Ribulose Bisphosphate Carboxylase Activity Research Articles

Effect of Heat Stress During Grain Filling on Phosphoenolpyruvate Carboxylase and Ribulose-1,5-bisphosphate Carboxylase/Oxygenase Activities of Various Green Organs in Winter Wheat

In two winter wheat (Triticum aestivum L.) cultivars differing in their response to high temperature, JD8 (tolerant) and J411 (sensitive) we studied the effect of heat stress on the activities of phosphoenolpyruvate carboxylase (PEPC) and ribulose-1,5-bisphosphate carboxylase (RuBPC) in green organs during grain-filling. There were significantly higher PEPC activities and lower RuBPC activities in each of the non-leaf organs (awn, glume, lemma, peduncle, and sheath) than in the flag leaf blade. Under heat stress for 12 d, the activity of RuBPC quickly declined and the activity of PEPC first increased and later declined in all organs, resulting in a great increase of the PEPC/RuBPC ratios in the organs, particularly in non-leaf organs which had a higher PEPC/RuBPC than the flag leaf blade in all times. The PEPC activity and PEPC/RuBPC ratio in every organ of JD8 were higher than those in the same organ of J411. Thus the differences in PEPC activities and PEPC/RuBPC may be associated with the differences in photosynthetic heat tolerance among the organs of the same plant or between the two cultivars.

Role of white light in reversing UV-B-mediated effects in the N 2-fixing cyanobacterium Anabaena BT2

The effects of various irradiances of artificial UV-B (280–315 nm) in the presence or absence of visible light (photosynthetically active radiation) on growth, survival, 14CO 2 uptake and ribulose 1,5-bisphosphate carboxylase (RuBISCO) activity were studied in the N 2-fixing cyanobacterium Anabaena BT2. We tested the hypothesis whether or not visible radiation offers any protection against UV-B-induced deleterious effects on growth and photosynthesis in Anabaena BT2. Attempts were also made to determine the irradiances of UV-B where inhibitory effects could be mitigated by simultaneous irradiation with visible light. Exposure of cultures to 0.2 W m −2 or higher irradiance of UV-B caused inhibition of growth and survival and growth ceased above 1.0 W m −2. 14CO 2 uptake and RuBISCO activity were found to be more sensitive to UV-B and around 60% reduction in 14CO 2 uptake and RuBISCO activity occurred after exposure of cultures to 0.4 W m −2 for 1 h. However, growth, 14CO 2 uptake and RuBISCO activity were nearly normal when UV-B (0.4 W m −2) and visible light (14.4 W m −2) were given simultaneously. Blue radiation (450 nm) was found to be the most effective in photoreactivation against UV-B, better than UV-A or any other light wavelength band. Our results demonstrate that the studied cyanobacterium possesses active photoreactivation mechanism(s) against UV-B-mediated damage which in turn probably allow survival under natural conditions in spite of being continuously exposed to the UV-B component present in the solar radiation. Continued growth of many algae and cyanobacteria in the presence of intense solar UV-B radiation under natural conditions seems to be due to the active role of photoreactivation.

Activity of the enzymes of carbon metabolism in Sulfobacillus sibiricus under various conditions of cultivation

The thermoacidophilic iron-oxidizing chemolithotroph Sulfobacillus sibiricus N1T is characterized by steady growth and amplified cell yield when grown in vigorously aerated medium containing Fe2+, glucose, and yeast extract as energy sources. In this case, carbon dioxide, glucose, and yeast extract are used as carbon sources. Glucose is assimilated through the fructose-bisphosphate pathway and the pentose-phosphate pathway. Glyoxylate bypass does not function in S. sibiricus, and the tricarboxylic acid cycle is disrupted at the level of 2-oxoglutarate dehydrogenase. The presence of ribulose-bisphosphate carboxylase indicates that carbon dioxide fixation proceeds through the Calvin cycle. The activity of ribulose-bisphosphate carboxylase is highest in autotrophically grown cells. The cells also contain pyruvate carboxylase, phosphoenolpyruvate carboxylase, phosphoenolpyruvate carboxykinase, and phosphoenolpyruvate carboxytransphosphorylase.

