The compartmentation of carboxylating and decarboxylating enzymes in guard cell protoplasts

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...

Section: C02 Fixation In Guard Cell Protoplastscontrasting

confidence: 90%

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

Shimazaki

Zeiger²

1987

“…This Supported by grants from the Deutsche Forschungsgemeinschaft to K. R. (Ra 122/11) and R. S. (Sche 217/3). conclusion was confirmed by metabolite determinations in mechanically isolated pairs of guard cells (13) as well as in suspensions of guard-cell protoplasts (21).…”

mentioning

confidence: 60%

Rubisco Activity in Guard Cells Compared with the Solute Requirement for Stomatal Opening

Reckmann¹,

Scheibe²,

Raschke³

1990

110

We investigated whether the reductive pentose phosphate path in guard cells of Pisum sativum had the capacity to contribute significantly to the production of osmotica during stomatal opening in the light. Amounts of ribulose 1,5-bisphophate carboxylase/ oxygenase (Rubisco) were determined by the [14C]carboxyarabinitol bisphosphate assay. A guard cell contained about 1.2 and a mesophyll cell about 324 picograms of the enzyme; the ratio was 1:270. The specific activities of Rubisco in guard cells and in mesophyll cells were equal; there was no indication of a specific inhibitor of Rubisco in guard cells. Rubisco activity was 115 femtomol per guard-cell protoplast and hour. This value was different from zero with a probability of 0.99. After exposure of guard-cell protoplasts to 14C02 for 2 seconds in the light, about one-half of the radioactivity was in phosphorylated compounds and <10% in malate. Guard cells in epidermal strips produced a different labelling pattern; in the light, <10% of the label was in phosphorylated compounds and about 60% in malate. The rate of solute accumulation in intact guard cells was estimated to have been 900 femto-osmol per cell and hour. If Rubisco operated at full capacity in guard cells, and hexoses were produced as osmotica, solutes could be supplied at a rate of 19 femto-osmol per cell and hour, which would constitute 2% of the estimated requirement. The capacity of guard-cell Rubisco to meet the solute requirement for stomatal opening in leaves of Pisum sativum is insignificant.

“…PPDK is found also in seeds (1,12) and leaves (1) of wheat and in some other C3 plant tissues (for references, see 2). The role of PPDK in C3 leaf tissues is not clearly known, although it has been suggested that it might be involved in the control of stomatal opening (16). The occurrence of PPDK in developing wheat seeds does not correlate quantitatively with photosynthetic enzymes (2), and it was suggested that one role of the enzyme might be in the conversion of C3 amino acids, especially alanine, to C4 and C5 amino acids.…”

mentioning

confidence: 99%

Pyruvate Orthophosphate Dikinase mRNA Organ Specificity in Wheat and Maize

Aoyagi

Bassham²

1984

Polyadenylated RNA was isolated from leaves and seeds of a C3 pant (Triticum aestivum L. cv Cheyenne, CI 8885) and from a C4 plant (Zea mays L. cv Golden bantam). Each polyadenylated RNA preparation was translated in vitro with micrococcal nuclease-treated reticulocyte lysate. When the in vitro translation products were probed with antibodies to pyruvate orthophosphate dikinase (PPDK) (EC 2.7.9.1), two sizes of polypeptide were identified. A 110 kilodalton polypeptide was found in the in vitro translation products of mRNA isolated exclusively from leaves of both wheat and maize. A 94 kilodalton polypeptide, similar to the PPDK polypeptide which can be extracted after in vivo synthesis in maize and wheat leaves and seeds, was found in the in vitro translation products obtained from wheat seeds and maize kernels.These results indicate that the mRNAs for PPDK polypeptides are organ-specific in both a C4 and a C3 plant. Hague et al. (1983 Nucleic Acids Res 11: 4853-4865) proposed that the larger size polypeptide of the in vitro translation polypeptide from maize leaf RNA contains a 'transit sequence' which permits entry into the chloroplasts of a polypeptide synthesized in vivo in maize leaf cell cytoplasm. It appears that in wheat leaves also the transit of synthesized PPDK polypeptide through an intracellular membrane may be required, while such a transit sequence seems not to be required within cells of wheat and maize seeds.Pyruvate orthophosphate dikinase (EC 2.7.9.1) is an essential enzyme for photosynthetic carbon dioxide fixation in C4 (8) and in some CAM (11) plants, where it catalyzes formation of phosphoenolpyruvate, the substrate for the initial carboxylation reaction. In C4 leaves, PPDK2 is found in the chloroplasts of the mesophyll cells (8). PPDK is found also in seeds (1, 12) and leaves (1) of wheat and in some other C3 plant tissues (for references, see 2). The role of PPDK in C3 leaf tissues is not clearly known, although it has been suggested that it might be involved in the control of stomatal opening (16). The occurrence of PPDK in developing wheat seeds does not correlate quantitatively with photosynthetic enzymes (2), and it was suggested that one role of the enzyme might be in the conversion of C3 amino acids, especially alanine, to C4 and C5 amino acids.Hague et al. (9) translation products of RNA extracted from maize leaf. Since the in vivo PPDK polypeptide from maize has a mol wt of about 94 to 97 kD, it was proposed that the polypeptide is nuclear encoded and is synthesized on cytoplasmic ribosomes as a 110 kD polypeptide with the extra 16 kD section serving as a 'transit' sequence to facilitate entry into the chloroplasts. Evidence that PPDK polypeptide is synthesized on cytoplasmic ribosomes in maize was found when PPDK was detected by an immunoprecipitation probe in the maize mutant iojap which is devoid of chloroplast ribosomes (6).The sizes of PPDK polypeptide(s) formed by in vitro translation of polyadenylated RNA from C3 leaf cells and from seed cells of C4 and C3 plants...