Malate Decarboxylation by Kalanchoë daigremontiana Mitochondria and Its Role in Crassulacean Acid Metabolism

Mitochondria isolated from Kalanchoë daigremontiana, a Crassulacean acid metabolism plant, decarboxylate added malate to pyruvate at rates of up to 100 micromoles per hour per milligram original chlorophyll in the presence of ADP. Omitting ADP reduces this rate by approximately 50%. Antimycin A inhibits malate decarboxylation and this inhibition could be relieved by addition of aspartate and α-ketoglutarate to the mitochondria. Increasing the pH of the external medium inhibited malate decarboxylation; a dramatic decrease in pyruvate production was observed between pH 7.2 and pH 7.4. It is suggested that cytoplasmic pH changes may regulate the contribution of mitochondria to malate decarboxylation in the light in vivo.     

Malate Metabolism in the Dark After CO(2) Fixation in the Crassulacean Plant Kalanchoë tubiflora

The metabolism of [¹³C]malate was studied in the Crassulacean plant Kalanchoë tubiflora following exposure to ¹³CO₂ for 2 hour intervals during a 16 hour dark cycle. Nuclear magnetic resonance spectroscopy of [¹³C]malate extracted from labeled tissue revealed that the transient flux of malate to the mitochondria, estimated by the randomization of [4-¹³C]malate to [1- ¹³C]malate by fumarase, varied substantially during the dark period. At both 15 and 25°C, the extent of malate label randomization in the mitochondria was greatest during the early and late parts of the dark period and was least during the middle of the night, when the rate of ¹³CO₂ uptake was highest. Randomization of labeled malate continued for many hours after malate synthesis had initially occurred. Internally respired ¹²CO₂ also served as a source of carbon for malate formation. At 15°C, 15% of the total malate was formed from respired ¹²CO₂, while at 25°C, 49% of the accumulated malate was derived from respired ¹²CO₂. Some of the malate synthesized from external ¹³CO₂ was also respired during the night. The proportion of the total [¹³C]malate respired during the dark period was similar at 15 and 25°C, and respiration of newly formed [¹³C]malate increased as the night period progressed. These data are discussed with regard to the relative fluxes of malate to the mitochondria and the vacuole during dark CO₂ fixation.     

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.     

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.     

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.     

Diurnal Changes in Metabolite Levels and Crassulacean Acid Metabolism in Kalanchoë daigremontiana Leaves

Diurnal changes in levels of selected metabolites associated with glycolysis, the C₃ cycle, C₄-organic acids, and storage carbohydrates were analyzed in active Kalanchoë daigremontiana Crassulacean acid metabolism leaves. Three metabolic transition periods occurred each day. During the first two hours of light, nearly all of the metabolite pools underwent transient changes. Beginning at daylight, stomata opened transiently and closed again within 30 minutes; malate synthesis continued for about 1 hour into the light; C₃ photosynthesis began within 30 minutes; and net quantities of starch and glucan began to accumulate after 2 hours, continuing linearly throughout the rest of the day.     

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.     

Changes in Metabolite Levels in Kalanchoë daigremontiana and the Regulation of Malic Acid Accumulation in Crassulacean Acid Metabolism

Changes in glucose-6-P, fructose-6-P, fructose-1,6-diP, 6-phospho-gluconate, phosphoenolpyruvate, 3-phosphoglycerate, and pyruvate levels in the leaves of the Crassulacean plant Kalanchoë daigremontiana Hammet et Perrier were measured enzymically during transitions from CO₂-free air to air, air to CO₂-free air, and throughout the course of acid accumulation in darkness. The data are discussed in terms of the involvement of phosphoenolpyruvate carboxylase in malic acid synthesis and in terms of the regulation of the commencement of malic acid synthesis and accumulation through the effects of CO₂ on storage carbohydrate mobilization and its termination through the effects of malic acid on phosphoenolpyruvate carboxylase activity.     

Nitrate Assimilation and Crassulacean Acid Metabolism in Leaves of Kalanchoë fedtschenkoi Variety Marginata

The enzymes necessary to assimilate ammonia either via glutamine synthetase and glutamate synthase or via the glutamate dehydrogenase pathways are present in both green and white leaf tissues of Kalanchoë fedtschenkoi. Nitrate reductase activity develops to a maximum in a Crassulacean acid metabolism (CAM) plant canopy before either ribulose 1,5-bisphosphate carboxylase, or phosphoenolpyruvate carboxylase, or CAM. Nitrate reductase also is activated each morning and is inactivated late in the day as in other plants. However, there does not appear to be any direct relationship between nitrate reductase activity and the level of acid, its daily pattern or the amplitude of CAM. Though nitrate reductase is activated maximally each day by light, in Kalanchoë leaves for six days the activity followed a precise daily pattern independent of continuous light or dark.     

