Characterization of chloroplastic fructose 1,6-bisphosphate aldolases as lysine-methylated proteins in plants

In pea (Pisum sativum), the protein-lysine methyltransferase (PsLSMT) catalyzes the trimethylation of Lys-14 in the large subunit (LS) of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), the enzyme catalyzing the CO(2) fixation step during photosynthesis. Homologs of PsLSMT, herein referred to as LSMT-like enzymes, are found in all plant genomes, but methylation of LS Rubisco is not universal in the plant kingdom, suggesting a species-specific protein substrate specificity of the methyltransferase. In this study, we report the biochemical characterization of the LSMT-like enzyme from Arabidopsis thaliana (AtLSMT-L), with a focus on its substrate specificity. We show that, in Arabidopsis, LS Rubisco is not naturally methylated and that the physiological substrates of AtLSMT-L are chloroplastic fructose 1,6-bisphosphate aldolase isoforms. These enzymes, which are involved in the assimilation of CO(2) through the Calvin cycle and in chloroplastic glycolysis, are trimethylated at a conserved lysyl residue located close to the C terminus. Both AtLSMT-L and PsLSMT are able to methylate aldolases with similar kinetic parameters and product specificity. Thus, the divergent substrate specificity of LSMT-like enzymes from pea and Arabidopsis concerns only Rubisco. AtLSMT-L is able to interact with unmethylated Rubisco, but the complex is catalytically unproductive. Trimethylation does not modify the kinetic properties and tetrameric organization of aldolases in vitro. The identification of aldolases as methyl proteins in Arabidopsis and other species like pea suggests a role of protein lysine methylation in carbon metabolism in chloroplasts.     

Brefeldin a inhibits circadian remodeling of chloroplast structure in the dinoflagellate gonyaulax

Circadian increases in the rate of carbon fixation in the dinoflagellate Gonyaulax are correlated with extensive plastid remodeling. One marker for this remodeling is mobilization of ribulose bisphosphate carboxylase/oxygenase (Rubisco) from the plastid periphery to plastid regions nearer the cell center called pyrenoids. Nuclear-encoded proteins such as Rubisco transit through the Golgi in dinoflagellates; hence, we blocked protein import into the plastids using Brefeldin A (BFA) to explore the mechanism for plastid remodeling. We find that pyrenoid formation normally occurs concurrently with increased Rubisco synthesis rates in vivo, and when BFA is given prior to the onset of Rubisco synthesis, pyrenoid formation is partially or completely inhibited by 0.1 or 0.3 microg/mL BFA, respectively. Rubisco synthesis itself is not affected, and BFA-treated cells accumulate Rubisco in novel structures we term BFA bodies. Interestingly, when given just after the onset of Rubisco synthesis, BFA delays but does not block Rubisco mobilization, suggesting that a timing signal for plastid remodeling is delivered to the organelles at the same time as newly synthesized Rubisco. BFA also inhibits the circadian increases in carbon fixation rates, supporting the hypothesis that the biochemical basis for this circadian rhythm may be Rubisco distribution within the plastid.     

Feedforward non-Michaelis-Menten mechanism for CO(2) uptake by Rubisco: contribution of carbonic anhydrases and photorespiration to optimization of photosynthetic carbon assimilation

Rubisco, the most abundant protein serving as the primary engine generating organic biomass on Earth, is characterized by a low catalytic constant (in higher plants approx. 3s(-1)) and low specificity for CO(2) leading to photorespiration. We analyze here why this enzyme evolved as the main carbon fixation engine. The high concentration of Rubisco exceeding the concentration of its substrate CO(2) by 2-3 orders of magnitude makes application of Michaelis-Menten kinetics invalid and requires alternative kinetic approaches to describe photosynthetic CO(2) assimilation. Efficient operation of Rubisco is supported by a strong flux of CO(2) to the chloroplast stroma provided by fast equilibration of bicarbonate and CO(2) and forwarding the latter to Rubisco reaction centers. The main part of this feedforward mechanism is a thylakoidal carbonic anhydrase associated with photosystem II and pumping CO(2) from the thylakoid lumen in coordination with the rate of electron transport, water splitting and proton gradient across the thylakoid membrane. This steady flux of CO(2) limits photosynthesis at saturating CO(2) concentrations. At low ambient CO(2) and correspondingly limited capacity of the bicarbonate pool in the stroma, its depletion at the sites of Rubisco is relieved by utilizing O(2) instead of CO(2), i.e. by photorespiration, a process which supplies CO(2) back to Rubisco and buffers the redox state and energy level in the chloroplast. Thus, the regulation of Rubisco function aims to keep steady non-equilibrium levels of CO(2), NADPH/NADP and ATP/ADP in the chloroplast stroma and to optimize the condition of homeostatic photosynthetic flux of matter and energy.     

