Phosphorylation of a Renatured Protein from Etiolated Wheat Leaf Protoplasts Is Modulated by Blue and Red Light

Abstract

Red-light irradiation of etiolated wheat (Triticum aestivum L.) leaf protoplasts rapidly increases calcium-dependent phosphorylation in vivo of 70- and 60-kD peptides, and the phosphorylation is attenuated by simultaneous far-red light (K.M. Fallon, P.S. Shacklock, A.J. Trewavas [1993] Plant Physiology 101:1039-1045). When these protoplasts were solubilized in sodium dodecyl sulfate and protein kinase was renatured in situ after gel electrophoresis, a single 60-kD protein kinase was detected. In situ phosphorylation was inhibited by prior exposure of etiolated protoplasts to 30 to 60 s of white, 1 to 2 min of blue, or 2 to 5 min of red light. The effect of red light was attenuated by concomitant far-red light. The inhibition of in situ phosphorylation by light was lost after a further prolonged incubation of protoplasts in darkness. In situ phosphorylation was calcium dependent, and the electrophoretic mobility of the protein kinase was increased in the presence of calcium ions. Although treatment of protoplasts with ionophores and channel blockers produced data consistent with in vivo regulation of phosphorylation by cytosol calcium, additional light-activated transduction pathways have to be invoked to explain all the observations.