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C4 photosynthesis is thought to have evolved via intermediate stages, with changes towards the C4 phenotype gradually enhancing photosynthetic performance. This hypothesis is widely supported by modelling studies, but experimental tests are missing. Mixing of C4 components to generate artificial intermediates can be achieved via crossing, and the grass Alloteropsis semialata represents an outstanding system since it includes C4 and non-C4 populations. Here, we analyse F1 hybrids between C3 and C4, and C3+C4 and C4 genotypes to determine whether the acquisition of C4 characteristics increases photosynthetic performance. The hybrids have leaf anatomical characters and C4 gene expression profiles that are largely intermediate between those of their parents. Carbon isotope ratios are similarly intermediate, which suggests that a partial C4 cycle coexists with C3 carbon fixation in the hybrids. This partial C4 phenotype is associated with C4-like photosynthetic efficiency in C3+C4 x C4, but not in C3 x C4 hybrids, which are overall less efficient than both parents. Our results support the hypothesis that the photosynthetic gains from the upregulation of C4 characteristics depend on coordinated changes in anatomy and biochemistry. The order of acquisition of C4 components is thus constrained, with C3+C4 species providing an essential step for C4 evolution.

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.

The photorespiratory pathway, in short photorespiration, is an essential metabolite repair pathway that allows the photosynthetic CO 2 fixation of plants to occur in the presence of oxygen. It is necessary because oxygen is a competing substrate of the CO 2 -fixing enzyme ribulose 1,5-bisphosphate carboxylase, forming 2-phosphoglycolate that negatively interferes with photosynthesis. Photorespiration very efficiently recycles 2-phosphoglycolate into 3-phosphoglycerate, which re-enters the Calvin–Benson cycle to drive sustainable photosynthesis. Photorespiration however requires extra energy and re-oxidises one quarter of the 2-phosphoglycolate carbon to CO 2 , lowering potential maximum rates of photosynthesis in most plants including food and energy crops. This review discusses natural and artificial strategies to reduce the undesired impact of air oxygen on photosynthesis and in turn plant growth.

Aquatic photosynthetic microorganisms, cyanobacteria and microalgae, account for almost half of the world's photosynthesis. They absorb carbon di oxide (CO2) as the major substrate to support photosynthesis, the beginning of energy flow into living organisms and one of the primary processes comprising the global carbon cycle. Among all photosynthetic mechanisms, inorganic carbon transport into ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is one of the major limiting steps in photosynthetic carbon fixation which involves active transport of HCO3−, CO2 and/or H+, or an energized biochemical mechanism. In fact, a unique system “carbon concentrating mechanism (CCM)” manages the inorganic carbon assimilation, accumulation of CO2 around RuBisCO, and utilization in algal cells. However, the information on mechanism of CO2 uptake and fixation inside the algal cells is limited. In order to make strategies for enhancement of CO2 fixation, understanding of CCM is crucial. Thus, this review provi...

Resume :Ribulose-1, 5-bisphosphate carboxylase-oxygenase Glyceraldehyde-3-phosphate dehydrogenase Fructose-1, 6-bisphosphatase and sedoheptulose-1, 7-bisphosphatase 197 Phosphoribulokinase Other important enzymes in the Calvin cycle Supramolecular complexes of the Calvin cycle

Lehnherr, B., Mächler, F. and Nösberger, J. 1985. Influence of temperature on the ratio of ribulose bisphosphate carboxylase to oxygenase activities and on the ratio of photosynthesis to photorespiration of leaves.—J. exp. Bot. 36: 1117-1125. Rates of net and gross photosynthesis of intact white clover leaves were measured by infrared gas analysis and by short term uptake of 14CO2 respectively. Ribulose bisphosphate carboxylase oxygenase (RuBPCO) was purified from young leaves and kinetic properties investigated in combined and separate assays. The ratio of carboxylase to oxygenase activities was compared with the ratio of photosynthesis to photorespiration at various temperatures and CO2 concentrations. The ratio of photosynthesis to photorespiration at 30 Pa p(CO2) was consistent with the ratio of carboxylase activity to oxygenase activity when each was measured above 20 °C. However, the ratio of photosynthesis to photorespiration increased with decreasing temperature, whereas the ratio of carboxylase to oxygenase activity was independent of temperature. This resulted in a disagreement between the measurements on the purified enzyme and intact leaf at low temperature. No disagreement between enzyme and leaf at low temperature occurred, when the ratio of photosynthesis to photorespiration was determined at increased CO2 concentrations. The results suggest an effect of low temperature and low CO2 concentration on the ratio of photosynthesis to photorespiration independent of the enzyme

C3 carbon fixation has a bad reputation, primarily because it is associated with photorespiration, a biochemical pathway thought to waste a substantial amount of the carbohydrate produced in a plant. This review presents evidence collected over nearly a century that (1) Rubisco when associated with Mn(2+) generates additional reductant during photorespiration, (2) this reductant participates in the assimilation of nitrate into protein, and (3) this nitrate assimilation facilitates the use of a nitrogen source that other organisms tend to avoid. This phenomenon explains the continued dominance of C3 plants during the past 23 million years of low CO2 atmospheres as well as the decline in plant protein concentrations as atmospheric CO2 rises.

