Cloning, expression analysis, and functional characterization of two secretory phospholipases A2 in durum wheat (Triticum durum Desf.).

We previously isolated four cDNAs in durum wheat, TdsPLA2I, TdsPLA2II, TdsPLA2III and TdsPLA2IV, that encode proteins with homology to plant secretory phospholipases A2 (sPLA2s) (Verlotta et al., Int. J. Mol. Sci., 14, 2013, 5146-5169). In this study, we have further characterized TdsPLA2II and TdsPLA2III sequences that, on the basis of our previous findings, might encode sPLA2 isoforms with different features. Functional analysis revealed that, similarly to other known sPLA2s, TdsPLA2II and TdsPLA2III have an optimum at pH 9.0, require Ca(2+), are heat stable, and are inhibited by the disulfide-bond-reducing agent dithiothreitol. However, differences emerged between these TdsPLA2 isoforms. Transcript analysis revealed that the TdsPLA2III gene is highly up-regulated under different environmental stresses; conversely, the TdsPLA2II gene is expressed at constant levels under almost all of the stress conditions examined. Moreover, TdsPLA2II is saturated at micromolar substrate and Ca(2+) concentrations, whereas TdsPLA2III requires millimolar concentrations to reach maximal activity. This suggests that TdsPLA2II normally functions under optimal conditions in vivo, whereas TdsPLA2III is only partially activated, depending on the specific phospholipid and Ca(2+) levels. Altogether these data lead to the hypothesis that in vivo TdsPLA2II and TdsPLA2III are differently regulated at both molecular and biochemical level and that TdsPLA2III plays a major role in durum wheat response to adverse environmental conditions.