Why abaxial illumination limits photosynthetic carbon fixation in spinach leaves

Limitations of carbon fixation within spinach leaves due to light and CO2 were investigated. Under equivalent photon fluxes, carbon fixation was higher when leaves were irradiated adaxially compared to abaxially. Maximal carbon fixation occurred in the middle of the palisade mesophyll under adaxial illumination, whereas, maximal carbon fixation occurred in the spongy mesophyll under abaxial illumination. Total carbon fixation and the pattern of carbon fixation across leaves were similar, when leaves were irradiated with 800 micromol quanta m(-2) s(-1) either adaxially alone or adaxially plus abaxially (1,600 micromol quanta m(-2) s(-1)). In contrast, when both leaf surfaces were irradiated simultaneously with 200 micromol quanta m(-2) s(-1), total carbon fixation increased and carbon fixation in the middle of the leaf was higher compared to leaves irradiated unilaterally with the low light. Feeding 14CO2 through either the adaxial or abaxial leaf surface did not change the pattern of carbon fixation across the leaf. Increasing 14CO2 pulse-feeding times from 5 s to 60 s allowed more 14CO2 to be fixed but did not change the pattern of 14CO2 fixation across the leaf. We concluded that in spinach, the distribution of both light and Rubisco activity within leaves has significant effects on the patterns of carbon fixation across leaves; whereas CO2 diffusion does not appear to affect the carbon fixation pattern within spinach leaves.     

Structural and biochemical characterization of the C₃-C₄ intermediate Brassica gravinae and relatives, with particular reference to cellular distribution of Rubisco

On the basis of its CO(2) compensation concentration, Brassica gravinae Ten. has been reported to be a C(3)-C(4) intermediate. This study investigated the structural and biochemical features of photosynthetic metabolism in B. gravinae. The cellular distribution of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) was also examined in B. gravinae, B. napus L. (C(3)), Raphanus sativus L. (C(3)), and Diplotaxis tenuifolia (L.) DC. (C(3)-C(4)) by immunogold electron microscopy to elucidate Rubisco expression during the evolution from C(3) to C(3)-C(4) intermediate plants. The bundle sheath (BS) cells of B. gravinae contained centrifugally located chloroplasts as well as centripetally located chloroplasts and mitochondria. Glycine decarboxylase P-protein was localized in the BS mitochondria. Brassica gravinae had low C(4) enzyme activities and high activities of Rubisco and photorespiratory enzymes, suggesting that it reduces photorespiratory CO(2) loss by the glycine shuttle. In B. gravinae, the labelling density of Rubisco was higher in the mesophyll chloroplasts than in the BS chloroplasts. A similar cellular pattern was found in other Brassicaceae species. These data demonstrate that, during the evolution from C(3) to C(3)-C(4) intermediate plants, the intercellular pattern of Rubisco expression did not change greatly, although the amount of chloroplasts in the BS cells increased. It also appears that intracellular variation in Rubisco distribution may occur within the BS cells of B. gravinae.     

Photosynthesis and ribulose 1,5-bisphosphate levels in intact chloroplasts

The response of ribulose 1,5-bisphosphate levels and CO(2) fixation rates in isolated, intact spinach chloroplasts to pyrophosphate, triose phosphates, dl-glyceraldehyde, O(2), catalase, and irradiance during photosynthesis has been studied. Within 1 minute in the light, a rapid accumulation of ribulose bisphosphate was measured in most preparations of intact chloroplasts, and this subsequently dropped as CO(2) fixation increased. Pyrophosphate, triose phosphates, and catalase increased CO(2) fixation and also the levels of ribulose bisphosphate. CO(2) fixation was inhibited by dl-glyceraldehyde and O(2) with corresponding decreases in ribulose bisphosphate. When the rate of photosynthesis decreased at limiting irradiances (low light), the level of ribulose bisphosphate in the chloroplast did not always decrease, suggesting that ribulose bisphosphate was not limiting CO(2) fixation under these conditions. When triose phosphates (fructose bisphosphate plus aldolase) were added to suspensions of chloroplasts at low irradiances, ribulose bisphosphate increased while CO(2) fixation decreased. These observations provide considerable evidence that high ribulose bisphosphate levels clearly are not solely sufficient to permit rapid rates of CO(2) fixation, but that factors other than ribulose bisphosphate concentration are overriding the control of photosynthesis.Isolated chloroplasts are capable of using carbon reserves to produce considerable ribulose bisphosphate. Upon illumination in the absence of CO(2) and O(2), intact chloroplasts produced up to 13 millimolar ribulose bisphosphate.     