Plants rely on the Calvin-Benson (CB) cycle for CO(2) fixation. The key carboxylase of the CB cycle is ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO). Efforts to enhance carbon fixation in plants have traditionally focused on RubisCO or on approaches that can help to remedy RubisCO's undesirable traits: its low catalytic efficiency and photorespiration. Towards reaching the goal of improving plant photosynthesis, cyanobacteria may be instrumental. Because of their evolutionary relationship to chloroplasts, they represent ideal model organisms for photosynthesis research. Furthermore, the molecular understanding of cyanobacterial carbon fixation provides a rich source of strategies that can be exploited for the bioengineering of chloroplasts. These strategies include the cyanobacterial carbon concentrating mechanism (CCM), which consists of active and passive transporter systems for inorganic carbon and a specialized organelle, the carboxysome. The carboxysome encapsulates RubisCO together with carbonic anhydrase in a protein shell, resulting in an elevated CO(2) concentration around RubisCO. Moreover, cyanobacteria differ from plants in the isoenzymes involved in the CB cycle and the photorespiratory pathways as well as in mechanisms that can affect the activity of RubisCO. In addition, newly available cyanobacterial genome sequence data from the CyanoGEBA project, which has more than doubled the amount of genomic information available for cyanobacteria, increases our knowledge on the CCM and the occurrence and distribution of genes of interest.

Elements of C4 photosynthesis—a complex adaptation that increases photosynthetic efficiency—may have evolved first to correct an intercellular nitrogen imbalance, and only later evolved a central role in carbon fixation.

Carbon isotope (13C/12C) fractionation (CIF) occurring in cells of organisms of different types is considered. Three metabolic points are shown to have an exceptional significance for the observed carbon isotope distribution in a living matter: 1) ribulose bisphosphate carboxylation in CO2 photoassimilation; 2) glycine decarboxylation in photorespiration; and 3) pyruvate decarboxylation in respiration metabolism. Carbon isotope effects (CIE) in ribulose bisphosphate (RuBP) carboxylation is characteristic of photosynthesizing organisms. It is responsible for the observed 12C enrichment of photosynthesizing organism biomass relative to environmental CO2. According to proposed mechanism of CIF, CO2 assimilation is a discrete process, which can be described as a flow of CO2 batches into a cell. Discreteness of the flow arises due to ability of Rubisco (the key enzyme of photosynthesis) periodically operate both as carboxylase and oxygenase depending on CO2/O2 ratio in a cell. It provides periodic filling/depl...

Nearly 50 years ago, inorganic carbon was shown to be fixed in microalgae as the C3 compound phosphoglyceric acid. The enzyme responsible for C3 carbon fixation, ribulose-1,5-bisphosphate carboxylase (Rubisco), however, requires inorganic carbon in the form of CO2 (ref. 2), and Rubisco enzymes from diatoms have half-saturation constants for CO2 of 30-60 microM (ref. 3). As a result, diatoms growing in seawater that contains about 10 microM CO2 may be CO2 limited. Kinetic and growth studies have shown that diatoms can avoid CO2 limitation, but the biochemistry of the underlying mechanisms remains unknown. Here we present evidence that C4 photosynthesis supports carbon assimilation in the marine diatom Thalassiosira weissflogii, thus providing a biochemical explanation for CO2-insensitive photosynthesis in marine diatoms. If C4 photosynthesis is common among marine diatoms, it may account for a significant portion of carbon fixation and export in the ocean, and would explain the greater enrichment of 13C in diatoms compared with other classes of phytoplankton. Unicellular C4 carbon assimilation may have predated the appearance of multicellular C4 plants.