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.     

TDZ induces shoot regeneration in various Kalanchoë blossfeldiana Poelln. cultivars in the absence of auxin

In order to optimize shoot regeneration in Kalancho? blossfeldiana, leaf and internode explants of seven cultivars including one inter-specific were studied. The effects of various combinations of α-naphthalene acetic acid (NAA) (0, 0.57 M) and thidiazuron (TDZ) (0, 0.45, 4.5, 22.5, 67.5 μM) on MS medium were examined. In all cultivars shoot regeneration frequency and number of shoots per explant were enhanced by increasing TDZ concentration. Supplementing the media with NAA did not improve shoot regeneration. Maximum regeneration frequency and optimum concentration of TDZ for shoot regeneration depended significantly on the cultivar. Internode explants, but not leaf explants, of some cultivars, were able to produce adventitious shoots without treatment with growth regulator.     

C Nuclear Magnetic Resonance Studies of Crassulacean Acid Metabolism in Intact Leaves of Kalanchoë tubiflora

¹³C nuclear magnetic resonance spectroscopy of intact leaves of Kalanchoë tubiflora was used to observe Crassulacean acid metabolism in vivo. ¹³C signals from C-4 of malate were observed after overnight exposure of leaves to ¹³CO₂. Illumination of the labeled leaves resulted in a gradual decrease in the malate signals. After a period of darkness in normal air, ¹³C signals were detected in all four carbons of malate in the previously labeled leaves. The ¹³C nuclear magnetic resonance spectrum of malate in solution was pH dependent, which allowed an estimation of the vacuolar pH from the whole leaf spectrum. The pH was 4.0 following a 14-hour dark period, but rose to greater than 6.0 after 6 hours of illumination.     

Long-Lasting Light Effects in Imbibed Kalanchoë blossfeldiana Seeds in the Presence of Gibberellic Acid

Two brief red (R) irradiations, separated by 24 hours, given to Kalanchoë blossfeldiana Poelln. cv Feuerblüte seeds, made secondarily dormant by a prolonged dark incubation period on water and transferred to GA₃, induce very low germination. Some effect of these irradiations is preserved, however, during a long dark interval in fully imbibed seeds and greatly increases the germination induced by another brief R exposure. This long-lasting light effect is, at 20°C, only lost after a dark interval of about 1 month. It can also be induced by two brief far-red (FR) exposures. Its preservation is temperature-dependent, low temperatures being favorable. Light-induced changes in the ATP-content were demonstrated during preservation and expression of the long-lasting light effect, indicating a long-lasting metabolic change. In seeds with primary dormancy sown on GA₃, an analogous long-lasting light effect is induced by one or two brief R or FR irradiations, even when they are given before germination can take place. The presence of GA₃, which was shown to induce a very low fluence germination response in Kalanchoë seeds, is required for the occurrence of the long-lasting light effect. The data suggest long-term preservation of some effect(s) of Pfr rather than persistent presence of Pfr itself.     

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.     

Nicotinamide Adenine Dinucleotide-specific "Malic" Enzyme in Kalanchoë daigremontiana and Other Plants Exhibiting Crassulacean Acid Metabolism

NAD-specific “malic” enzyme (EC 1.1.1.39) has been isolated and purified 1200-fold from leaves of Kalanchoë daigremontiana. Kinetic studies of this enzyme, which is activated 14-fold by CoA, acetyl-CoA, and SO₄²⁻, suggest allosteric properties. Cofactor requirements show an absolute specificity for NAD and for Mn²⁺, which cannot be replaced by NADP or Mg²⁺. For maintaining enzyme activity in crude leaf extracts a thiol reagent, Mn²⁺, and PVP-40 were required. The latter could be omitted from purified preparations. By sucrose density gradient centrifugation NAD-malic enzyme could be localized in mitochondria. A survey of plants with crassulacean acid metabolism revealed the presence of NAD-malic enzyme in all 31 plants tested. Substantial levels of this enzyme (121-186 μmole/hr·mg of Chl) were detected in all members tested of the family Crassulaceae. It is proposed that NAD-malic enzyme in general supplements activity of NADP-malic enzyme present in these plants and may be specifically employed to increase internal concentrations of CO₂ for recycling during cessation of gas exchange in periods of severe drought.     

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.     

