Postillumination respiration of maize in relation to oxygen concentration and glycolic Acid metabolism

Prior illumination in CO₂-free air enhances a respiration from maize (Zea mays L.) leaves different in onset and duration from the postillumination burst of photorespiration. The course of respiration after brief illumination of attached leaves was measured as CO₂ efflux in darkness into CO₂-free atmospheres with four O₂ concentrations. The peak of CO₂ efflux following illumination was suppressed by 2.23% O₂, was completely eliminated by 0.04% O₂, and was not stimulated by 40% O₂ compared with air. Compared with air, steady dark respiration was suppressed by 0.04% O₂ but was not affected by 2.23% nor 40% O₂. Excision and subsequent uptake of distilled water through the vascular system nearly eliminated the enhanced respiration.     

Photosynthesis and Activity of Ribulose Bisphosphate Carboxylase of Wheat and Maize Seedlings during and following Exposure to O(2)-Low, CO(2)-Free N(2)

Seven day old wheat and maize seedlings were exposed to 1300 or 2000 microeinsteins per square meter per second photosynthetically active radiation in CO₂-free air for 3 hours with either 1% O₂ in N₂ or N₂-only and then returned to normal air of 340 microliters per liter CO₂, 21% O₂ in N₂. Activity of the ribulose bisphosphate carboxylase and amount of the substrate, ribulose 1,5-bisphosphate, were measured during and following the CO₂-free treatments as was photosynthetic CO₂ fixation. Photoinhibition of photosynthesis was observed only with wheat seedlings following the N₂ only treatment. During the CO₂-free treatments, the levels of RuBP rose during all experiments except when wheat was photoinhibited. The activity of the ribulose bisphophate carboxylase, measured directly upon grinding the leaves, declined during the CO₂-free conditions. The carboxylase total activity increased in minutes in the leaf during and following the CO₂-free treatments. The specific activities of the wheat carboxylase went from 0.16 to 1.06 micromoles CO₂ fixed per milligram protein per minute while the maize carboxylase varied from 0.05 to 0.36 micromole CO₂ fixed per millogram protein per minute. This suggests that in these seedlings considerable inactive carboxylase must be stored in a form not activatable in extracts by CO₂ and Mg²⁺. Possible mechanisms of regulation of photosynthesis by the ribulose bisphosphate carboxylase must consider not only the amount of active enzyme, but the amount of enzyme which the plant can make activatable upon demand.     

Effects of Atmospheric CO(2) Enrichment on Photosynthesis, Respiration, and Growth of Sour Orange Trees

Numerous net photosynthetic and dark respiratory measurements were made over a period of 4 years on leaves of 24 sour orange (Citrus aurantium) trees; 8 of them growing in ambient air at a mean CO₂ concentration of 400 microliters per liter, and 16 growing in air enriched with CO₂ to concentrations approaching 1000 microliters per liter. Over this CO₂ concentration range, net photosynthesis increased linearly with CO₂ by more than 200%, whereas dark respiration decreased linearly to only 20% of its initial value. These results, together with those of a comprehensive fine-root biomass determination and two independent aboveground trunk and branch volume inventories, suggest that a doubling of the air's current mean CO₂ concentration of 360 microliters per liter would enhance the growth of the trees by a factor of 3.8.     

Prechilling of Xanthium strumarium L. Reduces Net Photosynthesis and, Independently, Stomatal Conductance, While Sensitizing the Stomata to CO(2)

Greenhouse-grown plants of Xanthium strumarium L. were exposed in a growth cabinet to 10 C during days and 5 C during nights for periods of up to 120 hours. Subsequently, CO₂ exchange, transpiration, and leaf temperature were measured on attached leaves and in leaf sections at 25 or 30 C, 19 C dew point of the air, 61 milliwatts per square centimeter irradiance, and CO₂ concentrations between 0 and 1000 microliters per liter ambient air. Net photosynthesis and stomatal conductance decreased and dark respiration increased with increasing duration of prechilling. The reduction in net photosynthesis was not a consequence of decreased stomatal conductance because the intercellular CO₂ concentration in prechilled leaves was equal to or greater than that in greenhouse-grown controls. The intercellular CO₂ concentration at which one-half maximum net photosynthesis occurred remained the same in prechilled leaves and controls (175 to 190 microliters per liter). Stomata of the control plants responded to changes in the CO₂ concentration of the air only slightly. Prechilling for 24 hours or more sensitized stomata to CO₂; they responded to changes in CO₂ concentration in the range from 100 to 1000 microliters per liter.     