Carbon dioxide fixation is overwhelmingly carried out by reactions of the Calvin-Benson cycle of plants, green algae, and cyanobacteria. Three other carbon dioxide reduction pathways are known to allow autotrophic growth, but these are mainly limited to anaerobic organisms. In this chapter the anaerobic autotrophic carbon reduction pathways are briefly described followed by a more detailed look at the Calvin-Benson cycle and its regulation. The Calvin-Benson cycle reaction sequence is similar to the non-oxidative branch of the pentose phosphate pathway although the enzyme transaldolase of the pentose phosphate pathway is not used and a novel enzymatic activity, sedoheptulose 1,7-bisphosphatase is substituted. The carbon fixation enzyme of the Calvin-Benson cycle, Rubisco, varies in its properties and is currently the subject of much research aimed at improving the efficiency of photosynthesis. Rubisco exists in three different conformations and there is also a gene coding for a related protein that does not have Rubisco activity but could be the evolutionary progenitor. The Calvin-Benson cycle is often referred to as the dark reactions of photosynthesis but it does not proceed in darkness for several reasons. In addition to providing reducing power and ATP, photosynthetic electron transport causes increased pH and magnesium in the stroma plus reducing power for thioredoxin to activate Calvin-Benson cycle enzymes and inactivate enzymes that would lead to futile cycles when Calvin-Benson cycle enzymes are active. There is a surprising regulation of phosphoglucose isomerase that can shift the regulation of the rate of starch synthesis from ADPglucose pyrophosphorylase to phosphoglucose isomerase. This may control the amount of carbon that leaves the Calvin-Benson cycle, since excessive loss of carbon from the Calvin-Benson cycle intermediates can lead to a collapse of the cycle. Finally, a recently discovered regulation mechanism based on a small protein called CP12 is discussed.

Carbon dioxide fixation is the biological process through which carbon dioxide is converted to organic compounds. Organisms that fix carbon dioxide provide the organic carbon necessary to support the existence of all heterotrophic life on our planet. This article provides an introduction to the various mechanisms of carbon dioxide fixation utilised by microorganisms. The Calvin–Benson–Bassham cycle is shared among plants, algae and many photo- and chemoautotrophic bacteria, and is probably the most well known carbon dioxide-fixation pathway. However, a number of other pathways exist that are unique to the microbial world and the diverse chemistry and strategies they utilise are fascinating. Among the six carbon-fixing pathways known at present, three pathways harbouring novel enzymes have just been established in the past few years. With the number and diversity of microorganisms still expanding, the possibilities are high that further novel pathways will be identified in the near future. Key Concepts: Carbon dioxide fixation is the biological process through which carbon dioxide is converted to organic compounds. Organisms that fix carbon dioxide provide the organic carbon necessary to support the existence of all heterotrophic life on our planet. In addition to the Calvin–Benson–Bassham cycle, which is also found in plants and microorganisms harbour a number of unique carbon dioxide-fixing pathways. Keywords: carbon dioxide fixation; Calvin–Benson–Bassham cycle; reductive tricarboxylic acid cycle; reductive acetyl-CoA pathway; 3-hydroxypropionate/malyl-CoA cycle; 3-hydroxypropionate/4-hydroxybutyrate cycle; dicarboxylate/4-hydroxybutyrate cycle; archaea; rubisco; autotrophy

The C3 carbon reduction cycle is the primary pathway of carbon fixation in all photosynthetic organisms, reducing carbon dioxide from the atmosphere to form carbohydrates and in higher plants it takes place in the chloroplast stroma. Keywords: Calvin cycle; photosynthesis; light regulation; thioredoxin; Rubisco

The limitation to photosynthetic CO2 assimilation in C3 plants in hot, dry environments is dominated by ribulose 1.5-bisphosphate carboxylase/oxygenase (Rubisco) because CO2 availability is restricted and photorespiration is stimulated. Using a combination of genetic engineering and transgenic technology, three approaches to reduce photorespiration have been taken; two of these focused on increasing the carboxylation efficiency of Rubisco either by reducing the oxygenase reaction directly or by manipulating the Rubisco enzyme by concentrating CO2 in the region of Rubisco through the introduction of enzymes of the C4 pathway. The third approach attempted to reduce photorespiration directly by manipulation of enzymes in this pathway. The progress in each of these areas is discussed, and the most promising approaches are highlighted. Under saturating CO2 conditions, Rubisco did not limit photosynthesis, and limitation shifted to ribulose bisphosphate (RuBP) regeneration capacity of the C3 cycle. Transgenic analysis was used to identify the specific enzymes that may be targets for improving carbon fixation, and the way this may be exploited in the high CO2 future is considered.

