Timothy W. Short

A PHOTORECEPTOR SYSTEM REGULATING in vivo AND in vitro PHOSPHORYLATION OF A PEA PLASMA MEMBRANE PROTEIN

Abstract

Blue Light-Induced Phosphorylation of a Plasma Membrane-Associated Protein in Zea mays L

Blue light induces a variety of photomorphogenic responses in higher plants, among them phototropic curvature, the bending of seedlings toward a unidirectional light source. In dark-grown coleoptiles of maize (Zea mays L.) seedlings, blue light induces rapid phosphorylation of a 114-kD protein at fluence levels that are sufficient to stimulate phototropic curvature. Phosphorylation in response to blue light can be detected in vivo in coleoptile tips preincubated in 32Pi or in vitro in isolated membranes supplemented with [[gamma]-32P]ATP. Phosphorylation reaches a maximum level in vitro within 2 min following an inductive light pulse, but substantial labeling occurs within the first 15 s. Isolated membranes remain activated for several minutes following an in vitro blue light stimulus, even in the absence of exogenous ATP. Phosphoamino acid analysis of the 114-kD protein detected phosphoserine and a trace of phosphothreonine. The kinase involved in phosphorylating the protein in vitro is not dependent on calcium. The 114-kD protein itself has an apparent binding site for ATP, detected by incubating with the nonhydrolyzable analog, 5[prime]-p-fluorosulfonyl-benzoyladenosine. This result suggests that the 114-kD protein, which becomes phosphorylated in response to blue light, may also be capable of kinase activity.

A Pea Plasma Membrane Protein Exhibiting Blue Light-Induced Phosphorylation Retains Photosensitivity following Triton Solubilization.

Phosphorylation of a polypeptide of approximately 120 kD in pea (Pisum sativum L.) plasma membranes in response to blue light has been shown to be involved in phototropic curvature, but the relationship of this protein to the kinase and photoreceptor acting upon it is uncertain. Using two-phase aqueous partitioning to isolate right-side-out plasma membrane vesicles, we have obtained evidence suggesting that the photoreceptor, kinase, and substrate are localized to the plasma membrane fraction. Latent phosphorylation accessible through Triton X-100 or freeze/thaw treatments of purified plasma membrane vesicles indicates that at least the kinase moiety is present on the internal face of the plasma membrane. Effects of solubilization of vesicles on fluence-response characteristics and on phosphorylation levels provide evidence that the receptor, kinase, and protein substrate are present together in individual mixed detergent micelles, either as a stable complex or as domains of a single polypeptide. In vivo blue-light irradiation results in a small but significant decrease in mobility of the 120-kD phosphorylated protein on sodium dodecylsulfate gel electrophoresis. This mobility shift is evident on Coomassie-stained gels and on western blots probed with polyclonal antibodies raised against the 120-kD protein. Among the plasma membrane proteins bound to the reactive nucleotide analog fluorosulfonylbenzoyladenine (FSBA), a distinct protein band at 120 kD can be detected on blots probed with anti-FSBA antibodies. This band exhibits an in vivo light-dependent mobility shift identical to that observed for the protein band and antibodies specific for the 120-kD protein, implying that the 120-kD protein has an integral nucleotide binding site and consistent with the possibility that the substrate protein is also a kinase.

Correlation of Blue Light-Induced Phosphorylation to Phototropism in Zea mays L

The physiology of light-induced phototropic curvature has been studied extensively in coleoptiles of grasses, particularly in Avena and Zea mays L. In Z. mays L., we have found that, in addition to curvature, blue light also induces rapid phosphorylation of a 114-kD protein in the tips of coleoptiles, and, in a previous report, we reported several characteristics of the phosphorylated substrate protein and kinase (J.M. Palmer, T.W. Short, S. Gallagher, W.R. Briggs [1993] Plant Physiol 102: 1211–1218). Here, we compare the phosphorylation response to several known aspects of phototropism physiology. Blue light-induced phosphorylation occurs only in the upper portion of the coleoptile and is absent from the node and mesocotyl. The specific activity of phosphorylation is highest in the extreme apical portion of the tip, which is also the site of maximal sensitivity to phototropic stimuli (A. W. Galston [1959] In Physiology of Movements, Encyclopedia of Plant Physiology, Springer, Berlin). Fluence-response determinations indicate that light dosage levels that stimulate curvature also stimulate phosphorylation. However, the threshold for inducing detectable phosphorylation in maize cannot be matched to the threshold for curvature induction. The recovery of sensitivity to phototropic stimuli after exposure to high fluences of light occurs with kinetics that are very similar to those for recovery of the phosphorylation response after a previous high-fluence light exposure. In addition, wavelengths of light in the blue and near-ultraviolet regions of the spectrum that maximally stimulate phototropic curvature also maximally stimulate in vitro phosphorylation in maize. The pattern of stimulation matches the absorption spectra of flavoproteins, which have been proposed as candidates for blue light photoreceptors.