Activities of Carbonic Anhydrase and Ribulose-1,5-Bisphosphate Carboxylase, and Dry Mass Accumulation in Brassica juncea following Defoliation

The effect of 30 % defoliation of shaded leaves in lower layers of plant was studied on activities of carbonic anhydrase (CA) and ribulose-1,5-bisphosphate carboxylase (RuBPC), leaf dry mass per unit leaf area, and plant dry mass of mustard (Brassica juncea). Removal of 30 % of leaves resulted in increased CA and RuBPC activities of leaves, and leaf and plant dry masses.

Physiological study of declining Pinus cembra (L.) trees in southern France

Pinus cembra (L.) trees growing at an elevation of 1,800 m in the Mercantour Alps National Park in southern France have shown various degrees of damage (yellowing and needle loss) since the early 1980s. Two stands were selected, one populated by healthy and the other by damaged trees, and 1-year-old needles were analysed. The needles of the declining trees displayed chlorotic mottled patterns associated with a significantly lower level of total chlorophyll compared to healthy trees. Measurements of gas exchange showed a lower photosynthetic rate linked to a lower activity of ribulose bisphosphate carboxylase and a higher stomatal conductance in needles of declining trees compared to healthy trees. In contrast, phosphoenolpyruvate carboxylase activity was found to be higher in declining tree needles, reaching twice the level measured in healthy tree needles. A significant oxidative stress was also observed in the needles which presented a 68% higher malondialdehyde content than healthy tree needles. The atmospheric concentration of ozone was quite high (40 nl·l–1, monthly mean in summer) in both stands but preliminary results based on stomatal conductance and ozone concentration measurements suggested a higher ozone flux in needles of declining trees. Ozone is thus suspected as a contributing factor in the observed decline of Pinus cembra trees.

Effects of Fruit Load on Photosynthesis and Ribulose 1,5-Bisphosphate Carboxylase Activity in Satsuma Mandarin (Citrus unshiu Marc.) Trees under Long-term Elevated Carbon Dioxide.

The effects of long-term carbon-dioxide (CO2) enrichment on photosynthetic rate (Pn), ribulose 1, 5-bisphosphate carboxylase (RuBPCO) activity, chlorophyll content, and carbon accumulation in leaves were investigated in potted satsuma mandarin (Citrus unshiu Marc.) trees with and without fruit load. The Pn of whole satsuma mandarin trees at 1, 000 μ1 CO2·liter-1 for the first 13 days increased significantly, up to about 200%, compared to that at a normal atmospheric CO2 (360μ1 CO2·liter-1). After that, Pn began to decrease gradually until it reached 170% on the 35th day. The Pn of the bearing trees remained higher than those of non-bearing trees. Although there was no difference in the chlorophyll content, the bearing trees had smaller specific leaf weight (SLW) and carbon content in the leaves. It seemed that the decrease in Pn during the long-term exposure to the elevated CO2 concentration was partly due to a decrease in RuBPCO activity and/or an accumulation of photoassimilates in the leaves. The sink capacity of fruit may be important for the maintenance of Pn in long-term exposure to an elevated CO2 concentration because the non-bearing trees had a particularly rapid decrease in Pn.

The Enzymes of Carbon Metabolism in the Thermotolerant Bacillar Strain K1

To determine enzymatic activities in the thermotolerant strain K1 (formerly “Sulfobacillus thermosulfidooxidans subsp. thermotolerans”), it was grown in a mineral medium with (1) thiosulfate and Fe2+ or pyrite (autotrophic conditions), (2) Fe2+, thiosulfate, and yeast extract or glucose (mixotrophic conditions), and (3) yeast extract (heterotrophic conditions). Cells grown mixo-, hetero-, and autotrophically were found to contain enzymes of the tricarboxylic acid (TCA) cycle, as well as malate synthase, an enzyme of the glyoxylate cycle. Cells grown organotrophically in a medium with yeast extract exhibited the activity of the key enzymes of the Embden–Meyerhof–Parnas and Entner–Doudoroff pathways. The increased content of carbon dioxide (up to 5 vol %) in the auto- and mixotrophic media enhanced the activity of the enzymes involved in the terminal reactions of the TCA cycle and the enzymes of the pentose phosphate pathway. Carbon dioxide is fixed in the Calvin cycle. The highest activity of ribulose bisphosphate carboxylase was detected in cells grown autotrophically at the atmospheric content of CO2 in the air used for aeration of the growth medium. The activities of pyruvate carboxylase, phosphoenolpyruvate carboxylase, phosphoenolpyruvate carboxykinase, and phospho-enolpyruvate carboxytransphosphorylase decreased with increasing content of CO2 in the medium.