This paper presents my personal account of research on CO(2) fixation from when I began these studies as a postdoctoral student in the early 1970s. It traces interests in microbial ribulose bisphosphate carboxylase/oxygenase (Rubisco) and considers early breakthroughs on the isolation, characterization, and significance of this enzyme from nonsulfur purple photosynthetic bacteria and other phototrophic organisms. This article also develops a historical perspective as to how recent efforts may lead to an understanding of molecular mechanisms by which the synthesis of this enzyme and other proteins of the pathway are regulated at the molecular level. In addition, how these studies impinge on the interactive control of CO(2) fixation, along with nitrogen fixation and hydrogen metabolism, is also considered. Finally, CO(2)-fixation studies in green sulfur photosynthetic bacteria and the discovery of the rather surprising Rubisco-like protein are described.

CO(2) fixation during photosynthesis is regulated by the activity of ribulose bisphosphate carboxylase (Rubisco). This conclusion became more apparent to me after CO(2)-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 (14)CO(2) fixation in light. The rates of (14)CO (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 CO(2) 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 CO(2) or O(2).

Photosynthetic microorganisms like cyanobacteria and many eukaryotic algae acclimate to a limited availability of carbon dioxide (CO2) in their environment by inducing a process called the carbon dioxide concentrating mechanism. This process uses an active inorganic carbon (Ci; CO2 and/or HCO3−) uptake system that leads to the internal accumulation of Ci to levels significantly higher than extracellular levels. Carbonic anhydrase activity converts much of the accumulated hydrogen carbonate to CO2, concentrating this substrate around Rubisco and thereby optimising photosynthetic efficiency even under low CO2 conditions. The efficiency of the process is further improved by the sequestration of Rubisco into specialised structures like the cyanobacterial carboxysome or the pyrenoid in eukaryotic algae. The carbon dioxide concentrating mechanism enhances carbon dioxide fixation and growth in algae. With the increasing demands for sustainable energy sources, algae with efficient carbon dioxide concentrating mechanisms are attractive models for biotechnological and transgenic applications for biofuel and biomass production. Key Concepts: The carbon dioxide concentrating mechanism helps photosynthetic algae optimise photosynthesis under limiting CO2 conditions. Rubisco uses both carbon dioxide and oxygen as substrates. Rubisco is localised in the carboxysome in cyanobacteria and the pyrenoid in the green alga, Chlamydomonas reinhardtii. The charged hydrogen carbonate needs transporters to enter the cell and cross organellar membranes. Carbonic anhydrase is an efficient enzyme that carries out the reversible interconversion of carbon dioxide and hydrogen carbonate and this reaction proceeds at a much faster rate than the uncatalysed reaction. At higher pH levels most of the inorganic carbon is in the form of hydrogen carbonate. Most algae with carbon dioxide concentrating mechanisms can take up both carbon dioxide and hydrogen carbonate. Carbon dioxide can diffuse out of the cell so algae trap carbon dioxide in the form of the charged hydrogen carbonate anion. Algae are used for large-scale biomass production and are currently being explored as sources of bio-fuel. Keywords: algae; carbon concentrating mechanism; cyanobacteria; Chlamydomonas; photosynthesis; rubisco; pyrenoid; carboxysome; carbonic anhydrase; hydrogen carbonate transporter

All oxygenic organisms from the simplest prokaryotic cyanobacteria to the most complicated land plants reduce atmospheric CO2 to sugar phosphates by a pathway known as the reductive pentose phosphate (RPP) pathway. This pathway located in chloroplast is the primary carboxylating mechanism in plants and is comprised of thirteen reactions catalyzed by eleven enzymes distributed in three distinct phases namely, carboxylation, reduction and the regeneration. Ribulose-1, 5-bis phosphate carboxylase/oxygenase (Rubisco) catalyzes the primary carbon fixation reaction. This enzyme also catalyzes the fixation of oxygen in a process known as photorespiration, which competes directly with fixation of CO2. At air levels of CO2 and O2, for every three CO2 molecules fixed, approximately one molecule of O2 is fixed by Rubisco. This competition between CO2 and O2 and energy costs associated with photorespiration largely determine the efficiency of C3 photosynthesis in air. Although the RPP pathway is the fundamental carboxylating mechanism, a number of plants have evolved adaptations in which CO2 is first fixed by a supplementary pathway and then released in the cells in which the RPP cycle operates. One of the supplementary pathways, the C4 pathway involves special leaf anatomy, i.e. the Kranz anatomy. In this pathway, CO2 is initially fixed in the cytosol of mesophyll cells by the enzyme phosphoenol pyruvate (PEP) carboxylase to form four carbon compound (malate or aspartate) which is then translocated to the bundle sheath cells and gets decarboxylated in the chloroplasts. The CO2 produced is then refixed by Rubisco. Based on the mechanism of decarboxylation, the crops possessing this pathway have been subgrouped into NAD-ME type, NADP-ME type and PCK-type. Plants endorsed with this pathway have no or very little photorespiration, greater efficiency and are able to grow under conditions of high light intensity and elevated temperature. A second supplementary pathway found in species of the Crassulaceae is called Crassulacean acid metabolism (CAM). The plants possessing this pathway fix CO2 in the night into C4 acids. During the day, CO2 released from decarboxylation of C4 acids is converted to sugar phosphates by RPP cycle.

