A new model of uptake of multiple sugars by S. cerevisiae

Carrying on from our work with yeast in batch culture with mixed substrates of glucose and fructose, a model developed previously was modified to incorporate firstly the use of sucrose as substrate and secondly the ability to simulate continuous culture. Experiments using sucrose as the limiting substrate were simulated based on sugar uptake, biomass and ethanol production, carbon dioxide production and oxygen consumption. The new sugar uptake models were able to successfully simulate these experiments. On the basis of these experiments there is a strong suggestion that a proportion of the sucrose utilised is transported into the cell rather than hydrolysed prior to entry. Continuous culture experiments were also simulated by adapting the modelling program into the format of a multiple differential equation problem solver called SPEEDUP.     

Indirect estimation of cell mass and substrate concentration using a computer-coupled mass spectrometer

On-line estimation of cell mass and substrate concentration based on exhaust gas analysis was developed. The O₂, CO₂, H₂O, and N₂ contents at the inlet and outlet of fermentor, analyzed by a computer-coupled quadrupole mass spectrometer, were used to calculate the oxygen uptake rate and carbon dioxide evolution rate, and these rates were further used to evaluate cell mass and substrate concentration in a recombinant Escherichia coli fermentation. Cell mass, glucose concentration, specific growth rate, and specific consumption rate of glucose were well estimated by this method; the oxygen uptake rate gave more accurate estimates for these state variables than did the carbon dioxide evolution rate.     

Carbon dioxide inhibition of yeast growth in biomass production

Saccharomyces cerevisiae was grown under aerobic and substrate-limiting conditions for efficient biomass production. Under these conditions, where the sugar substrate was fed incrementally, the growth pattern of the yeast cells was found to be uniform, as indicated by a constant respiratory quotient during the entire growing period. The effect of carbon dioxide was investigated by replacing portions of the nitrogen in the air stream with carbon dioxide, while maintaining the oxygen content at the normal 20% level, so that identical oxygen transfer rate and atmospheric pressure were maintained for all experiments with different partial pressures of carbon dioxide. Inhibition of yeast growth was negligible below 20% CO₂ in the aeration mixture. Slight inhibition was noted at the 40% CO₂ level and significant inhibition was noted above the 50% CO₂, level, corresponding to 1.6 × 10^?2M of dissolved CO₂ in the fermentor broth. High carbon dioxide content in the gas phase also inhibited the fermentation activity of baker's yeast.     

Similar photosynthetic response to elevated carbon dioxide concentration in species with different phloem loading strategies

Species have different strategies for loading sugars into the phloem, which vary in the route that sugars take to enter the phloem and the energetics of sugar accumulation. Species with passive phloem loading are hypothesized to have less flexibility in response to changes in some environmental conditions because sucrose export from mesophyll cells is dependent on fixed anatomical plasmodesmatal connections. Passive phloem loaders also have high mesophyll sugar content, and may be less likely to exhibit sugar-mediated down-regulation of photosynthetic capacity at elevated CO₂ concentrations. To date, the effect of phloem loading strategy on the response of plant carbon metabolism to rising atmospheric CO₂ concentrations is unclear, despite the widespread impacts of rising CO₂ on plants. Over three field seasons, five species with apoplastic loading, passive loading, or polymer-trapping were grown at ambient and elevated CO₂ concentration in free air concentration enrichment plots. Light-saturated rate of photosynthesis, photosynthetic capacity, leaf carbohydrate content, and anatomy were measured and compared among the species. All five species showed significant stimulation in midday photosynthetic CO₂ uptake by elevated CO₂ even though the two passive loading species showed significant down-regulation of maximum Rubisco carboxylation capacity at elevated CO₂. There was a trend toward greater starch accumulation at elevated CO₂ in all species, and was most pronounced in passive loaders. From this study, we cannot conclude that phloem loading strategy is a key determinant of plant response to elevated CO₂, but compelling differences in response counter to our hypothesis were observed. A phylogenetically controlled experiment with more species may be needed to fully test the hypothesis.     

