Cloning and characterisation of an almond 9-lipoxygenase expressed early during seed development

Abstract

Lipoxygenases (LOXs) are a class of non-heme iron containing dioxygenases which catalyse the hydroperoxidation of polyunsaturated fatty acids such as linoleic and linolenic acids. Plant LOXs are commonly believed to play an important role in fundamental plant processes such as defence, development and senescence. However, the physiological role of these enzymes in seed development is still largely unknown. In a previous work we showed that LOXs are highly expressed in almond seed at early developmental stages and that 9-LOX accounts for most of the LOX activity detected in almonds. To elucidate the role of 9-LOXs in seed development and in influencing seed quality, we carried out the molecular cloning and characterisation of a cDNA encoding the almond ( Prunus dulcis) seed 9-LOX. The predicted protein comprises 862 amino acids with a calculated molecular mass of 97.7 kDa and an isoelectric point of 5.53. The almond LOX shows 79% identity towards a LOX previously reported from hazelnut seed. A high degree of identity was also found towards cotton (75%) and tobacco (73%) LOXs involved in plant defence responses. The cDNA was expressed in E. coli and produced an active enzyme showing a strict product specificity, 9-hydroperoxides being produced almost exclusively with either linoleic or linolenic acid as substrates. The biochemical characterisation of the main LOX isoforms detected at different seed physiological stages indicated that 9-LOX metabolism is specific for seed development and could have important implications for seed quality.