Light-Stimulated Burst of Carbon Dioxide Uptake following Nocturnal Acidification in the Crassulacean Acid Metabolism Plant Kalanchoë diagremontiana

CO₂ exchange characteristics were studied during the light-stimulated burst of CO₂ uptake (MB) immediately following a period of nocturnal CO₂ fixation in the Crassulacean acid metabolism plant Kalanchoë daigremontiana. During the early parts of the MB, stimulation of net CO₂ uptake by low ambient O₂ concentration (1.5%) was small, and leaves showed the capacity for net CO₂ uptake at low ambient CO₂ partial pressure (30 microbars) and when the MB was interrupted by darkness. During the later phase of the MB, stimulation of net CO₂ uptake by 1.5% O₂ was increased, and net CO₂ loss was recorded both at 30 microbars CO₂ and during dark interruptions. These results suggest that CO₂ fixation during the MB occurs simultaneously via phosphoenolpyruvate carboxylase (predominant during the early phase of the MB) and via ribulose bisphosphate carboxylase (predominant during the later phase of the burst). The magnitude and duration of the MB was increased by a reduction in the length of the dark period and by low (15°C) compared to high (30°C) leaf temperatures.     

Malate Metabolism in Leaf Mitochondria from the Crassulacean Acid Metabolism Plant Kalanchoë blossfeldiana Poelln

The mechanisms and the controlling factors of malate oxidation by mitochondria from leaves of Kalanchoë blossfeldiana Poelln. plants performing Crassulacean acid metabolism were investigated using Percollpurified mitochondria. The effects of pH and of various cofactors (ATP, NAD⁺, coenzyme A) on malate dehydrogenase (EC 1.1.1.37) and malic enzyme (EC 1.1.1.39) solubilized from these mitochondria were examined. The crucial role of cofactor concentrations in the mitochondrial matrix on the pathways of malate oxidation is shown. The distribution of the electrons originating from malate between the different electron transport pathways and its consequence on the phosphorylation yield was studied. It was found that, depending on the electron transport pathway used, malate oxidation could yield from 3 to 0 ATP. Assayed under conditions of high reducing power and high energy charge, the ability of malic enzyme to feed electrons to the cyanide-resistant nonphosphorylating alternative pathway was found to be higher than that of other dehydrogenases linked to the functioning of the Krebs cycle (pyruvate dehydrogenase, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, succinate dehydrogenase). The physiological significance of such a functional relationship between malic enzyme activity and the nonphosphorylating alternative pathway is discussed in relation to Crassulacean acid metabolism.     

Light Scattering as an Indicator of the Energy State in Leaves of the Crassulacean Acid Metabolism Plant Kalanchoë pinnata

Both transmittance changes in a weak beam of green light (light scattering) and the slow decay of chlorophyll a fluorescence were used as indicators of the energy state of leaves of a Crassulacean acid metabolism plant, Kalanchoë pinnata, at frequent intervals during 12-hour light/12-hour dark cycles. To induce light scattering and fluorescence changes, leaves were exposed to red light for 6 minutes. When measurements were made during the light period, the leaves were kept in darkness for 6 minutes before illumination. In the middle of the light period, when malic acid decarboxylation was very active and stomatal conductance was low, light scattering changes were small and indicated that the energy state of leaves was low. This result was supported by determination of adenylate levels. Light scattering and ATP/ADP ratios increased during the late light period when the tissue was deacidified. Illumination produced maximum light scattering changes between the 2nd and 5th hour of the dark period, when rates of dark CO₂ fixation were highest. Light scattering and fluorescence measurements taken from leaves, which were illuminated with red or far-red light in the presence or absence of O₂ showed that, in addition to linear electron transport, K. pinnata has the potential for both cyclic and pseudocyclic electron transport. The results are relevant with regard to the high ATP demand during Crassulacean acid metabolism.     