Cytoplasmic effects on photosynthesis and ribulose-1,5-bisphosphate carboxylase activity in Brassica species

Brassica genotypes representing four different species viz. B. nigra and B. campestris (both primary diploids), B. juncea and B. carinata (both allotetraploids) were examined for photosynthesis rate (Pn), ribulose-1,5-bisphosphate carboxylase (RuBPC) activity, leaf soluble protein and chlorophyll content. B. nigra is the male and female parent of B. juncea and B. carinata, respectively. B. campestris is the maternal parent of B. juncea. Pn was significantly higher in B. nigra and B. carinata compared to the other two species. These two species also sustained Pn of the leaves for a longer period during ontogeny. When B. nigra was the male parent, the higher Pn of B. nigra was not inherited in B. juncea. On the other hand, the Pn and pattern of ontogenetic changes in B. campestris and B. juncea were more or less similar. In specific RuBPC activity on soluble protein basis, chlorophyll and soluble protein content B. carinata also followed B. nigra whereas, B. juncea followed B. campestris. These results indicated the possible maternal(cytoplasmic) influence on the inheritance of these traits.

The mechanism to suppress photosynthesis through end-product inhibition in single-rooted soybean leaves during acclimation to CO(2) enrichment.

Single-rooted soybean leaves were used to investigate the suppression of photosynthesis through end-product inhibition during acclimation to CO(2 )enrichment. The photosynthetic activity was greater in leaves cultured at a CO(2) partial pressure of 70 Pa (high-CO(2)) than that in the leaves cultured at 35 Pa CO(2) (control) during the initial exposure to CO(2) enrichment but then decreased rapidly with a large accumulation of starch, to well below the level of the control leaves. The response curve of photosynthesis (A) to the intercellular CO(2) concentration (Ci) in the high-CO(2) leaves cultured long-term exhibited a significantly low initial gradient. However, on exposure to darkness for 48 h, the initial gradient of the A to Ci curve and rate of photosynthesis were completely restored, and almost all of the accumulated starch was expended. The ribulose bisphosphate carboxylase (RuBPcase) content and activation ratio in the high-CO(2) leaves remained high and roughly constant during the experiment, and were unchanged by the exposure, while this enzyme was slightly inactivated or inhibited after long-term exposure to CO(2) enrichment. The lower rate of photosynthesis in the high-CO(2) leaves could be linearly increased to a rate approaching the control level by increasing the external atmospheric [CO(2)], which thereby compensated for a reduced CO(2) transfer diffusion from the intercellular space to the stroma in chloroplasts. It is consequently concluded that, during the acclimation to CO(2 )enrichment, the suppression of photosynthesis through end-product inhibition was mainly caused by a lowering of the carboxylation efficiency of RuBPcase due to hindrance of CO(2) diffusion from the intercellular space to the stroma in chloroplasts brought about by the large accumulation of starch.

The Cause of the Difference in Leaf Net Photosynthetic Rate between Two Soybean Cultivars

To explore the cause of difference in photosynthetic performance between different cultivars of crops, leaf net photosynt rate (PN) and photosystem 2 (PS2) photochemical efficiency (Fv/Fm), apparent quantum yield of carbon assimilation (φc), electron transport rate, photophosphorylation activity, etc. were measured in two soybean cultivars, Heinong 42 and Heinong 37. At pod setting and filling, significant differences in PN between them were observed. The former with a higher PN (from 7 to 38 %) had a significantly higher leaf thickness, leaf dry mass/area (LMA), chlorophyll content, soluble protein content, apparent quantum yield of electron transport through PS2 (φe), carboxylation efficiency (CE), and ribulose-1,5-bisphosphate carboxylase (RuBPC) activity. The significantly higher PN of Heinong 42 is mainly due to its higher content and activity of RuBPC.