Circadian rhythms in the chemoreflex control of breathing

Mechanisms underlying the circadian rhythm in lung ventilation were investigated. Ten healthy male subjects were studied for 36 h using a constant routine protocol to minimize potentially confounding variables. Laboratory light, humidity, and temperature remained constant, subjects did not sleep, and their meals and activities were held to a strict schedule. Respiratory chemoreflex responses were measured every 3 h using an iso-oxic rebreathing technique incorporating prior hyperventilation. Subjects exhibited circadian rhythms in oral temperature and respiratory chemoreflex responses, but not in metabolic rate. Basal ventilation [i.e., at subthreshold end-tidal carbon dioxide partial pressure (PET(CO(2)))] did not vary with time of day, but the ventilatory response to suprathreshold PET(CO(2)) exhibited a rhythm amplitude of approximately 25%, mediated mainly by circadian variations in the CO(2) threshold for tidal volume. We conclude that the circadian rhythm in lung ventilation is not a simple consequence of circadian variations in arousal state and metabolic rate. By raising the chemoreflex threshold, the circadian timing system may increase the propensity for respiratory instability at night.     

Overexpression of a cyanobacterial fructose-1,6-/sedoheptulose-1,7-bisphosphatase in tobacco enhances photosynthesis and growth

Transgenic tobacco plants expressing a cyanobacterial fructose-1,6/sedoheptulose-1,7-bisphosphatase targeted to chloroplasts show enhanced photosynthetic efficiency and growth characteristics under atmospheric conditions (360 p.p.m. CO2). Compared with wild-type tobacco, final dry matter and photosynthetic CO2 fixation of the transgenic plants were 1.5-fold and 1.24-fold higher, respectively. Transgenic tobacco also showed a 1.2-fold increase in initial activity of ribulose 1,5 bisphosphate carboxylase/oxygenase (Rubisco) compared with wild-type plants. Levels of intermediates in the Calvin cycle and the accumulation of carbohydrates were also higher than those in wild-type plants. This is the first report in which expression of a single plastid-targeted enzyme has been shown to improve carbon fixation and growth in transgenic plants.     

Effects of O(2) and CO(2) Concentration on the Steady-State Fluorescence Yield of Single Guard Cell Pairs in Intact Leaf Discs of Tradescantia albiflora: Evidence for Rubisco-Mediated CO(2) Fixation and Photorespiration in Guard Cells

A procedure for following changes in the steady-state yield of chlorophyll a fluorescence (F_s) from single guard cell pairs in variegated leaves of Tradescantia albiflora is described. As an indicator of photosynthetic electron transport, F_s is a very sensitive indirect measure of the balance of adenosine 5′-triphosphate (ATP) and reduced nicotinamide adenine dinucleotide phosphate (NADPH), producing reactions with the sink reactions that utilize those light-generated products. We found that F_s under constant light is sensitive to manipulation of ambient CO₂ concentrations, as would be expected if either phosphoenolpyruvate carboxylase or ribulose-1, 5 bisphosphate carboxylase/oxygenase (Rubisco)-dependent CO₂ fixation is the sink for photosynthetic ATP and NADPH in guard cells. However, we also found that changing O₂ concentration had a strong effect on fluorescence yield, and that O₂ sensitivity was only evident when the concentration of CO₂ was low. This finding provides evidence that both O₂ and CO₂ can serve as sinks for ATP and NADPH produced by photosynthetic electron transport in guard cell chloroplasts. Identical responses were observed with mesophyll cell chloroplasts in intact leaves. This finding is difficult to reconcile with the view that guard cell chloroplasts have fundamentally different pathways of photosynthetic metabolism from other chloroplasts in C₃ plants. Indeed, Rubisco has been detected at low levels in guard cell chloroplasts, and our studies indicate that it is active in the pathways for photosynthetic carbon reduction and photorespiration in guard cells.     

