Red Light-Dependent CO₂ Uptake and Oxygen Evolution in Guard Cell Protoplasts of Vicia faba L.: Evidence for Photosynthetic CO₂ Fixation

Abstract

ABSTRACISuspensions of dark-adapted guard cell protoplasts of Vicia fab L. alkalinized their medium in response to irradiation with red light. The lkalinization peaked within about 50 minutes and reached steady state shortly thereafter. Simultaneous measurements of 02 concentrations and medium pH showed that oxygen evolved in parallel with the red lightinduced alkalinization. When the protoplasts were returned to darkness, they acidified their medium and consumed oxygen. Both oxygen evolution and medium alkalinization were inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). In photosynthetically competent preparations, light-dependent medium alkalinization is diagnostic for photosynthetic carbon fixation, indicating that guard cell chloroplasts have that capacity. The striking contrast between the responses of guard cell protoplasts to red light, which induces alkainization, and that to blue light, which activates proton extrusion, suggests that proton pumping and photosynthesis in guard cells are regulated by light quality.Suspensions of photosynthetically competent algae, mesophyll cells or their protoplasts, alkalinize their medium when illuminated (3,4,8,9). The pH increase ensues from CO2 uptake into the cells; a displacement ofthe equilibrium reaction: CO2 + H20 HC03-+ H+ removes protons from the medium and results in alkalinization (8). The rate and magnitude of the pH changes largely depend on experimental conditions (4, 6) but, qualitatively, light-dependent medium alkalinization has been found to be highly correlated with photosynthetic CO2 fixation (3,8).Guard cells are thought to be devoid of the photosynthetic carbon reduction pathway (PCRP,4 see Outlaw [12] MATERIALS AND METHODS Guard cell protoplasts were isolated enzymatically from abaxial epidermes of 4-to 5-week-old leaves of Vicia faba L., as described previously (20). Mesophyll contamination of the preparation of guard cell protoplasts was estimated to be less than 0.01% (23). Isolated guard cell protoplasts (about 1 x 106 cells) were suspended in a reaction mixture containing 0.35 M mannitol, 0.5 mm MES-NaOH buffer (pH 6.2), 1 mm CaCl2, and 10 mM KCI. The suspension was continuously stirred in an open glass chamber enclosed in a Plexiglas water jacket filled with circulating water at 25C. Medium pH was measured with a pH glass electrode (Beckmann 39522) connected to a Radiometer pH meter, the pH signal was displayed on a chart recorder. Rates and magnitude of changes in medium pH were determined by back titration with 10 neq of H+ at the end of each experiment.Oxygen concentration was measured with a Clark-type 02 microelectrode, according to Revsbech (16). 02 concentrations in the medium were calculated from measurements made with the electrode tip slightly submerged in the top layer ofthe incubation medium, which was assumed to be in equilibrium with air, and solubility values for oxygen in water at 25°C. Rates of CO2 uptake and evolution were calculated from measured rates of alkalinization and acidification, respectively, unde...

Light Quality and Osmoregulation in Vicia Guard Cells

“…Recent physiological and biochemical evidence for PCRP activity in guard cells (9,10,11,35,38) (see Ref. 42 for review) provides an alternative explanation for these observations, pointing to photosynthetically produced sugars as a source of osmoticum.…”

“…42 for review) provides an alternative explanation for these observations, pointing to photosynthetically produced sugars as a source of osmoticum. From data on the red light-induced alkalinization and 02 evolution by guard cell protoplasts irradiated with red light, maximal rates of photosynthetic CO2 fixation were estimated at 300 Amol CO2 mg Chl-' h-' (38). Over a 5 h period, PCRP activity would provide 0.25 pmol of sucrose or 0.5 pmol of glucose per guard cell, decreasing their osmotic potential by 0.14 and 0.28 MPa, respectively.…”

“…In Vicia, red light-dependent stomatal opening in peels saturates at 50 ,umol m-2 s-, is insensitive to KCN, and is completely inhibited by DCMU (33). Red light-induced stomatal opening has been correlated with oxygen evolution and photophosphorylation (36,38) and with synthesis of intermediates of the PCRP3 (11).…”

“…In Vicia, red light-dependent stomatal opening in peels saturates at 50 ,umol m-2 s-, is insensitive to KCN, and is completely inhibited by DCMU (33). Red light-induced stomatal opening has been correlated with oxygen evolution and photophosphorylation (36,38) and with synthesis of intermediates of the PCRP3 (11).The interconversion of photosynthetically produced sugars and starch was a fundamental hypothesis of stomatal movements that dominated plant physiology p6rior to its abandonment in favor of the K+ hypothesis (16,21 …”

The guard cell chloroplast: a perspective for the twenty-first century

“…These include, a demonstration of the incorporation of radiolabeled CO 2 in phosphorylated compounds of illuminated guard cell protoplasts (Reckmann et al , 1990), measurements of DCMU-sensitive, red light-stimulated medium alkalinization by guard cell protoplasts (Shimazaki & Zeiger, 1987), dichlorophenyl dimethyl urea (DCMU) sensitive, sucrose accumulation in guard cells from sonicated epidermal peels incubated under red light (Poffenroth et al , 1992), immunocytochemical localization of rubisco in guard cells of a large number of species (Madavhan & Smith, 1982;Zemel & Gepstein, 1985), and demonstration of CO 2 fixation and photorespiration in single guard cells by measurement of chlorophyll fluorescence kinetics (Cardon & Berry, 1992). The evidence notwithstanding, many researchers continue to consider the issue unresolved.…”