Ion Homeostasis in Chloroplasts under Salinity and Mineral Deficiency : I. Solute Concentrations in Leaves and Chloroplasts from Spinach Plants under NaCl or NaNO(3) Salinity

Spinach (Spinacia oleracea var “Yates”) plants in hydroponic culture were exposed to stepwise increased concentrations of NaCl or NaNO₃ up to a final concentration of 300 millimoles per liter, at constant Ca²⁺-concentration. Leaf cell sap and extracts from aqueously isolated spinach chloroplasts were analyzed for mineral cations, anions, amino acids, sugars, and quarternary ammonium compounds. Total osmolality of leaf sap and photosynthetic capacity of leaves were also measured. For comparison, leaf sap from salt-treated pea plants was also analyzed. Spinach plants under NaCl or NaNO₃ salinity took up large amounts of sodium (up to 400 millimoles per liter); nitrate as the accompanying anion was taken up less (up to 90 millimoles per liter) than chloride (up to 450 millimoles per liter). Under chloride salinity, nitrate content in leaves decreased drastically, but total amino acid concentrations remained constant. This response was much more pronounced (and occurred at lower salt concentrations) in leaves from the glycophyte (pea, Pisum sativum var “Kleine Rheinländerin”) than from moderately salt-tolerant spinach. In spinach, sodium chloride or nitrate taken up into leaves was largely sequestered in the vacuoles; both salts induced synthesis of quarternary ammonium compounds, which were accumulated mainly in chloroplasts (and cytosol). This prevented impairment of metabolism, as indicated by an unchanged photosynthetic capacity of leaves.     

Absorption of magnesium and chloride by excised corn root

Absorption characteristics of Mg²⁺ and Cl⁻ were investigated with 5-day-old excised corn (Zea mays) roots. Uptake from both 0.5 and 10 milliequivalents per liter MgCl₂ solutions occurred at steady state rates for the first 6 hours. Inhibition by dinitrophenol and low temperatures established that absorption during this period was metabolically mediated in the absence and presence of Ca²⁺. Absorption isotherms indicated dual mechanisms of Mg²⁺ and Cl⁻ absorption from solutions above 1 milliequivalent per liter. The effect of H⁺ on absorption of Mg²⁺ and Cl⁻ was typical of that generally reported for other plant roots and other ions. In the physiological pH range, Ca²⁺ greatly suppressed the rate of Mg²⁺ absorption but had little effect on Cl⁻. The influence of Ca²⁺ on Mg²⁺ appeared to be noncompetitive and independent of its effect on membrane permeability.     

Effect of Sugar Concentration in Jerusalem Artichoke Extract on Kluyveromyces marxianus Growth and Ethanol Production

The effect of inulin sugars concentration on the growth and ethanol production by Kluyveromyces marxianus UCD (FST) 55-82 was studied. A maximum ethanol concentration of 102 g/liter was obtained from 250 g of sugars per liter initial concentration. The maximum specific growth rate varied from 0.44 h⁻¹ at 50 g of sugar per liter to 0.13 h⁻¹ at 300 g of sugar per liter, whereas the ethanol yield remained almost constant at 0.45 g of ethanol per g of sugars utilized.     

Physiological and ecological characters studies on Aloe vera under soil salinity and seawater irrigation

Experiments were conducted in two Aloe vera cultivars (F0 and F50) to study the characters of physiology and ecology under salt stress. The results indicate decreases in tissue water, total soluble sugars and glucose, and increases in dry matter and membrane injury occurred both in F0 and F50 irrigated with 60% seawater. Less cell membrane injury were observed in F50. Moreover, total soluble sugars in F0 decreased obviously, however, no significant change in F50, while sucrose in plants had no significant change. Furthermore, F0 and F50 accumulated more inorganic cations in stems and roots. In addition, leaf K⁺ and Ca²⁺ contents were more in F50 than that in F0 to maintain normal plant growth though accumulation of Na⁺. F50 had a relative superiority in growth under salinity conditions due to higher K⁺/Na⁺ ratio and lower Na⁺/Ca²⁺ ratio than F0.     

Influence of some metal ions on the lipid content and arachidonic acid production byMortierella sp.