An investigation into the roles of photosynthesis and respiration in h efflux from aerated suspensions of asparagus mesophyll cells

Aerated and stirred suspensions of mechanically isolated Asparagus sprengeri Regel mesophyll cells were used to investigate the roles of respiration and photosynthesis in net H⁺ efflux. Rates varied between 0.12 and 1.99 nanomoles H⁺ per 10⁶ cells per minute or 3 and 40 nanomoles H⁺ per milligram chlorophyll per minute. The mean rate of H⁺ efflux was 10% greater in the dark. 3-(3,4-Dichlorophenyl)-l,l-dimethylurea, an inhibitor of noncyclic photophosphorylation, did not inhibit H⁺ efflux from illuminated cells. Bubbling with N₂ or addition of oligomycin, an inhibitor of mitochondrial ATP production, resulted in rapid and virtually complete inhibition of H⁺ efflux in light or dark. In the absence of aeration, H⁺ efflux came to a halt but resumed with aeration or illumination. When aeration was switched to CO₂-free air, rates of H⁺ efflux were reduced 43% in the dark and 57% in the light. Oligomycin eliminated dark CO₂ fixation but not photosynthetic CO₂ fixation. It is suggested that H⁺ efflux is dependent on respiration and dark CO₂ fixation, but independent of photosynthesis.     

Saturation Kinetics of the Velocity of Stomatal Closing in Response to CO(2)

Stomatal closing movements in response to changes from CO₂-free to CO₂-containing air were recorded in leaf sections of Zea mays using air flow porometers. The response to CO₂ was fast; the shortest lag between the application of 300 microliters CO₂ per liter of air and the beginning of a stomatal response was 3 seconds. The velocity of stomatal closing increased with CO₂ concentration and approached its maximal value between 10³ and 10⁴ microliters CO₂ per liter of air. The CO₂ concentration at which the closing velocity reached half its maximal value was approximately 200 microliters CO₂ per liter of air, both in the light and in darkness. This indicates that the mechanism of stomatal responses to CO₂ is the same in both light regimes and that the range of stomatal sensitivity to changes in CO₂ concentration coincides with the range of CO₂ concentrations known to occur in the intercellular spaces of illuminated leaves.     

Conversion of photosynthetic products in the light in CO2-free O2 and N2 in leaves of Zea mays L. and Phaseolus vulgaris L.

Summary After 10 min illumination of segments of bean (Phaseolus vulgaris L.) or maize (Zea mays L.) leaves in air with ¹⁴CO₂, the atmosphere was changed to CO₂-free O₂ or N₂ and conversion of photosynthetic products in the light was investigated. The experiments have shown that after the ¹⁴CO₂ assimilation period the bean leaves contain the pool of weakly fixed ¹⁴C (WF-¹⁴C) which is converted into stable products during the subsequent period of illumination in CO₂-free N₂. In O₂ atmosphere the WF-¹⁴C pool is initially the main source of CO₂ evolved. The marked decrease in radioactivity of sucrose and starch during illumination of bean leaves in O₂ atmosphere indicates that these compounds were also the source of CO₂ evolved in the light. The total amount of previously fixed ¹⁴C remained almost on the same level during illumination of maize leaves in N₂ as well as in O₂. However, oxygen changed the distribution of ¹⁴C in photosynthetic products, which is suggested to be the consequence of the photorespiration process in maize.Abbreviation WF-¹⁴C weakly fixed ¹⁴C     

Effects of CO(2) Enrichment and Carbohydrate Content on the Dark Respiration of Soybeans

During the period of most active leaf expansion, the foliar dark respiration rate of soybeans (Glycine max cv Williams), grown for 2 weeks in 1000 microliters CO₂ per liter air, was 1.45 milligrams CO₂ evolved per hour leaf density thickness, and this was twice the rate displayed by leaves of control plants (350 microliters CO₂ per liter air). There was a higher foliar nonstructural carbohydrate level (e.g. sucrose and starch) in the CO₂ enriched compared with CO₂ normal plants. For example, leaves of enriched plants displayed levels of nonstructural carbohydrate equivalent to 174 milligrams glucose per gram dry weight compared to the 84 milligrams glucose per gram dry weight found in control plant leaves. As the leaves of CO₂ enriched plants approached full expansion, both the foliar respiration rate and carbohydrate content of the CO₂ enriched leaves decreased until they were equivalent with those same parameters in the leaves of control plants. A strong positive correlation between respiration rate and carbohydrate content was seen in high CO₂ adapted plants, but not in the control plants.

