Detection in Vivo of Very Rapid Red Light-Induced Calcium-Sensitive Protein Phosphorylation in Etiolated Wheat (Triticum aestivum) Leaf Protoplasts

Abstract

Etiolated wheat (Triticum aesfivum cv Mercia) leaf protoplasts respond to brief red-light irradiation by increasing in volume over a 10-min incubation period (M.E. Bossen, H.A. Dassen, R. E. Kendrick, W.J. Vredenberg [1988] Planta 174: 94-100). When the calcium-sensitive dye Fluo-3 was incorporated into these protoplasts, red-light irradiation initiated calcium transients lasting about 2 min (P.S. Shacklock, N.D. Read, A.J. Trewavas [1992] Nature 358 153-155). Release of calcium in the protoplasts by photolysis of incorporated 1 -(2-amino-5-[1 -hydroxy-l-(2-nitro-4, 5-methylenedioxyphenyl)-methyl]-phenoxy}-2-(2'-amino-5'-methy1phenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetrasodium salt (caged calcium) or caged inositol trisphosphate frequently induced transient increases in intracellular calcium levels, although the kinetics of these changes showed variation between experiments. Upon exposure to red light, a pronounced increase in the phosphorylation of a 70-kD and to a lesser extent a 60-kD peptide was observed, commencing within 15 s and continuing for up to 2 min. Simultaneous far-red and red irradiation attenuated the response. Upon release of incorporated caged calcium by cage photolysis, the labeling of these two peptides was greatly increased. When incorporated caged inositol trisphosphate was photolyzed, only the labeling of the 70-kD peptide was enhanced. Phosphorylation of the 70-kD peptide was also increased when extracellular calcium was elevated, but it decreased with increasing extracellular ECTA. These data thus provide direct evidence for the operation of an in vivo transduction sequence involving red light-dependent, calcium-sensitive protein phosphorylation.Light initiates a diverse range of phenomena in plants (Smith, 19 75). Processes specifically controlled by R include the regulation of growth, reproduction, and time measurement. The effects of R are mediated by the family of photoreceptors designated phytochrome, and the biochemical mechanism of action of R has been of intense interest for many years (Smith, 1975; Kendrick and Kronenberg, 1986). A current hypothesis involves the mediation of R-induced changes in [Ca2+lC and was first introduced by Haupt and Weisenseel (1976). These authors pointed to the evidence that phytochrome regulated the passage of ions across the plasma membrane and noted that Caz+ had a key role in cell signaling in animal cells. With the detection of calmodulin in plant cells (Anderson and Cormier, 1978) and the discovery by Hetherington and Trewavas (1982) of Ca2+-regulated protein kinases in plants, Roux (1984) and Roux et al. (1986) ' Supported by the Science and Engineering Research Council.

Light-regulated modification and nuclear translocation of cytosolic G-box binding factors in parsley.

“…As shown very recently, however, G proteins are probably involved in primary light-induced transduction events, whereas Ca2+, calmodulin, and cGMP may be involved in the transfer of the primary signals to the transcription factors and cis-acting elements of light-regulated genes as second messengers Foucroy et al, 1990;Neuhaus et al, 1993;Romero and Lam, 1993). In addition, as shown by in vitro and in vivo short-time irradiation experiments, fast changes in protein phosphorylation often precede any effects on transcriptional activity of light-regulated genes (reviewed in Ranjeva and Boudet, 1987 and in Singh and Song, 1990;Reymond et al, 1992;Short et al, 1992;Fallon et al, 1993). An in vitro modification of DNA binding activity of different (putative) plant transcription factors by phosphorylation/dephosphorylation events has been reported (Datta and Cashmore, 1989;Harrison et al, 1991;Klimczak et al, 1992;Sarokin and Chua, 1992;Sun et al, 1993).…”

Second Messengers Mediate Increases in Cytosolic Calcium in Tobacco Protoplasts

“…Increases in [Ca 2ϩ ] cyt produced either by influx or release from internal Ca 2ϩ stores stimulate the phosphorylation of proteins within the cell (Poovaiah and Reddy, 1990). An increase in [Ca 2ϩ ] cyt detected by Ca 2ϩ microelectrodes, Ca 2ϩ -sensitive fluorescence probes, and extrinsic and intrinsic aequorin (Cobbold and Rink, 1987;Chae et al, 1990;Knight et al, 1991;Shacklock et al, 1992), followed by the alteration of the activities of intercellular targets, enzymes, genes, pumps, and channels, have all been demonstrated in plants (Fallon et al, 1993;Neuhaus et al, 1993;Bowler et al, 1994a). In some circumstances activated plant cells can display repeated Ca 2ϩ spikes or oscillations (McAinsh et al, 1995) and spreading Ca 2ϩ waves (Shacklock et al, 1992;Campbell et al, 1996).…”

Pr-Specific Phytochrome Phosphorylation in Vitro by a Protein Kinase Present in Anti-Phytochrome Maize Immunoprecipitates

“…Red light stimulates rapid phosphorylation or dephosphorylation of specific proteins in etiolated oat coleoptile tips (Otto and Schafer, 1988) and also stimulates calcium-dependent phosphorylation of proteins in isolated pea nuclei (Datta et al, 1985) and wheat protoplasts (Fallon et al, 1993). The phosphorylation-dependent binding of transcription factors to light-regulated promoter elements (Datta and Cashmore, 1989;Sarokin and Chua, 1992) is additional evidence that phytochrome responses are transduced by pathways employing reversible protein phosphorylation.…”

“…The far-red reversibility of red-light effects on protein phosphorylation in vivo further indicate that protein phosphorylation is involved in phytochrome responses (Datta et al, 1985;Otto and Schafer, 1988;Fallon et al, 1993). Red light stimulates rapid phosphorylation or dephosphorylation of specific proteins in etiolated oat coleoptile tips (Otto and Schafer, 1988) and also stimulates calcium-dependent phosphorylation of proteins in isolated pea nuclei (Datta et al, 1985) and wheat protoplasts (Fallon et al, 1993).…”

“…However, the maximum transcriptional activation by G-protein modulators is only 25% of that caused by light (Romero and Lam, 1993), indicating that G-protein-independent steps may also transduce light signals. Red light induces a transient increase in cytosolic calcium (Shacklock et al, 1992) and activates calcium-and calmodulin-dependent signal-transduction pathways (Datta et al, 1985;Lew et al, 1990;Fallon et al, 1993).…”