Characterization of a Rapid, Blue Light-Mediated Change in Detectable Phosphorylation of a Plasma Membrane Protein from Etiolated Pea (Pisum sativum L.) Seedlings

Abstract

When crude microsomal membranes from apical stem segments of etiolated Pisum sativum L. cv Alaska are mixed in vitro with 'y-[32P]ATP, a phosphorylated band of apparent molecular mass 120 kilodaltons can be detected on autoradiographs of sodium dodecyl sulfate electrophoresis gels. If the stem sections are exposed to blue light immediately prior to membrane isolation, this band is not evident. The response is observed most strongly in membranes from the growing region of the stem, but no 120 kilodalton radiolabeled band is detected in membranes from the developing buds. Fluence-response curves for the reaction show that the system responds to blue light above about 0.3 micromole per square meter, and the visible phosphorylation completely disappears above 200 micromoles per square meter. Reciprocity is valid for the system, because varying illumination time or fluence rate give similar results. If the stem segments are left in the dark following a saturating blue irradiation, the radiolabeled band begins to return after about 10 minutes and is as intense as that from the dark controls within 45 to 60 minutes. A protein that comigrates with the phosphorylated protein on polyacrylamide gels is also undetectable after saturating blue light irradiations. The fluence range in which the protein band disappears is the same as that for the disappearance of the phosphorylation band. Its dark recovery kinetics and tissue distribution also parallel those for the phosphorylation. In vitro irradiation of the isolated membranes also results in a phosphorylation change at that molecular mass, but in the opposite direction. Comparisons of the kinetics, tissue distribution, and dark recovery of the phosphorylation response with those published for blue lightmediated phototropism or rapid growth inhibition indicate that the phosphorylation could be linked to one or both of those reactions. However, the fluence-response relationships for the change in detectable phosphorylation match quite closely those reported for phototropism but not those for growth inhibition. Blue light has also been found to regulate the capacity for in vitro phosphorylation of a second protein. It has an apparent molecular mass of 84 kilodaltons and is localized primarily in basal stem sections.

Blue Light Activates a Specific Protein Kinase in Higher Plants

“…Each species shows a phosphorylation pattern very similar to that of sunflower (dicot) or oat (monocot) as presented in Figure 1. These results correspond closely to what has been reported for pea (16), Arabidopsis (12), and maize (10,15). However, the absolute level of phosphorylation varies from species to species.…”

“…Gallagher et al (6), studying the phosphorylation of membrane proteins extracted from etiolated pea epicotyls, discovered that light was affecting the phosphorylation of a 120-kD protein associated with the plasma membrane. Subsequently, Short et al (16,17) showed that in vitro irradiation induced a strong enhancement of the phosphorylation of a 120-kD protein in pea and a 114-kD protein in maize (10,15). The characteristics of the reaction (localization, kinetics, fluence requirement) indicated that this could be an early step in the transduction chain for phototropism (16).…”

“…Subsequently, Short et al (16,17) showed that in vitro irradiation induced a strong enhancement of the phosphorylation of a 120-kD protein in pea and a 114-kD protein in maize (10,15). The characteristics of the reaction (localization, kinetics, fluence requirement) indicated that this could be an early step in the transduction chain for phototropism (16). The same conclusion was strongly supported by a study with Arabidopsis mutants, where a mutant with altered phototropic sensitivity showed a dramatic reduction of the blue light-induced phosphorylation of a membrane protein near 124 kD (12).…”

Photopolarization of the Fucus sp. Zygote by Blue Light Involves a Plasma Membrane Redox Chain

“…BL also controls many aspects of plant development including growth, tropism, and sexual maturation (Dring, 1987;Kaufman, 1993). Although mutants of Arabidopsis defective in their response to BL have been characterized (Chory, 1993) and BL has been shown to affect the phosphorylation state of specific proteins (Short and Briggs, 1990;Reymond et al, 1992aReymond et al, , 1992bPalmer et al, 1993aPalmer et al, , 1993b, the mechanism of BL action is still largely unknown (Chory, 1993) and the nature of BL receptors is still under investigation (Song, 1987;Vani and Raghavendra, 1989;Ahmad and Cashmore, 1993;Quinones et al, 1993).…”

“…This phenomenon could be explained by BL-induced phosphorylation of specific proteins (Chory, 1993). Various proteins with molecular masses around 110 kD whose phosphorylation state is affected by BL have been characterized (Short and Briggs, 1990;Reymond et al, 1992aReymond et al, , 1992bPalmer et al, 1993a;Short et al, 1993), but their functions remain to be established. Tissue-specific control of phosphorylation of a 114-kD protein is involved in BL-induced phototropism of maize coleoptiles (Palmer et al, 1993b).…”