Mitochondria, isolated simultaneously from the leaves of CO₂ enriched and control plants, showed no difference in NADH or malate-glutamate dependent O₂ uptake, and there were no observed differences in the specific activities of NAD⁺ linked isocitrate dehydrogenase and cytochrome c oxidase. Since the mitochondrial O₂ uptake and total enzyme activities were not greater in young enriched leaves, the increase in leaf respiration rate was not caused by metabolic adaptations in the leaf mitochondria as a response to long term CO₂ enrichment. It was concluded, that the higher respiration rate in the enriched plant's foliage was attributable, in part, to a higher carbohydrate status.

     

Relationship between Photosynthesis and Respiration: The Effect of Carbohydrate Status on the Rate of CO(2) Production by Respiration in Darkened and Illuminated Wheat Leaves

The rate of dark CO₂ efflux from mature wheat (Triticum aestivum cv Gabo) leaves at the end of the night is less than that found after a period of photosynthesis. After photosynthesis, the dark CO₂ efflux shows complex dependence on time and temperature. For about 30 minutes after darkening, CO₂ efflux includes a large component which can be abolished by transferring illuminated leaves to 3% O₂ and 330 microbar CO₂ before darkening. After 30 minutes of darkness, a relatively steady rate of CO₂ efflux was obtained. The temperature dependence of steady-state dark CO₂ efflux at the end of the night differs from that after a period of photosynthesis. The higher rate of dark CO₂ efflux following photosynthesis is correlated with accumulated net CO₂ assimilation and with an increase in several carbohydrate fractions in the leaf. It is also correlated with an increase in the CO₂ compensation point in 21% O₂, and an increase in the light compensation point. The interactions between CO₂ efflux from carbohydrate oxidation and photorespiration are discussed. It is concluded that the rate of CO₂ efflux by respiration is comparable in darkened and illuminated wheat leaves.     

Laboratory-Produced CO(2) Calibration Gases

A simple, inexpensive apparatus for making mixtures of accurately known amounts of CO₂ and CO₂-free atmospheric air is described. Calibration gases with CO₂ contents of 200 to 1500 microliters per liter produced with the apparatus had concentrations which were within 10 microliters per liter of the target concentration.     

Investigation on photorespiration with a sensitive C-assay

A leaf disk assay for photorespiration has been developed based on the rate of release of recently fixed ¹⁴CO₂ in light in a rapid stream of CO₂-free air at 30° to 35°. In tobacco leaves (Havana Seed) photorespiration with this assay is 3 to 5 times greater than the ¹⁴CO₂ output in the dark. In maize, photorespiration is only 2% of that in tobacco.

The importance of open leaf stomata, rapid flow rates of CO₂-free air, elevated temperatures, and oxygen in the atmosphere in order to obtain release into the air of a larger portion of the ¹⁴CO₂ evolved within the tissue in the light was established in tobacco. Photorespiration, but not dark respiration, was inhibited by α-hydroxy-2-pyridinemethanesulfonic acid, an inhibitor of glycolate oxidase, and by 3-(4-chlorophenyl)-1,1-dimethylurea (CMU), an inhibitor of photosynthetic electron transport, under conditions which did not affect the stomata. These experiments show that the substrates of photorespiration and dark respiration differ and also provide additional support for the role of glycolate as a major substrate of photorespiration. It was also shown that at 35° the quantity of ¹⁴CO₂ released in the assay may represent only 33% of the gross ¹⁴CO₂ evolved in the light, the remainder being recycled within the tissue.

It was concluded that maize does not evolve appreciable quantities of CO₂ in the light and that this largely accounts for the greater efficiency of net photosynthesis exhibited by maize. Hence low rates of photorespiration may be expected to be correlated with a high rate of CO₂ uptake at the normal concentrations of CO₂ found in air and at higher light intensities.

