The Molecular and Functional Characterization of the Durum Wheat Lipoxygenase TdLOX2 Suggests Its Role in Hyperosmotic Stress Response

Abstract

In plants, lipoxygenases (LOXs) are involved in various processes, such as growth, development, and response to stress cues. In the present study, the expression pattern of six durum wheat LOX-encoding genes (TdLpx-B1.1, TdLpx-B1.2, TdLpx-A2, TdLpx-B2, TdLpx-A3 and TdLpx-B3) under hyperosmotic stress was investigated. With osmotic (0.42 M mannitol) and salt (0.21 M NaCl) stress imposed at the early stages of seedling growth, a strong induction of the TdLpx-A2 gene expression in the shoots paralleled an equally strong increase in the LOX activity. Enhanced levels of malondialdehyde (MDA) and increased rates of superoxide anion generation were also observed as a result of the stress imposition. Sequence analysis of the TdLOX2 encoded by the TdLpx-A2 gene revealed that it belonged to the type-1 9-LOX group. When overexpressed in E. coli, TdLOX2 exhibited normal enzyme activity, high sensitivity to specific LOX inhibitors, with 76% and 99% inhibition by salicylhydroxamic and propyl gallate, respectively, and a preference for linoleic acid as substrate, which was converted exclusively to its corresponding 13-hydroperoxide. This unexpected positional specificity could be related to the unusual TV/K motif that in TdLOX2 replaces the canonical TV/R motif of 9-LOXs. Treatment of seedlings with propyl gallate strongly suppressed the increase in LOX activity induced by the hyperosmotic stress; the MDA accumulation was also reduced but less markedly, whereas the rate of superoxide anion generation was even more increased. Overall, our findings suggest that the up-regulation of the TdLpx-A2 gene is a component of the durum wheat response to hyperosmotic stress and that TdLOX2 may act by counteracting the excessive generation of harmful reactive oxygen species responsible for the oxidative damages that occur in plants under stress.