Adaptive signals in algal Rubisco reveal a history of ancient atmospheric carbon dioxide

Rubisco, the most abundant enzyme on the Earth and responsible for all photosynthetic carbon fixation, is often thought of as a highly conserved and sluggish enzyme. Yet, different algal Rubiscos demonstrate a range of kinetic properties hinting at a history of evolution and adaptation. Here, we show that algal Rubisco has indeed evolved adaptively during ancient and distinct geological periods. Using DNA sequences of extant marine algae of the red and Chromista lineage, we define positive selection within the large subunit of Rubisco, encoded by rbcL, to occur basal to the radiation of modern marine groups. This signal of positive selection appears to be responding to changing intracellular concentrations of carbon dioxide (CO(2)) triggered by physiological adaptations to declining atmospheric CO(2). Within the ecologically important Haptophyta (including coccolithophores) and Bacillariophyta (diatoms), positive selection occurred consistently during periods of falling Phanerozoic CO(2) and suggests emergence of carbon-concentrating mechanisms. During the Proterozoic, a strong signal of positive selection after secondary endosymbiosis occurs at the origin of the Chromista lineage (approx. 1.1 Ga), with further positive selection events until 0.41 Ga, implying a significant and continuous decrease in atmospheric CO(2) encompassing the Cryogenian Snowball Earth events. We surmise that positive selection in Rubisco has been caused by declines in atmospheric CO(2) and hence acts as a proxy for ancient atmospheric CO(2).     

Relative association of Rubisco with manganese and magnesium as a regulatory mechanism in plants

Rubisco, the enzyme that constitutes as much as half of the protein in a leaf, initiates either the photorespiratory pathway that supplies reductant for the assimilation of nitrate into amino acids or the C3 carbon fixation pathway that generates carbohydrates. The relative rates of these two pathways depend both on the relative extent to which O₂ and CO₂ occupies the active site of Rubisco and on whether manganese or magnesium is bound to the enzyme. This study quantified the activities of manganese and magnesium in isolated tobacco chloroplasts and the thermodynamics of binding of these metals to Rubisco purified from tobacco or a bacterium. In tobacco chloroplasts, manganese was less active than magnesium, but Rubisco purified from tobacco had a higher affinity for manganese. The activity of each metal in the chloroplast was similar in magnitude to the affinity of tobacco Rubisco for each. This indicates that, in tobacco chloroplasts, Rubisco associates almost equally with both metals and rapidly exchanges one metal for the other. Binding of magnesium was similar in Rubisco from tobacco and a bacterium, whereas binding of manganese differed greatly between the Rubisco from these species. Moreover, the ratio of leaf manganese to magnesium in C3 plants increased as atmospheric CO₂ increased. These results suggest that Rubisco has evolved to improve the energy transfers between photorespiration and nitrate assimilation and that plants regulate manganese and magnesium activities in the chloroplast to mitigate detrimental changes in their nitrogen/carbon balance as atmospheric CO₂ varies.     

Tree stem phosphoenolpyruvate carboxylase (PEPC): lack of biochemical and localization evidence for a C4-like photosynthesis system

Here, the kinetic properties and immunolocalization of phosphoenolpyruvate carboxylase (PEPC) and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in young stems of Fagus sylvatica were investigated. The aim of the study was to test the hypothesis that there is a C4-like photosynthesis system in the stems of this C3 tree species. The activity, optimal pH and L-malate sensitivity of PEPC, and the Michaelis-Menten constant (Km) for phosphoenolpyruvate (PEP), were measured in protein extracts from current-year stems and leaves. A gel blot experiment and immunolocalization studies were performed to examine the isozyme complexity of PEPC and the tissue distribution of PEPC and Rubisco in stems. Leaf and stem PEPCs exhibited similar, classical values characteristic of C3 PEPCs, with an optimal pH of c. 7.8, a Km for PEP of c. 0.3 mM and a IC50 for L-malate (the L-malate concentration that inhibits 50% of PEPC activity at the Km for PEP) of c. 0.1 mM. Western blot analysis showed the presence of two PEPC subunits (molecular mass c. 110 kDa) both in leaves and in stems. Immunogold labelling did not reveal any differential localization of PEPC and Rubisco, neither between nor inside cells. This study suggests that C4-type photosynthesis does not occur in stems of F. sylvatica and underlines the importance of PEPC in nonphotosynthetic carbon fixation by most stem tissues (fixation of respired CO2 and fixation via the anaplerotic pathway).     

