Phosphatidic Acidc ELISA allows for the specific measurement of phosphatidic acid concentrations in plant tissue, cell culture supernates and other relevant liquid.
Introduction
Phosphatidic acid (PA) is a common phospholipid that is a major constituent of cell membranes. Phosphatidic acid is the smallest of the phospholipids. Phosphatidic acid is not an abundant lipid constituent of any living organism, but it is extremely important as an intermediate in the biosynthesis of triacylglycerols and phospholipids. Indeed, it is often over-estimated in tissues as it can arise by inadvertent enzymatic hydrolysis during inappropriate storage or extraction conditions during analysis.
Phospholipase D activity and the phosphatidic acid produced may be even more significant in plants. They have long been recognized as of importance during germination and senescence, and they appear to have a role in response to stress damage and pathogen attack. A high content of phosphatidic acid induced by phospholipase D action during wounding or senescence brings about a loss of the membrane bilayer phase, as a consequence of the conical shape of this phospholipid in comparison to the cylindrical shape of structural phospholipids. As a result, cells lose their viability. The phosphatidic acid generated in this way is broken down further by phosphatases, acylhydrolases and lipoxygenases into fatty acids and other small molecules, which are subsequently absorbed and recycled. In addition, phosphatidic acid is important in the response to other forms of stress, including osmotic stress (salinity or drought), cold, and oxidation, although much remains to be learned of the mechanism by which it exerts its effects.
Phosphatidic acid is of considerable importance in cellular signalling in plants, for example in promoting pollen-tube growth, decreasing peroxide-induced cell death, and mediating the signalling processes that lead to responses to the plant hormone abscisic acid. Thus in the ‘model’ plant Arabidopsis, which contains twelve distinct members of the phospholipase D family, phosphatidic acid generated by the action of the enzymes interacts with a protein phosphatase to signal the closure of stomata promoted by abscisic acid; it interacts also with a further enzyme to mediate the inhibition of stomatal opening effected by abscisic acid. Together these reactions constitute a signalling pathway that regulates water loss from plants.
Test principle
Phosphatidic Acidc ELISA employs the quantitative sandwich enzyme immunoassay technique. A antibody specific for phosphatidic acid has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any phosphatidic acid present is bound by the immobilized antibody. An enzyme-linked antibody specific for phosphatidic acid is added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of phosphatidic acid bound in the initial step. The color development is stopped and the intensity of the color is measured.
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