The role of Ca<SUP>2+</SUP> in the interaction between lucerne (<i>Medicago sativa</i> L.) and the wilt fungus, <i>Verticillium albo-atrum</i> R & B has been examined. Elicitor prepared from culture-filtrates of <i>V.albo-antrum</i> reduced the incorporation of <SUP>45</SUP>Ca<SUP>2+</SUP> into protoplasts of lucerne. Elicitor and extracellular Ca<SUP>2+</SUP> caused an efflux of <SUP>45</SUP>Ca<SUP>2+</SUP> from protoplasts preloaded with <SUP>45</SUP>Ca<SUP>2+</SUP>, the elicitor causing the increase in efflux through the activity of an orthovanadate sensitive pump. Antagonists of Ca<SUP>2+</SUP> ion channels were used to show that a constant cycling of Ca<SUP>2+</SUP> occurs across the plasma membrane. Dibutyryl 3'5' cAMP and the calmodulin antagonist compound R24571 had no effect on the cycling of <SUP>45</SUP>Ca<SUP>2+</SUP> suggesing cAMP and calmodulin are not directly involved in the mechanism of cycling. A Ca<SUP>2+</SUP> dependent, calmodulin independent ATPase was identified which was sensitive to inhibition by sodium orthovanadate and the Ca<SUP>2+</SUP> ion channel blocker verapamil, which could be responsible for the efflux of <SUP>45</SUP>Ca<SUP>2+</SUP> across the plasma membrane. Tissues of lucerne were examined for the presence of receptor elements that could be components of a Ca<SUP>2+</SUP> second messenger system. Calmodulin and a calmodulin dependent 3',5' cAMP phosphodiesterase were identified. The 3',5' cAMP phosphodiesterase associated with a column of immobilised calmodulin, was inhibited by compound R24571 and was stimulated by bovine calmodulin. This enzyme provides a link between the Ca<SUP>2+</SUP> and the cAMP second messenger systems. Elicitor was shown to alter the pattern and increase the rate of phosphorylation of proteins from lucerne cell suspension cultures. A protein kinase activity that was stimulated by diacyl glycerol and phosphatidyl serine in the presence of Ca<SUP>2+</SUP> was associated with a protein fraction prepared by phosphatidyl serine affinity chromatography. The preparation cross-reacted with monoclonal antibodies raised to mammalian Protein kinase C. Two Ca<SUP>2+</SUP> dependent protein kinases and a calmodulin dependent protein kinase were identified in soluble extracts from lucerne. A Ca<SUP>2+</SUP> dependent protein kinase activity was also identified in a plasma membrane preparation. All four kinase activities phosphorylated proteins of lucerne.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:638697 |
| Date | January 1994 |
| Creators | Robinson, P. S. |
| Publisher | Swansea University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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