     

The Regulation of Photosynthesis in Leaves of Field-Grown Spring Wheat (Triticum aestivum L., cv Albis) at Different Levels of Ozone in Ambient Air

Wheat (Triticum aestivum L. cv Albis) was grown in open-top chambers in the field and fumigated daily with charcoal-filtered air (0.015 microliters per liter O₃), nonfiltered air (0.03 microliters per liter O₃), and air enriched with either 0.07 or 0.10 microliters per liter ozone (seasonal 8 hour/day [9 am-5 pm] mean ozone concentration from June 1 until July 10, 1987). Photosynthetic ¹⁴CO₂ uptake was measured in situ. Net photosynthesis, dark respiration, and CO₂ compensation concentration at 2 and 21% O₂ were measured in the laboratory. Leaf segments were freeze-clamped in situ for the determination of the steady state levels of ribulose 1,5-bisphosphate, 3-phosphoglycerate, triose-phosphate, ATP, ADP, AMP, and activity of ribulose, 1,5-bisphosphate carboxylase/oxygenase. Photosynthesis of flag leaves was highest in filtered air and decreased in response to increasing mean ozone concentration. CO₂ compensation concentration and the ratio of dark respiration to net photosynthesis increased with ozone concentration. The decrease in photosynthesis was associated with a decrease in chlorophyll, soluble protein, ribulose bisphosphate carboxylase/oxygenase activity, ribulose bisphosphate, and adenylates. No decrease was found for triose-phosphate and 3-phosphoglycerate. The ratio of ATP to ADP and of triosephosphate to 3-phosphoglycerate were increased suggesting that photosynthesis was limited by pentose phosphate reductive cycle activity. No limitation occurred due to decreased access of CO₂ to photosynthetic cells since the decrease in stomatal conductance with increasing ozone concentration did not account for the decrease in photosynthesis. Ozonestressed leaves showed an increased degree of activation of ribulose bisphosphate carboxylase/oxygenase and a decreased ratio of ribulose bisphosphate to initial activity of ribulose bisphosphate carboxylase/oxygenase. Nevertheless, it is suggested that photosynthesis in ozone stressed leaves is limited by ribulose bisphosphate carboxylation possibly due to an effect of ozone on the catalysis by ribulose bisphosphate carboxylase/oxygenase.     

Effects of temperature on the gas exchange of leaves in the light and dark

Summary Evolution of CO₂ into CO₂-free air was measured in the light and in the dark over a range of temperatures from 15 to 50°. Photosynthetic rates were measured in air and O₂-free air over the same range of temperatures. Respiration in the light had a different sensitivity to temperature compared with respiration in the dark. At the lower temperatures the rate of respiration in the light was higher than respiration in the dark, whereas at temperatures above 40° the reverse was observed. For any one species the maximum rates of photosynthesis and photorespiration occur at about the same temperature. The maximum rate for dark respiration generally is found at a temperature about 10° higher. Zea mays and Atriplex nummularia showed no enhancement of photosynthesis in O₂-free air nor any evolution of CO₂ in CO₂-free air at any of the temperatures.     

Effects of High Atmospheric CO(2) and Sink Size on Rates of Photosynthesis of a Soybean Cultivar

The effect of sink strength on photosynthetic rates under conditions of long-term exposure to high CO₂ has been investigated in soybean. Soybean plants (Merr. cv. Fiskeby V) were grown in growth chambers containing 350 microliters CO₂ per liter air until pod set. At that time, plants were trimmed to three trifoliolate leaves and either 21 pods (high sink treatment) or 6 pods (low sink treatment). Trimmed plants were either left in 350 microliters CO₂ per liter of air or placed in 1000 microliters CO₂ per liter of air (high CO₂ treatment) until pod maturity. Whole plant net photosynthetic rates of all plants were measured twice weekly, both at 350 microliters CO₂ per liter of air and 1000 microliters CO₂ per liter of air. Plants were also harvested at this time for dry weight measurements. Photosynthetic rates of high sink plants at both measurement CO₂ concentrations were consistently higher than those of low sink plants, and those of plants given the 350 microliter CO₂ per liter of air treatment were higher at both measurement CO₂ concentrations than those of plants given the 1000 microliters CO₂ per liter of air treatment. When plants were measured under treatment CO₂ levels, however, rates were higher in 1,000 microliter plants than 350 microliter CO₂ plants. Dry weights of all plant parts were higher in the 1,000 microliters CO₂ per liter air treatment than in the 350 microliters CO₂ per liter air treatment, and were higher in the low sink than in the high sink treatments.     