The influence of Ca²⁺, Mg²⁺, Mn²⁺, and Fe²⁺ ions on lipid accumulation, fatty acid composition and arachidonic acid (ARA) production byMortierella sp. S-17 was investigated. A beneficial effect of Mn²⁺ in the concentration range of 2–500 mg/L on lipogenesis was observed. The other elements at about 1 g/L repressed lipid accumulation and ARA yield. The highest yield of ARA (723 mg per liter or 148 mg per gram of dry mycelium) after incubation of the fungus in a glucose medium in the presence of 2 mg Mn²⁺ per liter was obtained. A strong inhibitory effect of Fe²⁺ (above 40 mg/L) on ARA formation was observed.     

Interactive Ion-Mediated Sap Flow Regulation in Olive and Laurel Stems: Physicochemical Characteristics of Water Transport via the Pit Structure

Sap water is distributed and utilized through xylem conduits, which are vascular networks of inert pipes important for plant survival. Interestingly, plants can actively regulate water transport using ion-mediated responses and adapt to environmental changes. However, ionic effects on active water transport in vascular plants remain unclear. In this report, the interactive ionic effects on sap transport were systematically investigated for the first time by visualizing the uptake process of ionic solutions of different ion compositions (K⁺/Ca²⁺) using synchrotron X-ray and neutron imaging techniques. Ionic solutions with lower K⁺/Ca²⁺ ratios induced an increased sap flow rate in stems of Olea europaea L. and Laurus nobilis L. The different ascent rates of ionic solutions depending on K⁺/Ca²⁺ ratios at a fixed total concentration increases our understanding of ion-responsiveness in plants from a physicochemical standpoint. Based on these results, effective structural changes in the pit membrane were observed using varying ionic ratios of K⁺/Ca²⁺. The formation of electrostatically induced hydrodynamic layers and the ion-responsiveness of hydrogel structures based on Hofmeister series increase our understanding of the mechanism of ion-mediated sap flow control in plants.     

Identification and Characterization of the Ca-ATPase which Drives Active Transport of Ca at the Plasma Membrane of Radish Seedlings

In microsomes from 24-hour-old radish (Raphanus sativus L.) seedlings ATP-dependent Ca²⁺ uptake occurs only in inside-out plasma membrane vesicles (F Rasi-Caldogno, MC Pugliarello, MI De Michelis [1987] Plant Physiol 83: 994-1000). A Ca²⁺-dependent ATPase activity can be shown in the same microsomes, when assays are performed at pH 7.5. The Ca²⁺-dependent ATPase is stimulated by the Ca²⁺ ionophore A₂₃₁₈₇ and is localized at the plasma membrane. Ca²⁺-dependent ATPase activity and ATP-dependent Ca²⁺ uptake present very similar saturation kinetics with erythrosin B (50% inhibition at about 0.1 micromolar), free Ca²⁺ (half-maximal rate at about 70 nanomolar), and MgATP (K_m 15-20 micromolar). Ca²⁺ uptake can be sustained by GTP or ITP at about 60% the rate measured in the presence of ATP; only very low Ca²⁺ uptake is sustained by CTP or UTP and none by ADP. These results indicate that the Ca²⁺-ATPase described in this paper is the enzyme which drives active transport of Ca²⁺ at the plasma membrane of higher plants.     

Calcium requirement of wheat in saline and non-saline conditions

Supplemental calcium (Ca²⁺) is used in hydroponic studies on salinity to lessen the potential for Ca²⁺ deficiency. However, the Ca²⁺ concentration and the sodium (Na⁺): Ca²⁺ ratio used vary considerably. The implications of using a wide range of Na⁺: Ca²⁺ ratios for studies of salinity tolerance in wheat are not known. Also, despite the risk of development of Ca²⁺ deficiency under salinity stress, there are few reliable reports on the critical level of Ca²⁺ which can be used to diagnose Ca²⁺ deficiency in wheat. Two experiments were conducted to examine Ca²⁺ requirements of wheat under saline and non-saline conditions and to derive a critical level for Ca²⁺. Four bread wheat genotypes (Triticum aestivum L.) and a durum wheat genotype [Triticum turgidum subsp. durum) (Desf.) Husn.] with known differences in salinity tolerance were grown at 100 mM NaCl for four weeks with varying levels of external Ca²⁺ which resulted in Na⁺:Ca²⁺ ratios of 30, 20, 15, 5 and 2. The critical Ca²⁺ concentration was defined in a second experiment by growing the same wheat genotypes at seven levels of Ca²⁺ (0.05, 0.1, 0.2, 0.5, 1, 2 and 10 mM) under non-saline conditions. When grown at 100 mM NaCl salinity tolerance was greatest when the Na⁺:Ca²⁺ ratio ranged from 5 to 15. Growing plants at lower or higher Na⁺:Ca²⁺ ratios induced nutrient imbalances and additional osmotic stress which reduced the growth of plants. Transient Ca²⁺ deficiency occurred at high Na⁺:Ca²⁺ ratios and low Mg²⁺ occurred at the lowest Na⁺:Ca²⁺ ratio. Adding NaCl raised the tissue Na⁺ concentration and reduced the Ca²⁺ concentration and the most appropriate Na⁺:Ca²⁺ ratio in the solution was that which resulted in tissue Ca²⁺ concentrations similar to those of non-salinised plants. The critical level of Ca²⁺ in the youngest fully emerged leaf blades was 15–23 mmol kg^-1 DW (600–900 mg kg^-1 DW).     

