Leaf anatomy and ultrastructure of the Crassulacean-acid-metabolism plant Kalanchoe daigremontiana.

Light-microscopic analysis of leaf clearings of the obligate Crassulacean-acid-metabolism (CAM) species Kalanchoe daigremontiana Hamet et Perr. has shown the existence of unusual and highly irregular venation patterns. Fifth-order veins exhibit a three-dimensional random orientation with respect to the mesophyll. Minor veins were often observed crossing over or under each other and over and under major veins in the mesophyll. Paraffin sections of mature leaves show tannin cells scattered throughout the mesophyll rather evenly spaced, and a distinct layer of tannin cells below the abaxial epidermis. Scanning electron microscopy showed that bundle-sheath cells are distinct from the surrounding mesophyll in veins of all orders. Transmission electron microscopy demonstrated developing sieve-tube elements in expanded leaves. Cytosolic vesicles produced by dictyosomes undergo a diurnal variation in number and were often observed in association with the chloroplasts. These vesicles are an interesting feature of cell ultrastructure of CAM cells and may serve a regulatory role in the diurnal malic-acid fluctuations in this species.

Plasmalemma- and tonoplast-ATPase activity in mesophyll protoplasts, vacuoles and microsomes of the Crassulacean-acid-metabolism plant Kalanchoe daigremontiana.

Adenosine-triphosphatase activity on the plasmalemma and tonoplast of isolated mesophyll protoplasts, isolated vacuoles and tonoplast-derived microsomes of the Crassulacean-acid-metabolism plant Kalanchoe daigremontiana Hamet et Perr., was localized by a cytochemical procedure using lead citrate. Enzyme activity was detected on the cytoplasmic surfaces of the plasmalemma and tonoplast. The identity of the enzymes was confirmed by various treatments differentiating the enzymes by their sensitivity to inhibitors of plasmalemma and tonoplast H(+)-ATPase. Isolated vacuoles and microsomes prepared from isolated vacuoles clearly exhibited single-sided deposition on membrane surfaces.

Characterization of the vacuolar ATPase activity of the crassulacean-acid-metabolism plant Kalanchoë daigremontiana. Receptor modulating.

Plants performing crassulacean acid metabolism show a large nocturnal accumulation of malic acid in the vacuole of the photosynthetic cells. It has been postulated that an H+-translocating ATPase energizes the transport of malic acid across the tonoplast into the vacuole. In the present work we have characterized the ATPase activity associated with vacuoles of the crassulacean-acid-metabolism plant Kalanchoë daigremontiana and compare it with other phosphohydrolases. Vacuoles were isolated by polybase-induced lysis of mesophyll-cell protoplasts. The vacuoles had a high activity of unspecific acid phosphatase (pH optimum 5.3). The acid phosphatase was strongly inhibited by ammonium molybdate (with 50% inhibition at about 0.5 mmol m-3), but was not completely inhibited even at much higher ammonium-molybdate concentrations. In contrast, the vacuolar ATPase activity, assayed in the presence of 100 mmol m-3 ammonium molybdate, had a pH optimum of 8.0. ATP was the preferred substrate, but GTP, ITP and ADP were hydrolyzed at appreciable rates. The mean ATPase activity at pH 8.0 was 14.5 nmol h-1 (10(3) vacuoles)-1, an average 13% of which was attributable to residual acid-phosphatase activity. Inorganic-pyrophosphatase activity could not be demonstrated unambiguously. The vacuolar ATPase activity was Mg2+-dependent, had an apparent Km for MgATP2- of 0.31 mol m-3, and was 32% stimulated by 50 mol m-3 KCl. Of the inhibitors tested, oligomycin slightly inhibited the vacuolar ATPase activity and diethylstilbestrol and NO-3 were both markedly inhibitory. Dicyclohexylcarbodiimide and tributyltin were also strongly inhibitory. Tributyltin caused a 50% inhibition at about 0.3 mmol m-3. This is taken as evidence that the vacuolar ATPase might function as an H+-translocating ATPase. It is shown that the measured activity of the vacuolar ATPase would be of the right order to account for the observed rates of nocturnal malic-acid accumulation in K. daigremontiana.