Activities of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase and Phosphoenolpyruvate Carboxylase, and Oxygen Evolution in Transgenic Tobacco Plants

Three clones of tobacco transformed with the T-DNA of Agrobacterium rhizogenes Ri plasmid A4b cultivated in vitro on a solid agar medium neither showed pronounced morphological diversity nor significantly differed in chlorophyll (Chl) contents from control plants. However, the transformation caused a 27 to 83 % decay in leaf oxygen evolution and in both ribulose-1,5-bisphosphate carboxylase (RuBPC) and phosphoenolpyruvate carboxylase (PEPC) activities. Therefore, the transformation brought about a reduction of active PEPC as well as activable RuBPC amounts in plant tissues. Individual clones did not mutually differ. In tobacco transformed with A. rhizogenes strain TR101 and grown in soil only the mean leaf area tended to reduce. Chl contents, Chl a/b ratio, oxygen evolution, and activities of both RuBPC and PEPC were insignificantly affected by the transformation.

Mulberry Leaf Metabolism under High Temperature Stress

Effects of high temperature on the activity of photosynthetic enzymes and leaf proteins were studied in mulberry (Morus alba L. cv. BC2-59). A series of experiments were conducted at regular intervals (120, 240 and 360 min) to characterize changes in activities of ribulose-1,5-bisphosphate carboxylase (RuBPC) and sucrose phosphate synthase (SPS), photosystem 2 (PS 2) activity, chlorophyll (Chl), carotenoid (Car), starch, sucrose (Suc), amino acid, free proline, protein and nucleic acid contents in leaves under high temperature (40 °C) treatments. High temperature markedly reduced the activities of RuBPC and SPS in leaf extracts. Chl content and PS 2 activity in isolated chloroplasts were also affected by high temperature, particularly over 360 min treatment. Increased leaf temperature affected sugar metabolism through reductions in leaf starch content and sucrose-starch balance. While total soluble protein content decreased under heat, total amino acid content increased. Proline accumulation (1.5-fold) was noticed in high temperature-stressed leaves. A reduction in the contents of foliar nitrogen and nucleic acids (DNA and RNA) was also noticed. SDS-PAGE protein profile showed few additional proteins (68 and 85 kDa) in mulberry plants under heat stress compared to control plants. Our results clearly suggest that mulberry plants are very sensitive to high temperature with particular reference to the photosynthetic carbon metabolism.

Carbon Fixation by the Peculiar Marine Diatom Haslea Ostrearia

During batch culture of Haslea ostrearia the highest carbon (14C) fixation rate was found in vivo in cells that did not accumulate the blue pigment marennine (green form). This fixation rate decreased concomitantly with the accumulation of marennine. In vitro, no phosphoenolpyruvate carboxylase (PEPC) activity was detected, but nearly equivalent activities of ribulose-1,5-bisphosphate carboxylase (RuBPC) and phosphoenolpyruvate carboxykinase (PEPCK) were found in the green form. However, the activity of RuBPC was lower than that of PEPCK during marennine accumulation. In vitro carboxylase activities were strongly inhibited by the addition of a marennine extract. A full description of this inhibition could not be confirmed within the cells because marennine accumulates in small cytoplasmic vesicles.

Effect of Iron Deficiency Induced Changes on Photosynthetic Pigments, Ribulose-1,5-Bisphosphate Carboxylase, and Photosystem Activities in Field Grown Grapevine (Vitis Vinifera L. cv. Pinot Noir) Leaves

The effect of iron deficiency on photosynthetic pigments, ribulose-1,5-bisphosphate carboxylase (RuBPC), and photosystem activities were investigated in field grown grapevine (Vitis vinifera L. cv. Pinot noir) leaves. The contents of chlorophyll (Chl) (a+b) and carotenoids per unit fresh mass showed a progressive decrease upon increase in iron deficiency. Similar results were also observed in content of total soluble proteins and RuBPC activity. The marked loss of large (55 kDa) and small (15 kDa) subunits of RuBPC was also observed in severely chlorotic leaves. However, when various photosynthetic electron transport activities were analysed in isolated thylakoids, a major decrease in the rate of whole chain (H2O → methyl viologen) electron transport was observed in iron deficient leaves. Such reduction was mainly due to the loss of photosystem 2 (PS2) activity. The same results were obtained when Fv/Fm was evaluated by Chl fluorescence measurements in leaves. Smaller inhibition of photosystem 1 (PS1) activity was also observed in both mild and severely chlorotic leaves. The artificial electron donors, diphenyl carbazide and NH2OH, markedly restored the loss of PS2 activity in severely chlorotic leaves. The marked loss of PS2 activity was evidently due to the loss of 33, 23, 28-25, and 17 kDa polypeptides in iron deficient leaves.