Metabolism of Radioactive Sugars by Tobacco leaf Disks

Destarched tobacco-leaf disks were floated on per cent. (w/v)solutions of sucrose uniformly labelled with ¹⁴C in either theglucose or fructose moiety, and on invert sugar in which onehexose only was so labelled. The experiments were carried outin an atmosphere of oxygen at 25° C. Seventy-five per cent,of the sugar lost from the external solutions was recoveredas starch, sucrose, fructose, glucose, and CO₂. With sucroseas the substrate, 30 per cent, of the material was recoveredas CO₂ and 17 per cent. each as starch, sucrose, fructose, andglucose. With invert sugar as the substrate, 30 per cent, wasagain recovered as CO₂ only 20 per cent. as the three sugarstogether, and 50 per cent. as starch. Whichever hexose was initiallylabelled and whether the sugar was supplied as sucrose or hexose,the relative specific activities of starch and sucrose in theleaf disks and of the CO₂ evolved were equal or nearly equalto that of the sugar supplied. With sucrose as the substratethe sucrose in the disks retained its asymmetry of label, andfree hexoses produced were similarly asymmetrically labelled.When invert sugar was the substrate the sucrose synthesizedwas strongly labelled in both moieties, as also were the freehexosea. It is concluded that fructose and glucose free or combinedin sucrose were equally available for starch synthesis and CO₂,formation, and that there can be no question of preferentialutilization of one or other hexose. Starch and CO₂ must arisefrom a common source in which readily formed derivatives ofthe hexoses are rapidly equilibrated. Free hexose cannot participatedirectly in either sucrose or starch synthesis. Accumulationof sugar not immediately metabolized and inversion of sucrosetake place at a site remote from the common pool. A scheme toaccommodate the results is discussed.     

Role of sugars in nitrate utilization by roots of dwarf bean

Nitrate uptake and in vivo, nitrate reductase activity (NRA) in roots of Phaseolus vulgaris, L. cv. Witte Krombek were measured in nitrogen-depleted plants of varying sugar status, Variation in sugar status was achieved at the start of nitrate nutrition by excision, ringing, darkness or administration of sugars to the root medium. The shape of the apparent induction pattern of nitrate uptake was not influenced by the sugar status of the absorbing tissue. When measured after 6 h of nitrate nutrition (0.1 mol m^?3), steady state nitrate uptake and root NRA were in the order intact>dark>ringed>excised. Exogenous sucrose restored NRA in excised roots to the level of intact plants. The nitrate uptake rate of excised roots, however, was not fully restored by sucrose (0.03–300 mol m^?3). When plants were decapitated after an 18 h NO₃^? pretreatment, the net uptake rate declined gradually to become negative after three hours. This decline was slowed down by exogenous fructose, whilst glucose rapidly (sometimes within 5 min) stimulated NG^?₃ uptake. Presumably due to a difference in NO₃^? due to a difference in NO₃^? uptake, the NRA of excised roots was also higher in the presence of glucose than in the presence of fructose after 6 h of nitrate nutrition. The sugar-stimulation of, oxygen consumption as well as the release of ¹⁴CO₂ from freshly absorbed (U-¹⁴C) sugar was the same for glucose and fructose. Therefore, we propose a glucose-specific effect on NO₃^? uptake that is due to the presence of glucose rather than to its utilization in root respiration. A differential glucose-fructose effect on nitrate reductase activity independent of the effect on NO₃^? uptake was not indicated. A constant level of NRA occurred in roots of NO₃^? induced plants. Removal of nutrient nitrate from these plants caused an exponential NRA decay with an approximate half-life of 12 h in intact plants and 5.5 h in excised roots. The latter value was also found in roots that were excised in the presence of nitrate, indicating that the sugar status primarily determines the apparent rate of nitrate reductase decay in excised roots.     

Analysis of steady state photosynthesis in alfalfa leaves

A method for carrying out kinetic tracer studies of steady state photosynthesis in whole leaves has been developed. An apparatus that exposes whole leaves to ¹⁴CO₂ under steady state conditions, while allowing individual leaf samples to be removed as a function of time, has been constructed. Labeling data on the incorporation of ¹⁴C into Medicago sativa L. metabolite pools are reported. A carbon dioxide uptake rate of 79 micromoles ¹⁴CO₂ per milligram chlorophyll per hour was observed at a CO₂ level slightly below that of air. Several actively turning over pools of early and intermediate metabolites, including 3-phosphoglyceric acid, glycerate, citrate, and uridine diphosphoglucose, showed label saturation after approximately 10 to 20 minutes of photosynthesis with ¹⁴CO₂ under steady state conditions. Alanine labeling increased more rapidly at first, and then at a lower rate as saturation was approached. Sucrose was a major product of photosynthesis and label saturation of the sucrose pool was not observed. Labeled carbon appeared rapidly in secondary metabolites. The steady state apparatus used has numerous advantages, including leaf temperature control, protection against leaf dehydration, high illumination, known ¹⁴CO₂ specific radioactivity, and provision for control and adjustment of ¹⁴CO₂ concentration. The apparatus allows for experiments of long duration and for sufficient sample points to define clearly the metabolic steady state.     