[Figure: see text] ▪ Abstract The C2 oxidative photosynthetic carbon cycle plus the C3 reductive photosynthetic carbon cycle coexist. Both are initiated by Rubisco, use about equal amounts of energy, must regenerate RuBP, and result in exchanges of CO2 and O2 to establish rates of net photosynthesis, CO2 and O2 compensation points, and the ratio of CO2 and O2 in the atmosphere. These concepts evolved from research on O2 inhibition, glycolate metabolism, leaf peroxisomes, photorespiration, 18O2/16O2 exchange, CO2 concentrating processes, and a requirement for the oxygenase activity of Rubisco. Nearly 80 years of research on these topics are unified under the one process of photosynthetic carbon metabolism and its self-regulation.

Photosynthesis, biological nitrogen fixation, and carbon dioxide assimilation are three fundamental biological processes catalyzed by photosynthetic bacteria. In the present study, it is shown that mutant strains of the nonsulfur purple photosynthetic bacteria Rhodospirillum rubrum and Rhodobacter sphaeroides , containing a blockage in the primary CO 2 assimilatory pathway, derepress the synthesis of components of the nitrogen fixation enzyme complex and abrogate normal control mechanisms. The absence of the Calvin–Benson–Bassham (CBB) reductive pentose phosphate CO 2 fixation pathway removes an important route for the dissipation of excess reducing power. Thus, the mutant strains develop alternative means to remove these reducing equivalents, resulting in the synthesis of large amounts of nitrogenase even in the presence of ammonia. This response is under the control of a global two-component signal transduction system previously found to regulate photosystem biosynthesis and the transcription of genes required for CO 2 fixation through the CBB pathway and alternative routes. In addition, this two-component system directly controls the ability of these bacteria to grow under nitrogen-fixing conditions. These results indicate that there is a molecular link between the CBB and nitrogen fixation process, allowing the cell to overcome powerful control mechanisms to remove excess reducing power generated by photosynthesis and carbon metabolism. Furthermore, these results suggest that the two-component system integrates the expression of genes required for the three processes of photosynthesis, nitrogen fixation, and carbon dioxide fixation.

Photosynthetic carbon fixation by phytoplankton is a key component of the global carbon cycle. Our understanding of the types of picoplankton and ultraphytoplankton involved in this process is evolving. However, mechanisms of regulation of photosynthetic carbon fixation in the oceans are poorly understood. All phytoplankton fix CO2 by reductive carboxylation employing the enzyme ribulose bisphosphate carboxylase (RuBPCase). The sequence of the gene encoding the large subunit of the enzyme (rbcL) has been relatively conserved, with two major evolutionary groups among oxygenic photoautrotrophs: the cyanobacteria/green algae/higher plants and the chromophytic algae. Gene probes made from representative members of these groups have been used to study the transcriptional regulation of RuBPCase in natural phytoplankton populations. Levels of rbcL mRNA correlated with rates of photosynthetic carbon fixation. A diel pattern in both carbon fixation and levels of rbcL mRNA was observed, with greatest values for both during daylight hours. This data supports transcriptional regulation as a major mechanism for regulation of carbon fixation in the oceans. This approach can be used to measure expression of conserved genes encoding other important geochemical functions.

The first stable product of photosynthetic carbon fixation by land plants is either the three-carbon molecule phosphoglycerate (in C3 plants) or the four-carbon compounds malate or aspartate (in C4 and CAM (crassulacean-acid metabolism) plants). Reinfelder et al. infer that a C4 biochemical pathway of carbon fixation also operates in marine diatoms1,2 on the basis of their discovery of the enzyme phosphoenolpyruvate (PEP) carboxylase and of their 14C-tracer results in the marine diatom Thalassiosira weissflogii. However, we consider that further analysis is called for to demonstrate that this marine diatom meets all the criteria for C4 photosynthesis.