Negative Effect of High pH on Biocidal Efficacy of Copper and Silver Ions in Controlling Legionella pneumophila

Copper-silver (Cu-Ag) ionization has effectively controlled Legionella spp. in the hot water systems of numerous hospitals. However, it was ineffective at controlling Legionella in one Ohio hospital despite the confirmation of adequate total concentrations of copper and silver ions. The pH of the water at this hospital was found to be 8.5 to 9.0. The purpose of this study was to investigate the impact of pH and other water quality parameters, including alkalinity (HCO₃⁻), hardness (Ca²⁺ and Mg²⁺), and amount of dissolved organic carbon (DOC), on the control of Legionella by Cu-Ag ionization. Initial concentrations of Legionella and copper and silver ions used in batch experiments were 3 × 10⁶ CFU/ml and 0.4 and 0.08 mg/liter, respectively. Changes in bicarbonate ion concentration (50, 100, and 150 mg/liter), water hardness (Ca²⁺ at 50 and 100 mg/liter; Mg²⁺ at 40 and 80 mg/liter), and level of DOC (0.5 and 2 mg/liter) had no significant impact on the efficacy of copper and silver ions in killing Legionella at a neutral pH. When the pH was elevated to 9 in these experiments, copper ions achieved only a 10-fold reduction in the number of Legionella organisms in 24 h, compared to a millionfold decrease at pH 7.0. Silver ions were able to achieve a millionfold reduction in 24 h at all ranges of water quality parameters tested. Precipitation of insoluble copper complexes was observed at a pH above 6.0. These results suggest that pH may be an important factor in the efficacy of copper-silver ionization in controlling Legionella in water systems.     

Effect of salinity on tomato fruit ripening

Tomato (Lycopersicon esculentum Mill) plants from various cultivars growing on half-strength Hoagland solution were exposed at anthesis to 3 or 6 grams per liter NaCl. Salinity shortened the time of fruit development by 4 to 15%. Fruits of salt-treated plants were smaller and tasted better than did fruits of control plants. This result was obtained both for ripe fruits tested on the day of picking and for those picked at 100% development and allowed to ripen at room temperature for 9 days. Percentage of dry weight, total soluble solids, and titratable acidity; content of reducing sugars, Cl⁻, Na⁺, and various pericarp pigments; and electrical conductivity of the juice were higher in fruits of saline-treated plants than they were in those of control plants, while the pH was lower. Ethylene and CO₂ evolution rates during ripening; as well as the activities of pectin methyl esterase, polymethylgalacturonase, and polygalacturonase; were also higher in fruits of the saline-treated plants. The treatment with 6 grams per liter NaCl shortened the fruit shelf life considerably.     

Metabolism of Separated Leaf Cells: III. Effects of Calcium and Ammonium on Product Distribution During Photosynthesis with Cotton Cells

Separated mesophyll cells from cotton (Gossypium hirsutum var. Stoneville 1613 Glandless) were isolated with pectinase and mechanical agitation. The separated cells had rates of light-dependent CO₂ fixation between 50 to 100 μmoles CO₂ per mg chlorophyll per hour. The presence of Ca²⁺ in the incubation medium did not significantly affect the type of photosynthetic products formed, but 2 mm Ca²⁺ did cause a 50% decrease in the appearance of photosynthetic products in the incubation medium. The movement of all types of products (sugars, organic, and amino acids) out of the cells was reduced similarly by the Ca²⁺. Light had no affect on the movement of products out of the cells, whereas 1 mm ethylenediaminetetra-acetate greatly increased the movement. The addition of 1.6 mm NH₄Cl to the cell suspensions caused a large increase in the amount of fixed ¹⁴C appearing in the amino acid fraction and a decrease in the sugar fraction. These metabolic changes in the cells were reflected in the movement of products out of the cells so that the incubation medium also contained a larger amount of label in amino acids and a smaller amount in sucrose. Although the cell plasma membrane restricted the movement of soluble products, it did not discriminate significantly between the types of products moved.     