Relationship between substrate concentration,growth rate,and respiration rate of Escherichia coli in continuous culture

Summary Using a continuous flow technique the relationship between growth rate and substrate concentration was investigated with glucose as the limiting factor of a culture of Escherichia coli. Graphical and numerical analysis of the experimental data demonstrated that the application of the Michaelis-Menten equation produced erroneous results, whereas, the constants obtained from the Teissier equation were in agreement with the experimental data. On this basis, new equations defining the steady state cell and substrate concentration in continuous flow cultures were developed and tested against experimental data.Comparison of the specific growth rates, substrate uptake rates and oxygen consumption rates demonstrated that all were directly proportional to each other and could be related to each other by mathematical equations. Specifically it was shown that as the growth rate increased from 0.06 to k _m =0.76 the substrate uptake rate increased from 134 to 1420 mg glucose per gram cell weight per hour and the oxygen consumption rate increased from 48.6 to 505 mg O₂ per gram cell weight per hour. Independent of the growth rate 37% of the carbohydrate consumed were oxidized. The yield factor varied from 0.44 at low growth rates to 0.54 at high growth rates. Analysis of the growth rate-substrate uptake rate relationship indicated that a minimum substrate uptake rate of 55 mg glucose per gram cell weight per hour existed below which cell reproduction would cease. This was supported by the fact that steady state conditions could not be maintained in the culture at D values below 0.02 when the substrate supply rate decreased below 45 mg glucose per gram cell weight per hour.Material contained in this paper was submitted as a thesis in partial fulfillment of the requirements for the Ph. D. degree of Dr. R. S. Lipe.     

Responses of CAM species to increasing atmospheric CO2 concentrations

Crassulacean acid metabolism (CAM) species show an average increase in biomass productivity of 35% in response to a doubled atmospheric CO₂ concentration. Daily net CO₂ uptake is similarly enhanced, reflecting in part an increase in chlorenchyma thickness and accompanied by an even greater increase in water‐use efficiency. The responses of net CO₂ uptake in CAM species to increasing atmospheric CO₂ concentrations are similar to those for C₃ species and much greater than those for C₄ species. Increases in net daily CO₂ uptake by CAM plants under elevated atmospheric CO₂ concentrations reflect increases in both Rubisco‐mediated daytime CO₂ uptake and phosphoenolpyruvate carboxylase (PEPCase)‐mediated night‐time CO₂ uptake, the latter resulting in increased nocturnal malate accumulation. Chlorophyll contents and the activities of Rubisco and PEPCase decrease under elevated atmospheric CO₂, but the activated percentage for Rubisco increases and the K_M(HCO₃ ^? ) for PEPCase decreases, resulting in more efficient photosynthesis. Increases in root:shoot ratios and the formation of additional photosynthetic organs, together with increases in sucrose‐Pi synthase and starch synthase activity in these organs under elevated atmospheric CO₂ concentrations, decrease the potential feedback inhibition of photosynthesis. Longer‐term studies for several CAM species show no downward acclimatization of photosynthesis in response to elevated atmospheric CO₂ concentrations. With increasing temperature and drought duration, the percentage enhancement of daily net CO₂ uptake caused by elevated atmospheric CO₂ concentrations increases. Thus net CO₂ uptake, productivity, and the potential area for cultivation of CAM species will be enhanced by the increasing atmospheric CO₂ concentrations and the increasing temperatures associated with global climate change.     

The influence of sucrose and an elevated CO2 concentration on photosynthesis of photoautotrophic peanut (Arachis hypogaea L.) cell cultures

Using photoautotrophic cells ofArachis hypogaea (L.) growing at ambient CO₂, it was shown that exogenous sucrose supplied to the liquid medium reduced¹⁴CO₂ fixation (supplied as NaH¹⁴CO₃). This was mostly due to a reduced labelling in P-esters, and to a lesser extent, in the serine/glycine moiety. However, radioactivity in the neutral sugar fraction was increased upon supplement of exogenous sucrose. The reduced labelling of P-esters and serine/glycine agrees with a lower concentration and specific activity of Rubisco in the sucrose supplied treatments as compared to the control. Following a transfer into a sugar free nutrient medium the concentration and activity of Rubisco is increased. The concentration of PEPCase was not influenced by sucrose application, although its specific activity was increased.At elevated CO₂ concentration (2.34% v/v) the Rubisco concentration and specific activity was at the same level as in the control (0.03% v/v CO₂). However, the concentration and the specific activity of PEPCase was increased and dry weight increase was about 8–9-fold higher than at ambient CO₂.     