Net photosynthesis (Pn) is inhibited by moderate heat stress. To elucidate the mechanism of inhibition, we examined the effects of temperature on gas exchange and ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) activation in cotton and tobacco leaves and compared the responses to those of the isolated enzymes. Depending on the CO2 concentration, Pn decreased when temperatures exceeded 35–40°C. This response was inconsistent with the response predicted from the properties of fully activated Rubisco. Rubisco deactivated in leaves when temperature was increased and also in response to high CO2 or low O2. The decrease in Rubisco activation occurred when leaf temperatures exceeded 35°C, whereas the activities of isolated activase and Rubisco were highest at 42°C and >50°C, respectively. In the absence of activase, isolated Rubisco deactivated under catalytic conditions and the rate of deactivation increased with temperature but not with CO2. The ability of activase to maintain or promote Rubisco activation in vitro also decreased with temperature but was not affected by CO2. Increasing the activase/Rubisco ratio reduced Rubisco deactivation at higher temperatures. The results indicate that, as temperature increases, the rate of Rubisco deactivation exceeds the capacity of activase to promote activation. The decrease in Rubisco activation that occurred in leaves at high CO2 was not caused by a faster rate of deactivation, but by reduced activase activity possibly in response to unfavorable ATP/ADP ratios. When adjustments were made for changes in activation state, the kinetic properties of Rubisco predicted the response of Pn at high temperature and CO2.

The activation states of a number of chloroplastic enzymes of the photosynthetic carbon reduction cycle and levels of related metabolites were measured in leaves of sugar beet (Beta vulgaris L., Klein E-type multigerm) under slowly changing irradiance during a day. The activation states of both phosphoribulokinase and NADP(+)-glyceraldehyde-3-phosphate dehydrogenase increased early in the light period and remained constant during the middle of the day. Initial ribulose 1,5-bisphosphate carboxylase activity was already about one third of the midday level, did not change for the first 2 hours, but then increased in parallel with the rate of carbon fixation. Because the activation states increased by turns, first phosphoribulokinase and NADP(+)-glyceraldehyde-3-phosphate dehydrogenase and later ribulose 1,5-bisphosphate carboxylase, the ratios of the activation states changed remarkably. Levels of ribulose bisphosphate and phosphoglycerate, which were high enough to affect enzyme reaction rates and changed in concert with activation state, indicate that these metabolites are involved in feedback/feedforward regulation of enzymes of carbon assimilation. This regulatory sequence is able to explain how the reaction rates for the enzymes of carbon assimilation are adjusted to maintain their activities in balance with each other and with the flux of carbon fixation.

The effects of ammonium assimilation on photosynthetic carbon fixation and O2 exchange were examined in two species of N-limited green algae, Chlorella pyrenoidosa and Selenastrum minutum. Under light-saturating conditions, ammonium assimilation resulted in a suppression of photosynthetic carbon fixation by S. minutum but not by C. pyrenoidosa. These different responses are due to different relationships between cellular ribulose bisphosphate (RuBP) concentration and the RuBP binding site density of ribulose bisphosphate carboxylase/oxygenase (Rubisco). In both species, ammonium assimilation resulted in a decrease in RuBP concentration. In S. minutum the concentration fell below the RuBP binding site density of Rubisco, indicating RuBP limitation of carboxylation. In contrast, RuBP concentration remained above the binding site density in C. pyrenoidosa. Compromising RuBP regeneration in C. pyrenoidosa with low light resulted in an ammonium-induced decrease in RuBP concentration below the RuBP binding site density of Rubisco. This resulted in a decrease in photosynthetic carbon fixation. In both species, ammonium assimilation resulted in a larger decrease in net O2 evolution than in carbon fixation. Mass spectrometric analysis shows this to be a result of an increase in the rate of mitochondrial respiration in the light.

When dark 14 CO 2 fixation in maize leaves was carried out under anaerobic conditions after preillumination in the absence of O 2 , the 14 C incorporation in aspartic acid was transient; its maximum level was very low compared with that of malic acid. The addition of 5% O 2 during the dark fixation period increased the total uptake of 14 CO 2 and the 14 C incorporation into aspartic acid. A study of the intramolecular distribution of radioactivity showed that 71 to 76% of the 14 C was located in the C 4 (β-carboxyl) of malate and aspartate and the remainder in the C 1 . This intramolecular labeling pattern did not change during the 5- to 60-second dark 14 CO 2 fixation period and was scarcely altered by the presence of O 2 . Three degradation techniques led to similar data. The significance of these results is discussed taking into account the known possible carboxylation pathways. It is concluded that ribulose 1,5-bisphosphate can be a primary acceptor of CO 2 when maize leaves are preilluminated in the absence of O 2 .

