Characterization of Arabidopsis secretory phospholipase A₂‐γ cDNA and its enzymatic properties¹

Abstract

Plant secretory phospholipases A 2 (sPLA 2 s) probably play important roles in phospholipid signaling based on the data reported from other organisms, but their functions are poorly understood because of the lack of cloned sPLA 2 genes. In this study, we cloned and characterized an Arabidopsis secretory phospholipase A 2 -Q Q (AtsPLA 2 -Q Q) cDNA, and examined its enzymatic properties. The recombinant protein of AtsPLA 2 -Q Q showed maximal enzyme activity at pH 8.0, and required Ca 2þ for activity. Moreover, AtsPLA 2 -Q Q showed sn-2 position speci¢city but no prominent acyl preference, though it showed head group speci¢city to phosphatidylethanolamine rather than to phosphatidylcholine. AtsPLA 2 -Q Q was found to predominate in the mature £ower rather than in other tissues, and subcellular localization analysis con¢rmed that AtsPLA 2 -Q Q is secreted into the intercellular space.

Kinetic characterization, optimum conditions for catalysis and substrate preference of secretory phospholipase A2 from Glycine max in model membrane systems

“…The effect of enzyme immobilization on the sPLA 2 kinetics was also reported [31]. Description and kinetics properties of sPLA 2 from plants have been more frequent in their recombinant counterpart after an appropriate expression, purification and folding protocols [3,10,11,27,32] compared with their equivalent found in animals sPLA 2 s. The reason of this is due to the relative high amounts of the latter proteins found in their respective natural sources (venoms and pancreatic juice) and, therefore it allows an efficient purification. However, few studies using purified plant enzymes were reported from seeds of elm [9] and of soybean [33].…”

“…Both GmsPLA 2 enzymes displayed an optimum at 13e16 mN/m, a rather medium surface pressure, similar to that found for pancreatic sPLA 2 [56], if it is compared with the equivalent surface pressure estimated for phospholipids packed in bilayers calculated to be more than 31 mN/m by using theoretical approach [57]; or indirectly whereby lipolytic enzymes that are able to hydrolyze phospholipids of intact erythrocyte membranes hydrolyze lipid monolayers at above 31 mN/m [58]. The secretrory nature of sPLA 2 in plants has been elegantly tested checking the subcellular localization of Arabidopsis PLA 2 s with constructs of AtsPLA 2 s-green fluorescent protein fusions introduced into onion epidermal cells indicating that AtsPLA 2 s are secreted and accumulated mainly in the interstices of the extracellular space [10]. On the other hand, the expression levels of AtsPLA 2 s in A. thaliana is rather low and depends on the secreted isoforms in the different parts of the plants [42].…”

Phospholipase A2 Is Required for PIN-FORMED Protein Trafficking to the Plasma Membrane in theArabidopsisRoot

“…Four Arabidopsis PLA 2 isoforms (PLA 2 a, b, g, and d) have been identified and biochemically characterized Bahn et al, 2003;Ryu, 2004;Ryu et al, 2005). While the PLA 2 a and PLA 2 b genes are actively expressed in the root, PLA 2 g is expressed at relatively low levels in the root, and PLA 2 d is expressed exclusively in floral tissues .…”

Lipid deacylating enzymes in plants: Old activities, new genes

“…AtsPLA 2 g and AtsPLA 2 d also seem to be expressed only in flowers [57]. The activity of the AtsPLA 2 b promoter has been analysed and it appears that the AtsPLA 2 b gene is expressed in all tissues of actively growing seedlings, whereas in older plants, expression was observed mainly in vascular tissues and in flowers [4,55].…”