Phospholipase Dα from sunflower ( Helianthus annuus): cloning and functional characterization

• Published in: Journal of plant physiology Vol. 167; no. 7; pp. 503 - 511
• Main Authors: Moreno-Pérez, A.J., Martínez-Force, E., Garcés, R., Salas, J.J.
• Format: Journal Article
• Language: English
• Published: Elsevier GmbH 01-01-2010
• Subjects: amino acid sequences; complementary DNA; enzymatic hydrolysis; enzyme activity; Escherichia coli; gene expression regulation; Helianthus annuus; Lipid degradation; molecular cloning; molecular sequence data; Phosphatidic acid; Phospholipase D; phospholipids; plant extracts; plant proteins; recombinant fusion proteins; sequence analysis; Signal transduction; Sunflower; ABA; Signal transduction; PA; Lipid degradation; PC; PE; PIP; PI; PLD; Sunflower; Phospholipase D; Phosphatidic acid
• ISSN: 0176-1617; 1618-1328
• DOI: 10.1016/j.jplph.2009.11.015

D type phospholipases (PLD) are enzymes that hydrolyze the head group of phospholipids to produce phosphatidic acid. This activity is ubiquitous in plant tissues, and has been isolated and characterized from different species and organs. Several families of these proteins have been described in plants on the basis of their gene sequences (PLD α, β, γ, δ, ζ and ε). They have been shown to be involved in many metabolic events, such as response to abiotic stress, signal transduction, and membrane lipid turnover and degradation. In the present study, PLD activity was measured in the soluble fractions isolated from different organs of this plant. A PLD of α type was cloned from leaf cDNA that was responsible for most of this activity. The gene encoding this 810 aa protein was heterologously expressed in
E. coli. This protein was not lethal for the eukaryotic host, although it altered its phospholipid profile. PLDα was purified to almost homogeneity by His-tag affinity chromatography, displaying an optimum pH of 6.5 and strong dependence on the presence of Ca
2+ and SDS in the assay medium. The enzyme was active towards phosphatidylcholine, Phosphatidylethanolamine and phosphatidylglycerol. Furthermore, the
HaPLDα gene was found to be expressed at high levels in leaf and stem tissues.