Blue Light Induces Phosphorylation at Seryl Residues on a Pea (Pisum sativum L.) Plasma Membrane Protein

We have partially characterized the blue-light-stimulated in vitro phosphorylation of a membrane protein from etiolated Pisum sativum L. stems. Properties of the response have implicated its involvement in signal transduction of phototropic stimuli (T.W. Short, W.R. Briggs [1990] Plant Physiol 92: 179–185; P. Reymond, T.W. Short, W.R. Briggs [1992] Proc Natl Acad Sci USA 89: 4718–4721). Analysis of proteolysis products and phosphoamino acidanalysis indicate that the substrate protein is phosphorylated on multiple seryl residues. Kinetics of the in vitro reaction show phosphorylation to be complete within 2 to 5 min at 30[deg]C in either light-exposed or dark-control plasma membrane preparations, regardless of whether the membranes were first solubilized in Triton X-100. Nucleotide competition assays show the kinase to be ATP specific. The pH optimum covers a broad range with a maximum near 7.5. A wide array of salts inhibits the phosphorylation at high concentrations, but millimolar concentrations of Mg2+ are required to form Mg.ATP complexes for maximal activity, whereas excess free Mg2+ or Ca2+ are not required for the reaction.

The Transduction of Light Signals in Plants: Responses to Blue Light

Although the majority of articles in this volume are devoted to studies of phytochrome, there has been considerable progress recently in understanding plant responses to blue light — mediated not by the blue light-absorbing bands of phytochrome, but rather by specific blue light photoreceptors. These inroads are being made at the cellular, biochemical, and molecular levels, and hold considerable promise of leading to an understanding of at least portions of the transduction chains set in action by blue light. While the systems under study do not involve phytochrome, they provide models for photoreceptor action that could apply to phytochrome, or phytochrome — blue light photoreceptor interaction.

On the Trail of the Photoreceptor for Phototropism in Higher Plants

Much of the photomorphogenesis of multicellular green plants is mediated by red, far red-reversible pigment phytochrome (Hendricks and Van der Woude, 1983) By contrast, only a few selected microorganisms respond to red light signals. However, both plants and microorganisms exhibit a wide range of responses to blue and ultraviolet light (Gressel and Rau, 1983; Senger, 1987), almost certainly mediated several different blue light photoreceptors (Briggs and lino, 1983; Gressel and Rau 1983; Iino, 1988; Palit et al, 1989). A class of photoreceptors showing action spectra that one would expect for flavoproteins are found both in higher plants and fungi (Briggs and Iino, 1983). This class is frequently given the general name “cryptochrome” We are presently working with a higher plant photoreceptor that we suspect to be in this class. We will describe our current studies on this pigment system here in the hopes that at least some of what we have found may be helpful in elucidating responses blue light in microorganisms.

Signal Transduction in Blue Light-Mediated Growth Responses

During the past decade there has appeared a large number of papers devoted to the effects of blue light on higher plants, algae, and fungi (see Senger, 1980, 1984, 1987; Senger and Schmidt, 1986). Despite intensive efforts, however, a specific blue light photoreceptor has yet to be rigorously characterized from these organisms. Indeed there is some evidence (Briggs and Iino, 1983) that there may be more than one blue light photoreceptor of physiological consequence, and there is strong evidence for an additional photoreceptor which absorbs in the UV-B region of the spectrum as well (Beggs et al., 1983; Wellmann, 1983). The absence of knowledge of the exact chemical nature of the photoreceptor(s) makes it difficult to say much either about the nature of signal perception or about early steps in the subsequent transduction chain leading to a physiological response.