonstrate the functional conservation of its signaling pathway, and show that JA-Ile and COI1 emergence in vascular plants required co-evolution of hormone biosynthetic complexity and receptor specificity