Effect of Potassium, Magnesium, and Calcium Deficiencies on Nitrogen Constituents and Chloroplast Components in Citrus Leaves

The chlorotic appearance of mineral-deficient citrus leaves presumably reflects degradative changes in chloroplast components, most of which have nitrogen as a principal constituent. To examine this assumption the size of some major nitrogen pools, the SDS-PAGE pattern of soluble and chloroplast membranal proteins, and the activities of nitrate reductase and ribulose bisphosphate carboxylase (RuBPcase) were determined in leaves of rough-lemon (Citrus volkameriana Ten. & Pasq) plants grown hydroponically for 3 to 10 months under K, Mg, and Ca deficiencies. Plants grown under minerally deficient conditions produced less biomass. Leaves developing under K, Mg, and Ca-deficient conditions had significantly reduced concentrations of the respective elements. Chlorophyll levels of the chlorotic Mg and Ca-deficient leaves were lower than those of control leaves but chlorophyll a/b ratios were not markedly different. Calcium deficiency caused significant decreases in total nitrogen, nitrates, and the free amino acid pool. Proline which is the major component of the free amino acid pool decreased by 82.5%. Calcium-deficient leaves had significantly lower nitrate reductase and RuBPcase activities. The level of RuBPcase holoenzyme and its subunits were also reduced. Protein levels of K, Mg, and Ca-deficient leaves were not significantly altered. The SDS-PAGE patterns of soluble and chloroplast membranal proteins did not reveal major qualitative changes. In conclusion, the data do not demonstrate a general close link between chlorosis of minerally deficient citrus leaves and nitrogen metabolism. Calcium deficiency seems to specifically interfere with early stages of nitrogen assimilation and free amino acid accumulation but the metabolic integrity of the leaf is apparently maintained even under severe nutritional stress conditions.

The photosynthetic characteristics of differently shaped leaves in Populus euphratica Olivier

There is a distinct leaf shape polymorphism within a single plant of P. euphratica Olivier. The anatomical structure, carbon isotope discrimination (Δ13C), and stomatal and photosynthetic behaviour were investigated in broad-ovate (BOL) and lanceolate (LL) leaves, located at the top and bottom in crown, respectively, of a mature Euphrates poplar growing in its native habitat. Both types of leaves had a non-Kranz anatomy and low Δ13C values. However, Δ13C of a LL was in average 3.2‰ larger than that of a BOL. In comparison with the LL, the BOL had a smaller stomatal conductance, causing subsequent decreases in transpiration rate and ratio of CO2 concentrations in intercellular spaces to air. Carbon assimilation rate and water use efficiency were higher in the BOLs than in the LLs. The BOL exhibited C4-like enzymological features, the activity of glycollate oxidase, and the ratio of activities of ribulose-1,5-bisphosphate carboxylase (RuBPC) to phosphoenolpyruvate carboxylase (PEPC) was lower in BOL than in LL throughout the whole growing season. The lowered ratio of RuBPC/PEPC in BOL was mainly associated with a marked decline in the activity of RuBPC, and only a slight increase in the activity of PEPC. These differences might contribute to microclimate adaptation in both types of leaves.

Photosynthetic acclimation to elevated CO2 in wheat cultivars

Wheat (T. aestivum) cvs. Kalyansona and Kundan grown under atmospheric (CA) and elevated CO2 concentrations (650±50 cm3 m-3 - CE) in open top chambers were examined for net photosynthetic rate (PN), stomatal limitation (l s) of P N, ribulose-1,5-bisphosphate carboxylase (RuBPC) activity, and saccharide content of the leaves. The P N values of both CA- and CE-grown plants compared at the same CO2 concentration showed a down regulation under CE at the post-anthesis stage. The negative acclimation of P N appeared to be due to both stomatal and mesophyll components, and the RuBPC activity got also adjusted. There was a decrease in activation state of RuBPC under CE. In connection with this, an increased accumulation of saccharides in wheat leaf under CE was observed. Kalyansona, owing to its larger sink potential in terms of the number of grains, showed a greater enhancement under CE in both post-ear emergence dry matter production and grain yield. Under CE, this cultivar also showed a lower down regulation of P N than Kundan.