The pathways of carbon flow during photosynthesis and photorespiration by marine microalgae are examined. It is concluded that, although in healthy, exponentially dividing cells of marine phytoplankton the bulk of carbon assimilation is via ribulose-1,5-bisphosphate carboxylase and the photosynthetic carbon reduction cycle, a significant proportion of carbon fixation may be by β-carboxylation (via phosphoenolpyruvate carboxylase, phosphoenolpyruvate carboxykinase or pyruvate carboxylase). However, such carbon assimilation does not represent a “C4 pathway” as found in certain higher plants. The fixation of carbon via β-carboxylation may, however, be a significant route of carbon assimilation in low irradiance environments. Although many microalgae, both marine and freshwater, posses “C3 biochemistry”, they nonetheless have physiological characteristics resembling those of C4 higher plants. The available evidence suggests that this is a consequence of the operation, in microalgae, of a “CO2 concentrating mechanism” which, effectively, suppresses photorespiration. However, the capacity for carrying out photorespiratory carbon metabolism is present in such cells even though photorespiratory activity may be reduced. The limited evidence available suggests that marine microalgae may possess the enzymatic machinery for both the glycolate pathway and, in diatoms at least, the tartronic semialdehyde cycle of photorespiratory carbon metabolism. The possession of a mechanism to accumulate CO2 and suppress photorespiration could confer a competitive advantage under conditions of CO2 limitation and, possibly, under nitrogen limitation.

The effect of N-assimilation on the partitioning of carbon fixation between phosphoenolpyruvate carboxylase (PEPcase) and ribulose bisphosphate carboxylase/oxygenase (Rubisco) was determined by measuring stable carbon isotope discrimination during photosynthesis by an N-limited green alga, Selenastrum minutum (Naeg.) Collins. This was facilitated by a two process model accounting for simultaneous CO(2) fixation and respiratory CO(2) release. Discrimination by control cells was consistent with the majority of carbon being fixed by Rubisco. During nitrogen assimilation however, discrimination was greatly reduced indicating an enhanced flux through PEPcase which accounted for upward of 70% of total carbon fixation. This shift toward anaplerotic metabolism supports a large increase in tricarboxylic acid cycle activity primarily between oxaloacetate and alpha-ketoglutarate thereby facilitating the provision of carbon skeletons for amino acid synthesis. This provides an example of a unique set of conditions under which anaplerotic carbon fixation by PEPcase exceeds photosynthetic carbon fixation by Rubisco in a C(3) organism.

The mechanism of photosynthetic carbon dioxide fixation in the green flagellate Dunaliella tertiolecta Butcher varies during growth in batch culture. Evidence for this change comes from three sources: i) algae from the stationary phase incorporated a greater proportion of the fixed carbon into amino arids and protein than did cells from the mid-exponential phase; ii) the activity of phosphoenolpyruvate carboxylase relative to that of ribulose-1, 5-di-phosphate carboxylase increased with age in batch culture; and, iii) cells from the stationary phase appeared to utilize the bicarbonate ion as the substrate for photosynthesis, whereas those from mid-exponential phase appeared to utilize fire carbon dioxide. These data suggest that a change of photosynthetic mechanism can occur within a single species of alga, depending on its physiological state.

Plants with the C 3 , C 4 , and crassulacean acid metabolism (CAM) photosynthetic pathways show characteristically different discriminations against 13 C during photosynthesis. For each photosynthetic type, no more than slight variations are observed within or among species. CAM plants show large variations in isotope fractionation with temperature, but other plants do not. Different plant organs, subcellular fractions and metabolises can show widely varying isotopic compositions. The isotopic composition of respired carbon is often different from that of plant carbon, but it is not currently possible to describe this effect in detail. The principal components which will affect the overall isotope discrimination during photosynthesis are diffusion of CO 2 , interconversion of CO 2 and HCO − 3 , incorporation of CO 2 by phosphoenolpyruvate carboxylase or ribulose bisphosphate carboxylase, and respiration. Theisotope fractionations associated with these processes are summarized. Mathematical models are presented which permit prediction of the overall isotope discrimination in terms of these components. These models also permit a correlation of isotope fractionations with internal CO 2 concentrations. Analysis of existing data in terms of these models reveals that CO 2 incorporation in C 3 plants is limited principally by ribulose bisphosphate carboxylase, but CO 2 diffusion also contributes. In C 4 plants, carbon fixation is principally limited by the rate of CO 2 diffusion into the leaf. There is probably a small fractionation in C 4 plants due to ribulose bisphosphate carboxylase.