Ca transport in membrane vesicles from pinto bean leaves and its alteration after ozone exposure

The influence of ozone on Ca²⁺ transport in plant membranes from pinto bean (Phaseolus vulgaris L. var Pinto) leaves was investigated in vitro by means of a filtration method using purified vesicles. Two transport mechanisms located at the plasma membrane are involved in a response to ozone: (a) passive Ca²⁺ influx into the cell and (b) active Ca²⁺ efflux driven by an ATP-dependent system, which has two components: a primary Ca²⁺ transport directly linked to ATP which is partially activated by calmodulin and a H⁺/Ca²⁺ antiport coupled to activity of a H⁺-ATPase. The passive Ca²⁺ permeability is increased by ozone. A triangular pulse of ozone stimulates a higher influx of Ca²⁺ than does a square wave, even though the total dose was the same (0.6 microliter per liter × hour). Leaves exposed to a square wave did not exhibit visible injury and were still able to recover from oxidant stress by activation of calmodulin-dependent Ca²⁺ extrusion mechanisms. On the other hand, leaves exposed to a triangular wave of ozone, exhibit visible injury and lost the ability of extruding Ca²⁺ out of the cell.     

A quench-flow kinetic investigation of calcium ion accumulation by isolated cardiac sarcoplasmic reticulum. Dependence of initial velocity on free calcium ion concentration and influence of preincubation with a protein kinase,MgATP, and cyclic AMP

Ca²⁺ accumulation at pH 6.8 by isolated rabbit heart microsomes derived chiefly from sarcoplasmic reticulum was investigated by a quench-flow technique. The reaction was terminated at preset times by addition to the reaction mixture of an equal volume of 10 to 50 mM ethyleneglycol-bis-(β-aminoethyl ether)-N,N′-tetraacetic acid buffered at pH 6.0. The initial velocity of Ca²⁺ accumulation by microsomal preparations exhibiting a steady state Ca²⁺ accumulation of 25.6 nmol Ca²⁺/mg increased from 3.67 to 33.4 nmol Ca²⁺/mg · s as the free Ca²⁺ concentration was raised from 0.2 to 18.9 μM. Preincubation of the cardiac microsomes with a partly purified soluble cardiac cyclic AMP-dependent protein kinase, MgATP, and cyclic AMP lead to a significant increase in the initial Ca²⁺ accumulation rate. The amounts of Ca²⁺ that were found to accumulate in the first 200 ms of the reaction are comparable to the quantities of the ion that according to literature data need to be removed from the myofilaments and the myoplasm for induction of relaxation of the myocardial fibers.     

Characteristics of Ca2+ transport by corn mitochondria

Mitochondria isolated from corn (Zea mays L.) coleoptiles by an improved procedure which yields functionally intact preparations are much more active in respiration-coupled Ca²⁺ accumulation than those employed in most earlier studies. Ca²⁺ uptake by these mitochondria is phosphate-dependent and is accompanied by decrease in Δψ, H⁺ extrusion and increase in the rate of respiration. A sigmoidal plot with a Hill coefficient of 2.22 was obtained when the rates of Ca²⁺ uptake were plotted as a function of free Ca²⁺ concentration. The K_0.5 for Ca²⁺ influx was about 31 μM and a V_max of 140 nmol Ca²⁺ per min per mg was attained at a free-Ca²⁺ concentration of about 120 μM. Ca²⁺ uptake is sensitive to inhibition by ruthenium red and Mg²⁺. The external free-Ca²⁺ concentration maintained at steady state was about 2 μM and was independent of the respiratory substrate and of external Na⁺, but was increased by exogenous Mg²⁺. In addition, this preparation of corn mitochondria has shown a much higher ability for Ca²⁺ retention in the presence of phosphate and NAD(P)H oxidants than liver mitochondria.     

