Lipid hydroperoxide levels in plant tissues

Hydroperoxides are the primary oxygenated products of polyunsaturated fatty acids and are key intermediates in the octadecanoid signalling pathway in plants. Lipid hydroperoxides (LHPO) were determined spectrophotometrically based on their reaction with an excess of Fe at low pH in the presence of the dye xylenol orange. Triphenylphosphine-mediated hydroxide formation was used to authenticate the signal generated by the hydroperoxides. The method readily detected lipid peroxidation in Phaseolus microsomes, senescing potato leaves and in a range of other plant tissues including Phaseolus hypocotyls (26 ± 5 nmol g FW), Alstroemeria floral tissues (sepals 66 ± 13 nmol g FW; petals 49 ± 6 nmol gFW), potato leaves (334 ± 75 nmol g FW), broccoli florets (568 ± 68 nmol g FW) and Chlamydomonas cells (602 ± 40 nmol g FW). Relative to the total fatty acid content of the tissues, the % LHPO was within the range of 0.6-1.7% for all tissue types (photosynthetic and non-photosynthetic) and represents the basal oxidation level of membrane fatty acids in plant cells. In order to relate the levels of LHPO to specific signalling pathways, transgenic potato plant lines were used in which lipoxygenase (LOX) (responsible for hydroperoxide biosynthesis) and hydroperoxide lyase (a route of hydroperoxide degradation) activities were largely reduced by an antisense-mediated approach. While the LHPO levels were similar to wild type in the individual LOX antisensed plants, basal LHPO levels, by contrast, were elevated by 38% in transgenic potato leaves antisensed in hydroperoxide lyase, indicating a role for this enzyme in the maintenance of cellular levels of LHPOs.