Gas Exchange Characteristics of the Submerged Aquatic Crassulacean Acid Metabolism Plant, Isoetes howellii

The submerged aquatic plant Isoetes howellii Engelmann possesses Crassulacean acid metabolism (CAM) comparable to that known from terrestrial CAM plants. Infrared gas analysis of submerged leaves showed Isoetes was capable of net CO₂ uptake in both light and dark. CO₂ uptake rates were a function of CO₂ levels in the medium. At 2,500 microliters CO₂ per liter (gas phase, equivalent to 1.79 milligrams per liter aqueous phase), Isoetes leaves showed continuous uptake in both the light and dark. At this CO₂ level, photosynthetic rates were light saturated at about 10% full sunlight and were about 3-fold greater than dark CO₂ uptake rates. In the dark, CO₂ uptake rates were also a function of length of time in the night period. Measurements of dark CO₂ uptake showed that, at both 2,500 and 500 microliters CO₂ per liter, rates declined during the night period. At the higher CO₂ level, dark CO₂ uptake rates at 0600 h were 75% less than at 1800 h. At 500 microliters CO₂ per liter, net CO₂ uptake in the dark at 1800 h was replaced by net CO₂ evolution in the dark at 0600 h. At both CO₂ levels, the overnight decline in net CO₂ uptake was marked by periodic bursts of accelerated CO₂ uptake. CO₂ uptake in the light was similar at 1% and 21% O₂, and this held for leaves intact as well as leaves split longitudinally. Estimating the contribution of light versus dark CO₂ uptake to the total carbon gain is complicated by the diurnal flux in CO₂ availability under field conditions.     

Effect of changes in shoot carbon-exchange rate on soybean root nodule activity

The effect of short- and long-term changes in shoot carbon-exchange rate (CER) on soybean (Glycine max [L.] Merr.) root nodule activity was assessed to determine whether increases in photosynthate production produce a direct enhancement of symbiotic N₂ fixation. Shoot CER, root + nodule respiration, and apparent N₂ fixation (acetylene reduction) were measured on intact soybean plants grown at 700 microeinsteins per meter per second, with constant root temperature and a 14/10-hour light/dark cycle. There was no diurnal variation of root + nodule respiration or apparent N₂ fixation in plants assayed weekly from 14 to 43 days after planting. However, if plants remained in darkness following their normal dark period, a significant decline in apparent N₂ fixation was measured within 4 hours, and decreasing CO₂ concentration from 320 to 90 microliters CO₂ per liter produced diurnal changes in root nodule activity. Increasing shoot CER by 87, 84, and 76% in 2-, 3-, and 4-week-old plants, respectively, by raising the CO₂ concentration around the shoot from 320 to 1,000 microliters CO₂ per liter, had no effect on root + nodule respiration or acetylene-reduction rates during the first 10 hours of the increased CER treatment. When the CO₂-enrichment treatment was extended in 3-week-old plants, the only measured parameter that differed significantly after 3 days was shoot CER. After 5 days of continuous CO₂ enrichment, root + nodule respiration and acetylene reduction increased, but such changes reflected an increase in root nodule mass rather than greater specific root nodule activity. The results show that on a 24-hour basis the process of symbiotic N₂ fixation in soybean plants grown under controlled environmental conditions functioned at maximum capacity and was not limited by shoot CER. Whether N₂-fixation capacity was limited by photosynthate movement to root nodules or by saturation of metabolic processes in root nodules is not known.     

Effect of CO(2), O(2), and Light on Photosynthesis and Photorespiration in Wheat

Unidirectional O₂ fluxes were measured with ¹⁸O₂ in a whole plant of wheat cultivated in a controlled environment. At 2 or 21% O₂, O₂ uptake was maximum at 60 microliters per liter CO₂. At lower CO₂ concentrations, it was strongly inhibited, as was photosynthetic O₂ evolution. At 2% O₂, there remained a substantial O₂ uptake, even at high CO₂ level; the O₂ evolution was inhibited at CO₂ concentrations under 330 microliters per liter. The O₂ uptake increased linearly with light intensity, starting from the level of dark respiration. No saturation was observed at high light intensities. No significant change in the gas-exchange patterns occurred during a long period of the plant life. An adaptation to low light intensities was observed after 3 hours illumination. These results are interpreted in relation to the functioning of the photosynthetic apparatus and point to a regulation by the electron acceptors and a specific action of CO₂. The behavior of the O₂ uptake and the study of the CO₂ compensation point seem to indicate the persistence of mitochondrial respiration during photosynthesis.     

Evaluation of the light/dark C assay of photorespiration: tobacco leaf disk studies with glycidate and glyoxylate

Preincubation of illuminated tobacco (Nicotiana tabacum L.) leaf disks in glycidate (2,3-epoxypropionate) or glyoxylate inhibited photorespiration by about 40% as determined by the ratio of ¹⁴CO₂ evolved into CO₂-free air in light and in darkness. However, under identical preincubation conditions used for the light/dark ¹⁴C assays, the compounds failed to reduce photorespiration or stimulate net photosynthesis in tobacco leaf disks based on other CO₂ exchange parameters, including the CO₂ compensation concentration in 21% O₂, the inhibitory effect of 21% O₂ on net photosynthesis in 360 microliters per liter of CO₂ and the rate of net photosynthetic ¹⁴CO₂ uptake in air.