ATP-dependent calcium transport and its correlation with Ca2+-ATPase activity in basolateral plasma membranes of rat duodenum

Isolated basolateral plasmamembrane vesicles from rat duodenum epithelial cells exhibit ATP-dependent calcium-accumulation and Ca²⁺-dependent ATPase activity. Calcium accumulation stimulated by ATP is prevented by the calcium ionophore A23187, inhibited 80% by 0.1 mM orthovanadate but is not effected by oligomycin. Calcium accumulation is not observed with the substrate β-γ-(CH₂)-ATP, ADP and p-nitrophenyl phosphate. Kinetic studies reveal an apparent K_m of 0.2 μM Ca²⁺ and a V_max of 5.3 nmol Ca²⁺/min per mg protein for the ATP-dependent calcium-uptake system. Calmodulin and phenothiazines have no effect on calcium accumulation in freshly prepared membranes, but small effects are inducable after a wash with a 5 mM EGTA. The kinetic parameters of Ca²⁺-ATPase are: K_m = 0.25 μM Ca²⁺ and V_max = 19.2 nmol P_i/min per mg protein. Three techniques, osmotic shock, treatment with Triton X-100 or the channel-forming peptide alamethacin, reveal that about 40% of the vesicles are resealed. Assuming that half of the resealed vesicles have an inside-out orientation, the V_max of ATP-dependent calcium uptake amounts to 25 nmol Ca²⁺/min per mg protein and of the Ca²⁺-ATPase to 23 nmol P_i/min per mg protein. The close correlation between kinetic parameters of Ca²⁺-ATPase and ATP-dependent calcium-transport strongly suggests that both systems are expressions of a Ca²⁺-pump located in duodenal basolateral plasma membranes.     

Effects of Acibenzolar-S-Methyl Application to Rotylenchulus reniformis and Meloidogyne javanica

Effects of acibenzolar-s-methyl, an inducer of systemic acquired resistance in plants, on Rotylenchulus reniformis and Meloidogyne javanica in vitro and in vivo were determined. A single foliar application of acibenzolar at 50 mg/liter (5 ml of solution per plant) to 7-day-old cowpea or soybean seedlings decreased R. reniformis and M. javanica egg production by 50% 30 days after inoculation. The mechanism of acibenzolar on plant-parasitic nematodes was then investigated. Acibenzolar at 50 to 200 mg/liter did not affect movement of R. reniformis and M. javanica or penetration of second-stage juveniles (J2) of M. javanica on cowpea. However, M. javanica development was slowed and fecundity was reduced in plants treated with acibenzolar. On average, 50% of J2 that penetrated acibenzolar-treated cowpeas developed into mature females with eggs, whereas the other 50% exhibited arrested development. The number of eggs per egg mass was 450 in water-treated cowpeas, whereas the number declined to 250 in acibenzolar-treated plants. Acibenzolar may be responsible for stimulating the plants to express some resistance to the nematodes.     

Calcium uptake into acini from rat pancreas: Evidence for intracellular ATP-dependent calcium sequestration

Summary Intracellular ATP-dependent Ca²⁺ sequestration mechanisms were studied in isolated dispersed rat pancreatic acini following treatment with saponin or digitonin to disrupt their plasma membranes. In the presence of⁴⁵Ca²⁺ concentrations <10^–6 mol/liter, addition of 5 mmol/liter ATP caused a rapid increase in⁴⁵Ca²⁺ uptake exceeding the control by fivefold. ADP mimicked the ATP effect by 50 to 60%, whereas other nucleotides such as AMP-PNP, AMP-PCP, CTP, UTP, ITP, GTP, cAMP and cGMP did not. Maximal ATP-promoted Ca²⁺ uptake was obtained at 10^–5 mol/liter Ca²⁺ uptake by mitochondrial inhibitors was dependent on the Ca²⁺ concentration, indicating the presence of different Ca²⁺ storage systems. Whereas the apparent half-saturation constant found for mitochondrial Ca²⁺ uptake was 4.5×10^–7 mol/liter, in the presence of antimycin and oligomycin (nonmitochondrial uptake) it was 1.4×10^–8 mol/liter. In the absence of Mg²⁺ both ATP- and ADP-promoted Ca²⁺ uptake was nearly abolished. The Ca²⁺ ionophore and mersalyl blocked Ca²⁺ uptake. Electron microscopy showed electrondense precipitates in the rough endoplasmic reticulum of saponintreated cells in the presence of Ca²⁺, oxalate and ATP, which were absent in intact cells and in saponin-cells without ATP or pretreated with A23187. The data suggest the presence of mitochondrial and nonmitochondrial ATP-dependent Ca²⁺ storage systems in pancreatic acini. The latter is likely to be located in the rough endoplasmic reticulum.     