Effects of CO2 and sugars on photosynthesis and composition of avocado leaves grown in vitro

The effects of micropropagation conditions on avocado (Persea americana Mill.) have been measured in leaves and plants cultured in vitro. The consequences of the type and concentration of sugar in the medium and of carbon dioxide concentration in the atmosphere on the rates of photosynthesis and amounts of ribulose 1,5-biphosphate carboxylase-oxygenase (EC 4.1.1.39; Rubisco) and total soluble protein (TSP) were measured. At the highest sucrose supply (87.6 mM), Rubisco content was substantially decreased in leaves, and even more when elevated CO₂ (1 000 μmol·mol⁻¹) was supplied. Maximum photosynthetic rate (P_max) was significantly decreased when plants developed in high sucrose and elevated CO₂. However, Rubisco concentration was significantly greater when glucose was supplied at the same molar concentration or when the concentration of sucrose was small (14.6 mM), and no differences were observed due to the CO₂ concentration in the air in these treatments. The ratio of Rubisco to total soluble protein (Rubisco/TSP) was dramatically decreased in plants grown in the highest concentration of sucrose and with elevated CO₂. Leaf area and ratio of leaf fresh weight/(stem + root) fresh weight, were greater in plants grown with CO₂ enriched air. However, upon transplanting, survival was poorer in plants grown on low sucrose/high CO₂ compared to those grown on high sucrose/high CO₂.     

Effects of CO2 enrichment on whole-plant carbon budget of seedlings of Fagus grandifolia and Acer saccharum in low irradiance

Carbon exchange rates (CER) and whole-plant carbon balances of beech (Fagus grandifolia) and sugar maple (Acer saccharum) were compared for seedlings grown under low irradiance to determine the effects of atmospheric CO₂ enrichment on shade-tolerant seedlings of co-dominant species. Under contemporary atmospheric CO₂, photosynthetic rate per unit mass of beech was lower than for sugar maple, and atmospheric CO₂ enrich ment enhanced photosynthesis for beech only. Aboveground respiration per unit mass decreased with CO₂ enrichment for both species while root respiration per unitmass decreased for sugar maple only. Under contemporary atmoapheric CO₂, beech had lower C uptake per plant than sugar maple, while C losses per plant to nocturnal aboveground and root respiration were similar for both species. Under elevated CO₂, C uptake per plant was similar for both species, indicating a significant relative increase in whole-seedling CER with CO₂ enrich ment for beech but not for sugar maple. Total C loss per plant to aboveground respiration was decreased for beech only because increase in sugar maple leaf mass counterbalanced a reduction in respiration rates. Carbon loss to root respiration per plant was not changed by CO₂ enrichment for either species. However, changes in maintenance respiration cost and nitrogen level suggest changes in tissue composition with elevated CO₂. Beech had a greater net daily C gain with CO₂ enrichment than did sugar maple in contrast to a lower one under contemporary CO₂. Elevated CO₂ preferentially enhances the net C balance of beech by increasing photosynthesis and reducing respiration cost. In all cases, the greatest C lost was by roots, indicating the importance of belowground biomass in net C gain. Relative growth rate estimated from biomass accumulation was not affected by CO₂ enrichment for either species possibly because of slow growth under low light. This study indicates the importance of direct effects of CO₂ enrichment when predicting potential change in species distribution with global climate change.     

Fermentation parameters of Peptostreptococcus productus on gaseous substrates (CO,H2/CO2)

Intrinsic growth and substrate uptake parameters were obtained for Peptostreptococcus productus, strain U-1, using carbon monoxide as the limiting substrate. A modified Monod model with substrate inhibition was used for modeling. In addition, a product yield of 0.25 mol acetate/mol CO and a cell yield of 0.034 g cells/g CO were obtained. While CO was found to be the primary substrate, P. productus is able to produce acetate from CO₂ and H₂, although this substrate could not sustain growth. Yeast extract was found to also be a growth substrate. A yield of 0.017 g cell/g yeast extract and a product yield of 0.14 g acetate/g yeast extract were obtained. In the presence of acetate, the maximum specific CO uptake rate was increased by 40% compared to the maximum without acetate present. Cell replication was inhibited at acetate concentrations of 30 g/l. Methionine was found to be an essential nutrient for growth and CO uptake by P. productus. A minimum amount of a complex medium such as yeast extract (0.01%) is, however, required.     

