Brady A. Vick

The biosynthesis of jasmonic acid: A physiological role for plant lipoxygenase☆

Linolenic acid was converted to a cyclic product, 12-oxo-phytodienoic acid, by lipoxygenase and hydroperoxide cyclase enzymes present in Vicia faba pericarp. Isotope labeling studies in which [U-14C] 12-[180] oxo-phytodienoic acid was incubated with thin sections of pericarp tissue showed that 12-oxo-phytodienoic acid is a biosynthetic precursor to jasmonic acid, a plant growth regulator which promotes senescence. Key enzymes proposed for this pathway are a reductase enzyme which reduces a double bond in the cyclopentenone ring, and beta-oxidation enzymes which remove six carbons from the carboxyl end of the molecule.

Oxidative Systems for Modification of Fatty Acids: The Lipoxygenase Pathway

Publisher Summary This chapter discusses the advances in plant lipoxygenase A special effort has been made to focus on physiological roles in which LOX might participate. A clear theme emerging from this effort is that a unique physiological role for LOX probably does not exist in plants. It likely has a more general function of responding to plant requirements by synthesizing fatty acid hydroperoxide, the central intermediate for a variety of diverging metabolic pathways. In this regard, the LOX pathway in plants can be similar to the mammalian LOX or cyclo-oxygenase pathways. In animals, the hydroperoxide or endoperoxide products of these two enzymes are transformed into a variety of leukotrienes and prostaglandins, depending on the nature of the cellular stimulus. Substrates for LOX are fatty acids that possess a cis , cis -1,4-pentadiene structure. The most common plant fatty acids that have this structure are linoleic and linolenic acids, but other fatty acids having this structure are also effective substrates to varying degrees.

A spectrophotometric assay for hydroperoxide lyase

Spectrophotometric assays for hydroperoxide lyase have traditionally measured the loss in absorption at 234 nm due to the disruption of conjugated diene in the fatty acid hydroperoxide. However, that assay does not distinguish between hydroperoxide lyase and hydroperoxide dehydrase activities, both of which cause a loss of conjugation in the substrate hydroperoxide. A new assay has been developed which is specific for hydroperoxide lyase. It is based on the ability of hydroperoxide lyase products, aldehydes and ω-oxoacids, to serve as substrates for yeast alcohol dehydrogenase. Thus, the new hydroperoxide lyase assay is a coupled enzyme assay, which is conducted spectrophotometrically at 340 nm and measures the oxidation of nicotinamide adenenine dinucleotide, reduced form (NADH). In addition to its specificity for hydroperoxide lyase, the coupled assay allows higher concentrations of both fatty acid hydroperoxide substrate and crude enzyme extracts than the assay conducted at 234 nm.

Construction of an RFLP linkage map for cultivated sunflower

Abstract An RFLP linkage map was constructed for cultivated sunflower Helianthus annuus L., based on 271 loci detected by 232 cDNA probes. Ninety-three F2 plants of a cross between inbred lines RHA 271 and HA 234 were used as the mapping population. These genetic markers plus a fertility restoration gene, Rf1, defined 20 linkage groups, covering 1164 cM of the sunflower genome. Of the 71 loci 202 had codominant genotypic segregation, with the rest showing dominant segregation. Thirty-two of the 232 probes gave multiple locus segregation. There were 39 clusters of tightly linked markers with 0 cM distance among loci. This map has an average marker-to-marker distance of 4.6 cM, with 11 markerless regions exceeding 20 cM.

Transformation of sunflower (Helianthus annuus L.) following wounding with glass beads.

A procedure was developed for transformation of Helianthus annuus (sunflower) using Agrobacterium tumefaciens. Cotyledons were removed from young seedlings, and the remaining tissue was uniformly wounded by shaking with glass beads. The wounded tissue was then co-cultivated with a hypervirulent strain of Agrobacterium tumefaciens harboring the binary plasmid pCNL56. Minimal use of defined medium was required, and no callus was observed. The polymerase chain reaction (PCR) followed by DNA hybridization demonstrated the presence of gusA DNA from pCNL56 in total leaf DNA of 6 primary transformants and 2 progeny plants. No Agrobacterium DNA was detected in total DNA from transformed sunflower leaves that was amplified with primers specific to the miaA chromosomal gene of Agrobacterium. Foreign DNA was also detected in the next generation. β-Glucuronidase (GUS) activity was demonstrated for 5 of the T2 transgenic plants. Grafting was used to increase the number of seeds present on plants that had undergone tissue culture manipulations.A procedure was developed for transformation of Helianthus annuus (sunflower) using Agrobacterium tumefaciens. Cotyledons were removed from young seedlings, and the remaining tissue was uniformly wounded by shaking with glass beads. The wounded tissue was then co-cultivated with a hypervirulent strain of Agrobacterium tumefaciens harboring the binary plasmid pCNL56. Minimal use of defined medium was required, and no callus was observed. The polymerase chain reaction (PCR) followed by DNA hybridization demonstrated the presence of gusA DNA from pCNL56 in total leaf DNA of 6 primary transformants and 2 progeny plants. No Agrobacterium DNA was detected in total DNA from transformed sunflower leaves that was amplified with primers specific to the miaA chromosomal gene of Agrobacterium. Foreign DNA was also detected in the next generation. β-Glucuronidase (GUS) activity was demonstrated for 5 of the T2 transgenic plants. Grafting was used to increase the number of seeds present on plants that had undergone tissue culture manipulations.

