Inhibition of Dark CO(2) Fixation and Photosynthesis in Leaf Discs of Corn Susceptible to the Host-specific Toxin Produced by Helminthosporium maydis, Race T

The host-specific toxin produced by Helminthosporium maydis, race T, causes 50% inhibition of dark fixation of ¹⁴CO₂ by leaf discs of susceptible (Texas male sterile) corn when it is diluted to approximately 1/10,000 of the volume of the original fungus culture filtrate. Dilutions of 1/10 or less are required for equivalent inhibition of discs prepared from resistant (N) corn. Root growth and photosynthesis were considerably less sensitive (dilution values 1/3000 and 1/1200, respectively), as was leakage of ¹⁴C induced by toxin from preloaded discs. Based on literature values for dilutions causing ion leakage or inhibition of mitochondrial oxidation, toxin dilutions several orders of magnitude greater bring about inhibition of dark CO₂ fixation. Preincubation of discs in light increased sensitivity of dark fixation to toxin and an effect of light on symptom development was shown. Phosphoenolypruvate carboxylase activity in extracts of roots or leaves was not affected by toxin nor was the enzyme level altered in excised leaves treated with toxin. Inhibition of dark fixation of CO₂ provides a bioassaay for race T toxin which is both reliable and rapid.     

Analogs of host-specific phytotoxin produced by Helminthosporium maydis,race T: II. Biological activities

Inhibition of dark CO₂ fixation by susceptible corn leaves was used to compare the relative toxicity of synthetic analogs with that of the host-specific phytotoxin produced by the fungal corn pathogen, Helminthosporium maydis, race T. Analogs with C₁₅, C₂₅, or C₂₆ chain lengths and 1,5-dioxo-3-hydroxy functions were only slightly less toxic (2–6 × 10^?7M) than native T toxin (C₃₅–C₄₅ chain lengths) or its individual components (3 × 10^?8M). Like native toxin, analogs were host-specific in that they did not inhibit dark CO₂ fixation in leaf tissue of resistant corn at concentrations 10²–10³ times greater than those effective with susceptible corn. These findings support the structures previously proposed for native T toxin.     

Biological Activity of the Isomeric Forms of Helminthosporium sacchari Toxin and of Homologs Produced in Culture

The effect of Helminthosporium sacchari (HS) toxin isomers and related, pathogen-produced compounds on dark CO₂ fixation in HS-susceptible sugar cane leaf slices was investigated. HS toxin consists of a mixture of three isomeric bis-5-O-(β-galactofuranosyl)-β-galactofuranosides (A, B, and C) differing in the position of one double bond in the sesquiterpene aglycone. Maximum inhibition of dark CO₂ fixation in susceptible sugar cane (CP52-68) occurred within 30 to 40 minutes, and amounts necessary to reach 50% inhibition values typically were approximately 1.7 micromolar for natural toxin mixture ( 2:3:5 mixture of isomers A:B:C) and 4, 6, and 0.7 micromolar for isomers A, B, and C, respectively. Other fractions from cultures of the pathogen consist of comparable mixtures of sesquiterpene isomers but have only 1, 2, or 3 galactofuranose units (HS₁, HS₂, HS₃) or two α-glucopyranose units as well as four β-galactofuranose units (HS₆). The lower toxin homologs were not toxic to clone CP52-68, but protected sugar cane from the effects of toxin. Minimum ratios of protectant: toxin giving 95% protection were approximately 50:1, 6:1, and 12:1 for HS₁, HS₂, and HS₃, respectively. HS₂ and HS₃ protected when added up to 12 minutes after toxin as well as when added with or before toxin. Some common plant galactopyranosides were not toxic and did not protect at 500:1 molar excess. The sample of HS₆ was toxic at 500 micromolar, and did not protect against HS toxin. With the availability of purified, homogeneous preparations of HS toxin, homologs, and chemically modified or synthetic analogs, the dark CO₂ fixation assay should prove to be a useful tool for understanding the mode of action of HS toxin.     