Continuous culture growth of photoautotrophic cell suspensions from Chenopodium rubrum

The construction and operation of a continuous culture system for the propagation of cell suspensions from Chenopodium rubrum under photoautotrophic conditions has been described. A dilution rate of 0.16/day gave an equilibrium culture density of 1,100,000 cells/ml and a mean doubling time of 150 hours. During continuous culture steady state conditions with respect to nutrient uptake, cell protein and chlorophyll content, starch accumulation, in vitro activities of enzymes related to different metabolic pathways could be established. Photosynthetic CO₂ assimilation of steady state cells was about 100 mol CO₂/mg chlorophyll x hour. Dark CO₂ fixation was 3% of the light values.     

Role of sucrose phosphate synthase in sucrose biosynthesis in ripening bananas and its relationship to the respiratory climacteric

During ripening of bananas (Musa spp. [AAA group, Cavendish subgroup]), there is a massive conversion of starch to sucrose. Also during ripening there is a rise in respiration known as the respiratory climacteric. In this study changes in carbohydrate content, activities of starch and sucrose metabolizing enzymes, and respiration were measured to assess their potential interrelationships. Sucrose phosphate synthase activity increased dramatically during the first 4 days after initiation of ripening by ethylene treatment. Starch concentration decreased and sucrose concentration increased during this time period. Developmental changes in sucrose phosphate synthase activity were measured with limiting substrate (plus Pi) and saturating substrate concentrations. Activities were not parallel under the two assay conditions, providing tentative evidence that kinetically different forms of the enzyme may exist at different stages of ripening. Sucrose accumulation rate was most highly correlated with sucrose phosphate synthase activity assayed with limiting substrate concentrations (plus Pi). The cumulative amount of CO₂ respired during ripening was positively correlated with sugar accumulation (R² = 0.97). From this linear regression it was calculated that a constant 0.605 millimoles of CO₂ was evolved per mole of sucrose formed throughout ripening. Using this quantity, the percentage of the total respiratory ATP produced which was required for the conversion of starch to sucrose was calculated assuming different models for carbon export from the amyloplast. The results suggest that sucrose biosynthesis during ripening constitutes a significant sink for respiratory ATP.     

Uptake and Metabolism of Carbohydrates by Bradyrhizobium japonicum Bacteroids

Bradyrhizobium japonicum bacteroids were isolated anaerobically and were supplied with ¹⁴C-labeled trehalose, sucrose, UDP-glucose, glucose, or fructose under low O₂ (2% in the gas phase). Uptake and conversion of ¹⁴C to CO₂ were measured at intervals up to 90 minutes. Of the five compounds studied, UDP-glucose was most rapidly absorbed but it was very slowly metabolized. Trehalose was the sugar most rapidly converted to CO₂, and fructose was respired at a rate at least double that of glucose. Sucrose and glucose were converted to CO₂ at a very low but measurable rate (<0.1 nanomoles per milligram protein per hour). Carbon Number 1 of glucose appeared in CO₂ at a rate 30 times greater than the conversion of carbon Number 6 to CO₂, indicating high activity of the pentose phosphate pathway. Enzymes of the Entner-Doudoroff pathway were not detected in bacteroids, but very low activities of sucrose synthase and phosphofructokinase were demonstrated. Although metabolism of sugars by B. japonicum bacteroids was clearly demonstrated, the rate of sugar uptake was only 1/30 to 1/50 the rate of succinate uptake. The overall results support the view that, although bacteroids metabolize sugars, the rates are very low and are inadequate to support nitrogenase.     

Effects of Calcium on Sugar Transport in Azotobacter vinelandii

A fast and environmentally safe procedure was used to study sugar uptake by Azotobacter vinelandii. Transport experiments were performed in a 24-well plate and aerated by rapid oscillatory vibration. Samples were washed by centrifugation and dissolved in biodegradable scintillation cocktail for counting. At cell concentrations up to 6 × 10⁸ cells per ml, the uptake of sucrose was a function of time and was proportional to the cell concentration. This modified uptake assay was used to test the effect of cations on sugar uptake in A. vinelandii. Results showed that Ca²⁺ at 1 to 2 mM stimulated sucrose uptake by decreasing the apparent K_m of sucrose transport. Higher Ca²⁺ concentrations inhibited sucrose uptake in this organism.     