Temperature Dependence of Photosynthesis in Agropyron smithii Rydb. : III. Responses of Protoplasts and Intact Chloroplasts.

Protoplasts and intact chloroplasts isolated from Agropyron smithii Rybd. were utilized in an effort to determine the limiting factor(s) for photosynthesis at supraoptimal temperatures. Saturated CO(2)-dependent O(2) evolution had a temperature optimum of 35 degrees C for both protoplasts and intact chloroplasts. A sharp decline in activity was observed as assay temperature was increased above 35 degrees C, and at 45 degrees C only 20% of the maximal rate remained. The temperature optimum for 3-phosphoglycerate reduction by intact chloroplasts was 35 degrees C. Above this temperature, 3-phosphoglycerate reduction was more stable than CO(2)-dependent O(2) evolution. Reduction of nitrite in coupled intact chloroplasts had a temperature optimum of 40 degrees C with only slight variation in activity between 35 degrees C and 45 degrees C. Reduction of nitrite in uncoupled chloroplasts had a temperature optimum of 40 degrees C, but increasing the assay temperature to 45 degrees C resulted in a complete loss of activity. Reduction of p-benzoquinone by protoplasts and intact chloroplasts had a temperature optimum of 32 degrees C when measured in the presence of dibromothymoquinone. This photosystem II activity exhibited a strong inhibition of O(2) evolution as assay temperature increased above the optimum. It is concluded that, below the temperature optimum, ATP and reductant were not limiting photosynthesis in these systems or intact leaves. Above the temperature optimum, photosynthesis in these systems is limited in part by the phosphorylation potential of the stromal compartment and not by the available reductant.

ATPase activity associated with isolated vacuoles of the crassulacean acid metabolism plant Kalanchoë daigremontiana.

A technique is described that allows a relatively rapid and controlled isolation of vacuoles from leaves of the crassulacean acid metabolism (CAM) plant Kalanchoë daigremontiana. The method involves polybase-induced lysis of mesophyllcell protoplasts and isolation of vacuoles on a discontinuous density gradient. ATPase activity is associated with the isolated vacuoles and is not attributable to contamination by cytoplasmic constituents. It is suggested that this ATPase is responsible for the energization of malic-acid accumulation in the vacuole in CAM plants.

Active, Irreversible Accumulation of Extreme Levels of H(2)SO(4) in the Brown Alga, Desmarestia.

The brown algae Desmarestia ligulata var. ligulata (Lightf.) Lamour., and D. viridis (Mull.) Lamour., accumulate H(2)SO(4) until their average internal pH is 0.5 to 0.8. A related species, D. aculeata (L.) Lamour., does not accumulate acid. The H(2)SO(4) accumulation is accompanied by a reduction in the K(+) and Cl(-) content, presumedly to maintain osmotic balance. Measurements of the membrane potential and H(+) and SO(4) (2-) concentrations indicate that both ions are accumulated in the vacuole against their electrochemical potential gradients.The internal pH remains constant in all three species over the growing season, despite striking changes in the algal morphology. The pH is not affected by periods of darkness of up to 34 hours. Sulfate accumulated in the vacuoles appears to be trapped there since incubation of D. ligulata for up to 10 days in sulfate-free medium resulted in little loss of either vacuolar sulfate or H(+). Although the uptake of H(2)SO(4) into the vacuole must require energy, the maintenance of the vacuolar H(2)SO(4) may be due to the impermeability of the tonoplast, with little necessity for continued expenditure of energy.

Inhibition of Photosynthetic Energy Conversion by Cupric Ion : EVIDENCE FOR Cu-COUPLING FACTOR 1 INTERACTION.