Calcium transport by ehrlich ascites cell mitochondria in vitro and in situ

The rate, maximum extent of accumulation, and passive release of Ca²⁺ by mitochondria within Ehrlich ascites tumor cells treated with digitonin and by isolated tumor mitochondria have been compared. The mitochondrial protein content of Ehrlich cells was determined by cytochrome and cytochrome oxidase analyses. The Ca²⁺ uptake rate in situ is approximately one-half the rate in vitro whereas maximum Ca²⁺ accumulation by mitochondria within the cell is about twice the value for isolated mitochondria. When isolated tumor mitochondria were supplemented with exogenous ATP the maximum uptake (approximately 3.0 μeq Ca²⁺/mg protein) was about the same as in situ. Adenine nucleotides retained in digitonized cells may account for the observed differences. The rate of uncoupler stimulated Ca²⁺ release from mitochondria within the cell (ca. 10 neq Ca²⁺/min · mg mitochondrial protein for Ca²⁺ loads up to 800 neq Ca²⁺/mg protein) agrees exceptionally well with previous estimates for isolated tumor mitochondria. Therefore the capacity for extensive Ca²⁺ accumulation without uncoupling and attenuation of Ca²⁺ efflux are virtually the same in the cell as in vitro.     

The isolation of coupled mitochondria from Physarum polycephalum and their response to Ca2+

A method for the isolation of coupled mitochondria from the acellular slime mould Physarum polycephalum is described. The mitochondria oxidize respiratory substrates at rates comparable to those of mitochondria from other micro-organisms and show similar responses to respiratory inhibitors. ADP/O values approach similar values to those obtained with mitochondria from higher organisms: 3 with NAD-linked substrates, 2 with succinate, and 1 with ascorbate-TMPD.Mitochondria actively take up low concentrations of Ca²⁺ with stimulation of their respiration. With succinate or pyruvate-malate as substrates respiratory responses are depressed by Ca²⁺ concentrations in excess of 200 μM in the presence or absence of phosphate.Exogenous NADH is unique in supporting the uptake of large amounts of Ca²⁺ in the presence of phosphate and in showing an unusual ‘uncoupled’ response in the absence of phosphate.A sigmoidal relationship occurs between initial velocity of Ca²⁺ uptake and Ca²⁺ concentration with a maximum velocity of approx. 15 nmol/s per mg protein and half maximum velocity occurring at approx. 50 μM Ca²⁺.     

Acclimation of Two Tomato Species to High Atmospheric CO(2): II. Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase and Phosphoenolpyruvate Carboxylase

Lycopersicon esculentum Mill. cv Vedettos and Lycopersicon chmielewskii Rick, LA 1028, were exposed to two CO₂ concentrations (330 or 900 microliters per liter) for 10 weeks. The elevated CO₂ concentrations increased the initial ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity of both species for the first 5 weeks of treatment but the difference did not persist during the last 5 weeks. The activity of Mg²⁺-CO₂-activated Rubisco was higher in 900 microliters per liter for the first 2 weeks but declined sharply thereafter. After 10 weeks, leaves grown at 330 microliters per liter CO₂ had about twice the Rubisco activity compared with those grown at 900 microliters per liter CO₂. The two species showed the same trend to Rubisco declines under high CO₂ concentrations. The percent activation of Rubisco was always higher under high CO₂. The phosphoenolpyruvate carboxylase (PEPCase) activity measured in tomato leaves averaged 7.9% of the total Rubisco. PEPCase showed a similar trend with time as the initial Rubisco but with no significant difference between nonenriched and CO₂-enriched plants. Long-term exposure of tomato plants to high CO₂ was previously shown to induce a decline of photosynthetic efficiency. Based on the current study and on previous results, we propose that the decline of activated Rubisco is the main cause of the acclimation of tomato plants to high CO₂ concentrations.