Competing carboxylases: circadian and metabolic regulation of Rubisco in C3 and CAM Mesembryanthemum crystallinum L

The temporal co-ordination of ribulose 1·5-bisphosphate carboxylase/oxygenase (Rubisco) and phosphoenolpyruvate carboxylase (PEPc) activities by Mesembryanthemum crystallinum L. in C(3) and crassulacean acid metabolism (CAM) modes was investigated under conventional light-dark (LD) and continuous light (LL) conditions. When C(3) , net CO(2) assimilation rate increased during each subjective night under LL with maximum carboxylation unrelated to Rubisco activation state. The CAM circadian rhythm of CO(2) uptake was more pronounced, with CO(2) assimilation rate maximal towards the end of each subjective night. In vivo and in vitro techniques were integrated to map carboxylase enzyme regulation to the framework provided by CAM LL gas exchange activity. Rubisco was activated in vitro throughout each subjective dark period and consistently deactivated at each subjective dawn, similar to that observed at true dawn in constitutive CAM species. Instantaneous carbon isotope discrimination showed in vivo carboxylase co-dominance during the CAM subjective night, initially by Rubisco and latterly C(4) (PEPc), despite both enzymes seemingly activated in vitro. The circadian rhythm in titratable acidity accumulation was progressively damped over successive subjective nights, but maintenance of PEPc carboxylation capacity ensures that CAM plants do not become progressively more 'C(3) -like' with time under LL.     

Early iron deficiency stress response in leaves of sugar beet.

Iron nutrient deficiency was investigated in leaves of hydroponically grown sugar beets (Beta vulgaris) to determine how ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) gene expression is affected when thylakoid components of photosynthesis are diminished. Rubisco polypeptide content was reduced by 60% in severely iron-stressed leaves, and the reduction was directly correlated to chlorophyll content. The concentration of Rubisco protein in iron-stressed leaves was found to be regulated by availability of mRNAs, and CO2 fixation by Rubisco was reduced from 45 mumol CO2 m-2 s-1 in extracts from iron-sufficient leaves to 20 mumol CO2 m-2 s-1 in extracts from severely stressed leaves. The rate of CO2 fixation was directly correlated to leaf chlorophyll content. Rubisco in iron-sufficient control leaves was 59% activated, whereas in severely stressed leaves grown under the same light, Rubisco was 43% activated. RNA synthesis was reduced by about 50% in iron-deficient leaves, but 16S and 25S rRNA and ctDNA were essentially unaffected by iron stress.     

Site of action of inhibitors of carbon dioxide assimilation by whole lettuce chloroplasts

The sites of action of several compounds, reported to inhibit CO(2) fixation by chloroplast preparations were located by developing assays in lettuce chloroplasts to test their effect on partial reactions of the carbon cycle and on carbonic anhydrase. The results indicated that: d, l-glyceral-dehyde and 5'-AMP inhibit phosphoribulose kinase or isomerase. 3-Phosphoglyceric acid and 6-phosphogluconate inhibit ribulose diphosphate carboxylase. Azide, Mg(2+), and nitrite inhibit the activity of carbonic anhydrase of lettuce chloroplasts and light-dependent CO(2) fixation by intact chloroplasts with similar sensitivities. None of these inhibited CO(2) fixation in ruptured chloroplasts. It is suggested that the inhibition by azide, nitrite, and magnesium ions of CO(2) fixation by intact chloroplasts is due to their inhibition of the activity of carbonic anhydrase.     

