The Confocal Laser Scanning Microscopy for Detection of Plant Tyrosine Phosphorylation

A significant number of protein phosphotyrosine sites of the cells of Pisum sativum callus culture and the cells of marine halophyte green alga Dunaliella maritima was shown using the monoclonal primary antyphosphotyrosine antibodies PY20 and FITC-conjugated secondary antibodies by the confocal laser scanning microscopy. A high specificity (mu M) of the binding of the antibodies used to protein phosphotyrosine sites in plants was detected. The protein tyrosine phosphatase inhibitor sodium orthovanadate increased (in 10-12 times) in the number of plant protein phosphotyrosine sites. When treated in vivo during 10 min with antioxidant ascorbic acid and reducing agent DTT, the number of protein phosphotyrosine sites decreased 2-fold, whereas it increased 1.5-3-fold (in average from 4 experiments) under effect of the catalase inhibitor aminotriazol. The results obtained were discussed in view of the redox regulation of protein tyrosine phosphatases activity.