Cyclic nucleotide phosphodiesterase activity of Medicago sativa L.

The hypersensitve response (HR) is an expression of resistance in plants that occurs at the point of infection by a microorganism. It involves expression of a number of active defence responses and synthesis of defence related molecules. Phytoalexin synthesis is one such defence response, and its occurrence in Medicago satjva (lucerne) tissues is the subject of our present studies. Phytoalexin synthesis induced in lucerne by the phytopathogen Verticillium albo-atrum involves interaction of a component (elicitor) from the fungus with a receptor in the host and results in increased synthesis of enzymes from the biosynthetic pathway. Previously we have identified some elements of the signal transduction mechanism that mediate the response in lucerne and adenosine 3’,5’-cyclic monophosphate (CAMP) was implicated as a second messenger [l]. CAMP is known to bring about its effects by regulating the activities of intracellular kinases and ion channels, however, as a second messenger it does not always operate in isolation and its interaction with its targets can be regulated by fluxes in intracellular Ca2’. For example, Ca” is known to act directly on enzymes such as cAh4P-phosphodiestere, or indirectly via the calcium binding protein calmodulin. e.g. via adenylyl cyclase [2]. As part of our examination of the role of CAMP in the phytoalexin response in lucerne we report here the presence of CAMP-phosphodiestere activity and evidence of its regulation by Ca” and calmodulin. Cells from 4-day suspension cultures of lucerne were harvested by centrifugation (800g, 6 min) and disrupted with a pestle and mortar with the aid of a little acid washed sand, in 50 mM TrisMCI buffer, pH 7.5 (0.5ml buffer/g. fresh wt.), containing lOmM Bmercaptoethanol, 1 mM EDTA, 1 pm leupeptin, 1 pM pepstatin A and 0.5 mM phenylmethylsulphonyl fluoride (PMSF). The homogenate was filtered through 2 layers of Miracloth (Calbiochem), centrifuged (31,OOOg, 10 min) and the supernatant dialysed for 16 hours at 4 “C against 20 volumes of homogenising buffer without EDTA. The dialysate was freeze-dried, and resuspended in the same buffer at a concentration of 6.76 mg proteidml. Cyclic nucleotide phosphodiesterase activity was determined by incubation of the extract (100-200 pg protein.) in 50 mM Tris/HCI pH 7.5 containing ImM CaCI,: 10 mM MgCI2; and either lOmM 3’,5’-cyclic AMP or lOmM 2’,3’-cyclic AMP as substrate, in a total volume of 600 ul. At the end of 10 min at 37 “C, the reaction was terminated by heating for 2 min at 90 “C. After cooling, the reaction mixture was incubated with 0.01 U (equivalent to 0.01 mmol substrate transformed per min) 5’-nucleotidase (Crotalus adamantus venom-Sigma) and/or 0.01 U 3’-nucleotidase (Rye Grass -Sigma), for 1 hour at 37°C. The reaction was terminated by heating at 90 “C for 2 min. The phosphate content of a 600 p1 aliquot of the sample was determined following centrifugation by incubation with 150 p1 of freshly made malachite green solution [3]. The absorbance was measured after 10 min at 630 nm. A standard curve was used to determine the concentration of phosphate. Lucerne extract, under the assay conditions described and in the presence of 10 mM 3’,5’-cyclic AMP, was found to contain phosphodiesterase activity equivalent to 36 nmol phosphatehg proteidmin Activity was not restricted to the 3 ’ 3 ’ nucleotide, however. When lOmM 2’,3’-cyclic AMP was employed as substrate an activity of 1586 nmol phosphate/mg proteidmin. was observed. Repeating the incubation with the 3’,5’-nucleotide but leaving out either the 3’or 5’nucleotidase indicated the ratio of 3 ’to 5’-AMP products to be 1: 1. The two activities may represent a single enzyme with multisubstrate specificity or at least two separate phosphodiesterases. To establish their dependency on Ca”, the effect of 2mM EGTA on activity was determined. The results, (Table 1) show that chelating Ca” had more effect upon 3’,5’activity (44.9% inhibition) than on the 2’,3’-activity (23.6%).