An external delta-carbonic anhydrase in a free-living marine dinoflagellate may circumvent diffusion-limited carbon acquisition

The oceans globally constitute an important sink for carbon dioxide (CO(2)) due to phytoplankton photosynthesis. However, the marine environment imposes serious restraints to carbon fixation. First, the equilibrium between CO(2) and bicarbonate (HCO(3)(-)) is pH dependent, and, in normal, slightly alkaline seawater, [CO(2)] is typically low (approximately 10 mum). Second, the rate of CO(2) diffusion in seawater is slow, so, for any cells unable to take up bicarbonate efficiently, photosynthesis could become carbon limited due to depletion of CO(2) from their immediate vicinity. This may be especially problematic for those dinoflagellates using a form II Rubisco because this form is less oxygen tolerant than the usually found form I enzyme. We have identified a carbonic anhydrase (CA) from the free-living marine dinoflagellate Lingulodinium polyedrum that appears to play a role in carbon acquisition. This CA shares 60% sequence identity with delta-class CAs, isoforms so far found only in marine algae. Immunoelectron microscopy indicates that this enzyme is associated exclusively with the plasma membrane. Furthermore, this enzyme appears to be exposed to the external medium as determined by whole-cell CA assays and vectorial labeling of cell surface proteins with (125)I. The fixation of (14)CO(2) is strongly pH dependent, suggesting preferential uptake of CO(2) rather than HCO(3)(-), and photosynthetic rates decrease in the presence of 1 mm acetazolamide, a non-membrane-permeable CA inhibitor. This constitutes the first CA identified in the dinoflagellates, and, taken together, our results suggest that this enzyme may help to increase CO(2) availability at the cell surface.     

Contribution of fructose-1,6-bisphosphatase and sedoheptulose-1,7-bisphosphatase to the photosynthetic rate and carbon flow in the Calvin cycle in transgenic plants

To clarify the contributions of fructose-1,6-bisphosphatase (FBPase) and sedoheptulose-1,7-bisphosphatase (SBPase) separately to the carbon flux in the Calvin cycle, we generated transgenic tobacco plants expressing cyanobacterial FBPase-II in chloroplasts (TpF) or Chlamydomonas SBPase in chloroplasts (TpS). In TpF-11 plants with 2.3-fold higher FBPase activity and in TpS-11 and TpS-10 plants with 1.6- and 4.3-fold higher SBPase activity in chloroplasts compared with the wild-type plants, the amount of final dry matter was approximately 1.3-, 1.5- and 1.5-fold higher, respectively, than that of the wild-type plants. At 1,500 micromol m(-2) s(-1), the photosynthetic activities of TpF-11, TpS-11 and TpS-10 were 1.15-, 1.27- and 1.23-fold higher, respectively, than that of the wild-type plants. The in vivo activation state of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and the level of ribulose-1,5-bisphosphate (RuBP) in TpF-11, TpS-10 and TpS-11 were significantly higher than those in the wild-type plants. However, the transgenic plant TpF-9 which had a 1.7-fold higher level of FBPase activity showed the same phenotype as the wild-type plant, except for the increase of starch content in the source leaves. TpS-11 and TpS-10 plants with 1.6- and 4.3-fold higher SBPase activity, respectively, showed an increase in the photosynthetic CO(2) fixation, growth rate, RuBP contents and Rubisco activation state, while TpS-2 plants with 1.3-fold higher SBPase showed the same phenotype as the wild-type plants. These data indicated that the enhancement of either a >1.7-fold increase of FBPase or a 1.3-fold increase of SBPase in the chloroplasts had a marked positive effect on photosynthesis, that SBPase is the most important factor for the RuBP regeneration in the Calvin cycle and that FBPase contributes to the partitioning of the fixed carbon for RuBP regeneration or starch synthesis.     

Activation of Rubisco controls CO<Subscript>2</Subscript> assimilation in light: a perspective on its discovery

CO₂ fixation during photosynthesis is regulated by the activity of ribulose bisphosphate carboxylase (Rubisco). This conclusion became more apparent to me after CO₂-fixation experiments using isolated spinach chloroplasts and protoplasts, purified Rubisco enzyme, and intact leaves. Ribulose bisphosphate (RuBP) pools and activation of Rubisco were measured and compared to ¹⁴CO₂ fixation in light. The rates of ¹⁴CO ₂ assimilation best followed the changes in Rubisco activation under moderate to high light intensities. RuBP pool sizes regulated ¹⁴ ₂ assimilation only in very high CO₂ levels, low light and in darkness. Activation of Rubisco involves two separate processes: carbamylation of the protein and removal of inhibitors blocking carbamylation or blocking RuBP binding to carbamylated sites before reaction with CO₂ or O₂. This revised version was published online in June 2006 with corrections to the Cover Date.