Modulation of Rubisco Activity during the Diurnal Phases of the Crassulacean Acid Metabolism Plant Kalanchoë daigremontiana

The regulation of Rubisco activity was investigated under high, constant photosynthetic photon flux density during the diurnal phases of Crassulacean acid metabolism in Kalanchoë daigremontiana Hamet et Perr. During phase I, a significant period of nocturnal, C₄-mediated CO₂ fixation was observed, with the generated malic acid being decarboxylated the following day (phase III). Two periods of daytime atmospheric CO₂ fixation occurred at the beginning (phase II, C₄–C₃ carboxylation) and end (phase IV, C₃–C₄ carboxylation) of the day. During the 1st h of the photoperiod, when phosphoenolpyruvate carboxylase was still active, the highest rates of atmospheric CO₂ uptake were observed, coincident with the lowest rates of electron transport and minimal Rubisco activity. Over the next 1 to 2 h of phase II, carbamylation increased rapidly during an initial period of decarboxylation. Maximal carbamylation (70%–80%) was reached 2 h into phase III and was maintained under conditions of elevated CO₂ resulting from malic acid decarboxylation. Initial and total Rubisco activity increased throughout phase III, with maximal activity achieved 9 h into the photoperiod at the beginning of phase IV, as atmospheric CO₂ uptake recommenced. We suggest that the increased enzyme activity supports assimilation under CO₂-limited conditions at the start of phase IV. The data indicate that Rubisco activity is modulated in-line with intracellular CO₂ supply during the daytime phases of Crassulacean acid metabolism.     

Water-relation Parameters of Individual Mesophyll Cells of the Crassulacean Acid Metabolism Plant Kalanchoë daigremontiana

Water-relation parameters of leaf mesophyll cells of the CAM plant Kalanchoë daigremontiana have been determined directly in cells of tissue slices using the pressure-probe technique. Turgor pressures measured in cells of the second to fourth layer from the cut surface showed an average of 1.82 ± 0.62 bar (mean ± sd; n = 157 cells). This was lower than expected from measurements of the osmotic pressure of the cell sap. The half-time (T_1/2) for water-flux equilibration of individual cells was 2.5 to 8.8 seconds. This is the fastest T_1/2 found so far for higher-plant cells. The calculated values of the hydraulic conductivity were in the range of 0.20 to 1.6 × 10⁻⁵ centimeters second⁻¹ bar⁻¹, with an average of (0.69 ± 0.46) × 10⁻⁵ centimeters second⁻¹ bar⁻¹ (mean ± sd; n = 8 cells). The T_1/2 values of water exchange of individual cells are consistent with the overall rates of water-flux equilibration measured for tissue slices.The volumetric elastic moduli (∈) of individual cells were in the range 13 to 128 bar for turgor pressures between 0.0 and 3.4 bar; the average ∈ value was 42.4 ± 27.7 bar (mean ± sd; n = 21 cells). This ∈ value is similar to that observed for other higher-plant cells.The water-storage capacity of individual cells, calculated as C_c = V/(∈ + πⁱ) (where V = cell volume and πⁱ = internal osmotic pressure) was 9.1 × 10⁻⁹ cubic centimeters bar⁻¹ per cell, and the capacity for the tissue was 2.2 × 10⁻² cubic centimeters bar⁻¹ gram⁻¹ fresh weight. The significance of the water-relation parameters determined at the cellular level is discussed in terms of the water relations of whole leaves and the high water-use efficiency characteristic of CAM plants.     

Reduction State of Q and Nonradiative Energy Dissipation during Photosynthesis in Leaves of a Crassulacean Acid Metabolism Plant, Kalanchoë daigremontiana Hamet et Perr