This study describes a specific Cu(2+) and light-dependent inhibition of spinach (Spinacia oleracea L.) chloroplast reactions involving coupling factor 1 function. A primary effect is an inhibition of photophosphorylation induced by illumination of Class II chloroplasts with micromolar Cu(2+) and pyocyanine in the absence of ADP, Mg(2+), and HPO(4) (2-). The inhibition, which is dependent on free Cu(2+) as indicated by protection by ethylene diamine tetraacetic acid and dithiothreitol, requires illumination (electron flow) for establishment of the specific inhibition to be noted. Protection is also afforded by uncouplers and some partial protection is provided by micromolar concentrations of ADP and ATP. The data strongly suggest that Cu(2+) causes an O(2)-independent oxidation of sulfhydryl groups on coupling factor 1, which are essential to catalytic function. This conclusion is supported by the reduction of energy-dependent (3)H-N-ethylmaleimide labeling of the gamma subunit of coupling factor 1 by the Cu(2+)-light pretreatment.

Temperature Dependence of Photosynthesis in Agropyron smithii Rydb. : I. FACTORS AFFECTING NET CO(2) UPTAKE IN INTACT LEAVES AND CONTRIBUTION FROM RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE MEASURED IN VIVO AND IN VITRO.

As part of an extensive analysis of the factors regulating photosynthesis in Agropyron smithii Rydb., a C(3) grass, we have examined the response of leaf gas exchange and ribulose-1,5-bisphosphate (RuBP) carboxylase activity to temperature. Emphasis was placed on elucidating the specific processes which regulate the temperature response pattern. The inhibitory effects of above-optimal temperatures on net CO(2) uptake were fully reversible up to 40 degrees C. Below 40 degrees C, temperature inhibition was primarily due to O(2) inhibition of photosynthesis, which reached a maximum of 65% at 45 degrees C. The response of stomatal conductance to temperature did not appear to have a significant role in determining the overall temperature response of photosynthesis. The intracellular conductance to CO(2) increased over the entire experimental temperature range, having a Q(10) of 1.2 to 1.4. Increases in the apparent Michaelis constant (K(c)) for RuBP carboxylase were observed in both in vitro and in vivo assays. The Q(10) values for the maximum velocity (V(max)) of CO(2) fixation by RuBP carboxylase in vivo was lower (1.3-1.6) than those calculated from in vitro assays (1.8-2.2). The results suggest that temperature-dependent changes in enzyme capacity may have a role in above-optimum temperature limitations below 40 degrees C. At leaf temperatures above 40 degrees C, decreases in photosynthetic capacity were partially dependent on temperature-induced irreversible reductions in the quantum yield for CO(2) uptake.

Temperature Dependence of Photosynthesis in Agropyron smithii Rydb. : II. CONTRIBUTION FROM ELECTRON TRANSPORT AND PHOTOPHOSPHORYLATION.

As part of an analysis of the factors regulating photosynthesis in Agropyron smithii Rydb., a C(3) grass, the response of electron transport and photophosphorylation to temperature in isolated chloroplast thylakoids has been examined. The response of the light reactions to temperature was found to depend strongly on the preincubation time especially at temperatures above 35 degrees C. Using methyl viologen as a noncyclic electron acceptor, coupled electron transport was found to be stable to 38 degrees C; however, uncoupled electron transport was inhibited above 38 degrees C. Photophosphorylation became unstable at lower temperatures, becoming progressively inhibited from 35 to 42 degrees C. The coupling ratio, ATP/2e(-), decreased continuously with temperature above 35 degrees C. Likewise, photosystem I electron transport was stable up to 48 degrees C, while cyclic photophosphorylation became inhibited above 35 degrees C. Net proton uptake was found to decrease with temperatures above 35 degrees C supporting the hypothesis that high temperature produces thermal uncoupling in these chloroplast thylakoids. Previously determined limitations of net photosynthesis in whole leaves in the temperature region from 35 to 40 degrees C may be due to thermal uncoupling that limits ATP and/or changes the stromal environment required for photosynthetic carbon reduction. Previously determined limitations to photosynthesis in whole leaves above 40 degrees C correlate with inhibition of photosynthetic electron transport at photosystem II along with the cessation of photophosphorylation.