The effects of both glycidate and glyoxylate on the ¹⁴C assay are inconsistent with other measures of photorespiratory CO₂ exchange in tobacco leaf disks, and thus these data question the validity of the light to dark ratio of ¹⁴CO₂ efflux as an assay for relative rates of photorespiration (Zelitch 1968, Plant Physiol 43: 1829-1837). The results of this study specifically indicate that neither glycidate nor glyoxylate reduces photorespiration or stimulates net photosynthesis by tobacco leaf disks under physiological conditions of pO₂ and pCO₂, contrary to previous reports.

     

Effects of CO(2)-Enrichment and of Aminoacetonitrile on Growth and Photosynthesis of Photoautotrophic Calli of Nicotiana plumbaginifolia

Photoautotrophic calli of Nicotiana plumbaginifolia were grown for 3 weeks under two CO₂ concentrations (500 and 20,000 microliters of CO₂ per liter). Calli cultured at high CO₂ exhibited a two-fold higher rate of growth. At CO₂ test levels, these calli were characterized by a lower net photosynthetic capacity than calli cultured at low CO₂. This diminution due to CO₂ adaptation could be ascribed to a 170% stimulation of dark respiration, a 40% decrease in total ribulose-1,5-bisphosphate carboxylase (Rubisco) activity, and also to a feedback inhibition of photosynthesis: high CO₂ grown calli contained about 5.5-fold more sucrose and three-fold less orthophosphate (Pi) than low CO₂ grown calli. Whether the decrease in Rubisco activity is related to the accumulation of sucrose and to the Pi limitation is discussed. Both calli exhibited a Warburg-effect showing the existence of active photorespiration at low CO₂. In calli grown at low CO₂ with 5 millimolar aminoacetonitrile (AAN), an inhibitor of the glycolate pathway, fresh weight decreased by 25% and chlorophyll content by 40%, dark respiration increased by 50% and net CO₂ uptake decreased by about 60% at 340 microliters of CO₂ per liter and 35% at 10,000 microliters of CO₂ per liter. In these calli, glutamine and glutamate contents were half of control calli. In contrast, AAN did not provoke any noticeable effect in calli grown at high CO₂. In photoautotrophic calli, the inhibition of the glycolate pathway by AAN results in severe perturbations in glutamate metabolism and in chlorophyll biosynthesis.     

Changes in Levels of Intermediates of the C(4) Cycle and Reductive Pentose Phosphate Pathway under Various Light Intensities in Maize Leaves

The rate of CO₂ assimilation and levels of metabolites of the C₄ cycle and reductive pentose phosphate pathway in attached leaves of maize (Zea mays L.) were measured over a range of light intensity from 0 to 1,900 microEinsteins per square meter per second under a saturated CO₂ concentration of 350 microliters per liter and a limiting CO₂ concentration of 133 microliters per liter. The level of ribulose 1,5-bisphosphate (RuBP) stayed almost constant (around 60 nanomoles per milligram chlorophyll [Chl]) from low to high light intensities under 350 microliters per liter. Levels of 3-phosphoglycerate (PGA) increased from 100 to 650 nanomoles per milligram Chl under 350 microliters per liter CO₂ with increasing light intensity. The calculated RuBP concentration of 6 millimolar (corresponded to 60 nanomoles per milligram Chl) was about two times above the estimated RuBP binding-site concentration on ribulose bisphosphate carboxylase-oxygenase (Rubisco) of ~2.6 millimolar in maize bundle sheath chloroplasts in the light. The ratio of RuBP/PGA increased with decreasing light intensity under 350 microliters per liter CO₂. These results suggest that RuBP carboxylation is under control of light intensity possibly due to a limited supply of CO₂ to Rubisco through the C₄ cycle whose activity is highly dependent on light intensity. Pyruvate level increased with increasing light intensity as long as photosynthesis rate increased. A positive relationship between levels of PGA and those of pyruvate during steady-state photosynthesis under various conditions suggests that an elevated concentration of PGA increases the carbon input into the C₄ cycle through the conversion of PGA to PEP and consequently the level of total intermediates of the C₄ cycle can be raised to mediate higher photosynthesis rate.