Fluorescence was measured in leaves of the CAM plant Kalanchoë daigremontiana using a pulse modulation technique at room temperature. During a 12-h light period at 500 micromole photons per square meter per second (400-700 nanometers) in air containing 350 microbar CO₂, the component of fluorescence quenching related to the reduction state of Q, the primary electron transport acceptor of PSII, remained fairly constant and showed that only 20% of Q were in the reduced form. The reduction state was slightly increased at the onset and at the end of the light period. By contrast, the nonphotochemical component of fluorescence quenching which is a measure of the fraction of nonradiative deexcitation underwent marked diurnal changes. Nonradiative energy conversion was low during the phase of most active malic acid decarboxylation in the middle of the light period when uptake of atmospheric CO₂ was negligible, and when internal CO₂ partial pressures were higher than in air; this allowed for high rates of CO₂ reduction in the chloroplasts. Nonradiative energy conversion was high during the early and the late light period when atmospheric CO₂ was taken up and internal CO₂ partial pressures were below air level. Manipulation of the internal CO₂ partial pressure during the late light period by increasing or decreasing the external CO₂ partial pressure to 1710 and 105 microbar, respectively, led to changes in the magnitude of energy dependent fluorescence quenching which were consistent with the relationship between nonradiative energy dissipation and internal CO₂ partial pressure observed during the diurnal cycle. Again, the reduction state of Q was hardly affected by these treatments. Thus, changes in electron transport rate during the diurnal CAM cycle at a given photon flux density lead primarily to alterations in the rate of nonradiative energy dissipation, with the reduction state of Q being maintained at a relatively low and constant level. Conditions are described under which nonphotochemical dissipation of excitation energy reaches a maximum value and the reduction state of Q is increased.     

Measurements of the Engagement of Cyanide-Resistant Respiration in the Crassulacean Acid Metabolism Plant Kalanchoë daigremontiana with the Use of On-Line Oxygen Isotope Discrimination

Discrimination against ¹⁸O during dark respiration in tissues of Kalanchoë daigremontiana, Medicago sativa, and Glycine max was measured using an on-line system that enabled direct measurements of the oxygen fractionation of samples in a gas-phase leaf disk electrode unit. Discrimination factors for cytochrome pathway respiration were 18.6 to 19.8%_o for all tissues. However, discrimination in cyanide-resistant respiration was significantly higher in green tissues (30.4-31.2%_o) compared with nongreen tissues (25.3-25.9%_o). Using these discrimination factors, the partitioning of electron transport to these pathways was calculated from measurements of discrimination in the absence of inhibitors. Changes in flux through the alternative pathway were measured during the light and dark phases of Crassulacean acid metabolism in leaf disks of K. daigremontiana. The flux of electrons through the alternative pathway was higher during deacidification than during the other phases of Crassulacean acid metabolism. The increase in alternative pathway electron flux accounted for all of the increased respiration in the light phase. Despite this increase, simultaneous measurements of malate concentration and respiratory flux confirm that only a small proportion of the total malate decarboxylation occurs in the mitochondria.     

Effect of Varying CO(2) Partial Pressure on Photosynthesis and on Carbon Isotope Composition of Carbon-4 of Malate from the Crassulacean Acid Metabolism Plant Kalanchoë daigremontiana Hamet et Perr

Intact leaves of Kalanchoë daigremontiana were exposed to CO₂ partial pressures of 100, 300, and 1000 microbars. Malic acid was extracted, purified, and degraded in order to obtain isotopic composition of carbon-1 and carbon-4. From these data, it is possible to calculate the carbon isotope composition of newly fixed carbon in malate. In all three treatments, the isotopic composition of newly introduced carbon is the same as that of the CO₂ source and is independent of CO₂ partial pressures over the range tested. Comparison with numerical models described previously (O'Leary 1981 Phytochemistry 20: 553-567) indicates that we would expect carbon 4 of malate to be 4‰ more negative than source CO₂ if diffusion is totally limiting or 7‰ more positive than source CO₂ if carboxylation is totally limiting. Our results demonstrate that stomatal aperture adjusts to changing CO₂ partial pressures and maintains the ratio of diffusion resistance to carboxylation resistance approximately constant. In this study, carboxylation and diffusion resistances balance so that essentially no fractionation occurs during malate synthesis. Gas exchange studies of the same leaves from which malate was extracted show that the extent of malate synthesis over the whole night is nearly independent of CO₂ partial pressure, although there are small variations in CO₂ uptake rate. Both the gas exchange and the isotope studies indicate that the ratio of external to internal CO₂ partial pressure is the same in all three treatments. Inasmuch as a constant ratio will result in constant isotope fractionation, this observation may explain why plants in general have fairly invariable ¹³C contents, despite growing under a variety of environmental conditions.