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.

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Changes in polyamine metabolism were investigated in relation to growth of cell suspension cultures of carrot (Daucus carota, cv Chantenay). Changes in levels of the major amines putrescine and spermidine throughout the culture period correlated poorly with changes in fresh weight, but a closer correlation with the minor component spermine was observed. The arginine decarboxylase (ADC) inhibitor difluoromethylarginine (DFMA) strongly and specifically inhibited ADC activity in the supernatant, reduced the major amine (putrescine) by 95% and the total amine content by 80%. It had no effect on cell number and stimulated fresh weight by over 25% through increased cell expansion. Spermine content, in contrast, increased with DFMA concentration in parallel with fresh weight increases. Difluoromethylornithine strongly inhibited ornithine decarboxylase activity in the pellet, but had little effect on either polyamine levels or culture growth. It was concluded that little evidence for a correlation between free polyamines and cell number in carrot cultures could be detected, but that a possible correlation between spermine content and cell expansion was observed.The polyamines occur ubiquitously in plants, animals, and prokaryotes, and their role in growth, development, and stress metabolism has received active investigation (4, 21 24 carota) have been used to investigate the relationship between growth rate and free polyamines, both by measurement of endogenous levels over the growth period and by the use of inhibitors (15) that lead to large alterations in polyamine content. MATERIALS AND METHODSCell Culture. Suspension cultures of Daucus carota (cv Chantenay) isolated 3 years previously were routinely maintained on Murashige and Skoog (20) medium (Flow Labs, Irvine, Scotland), supplemented with 2,4-dichlorophenoxyacetic acid (0.2 mg/L), kinetin (0.2 mg/L), and sucrose (2%), in 250-mL Erlenmeyer flasks at 25°C in diffuse fluorescent light at a shaker speed of 90 rpm and subcultured at 14-d intervals by inoculating 5 mL of suspension into 85 mL of fresh medium. For determination of fresh weight, aliquots of suspension were filtered through preweighed plastic syringe barrels fitted with sintered polypropylene discs (40-,um pore size). After centrifugation at 200g for 5 min to remove surface liquid, the tubes were re-weighed. The pad of weighed tissue was removed from the tube by air pressure and immediately used for amine and enzyme activity determinations.Cell counts were made on weighed tissue by a modification of the method of Brown and Rickless (6). Portions (100 mg) of tissue were macerated in 5% chromium trioxide in 5% HCI, separated by drawing through a hypodermic needle, and counted with a hemocytometer after appropriate dilution.Estimation of Free Amines. For amine determination, triplicate samples of 1 g fresh weight tissue were extracted overnight at 0°C in 5 mL of 5% perchloric acid to which were added 50 ,uL of 10 mm diaminooctane as internal standard, after Flores and Galston (11). After centrifuga...

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The central role played by light in the coordination of plant growth and development has led to intense interest in the detection and transduction of light signals (Chory, 1993). The primary receptor of R is phytochrome (Hart, 1988) and multiple forms are known to exist, which may control different cellular processes. Increasing evidence suggests that one receptor for B may be a protein kinase (Gallagher et al., 1988). Since both R and B initiate separate but often interrelated physiological responses, it has been assumed that the transduction pathways may be distinct, but the identity of either remains unclear (Trewavas and Gilroy, 1991).It has long been thought that [Ca2+i] may act in transduction of photomorphogenetic signals resulting from R due to its important role in animal cells (Haupt and Weisenseel, 1976). Early studies involving 45Ca2+ flux measurements and autoradiography (Weisenseel and Ruppert, 1977; Dreyer and Weisenseel, 1979; Hale and Roux, 1980) provided indirect evidence to support this hypothesis. Following the detection of a plasma membrane-located Ca-dependent protein kinase in plant cells (Hetherington and Trewavas, 1982), Roux (1984) Grimm et al. (1989), who showed that phytochrome often co-purifies with a 60-kD Ca-dependent protein kinase. Thummler et al. (1992) demonstrated that Ceratodon phytochrome, unlike other plant phytochromes, has a conserved protein kinase sequence.When etiolated wheat (Tviticum aestivum L.) leaf protoplasts are irradiated with R, their volume increases by up to 20% (Bossen et al., 1988). It is believed that R-induced protoplast swelling employs the same molecular mechanism as R-induced leaf unrolling (Zhou et al., 1990). Using Casensitive fluorescent dyes and laser confocal scanning imaging, we have been able to show that R induces [Ca2+], transients lasting several minutes in single etiolated wheat leaf protoplasts (Shacklock et al., 1992). We mimicked these transients by photolysis of incorporated caged Ca (nitr-5) and incorporated caged inositol trisphosphate and showed that these artificially produced transients increased the volume of the protoplasts, thus mimicking the physiological effects of R. By incubating etiolated wheat leaf protoplasts in 32Pi for time periods between 15 s and 15 min, we detectedR-induced changes in the phosphorylation of two peptides with approximate electrophoretic mobilities of 60 and 70 kD (Fallon et al., 1993). Phosphorylation of these two peptides increased during 45 s of R irradiation and showed kinetics similar to those of the detected [Ca2+], transients. Again, we were able to mimic the R-increased phosphorylation of these two peptides by photolyzing incorporated caged Ca.The 60-and 70-kD peptides were the primary species labeled in these short incubation periods; only after several hours of incubation in 32Pi was the labeling of numerous other peptides detected. This suggested the possibility that the very rapid labeling of the 60-and 70-kD peptides was the result of autophosphorylation, which is often faster t...