Cell division arrest by gamma-irradiation in photoautotrophic suspension culture of Euphorbia characias: Maintenance of photosynthetic capacity and overaccumulation of sucrose

Gamma-irradiation (250 Gy) applied to photoautotrophic cell suspensions of Euphorbia characias L. in the exponential growth phase led to the arrest of cell division and to a subsequent overaccumulation of sucrose and dry matter. From the fourth day of culture, the chlorophyll content and gross photosynthesis were not depressed by gamma-treatment nor by sugar accumulation. In both cultures, no difference was observed between oxygen uptake in the light at CO₂ saturating concentration and in the dark, suggesting that no change in energy-dissipative reactions took place after irradiation. A slight increase in oxygen uptake in both light and dark was observed in irradiated cells during the first four days. However, in the absence of limiting factors, the photosynthetic capacities of the dividing and irradiated non-dividing photoautotrophic cells were identical but higher than that of the non-dividing cells in the stationary growth phase. This suggests that gamma-irradiation arrests cell division by a mechanism different to that occuring in stationary-phase cultures. This may be of value in investigating the metabolism of secondary products.     

Sources of sucrose translocated from illuminated sugar beet source leaves

A search for source leaf sucrose pools that differed in their relation to export was carried out in photosynthesizing leaves of Beta vulgaris L. The time course of depletion of [¹⁴C]sucrose in a leaf in unlabeled CO₂ following steady state labeling provided evidence for two distinct sucrose pools. After the start of the light period, leaf blade sucrose remained constant although it exchanged between the two pools. Newly synthesized sucrose destined for export passed through one pool more rapidly than through the other. All of the leaf blade sucrose appeared to exchange with export sucrose. Modeling and regression analysis of [¹⁴C]sucrose data provided a means for estimating the size of the two pools. From 20 to 40% of the sucrose was calculated to be present in the pool that provided the less direct path to export; this was likely vacuolar sucrose. The remainder of the sucrose in the blade was probably in the cytoplasm and veins. Added amounts of leaf blade sucrose, produced in response to elevated CO₂, appeared to be stored mainly in the vacuolar compartment.     

Growth Kinetics, Carbohydrate, and Leaf Phosphate Content of Clover (Trifolium subterraneum L.) after Transfer to a High CO(2) Atmosphere or to High Light and Ambient Air

Intact air-grown (photosynthetic photon flux density, 400 microeinsteins per square meter per second) clover plants (Trifolium subterraneum L.) were transfered to high CO₂ (4000 microliters CO₂ per liter; photosynthetic photon flux density, 400 microeinsteins per square meter per second) or to high light (340 microliters CO₂ per liter; photosynthetic photon flux density, 800 microeinsteins per square meter per second) to similarly stimulate photosynthetic net CO₂ uptake. The daily increment of net CO₂ uptake declined transiently in high CO₂, but not in high light, below the values in air/standard light. After about 3 days in high CO₂, the daily increment of net CO₂ uptake increased but did not reach the high light values. Nightly CO₂ release increased immediately in high light, whereas there was a 3-day lag phase in high CO₂. During this time, starch accumulated to a high level, and leaf deterioration was observed only in high CO₂. After 12 days, starch was two- to threefold higher in high CO₂ than in high light, whereas sucrose was similar. Leaf carbohydrates were determined during the first and fourth day in high CO₂. Starch increased rapidly throughout the day. Early in the day, sucrose was low and similar in high CO₂ and ambient air (same light). Later, sucrose increased considerably in high CO₂. The findings that (a) much more photosynthetic carbon was partitioned into the leaf starch pool in high CO₂ than in high light, although net CO₂ uptake was similar, and that (b) rapid starch formation occurred in high CO₂ even when leaf sucrose was only slightly elevated suggest that low sink capacity was not the main constraint in high CO₂. It is proposed that carbon partitioning between starch (chloroplast) and sucrose (cytosol) was perturbed by high CO₂ because of the lack of photorespiration. Total phosphate pools were determined in leaves. Concentrations based on fresh weight of orthophosphate, soluble esterified phosphate, and total phosphate markedly declined during 13 days of exposure of the plants to high CO₂ but changed little in high light/ambient air. During this time, the ratio of orthophosphate to soluble esterified phosphate decreased considerably in high CO₂ and increased slightly in high light/ambient air. It appears that phosphate uptake and growth were similarly stimulated by high light, whereas the coordination was weak in high CO₂.