An Analysis of the Relationship between Respiration and the Transmembrane Potential in Corn Roots.

The effects of cyanide, anoxia, and temperatures varying from 2 to 42 C on the cell membrane electropotential difference (PD) of washed and freshly excised corn roots have been determined. Respiration rates of freshly excised root segments in response to cyanide and to varying temperatures were also measured. The cell membrane PD of roots which had been washed for 12 to 15 hours was almost insensitive to cyanide and anoxia but sensitive to low temperature. In contrast, the cell membrane PD of freshly excised roots was reversibly depolarized by all three treatments, cyanide depolarized from -117 to -86 millivolts and the sequential imposition of anoxia further lowered the PD to -69 millivolts. Anoxia applied first depolarized maximally and the PD was not further lowered by sequential cyanide treatment. Arrhenius plot analysis of the temperature response of respiration showed an apparent transition at 13 C with an activation energy of 20.0 kilocalories per mole below and 8.8 kilocalories per mole above the transition temperatures. The energy of activation for repolarization of PD is much higher; 53.4 kilocalories per mole below 7 to 8 C and 25.4 kilocalories per mole above this apparent transition. The energy requirement for polarization of the cell membrane PD was calculated based on the temperature responses of the cell membrane PD and respiration. It was estimated that 3.5% of the energy output from respiration at 22 C is required for cell polarization. It is unlikely that ion transport is limited by energy availability below the 8 C transition in this chill sensitive species.

Temperature dependence of the concentration kinetics of absorption of phosphate and potassium in corn roots.

The effect of temperature on respiration and kinetics of H(2)PO(4) (-) and K(+) uptake in corn roots was determined in the range of 2 to 42 C. The response of uptake to temperature, determined from Q(10) and activation energy (Ea) data, for the anion and the cation differ significantly, especially in the range of uptake mechanism (Mech.) I. At 2.5 micromolar the Ea for K(+) uptake below the 13 C transition is 29.3 kilocalories per mole. As the K(+) concentration is increased, Ea declines and at 0.25 millimolar is 21.6 kilocalories per mole. Accompanying this change in Ea is a shifting of the apparent transition temperature from 13 to 17 C. Above the temperature transition the Ea's for K(+) uptake in the Mech. I range are quite low (3.0) and this value is unchanged by increases of K(+) concentration to 0.25 millimolar. In the range of Mech. II above 1 millimolar K(+) the temperature transitions are not seen and plots become linear. The Ea's show an increasing trend from 4.7 at 1 millimolar to 6.1 at 50 millimolar. The uptake of H(2)PO(4) (-) is much more temperature sensitive having a constant Ea at concentrations in the Mech. I range below the 13 C temperature transition. The Arrhenius plots reveal a second transition at 22 C and the Ea for this segment is 21.0. Above the second transition the Ea remains high (10.0) and is constant in the range of Mech. I. In the range of Mech. II there is a concentration dependent decline in Ea for H(2)PO(4) (-) uptake (22.7 at 1.0 millimolar to 1.0 at 50 millimolar). There is no definable low temperature transition at these concentrations. Ion uptake is found to be much more sensitive to low temperature than respiration in this chill-sensitive species. The data suggest that the low temperature reduction of ion transport is more closely related to restriction of function of active transport systems than to either respiration or membrane permeability.

Effects of temperature on the hill reaction and photophosphorylation in isolated cactus chloroplasts.

Chloroplasts isolated from Opuntia polyacantha Haw. (Cactaceae) are capable of noncyclic electron transport and ATP synthesis. Hill reaction rates, measured by O(2) evolution or by ferricyanide reduction, increase with increasing temperature to approximately 40 C. The temperature optimum of NADP reduction is 42 C while the optimum for noncyclic photophosphorylation is 35 C. NADP-linked phosphorylation exhibits a higher coupling ratio (P/e(2)) than ferricyanide-linked photophosphorylation. The temperature optima for photochemical energy production correlate with photosynthetic properties of Crassulacean acid metabolism (CAM) plants and are discussed in relation to the operation of CAM at high tissue temperature.