Phospholipases Dα and δ are involved in local and systemic wound responses of cotton (G. hirsutum)

Abstract

Phospholipases D (PLDs) catabolize structural phospholipids to produce phosphatidic acid (PtdOH), a lipid playing central role in signalling pathways in animal, yeast and plant cells. In animal cells two PLD genes have been studied while in model plant Arabidopsis twelve genes exist, classified in six classes (α-ζ). This underlines the role of these enzymes in plant responses to environmental stresses. However, information concerning the PLD involvement in the widely cultivated and economically important cotton plant responses is very limited. The aim of this report was to study the activity of conventional cotton PLD and its participation in plant responses to mechanical wounding, which resembles both biotic and abiotic stresses. PLDα activity was identified and further characterized by transphosphatidylation reaction. Upon wounding, cotton leaf responses consist of an acute in vitro increase of PLDα activity in both wounded and systemic tissue. However, determination of the in vivo PtdOH levels under the same wounding conditions revealed a rapid PtdOH formation only in wounded leaves and a late response of a PtdOH increase in both tissues. Εxpression analysis of PLDα and PLDδ isoforms showed mRNA accumulation of both isoforms in the wounded tissue, but only PLDδ exerts a high and sustainable expression in systemic leaves, indicating that this isoform is mainly responsible for the systemic wound-induced PtdOH production. Therefore, our data suggest that PLDα and PLDδ isoforms are involved in different steps in cotton wound signalling.

Keywords: Cotton (Gossypium hirsutum); HKD, catalytic motif; HRM, High Resolution Melting; PLD substrates; PLD, catalytic motif containing Histidine-Lysine-Aspartic acid residues; PLD, phospholipase D; PLDα and PLDδ; Phosphatidic acid; Phospholipase D activity; PtdCho, phosphatidylcholine; PtdEtOH, phosphatidylethanol; PtdEth, phosphatidylethanolamine; PtdOH, phosphatidic acid.; Wounding.

Fig. 1

Purification of G. hirsutum PLDα activity. PLDα activity was isolated from the aerial part of 6-w-old cotton plants and fractionated by centrifugation as described in Materials and methods. In the resulting fractions, activity was assayed using unlabelled PtdCho as substrate. For the estimation of the PLDα specific activity, the PtdOH formed was quantified by phosphorus determination after TLC separation. Results are means±S.D. of three experiments.

Fig. 2

Effect of mechanical wounding on PLDα activity of wounded and systemic leaves. Wounded (local response) or neighboring non-wounded (systemic response) cotton leaves subjected to mechanical wounding for the indicated time points were homogenized and fractionated as described in Materials and methods. The resulting microsomal fractions were assayed for PLDα activity using [³H]PtdCho as substrate. For the estimation of PLDα specific activity, the [³H]PtdOH formed was quantified by scintillation counting after TLC separation. Results are shown as percentage increases of the PLDα specific activities over control and are means±S.D. of three experiments. Percentage changes in activity are significantly different (P<0.01).

Fig. 3

Effect of wounding on PtdOH levels of cotton leaves. Wounded (local response) or neighboring non-wounded (systemic response) cotton leaves from plants subjected to mechanical wounding for the indicated time points were detached and immediately immersed in hot isopropanol. Lipid extraction was then performed as described in Materials and methods, followed by TLC separation and phosphorus determination of the isolated lipids. PtdOH levels of wounded and systemic leaves are shown as μmol PtdOH/g dry weight of tissue (leaves). Results are means±S.D. of four samples from two independent experiments (*P<0.01 compared to control).

Fig. 4

Effects of wounding on phospholipid content of cotton leaves. Wounded (local response) or neighboring non-wounded (systemic response) cotton leaves from plants subjected to mechanical wounding for the indicated time points were detached and immediately immersed in hot isopropanol. The lipid extraction was performed as described in Materials and methods, followed by TLC separation and phosphorus determination of the isolated lipids. (A) Basal levels of major phospholipids (PtdCho and PtdEth) and PtdOH in cotton leaves are presented as percentage of total (PtdCho+PtdEth+PtdOH) lipid content (B) PtdOH, PtdCho and PtdEth levels of wounded leaves are shown as percentage changes of the corresponding basal ratio. (C) PtdOH, PtdCho and PtdEth levels of systemic leaves are shown as percentage changes of the corresponding basal ratio. (A, B and C), results are means±S.D. of four samples from two independent experiments.

Fig. 5

Effects of wounding on cotton PLDα and PLDδ expression. Wounded (local response) or neighboring non-wounded (systemic response) cotton leaves subjected to mechanical wounding for the indicated time points were collected. After leaf RNA extraction and reverse transcription, the expression levels of (A) GhPLDα1-2 (GU569956.1), (B) GhPLDα1-1 (GU569957.1), (C) GhPLDα2-2 (GU569958.1), (D) GhPLDα2-1 (GU569953.1) and (E) GhPLDδ (AY138251.1 and AY138252.1) isoforms were analyzed by Real-Time PCR. (A–E) Results are shown as fold changes of basal values (non-wounded leaves, t=0) and are means±S.D. of triplicate reactions.