Photosynthesis and growth of Erythrina variegata as affected by water stress and triacontanol

Erythrina variegata Lam. seedlings were grown under water stress (Ψ = -3.2 MPa) and subsequently sprayed with triacontanol (Tria). Water stress significantly reduced shoot growth rate, while roots continued to grow. Content of chlorophyll (Chl) a decreased more than that of Chl b. Water stress also reduced photosynthetic activity of chloroplasts as measured by Chl fluorescence induction. Stress effect was identified at the oxidation site of photosystem (PS) 2 prior to the hydroxylamine donating site and perhaps close to or after the diphenylcarbazide donating site. The loss of O2 evolving thylakoid polypeptides (33, 23, 17 kDa) and the large (55 kDa) and small (15 kDa) subunits of ribulose-1,5-bisphosphate carboxylase (RuBPC) were found in water stressed seedlings. The reduction in RuBPC activity was accompanied by reduction of CO2 fixation and stomatal conductance. All photosynthetic parameters were improved by Tria.

Impacts of ultraviolet-B irradiation on nitrogen-fixing cyanobacteria of rice paddy fields

Summary The impact of ultraviolet-B (UV-B; 280–315 nm) irradiation on ribulose 1,5-bisphosphate carboxylase (RuBISCO) activity, membrane permeability, NaH14CO3 uptake and isolated phycocyanin has been studied in five N2-fixing cyanobacterial strains, viz., Anabaena sp., Nostoc sp., Nostoc carmium, Nostoc commune and Scytonema sp., isolated from Indian rice paddy fields. UV-B exposure for as little as 30 min resulted in a considerable decrease in the RuBISCO activity, which further decreased with increasing exposure time. Similarly, NaH14CO3 uptake was also severely affected by UV-B irradiation in all cyanobacterial strains studied so far. Isolated phycocyanin (λmax 620 nm) of Anabaena sp. showed a drastic decline in absorbance with increasing exposure time. Fluorescence excitation at 620 nm resulted in an emission at 640 nm that decreased as well and showed a gradual shift towards shorter wavelengths with increasing UV-B exposure time, indicating an impaired energy transfer from the accessory pigment, phycocyanin, to the photosynthetic reaction centers. SDS-PAGE analyses of the isolated phycocyanin revealed a loss in the αβ monomers (20kDa), the rod-core linker polypeptides (45kDa) and the core-membrane linker polypeptides (70kDa), denoting a disintegration of the aggregation state of phycocyanin following UV-B exposure and thus the uncoupling of the energy transfer pathway. Cyanobacterial membrane disruption occurs during UV-B irradiation, leading to changes in membrane permeability and release of l4C labelled compounds.

The Effects of CO2 on the Photosynthetic Fixation of CO2 and the Activity of Ribulose-1,5-Bisphosphate Carboxylase in Single-Rooted Soybean Leaves under Sink-Limited Conditions

Ribulosebisphosphate carboxylase activity of single- rooted soybean leaves was lowered under sink-limited con- ditions. The low photosynthetic and enzymatic activities of the sink-limited leaves returned to the high levels, similar to those of the control leaves, within 30 min of the treatment with high CO2. turing them under continuous light or with cooling of the root system. These results provided good evidence for the end-product inhibition of photosynthesi s, a concept that was first proposed by Boussingault (1868). A subsequent study using this model plant showed that the sink-limited condition caused a reduction in the ac- tivity of ribulose-1,5-bisphosphate carboxylase (RuBPcase) that could be extracted from the leaves. This reduction in activity was reversed by incubation of the extract with bi- carbonate in the presence of MgCl 2 (Sawada et al. 1990). Inorganic phosphate was also effective in reversing the re- duction in the activity of RuBPcase in the presence of ex- tremely low levels of endogenous CO2 (Sawada et al. 1990, 1992). It was suggested that the reduction in photosynthetic activity in the sink-limited leaves might have been due to the deactivation of RuBPcase, caused by a decrease in the level of Pj in the chloroplast stroma as a consequence of a significant increase in levels of various phosphorylated in- termediates in the photosynthetic carbon cycle. This sugges- tion is supported by the recently reported result that Pj stim- ulated the binding of activator CO2 to RuBPcase that had been purified from spinach leaves and activated this en- zyme at a physiological concentration (5-7 fjM) of CO 2 in chloroplast stroma that is in equilibrium with atmospheric CO2 (Anwaruzzaman et al. 1995).