Effects of Water and Turgor Potential on Malate Efflux from Leaf Slices of Kalanchoë daigremontiana

Malate efflux from leaf cells of the Crassulacean acid metabolism plant Kalanchoë daigremontiana Hamet et Perrier was studied using leaf slices submerged in experimental solutions. Leaves were harvested at the end of the dark phase and therefore contained high malate levels. Water potentials of solutions were varied between 0 and −5 bar using mannitol (a slowly permeating solute) and ethylene glycol (a rapidly permeating solute), respectively. Mannitol solutions of water potentials down to −5 bar considerably reduced malate efflux. The slowly permeating solute mannitol reduces both water potential and turgor potential of the cells. The water potential of a mannitol solution of −5 bar is just above plasmolyzing concentration. Malate efflux in ethylene glycol at −5 bar was only slightly smaller than at 0 bar, and much higher than in mannitol at −5 bar. Tissues in rapidly permeating ethylene glycol would have turgor potentials similar to tissues in 0.1 mm CaSO₄. The results demonstrate that malate efflux depends on turgor potential rather than on water potential of the cells.     

Flavonoids in flowers of 16 Kalanchoë blossfeldiana varieties

Kalancho? blossfeldiana varieties with orange, pink, red and magenta flowers were found to contain 3,5-O-beta-D-diglucosides of pelargonidin, cyanidin, peonidin, delphinidin, petunidin and malvidin. Pink, red and magenta varieties contained relatively high amounts of quercetin based flavonols. Four distinct quercetin flavonols were identified, namely quercetin 3-O-beta-D-glucoside and three that were quercetin 3-O-alpha-L-rhamnoside based, with either glucose, xylose or arabinose attached to position 2 of the rhamnose. In addition, the presence of at least three kaempferol based diglycosides was suggested from LC-MS analyses. Orange varieties contained very low amounts of flavonol co-pigments and of delphinidin derivatives. The flower extracts of the varieties 'Diva' (magenta) and 'Molly' (red) had identical anthocyanin ratios but differed significantly in flavonol content. The magenta variety contained four times as much quercetin relative to anthocyanidin as the red variety. This difference was mainly due to a larger content of quercetin 3-O-(2'-O-beta-D-glucopyranosyl-alpha-L-rhamnopyranoside). Based on pigment and co-pigment analyses, approaches for molecular breeding towards blue flower colour are discussed.     

A search for growth related genes in Kalanchoë blossfeldiana

Differential display of mRNA from four sets of contrasting phenotypes were carried out in order to identify and isolate genes associated with elongating growth of Kalanchoë blossfeldiana. A total of 17 unique differential expressed cDNA fragments were sequenced and 12 showed homology to genes in other plant species. Three genes were subsequently tested for growth related activity by Virus Induced Gene Silencing (VIGS) in Nicotiana benthamiana. One gene fragment (13C) resulted in plants with significantly reduced growth (N = 20, P = 0.05, one-tailed students t-test) from day 25 after virus infection. Full-length cDNA and genomic DNA sequences were obtained by inverse PCR and thermal asymmetric interlaced (TAIL) PCR and the gene was named KbORF1. The predicted gene is 2244 bp long with three exons of 411 bp in total encoding a protein of 137 amino acid residues with homologs widespread among plants. The protein has no known function, but its expression has been confirmed in a proteomic study of Arabidopsis. Southern blot analysis shows two hybridizing fragments in agreement with the tetraploid nature of K. blossfeldiana. Fragment 13C comprises 446 bp of the gene, and the portion of 13C conferring growth retardation by VIGS is located 10 bp into the second intron indicating a regulatory function of this part of the KbORF1 mRNA. Differential display in combination with VIGS as a screening method proved to be a good functional approach not only to search for genes of interest, but also to isolate expressed genetic regulatory domains.     

Carbon Dioxide and Water Vapor Exchange in the Crassulacean Acid Metabolism Plant Kalanchoë pinnáta during a Prolonged Light Period: METABOLIC AND STOMATAL CONTROL OF CARBON METABOLISM

Net CO₂ and water vapor exchange were studied in the Crassulacean acid metabolism plant Kalanchoë pinnáta during a normal 12-hour light/12-hour dark cycle and during a prolonged light period. Leaf temperature and leaf-air vapor pressure difference were kept constant at 20 C and 9 to 10 millibar. There was a 25% increase in the rate of CO₂ fixation during the first 6 hours prolonged light without change in stomatal conductance. This was associated with a decrease in the intracellular partial pressure of CO₂, a decrease in the stimulation of net CO₂ uptake by 2% O₂, and a decrease in the CO₂ compensation point from 45 to 0 microbar. In the normal light period after deacidification, leaves showed a normal light dependence of CO₂ uptake but, in prolonged light, CO₂ uptake was scarcely light-dependent. The increase in titratable acidity in prolonged light was similar to that in the dark.