Thermal alteration of a cyclic fatty acid produced by a flaxseed extract

Methyl 8-[2-(cis-pent-2′-enyl)-3-oxo-cis-cyclopent-4-enyl] octanoate (I) is the methyl ester of a cyclic fatty acid synthesized enzymically from an incubation of linolenic acid with an extract of flaxseed (Linum usitatissimum L.). A proposed trivial name for I is methyl 12-oxo-cis-10, 15-phytodienoate (12-oxo-PDA). The evidence presented indicated that compound I has thecis configuration of the carbon chains with respect to the cyclopentenone ring. Treatment with acid, base, or heat isomerized I to a second product (II) that has thetrans configuration of the carbon chains. Prolonged heat treatment of II yielded a third cyclic product, methyl 12-oxo-9(13),cis-15-phytodienoate (III).

Formation of 12-[18O]Oxo-cis-10,cis-15-phytodienoic acid from 13-[18O]hydroperoxylinolenic acid by hydroperoxide cyclase

Abstract13-[18O] Hydroperoxylinolenic acid was permitted to react with an extract of flaxseed acetone powder containing hydroperoxide cyclase activity. The resulting product, 12-oxo-cis-10,cis-15-phytodienoic acid (12-oxo-PDA), contained18O in the carbonyl oxygen at carbon 12, suggesting that an epoxide was an intermediate in the hyderoperoxide cyclase reaction. A substrate specificity study showed that acis double bond β,γ to the conjugated hydroperoxide group was essential for the substrate to be converted to a cyclic product by hydroperoxide cyclase.

The purification and characterization of fatty acid hydroperoxide lyase in sunflower

Two fatty acid hydroperoxide lyases (HPO lyase I and II) were purified to apparent homogeneity from etiolated hypocotyls of sunflower (Helianthus annuus L.) by a combination of ion-exchange, hydrophobic interaction, and gel filtration chromatography. The two HPO lyases were separated during the hydrophobic interaction chromatography step, with HPO lyase I more hydrophilic than HPO lyase II. The estimated M(r) of both native HPO lyases was determined by gel filtration to be 200,000 and SDS-PAGE in the presence of 100 mM dithiothreitol showed that the enzyme was composed of a single 53 kDa peptide, suggesting that the enzyme exists as a tetramer in vivo. HPO lyase was also abundant in the cotyledons and green leaves. HPO lyases I and II from hypocotyl metabolized 13-hydroperoxylinoleic acid and 13-hydroperoxylinolenic acid to the same extent, but the green leaf enzyme was more than ten-fold more active with 13-hydroperoxylinolenic acid than 13-hydroperoxylinoleic acid. A difference spectrum between CO-bound and CO-unbound dithionite-reduced HPO lyase I showed an absorbance maximum at 452 nm, indicating that it was a cytochrome P450-type enzyme. The activities of HPO lyase I and II were significantly inhibited by nordihydroguaiaretic acid, sulfhydryl reagents, and piperonylbutoxide, which is a cytochrome P450 inhibitor.

The Lipoxygenase Pathway

After more than fifty years since its discovery, the study of the lipoxygenase pathway remains one of the most engaging, yet elusive areas of plant lipid research. Since 1932 when Andre and Hou (1) first reported on the existence of oxidizing enzymes which alter soybean oil, Chemical Abstracts has cited more than 1000 research articles under the subject of plant lipoxygenase. Despite the abundance of research effort on this topic, reviews (2–6) over the decades have been forced to report that the physiological role of lipoxygenase in plants is unknown.

Formation of γ-ketols from 13- and 9-hydroperoxides of linolenic acid by flaxseed hydroperoxide isomerase

A reexamination of the flaxseed hydroperoxide isomerase reaction showed that a minor enzymic product (ca. 5%), identified as a γ-ketol, was present. The substrates were the 13- or 9-hydroperoxides of linolenic acid, which were converted to 9-hydroxy-12-oxo-cis-15-trans-11-octadecadienoic acid, respectively. These compounds were formed in addition to the major products reported earlier: a 12,13-α-ketol and 12-oxo-cis-10,15-phytodienoic acid from the 13-isomer, and a 9,10-α-ketol from the 9-isomer.