Expression, purification and refolding of active durum wheat (Triticum durum Desf.) secretory phospholipase A2 from inclusion bodies of Escherichia coli.

Recently, a durum wheat (Triticum durum Desf.) secretory phospholipase A2 (TdsPLA2III) was identified in leaves as potentially involved in plant responses to conditions of limiting water supply. Therefore, to allow future functional studies on TdsPLA2III and shed further light on the involvement of sPLA2 isoforms in specific plant functions, here we report a protocol for the overexpression of TdsPLA2III in Escherichia coli in the form of inclusion bodies, and for its purification and refolding. The use of the Gateway system (Invitrogen) allows the expression of a large quantity of the mature form (without the signal peptide) of TdsPLA2III with an N-terminal 6×His-tag, for purification using Ni-affinity chromatography. The purified recombinant 6×His-TdsPLA2III fusion protein is then refolded using a step-wise dialysis approach. About 40mg purified and active protein was obtained from 1L of cell culture. This recombinant 6×His-TdsPLA2III protein shows PLA2 activity, as it can hydrolyze linoleate from the sn-2 position of 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine. Moreover, it has some features that are typical of other known plant sPLA2s: Ca(2+)-dependence, inhibition by the disulfide bond reducing agent dithiothreitol, and resistance to high temperature.

Secretory Phospholipases A2 in Durum Wheat (Triticum durum Desf.): Gene Expression, Enzymatic Activity, and Relation to Drought Stress Adaptation.

Phospholipases A2 (PLA2s) are known to mediate signaling cascades during plant growth and development, as well as biotic and abiotic stress responses. In this context, the present study provides extensive characterization of specific PLA2s in durum wheat, and assesses their involvement in durum wheat response to drought stress. In durum wheat leaves, four full-length expressed sequences encoding putative PLA2s were isolated and characterized as belonging to the class of secretory PLA2s (sPLA2s): TdsPLA2I, TdsPLA2II, TdsPLA2III and TdsPLA2IV. PLA2 activity was also detected, the characteristics of which resemble those of previously characterized plant sPLA2s: strong preference for phospholipids; requirement for millimolar Ca2+ concentrations; optimal activity at basic pH; heat stability; and inhibition by the reducing agent dithiothreitol. With drought stress imposed at both the vegetative and reproductive stages, accumulation of TdsPLA2I and TdsPLA2III transcripts, and to a lesser extent of TdsPLA2IV transcript, paralleled increased PLA2 activity; both transcript levels and enzymatic activity decreased as a consequence of stress recovery. Consistently, free fatty acid analysis of drought-stressed leaves revealed increased linoleate, linolenate and palmitate contents, which were reversed by plant re-watering. Overall, these findings strongly suggest that there are inducible sPLA2 isoforms in durum wheat that have roles in orchestrating the plant response to drought stress.

Insight into durum wheat Lpx-B1: a small gene family coding for the lipoxygenase responsible for carotenoid bleaching in mature grains.

BACKGROUND: The yellow colour of pasta products is one of the main criteria used by consumers to assess pasta quality. This character is due to the presence of carotenoid pigments in semolina. During pasta processing, oxidative degradation of carotenoid pigments occurs mainly due to lipoxygenase (LOX). In durum wheat (Triticum durum Desf.), two Lpx-1 genes have been identified on chromosome 4B, Lpx-B1.1 and Lpx-B1.2, and evidences have been reported that the deletion of Lpx-B1.1 is associated with a strong reduction in LOX activity in semolina. In the present study, we characterised the Lpx-B1 gene family identified in a durum wheat germplasm collection and related the distribution and expression of the Lpx-B1 genes and alleles to variations in LOX activity in the mature grains. RESULTS: In addition to the already known Lpx-B1.1 and Lpx-B1.2 genes, a new gene was identified, Lpx-B1.3, along with three different Lpx-B1.1 alleles, Lpx-B1.1a, Lpx-B1.1b and the partially deleted Lpx-B1.1c. Screening of the germplasm collection showed that all of the genotypes have one of the three Lpx-B1.1 alleles, associated with either Lpx-B1.2 or Lpx-B1.3, thus showing that in this collection the two genes are alternatives. Therefore, based on Lpx-B1 distribution, three different haplotypes were distinguished: haplotype I, carrying Lpx-B1.3 and the Lpx-B1.1b allele; haplotype II carrying Lpx-B1.2 and the Lpx-B1.1a allele; and haplotype III carrying Lpx-B1.2 and the Lpx-B1.1c allele. Determination of Lpx-B1 transcript abundance and total LOX activity in mature grains revealed differences among these three haplotypes: haplotypes I, II and III showed high, intermediate and low levels, respectively, of functional Lpx-B1 transcripts and enzymatic activity. CONCLUSIONS: In this germplasm collection, the Lpx-B1 gene family accounts for most of the total LOX activity in the mature grains. Information on these Lpx-B1 haplotypes provides significant improvement for prediction of LOX-1 activity levels in mature grains, and will therefore help in breeding programmes aimed at selection of new durum wheat genotypes with higher carotenoid contents in their end products.

Nitrogen metabolism in durum wheat under salinity: accumulation of proline and glycine betaine.

We studied the effect of salinity on amino acid, proline and glycine betaine accumulation in leaves of different stages of development in durum wheat under high and low nitrogen supply. Our results suggest that protective compounds against salt stress are accumulated in all leaves. The major metabolites are glycine betaine, which preferentially accumulates in younger tissues, and proline, which is found predominantly in older tissues. Proline tended to accumulate early, at the onset of the stress, while glycine betaine accumulation was observed during prolonged stress. Nitrate reductase (NR) and glutamate synthase (GOGAT) are positively correlated with these compatible solutes: proline is associated with NR in the oldest leaves of high-nitrate plants and glycine betaine is associated with GOGAT in the youngest leaves of both low- and high-nitrate plants. In high-nitrate conditions proline accounts for more than 39% of the osmotic adjustment in the cytoplasmic compartments of old leaves. Its nitrogen-dependent accumulation may offer an important advantage in that it can be metabolised to allow reallocation of energy, carbon and nitrogen from the older leaves to the younger tissues. The contribution of glycine betaine is higher in young leaves and is independent of nitrogen nutrition.