The Calvin cycle of carbon dioxide fixation constitutes a biosynthetic pathway for the generation of (multi-carbon) intermediates of central metabolism from the one-carbon compound carbon dioxide. The product of this cycle can be used as a precursor for the synthesis of all components of cell material. Autotrophic carbon dioxide fixation is energetically expensive and it is therefore not surprising that in the various groups of autotrophic bacteria the operation of the cycle is under strict metabolic control. Synthesis of phosphoribulokinase and ribulose-1,5-bisphosphate carboxylase, the two enzymes specifically involved in the Calvin cycle, is regulated via end-product repression. In this control phosphoenolpyruvate most likely has an alarmone function. Studies of the enzymes isolated from various sources have indicated that phosphoribulokinase is the target enzyme for the control of the rate of carbon dioxide fixation via the Calvin cycle through modulation of existing enzyme activity. In general, this enzyme is strongly activated by NADH, whereas AMP and phosphoenolpyruvate are effective inhibitors. Recent studies of phosphoribulokinase in Alcaligenes eutrophus suggest that this enzyme may also be regulated via covalent modification.

It is demonstrated that carbon fixation in photosynthesis is regulated in two kinetically coupled pathways involving the specialized pair of non-equivalent, enzyme-bound glycerate-3-P (3-PGA) molecules obtained from ribulose 1,5-bisphosphate (RuBP) carboxylation in the light.A non-cyclic pathway is suggested (reaction 2) for the direct biosynthesis of sucrose from the 3-PGA obtained from C-3, C-4 and C-5 of the six-carbon carboxylation adduct. Concomitant to the appearance of sucrose as the principal product, the Mg 2+ -bound 3-PGA molecule formed from C-1, C-2 and C-2′ of the C 6 intermediate is released and subsequently reduced in regenerating the RuBP. It is proposed that the nocturnal inhibitor, 2-carboxyarabinitol-1-phosphate (1-PCA) is obtained from a condensation of 3-PGA and glyceraldehyde.

The release of CO 2 during photosynthesis that is due to the production and metabolism of glycollic acid is usually regarded as outward evidence for the wasteful process of photorespiration in plants. In the light, glycollic acid is produced almost entirely as a result of the oxygenase activity of ribulose bisphosphate carboxylaseoxygenase (Rubisco). Metabolism of the glycollic acid not only releases recently assimilated carbon back into the atmosphere but also uses a considerable amount of energy to recycle remaining carbon from the glycollate to intermediates of the photosynthetic carbon reduction cycle. Furthermore, nitrogen from amino acids is released as ammonia during the metabolism of glycollate; some further energy is needed for this ammonia to be reassimilated. The oxygenation of ribulose bisphosphate is competitive with carboxylation and it appears to be the relative concentrations of oxygen and carbon dioxide present in cells containing the enzyme that mainly determine the relative rates of the two reactions in leaves. Systems which concentrate carbon dioxide in photosynthetic cells decrease the extent of photorespiration in C 4 species, certain algae and cyanobacteria. However, carboxylases from different species also vary considerably in their relative capacities to catalyse carboxylation and oxygenation of ribulose bisphosphate under standard conditions. This variation allows some hope that photorespiration might be decreased without recourse to energydependent systems for increasing cellular CO 2 concentrations.

In experiments with the facultative Crassulacean acid metabolism (CAM) species, Mesembryanthemum crystallinum , only plants which received high levels of inorganic salts fixed substantial amounts of CO 2 by the CAM pathway. Equivalent osmolarities of polyethylene glycol 6000 did not yield any CAM fixation. Plant water potential and turgor pressure had no detectable influence on the amount of CAM fixation. These observations rule out the possibility that the inorganic ions were acting as osmotic agents. Carbon dioxide and water exchange analysis showed that when water supply was not limiting, salt-deprived plants sustained higher reductive pentose phosphate cycle carbon fixation rates than salt-treated plants. Under water stress conditions, salt-deprived plants using only the reductive pentose phosphate cycle pathway assimilated less carbon and were less efficient in their water use than salt-treated plants using predominately the CAM pathway. These results support the hypothesis that the ability to use the CAM pathway reduces the capacity for reductive pentose phosphate cycle fixation but permits higher productivity in water-limited environments.

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.

SUMMARY Anatomical, physiological and biochemical characteristics of Verbascum thapsus demonstrate that this species is a C3 carbon fixation type rather than C4 as suggested by Parkhurst (1976). Leaf anatomy, net photosynthesis rate, carbon dioxide compensation point, photorespiration rate, temperature optimum for photosynthesis and ratio of ribulose-1,5-diphosphate carboxylase to phosphoenolpyruvate carboxylase activity for V. thapsus are all typical of C3 rather than C4 species. The data presented illustrate the danger of assuming a species is either C3or C4 based on a single characteristic. Parkhurst (1976) predicted that optimum photosynthesis for V. thapsus would occur at high temperature and would increase faster than transpiration with increased temperature. Our data are contrary to these predictions.