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Title: The role of Ca2+ as a second messenger in an inducible defence response in cells of Medicago sativa L.
Author: Robinson, P. S.
Awarding Body: University College of Swansea
Current Institution: Swansea University
Date of Award: 1994
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The role of Ca2+ in the interaction between lucerne (Medicago sativa L.) and the wilt fungus, Verticillium albo-atrum R & B has been examined. Elicitor prepared from culture-filtrates of V.albo-antrum reduced the incorporation of 45Ca2+ into protoplasts of lucerne. Elicitor and extracellular Ca2+ caused an efflux of 45Ca2+ from protoplasts preloaded with 45Ca2+, the elicitor causing the increase in efflux through the activity of an orthovanadate sensitive pump. Antagonists of Ca2+ ion channels were used to show that a constant cycling of Ca2+ occurs across the plasma membrane. Dibutyryl 3'5' cAMP and the calmodulin antagonist compound R24571 had no effect on the cycling of 45Ca2+ suggesing cAMP and calmodulin are not directly involved in the mechanism of cycling. A Ca2+ dependent, calmodulin independent ATPase was identified which was sensitive to inhibition by sodium orthovanadate and the Ca2+ ion channel blocker verapamil, which could be responsible for the efflux of 45Ca2+ across the plasma membrane. Tissues of lucerne were examined for the presence of receptor elements that could be components of a Ca2+ 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 Ca2+ 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 Ca2+ 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 Ca2+ dependent protein kinases and a calmodulin dependent protein kinase were identified in soluble extracts from lucerne. A Ca2+ dependent protein kinase activity was also identified in a plasma membrane preparation. All four kinase activities phosphorylated proteins of lucerne.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available