Abstract
Background: Plant lipoxygenases (LOXs, EC 1.13.11.12) are involved in lipid degradation, regulation of growth and development, senescence, and defence reactions. LOX represents the starting enzyme of the octadecanoid pathway. The aim of the work was to purify LOX from California poppy (Eschscholtzia californica Cham.), to determine its biochemical properties and to identify and quantify the products of LOX reaction with unsaturated fatty acids. Methods: LOX from California poppy seedlings was purified by hydrophobic chromatography (Phenyl-Sepharose CL-4B) and by ion-exchange chromatography (Q-Sepharose). The isolated LOX was incubated with linoleic acid used as a substrate. The HPLC experiments were performed with the Agilent Technologies 1050 series HPLC system. For the preparative separation of a mixture of hydroxy fatty acids from the sample matrix, the RP-HPLC method was used (column 120-5 Nucleosil C18). Then, the NP-HPLC analysis (separation, identification, and determination) of hydroxy fatty acid isomers was carried out on a Zorbax Rx-SIL column. Results: The purified LOX indicates the presence of a nontraditional plant enzyme with dual positional specificity (a ratio of 9- and 13-hydroperoxide products 1:1), a relative molecular mass of 85 kDa, a pH optimum of 6.5, an increasing activity stimulation by CaCl2 till 2 mM, and a high substrate reactivity to linoleic acid with kinetic values of KM 2.6 mM and Vmax 3.14 μM/min/mg. Conclusions: For the first time, the LOX from California poppy seedlings was partially purified and the biochemical properties of the enzyme were analyzed. A dual positional specificity of the LOX found from California poppy seedlings is in agreement with the results obtained for LOXs isolated from other Papaveraceaes. A 1:1 ratio of 9-/13-HODE is attractive for the simultaneous investigation of both biotic stress responses (indicated by the 9-HODE marker) and the biosynthesis of jasmonic acid and jasmonates (indicated by the 13-HODE marker).
Highlights
California poppy (Eschscholtzia californica Cham.) of the Papaveraceae family is an annual plant originating from California [1]
Changes in LOX activities during the germination of California poppy were analyzed in 1–7 day(s)-old seedlings (Figure 1)
All of these results suggest that high levels found during the dark, the maximal accumulation of oxylipins was observed on the 3rd day of germination
Summary
California poppy (Eschscholtzia californica Cham.) of the Papaveraceae family is an annual plant originating from California [1]. The entire plant produces a mixture of tertiary and quaternary isoquinoline alkaloids. In traditional medicine, it is used for its sedative, anxiolytic, and spasmolytic effects. Among the alkaloids present in California poppy, sanguinarine has attracted increasing. This benzophenanthridine alkaloid has antimicrobial activity and plays a role in plant defence [2]. The aim of the work was to purify LOX from California poppy (Eschscholtzia californica Cham.), to determine its biochemical properties and to identify and quantify the products of LOX reaction with unsaturated fatty acids. The HPLC experiments were performed with the Agilent Technologies 1050 series
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