Reactions in chloroplasts,cytoplasm and mitochondria of leaf slices under osmotic stress

The effect of osmotic dehydration on metabolic reactions in three different subcellular compartments (chloroplast, cytoplasm and mitochondria) was studied in vacuum-infiltrated thin leaf slices from various plants, in the absence of stomatal control. The reactions tested were CO₂ fixation in the light (chloroplast), CO₂ fixation in the dark (cytoplasm), and O₂ uptake in the dark (mitochondria). In most plants, the sensitivity of dark CO₂ fixation to dehydration was similar to the sensitivity of photosynthesis. In leaf slices from a plant with Crassulacean acid metabolism (Kalanchoe pinnata), dark CO₂ fixation (which reached similar rates as light fixation) was slightly more sensitive to osmotic stress than photosynthesis. Dark respiration (measured as O₂ uptake) was significantly more resistant to hypertonic stress than both types of CO₂ fixation. In crude leaf extracts from spinach, the response of soluble enzymes from the three different subcellular compartments to high concentrations of various electrolytes and neutral compounds was examined and compared with the in-vivo data.     

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.     

Cytoplasm-specific Effects of Helminthosporium maydis Race T Toxin on Survival of Corn Mesophyll Protoplasts

High yields of mesophyll protoplasts were obtained from leaves of corn (Zea mays L., inbred W64A). Many protoplasts survived a week in the dark in a simple osmoticum. Culture filtrate from Helminthosporium maydis race T at dilutions of 1:10,000 to 1:20,000 destroyed protoplasts with Texas male-sterile (T) cytoplasm. Substantial damage to protoplasts with nonmale-sterile (N) cytoplasm occurred only at a 1:20 dilution. High concentrations of partially purified H. maydis race T (HMT) toxin (32.5-130 μg dry weight/ml) did not reduce survival of protoplasts with N cytoplasm or C or S male-sterile cytoplasms after 6 days of exposure. Protoplasts with T or TRf (fertility restored) cytoplasm collapsed within 1 to 3 days after treatment with 0.13 μg of HMT toxin/ml, which was one-fifth the level causing 50% inhibition of T cytoplasm seedling root growth. Protoplasts with T cytoplasm which were washed after 30 minutes or more of exposure to HMT toxin also collapsed within a few days. Cultured W64A T protoplasts and freshly isolated protoplasts from inbreds C103 and Mo17 with T cytoplasm were less sensitive to HMT toxin than freshly isolated W64A T protoplasts. Toxin-treated protoplasts survived longer in the light than in the dark. The sensitivity and specificity of the system described will facilitate physiological, ultrastructural, and genetic studies of toxin action.     

Molecular Features Affecting the Biological Activity of the Host-Selective Toxins from Cochliobolus victoriae

The structures of the toxins produced by Cochliobolus victoriae, victorin B, C, D, E, and victoricine, have recently been established. These toxins and modified forms of victorin C were tested for their effect on dark CO₂ fixation in susceptible oat (Avena sativa) leaf slices. Half-maximal inhibition of dark CO₂ fixation occurred with the native toxins in the range of 0.004 to 0.546 micromolar. An essential component for the inhibitory activity of victorin is the glyoxylic acid residue, particularly its hydrated aldehyde group. Removal of glyoxylic acid completely abolished the inhibitory activity of victorin, and the reduction of the aldehydo group transformed the toxin into a protectant. Conversion of victorin to its methyl ester resulted in diminution of inhibitory activity to 10% of the original activity of the toxin, whereas derivatization of the ε-amino group of the β-hydroxylysine moiety resulted in a decrease of inhibitory activity to 1% of that of victorin C. However, the derivatized toxin retained its host selectivity. In addition, the opening of the macrocyclic ring of the toxin drastically reduced the inhibitory activity.     

Reversible light-activation of ribulose bisphosphate carboxylase/oxygenase in isolated barley protoplasts and chloroplasts

The enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase displayed near-maximal activity in isolated, intact barley (Hordeum vulgare L. cv. Pennrad) mesophyll protoplasts. The carboxylase deactivated 40 to 50% in situ when protoplasts were dark-incubated 20 minutes in air-equilibrated solutions. Enzyme activity was fully restored after 1 to 2 minutes of light. Addition of 5 millimolar NaHCO₃ to the incubation medium prevented dark-inactivation of the carboxylase. There was no permanent CO₂-dependent activation of the protoplast carboxylase either in light or dark. Activation of the carboxylase from ruptured protoplasts was not increased significantly by in vitro preincubation with CO₂ and Mg²⁺. In contrast to the enzyme in protoplasts, the carboxylase in intact barley chloroplasts was not fully reactivated by light at atmospheric CO₂ levels. The lag phase in carbon assimilation was not lengthened by dark-adapting protoplasts to low CO₂ demonstrating that light-activation of the carboxylase was not involved in photosynthetic induction. Irradiance response curves for reactivation of the the carboxylase and for CO₂ fixation by isolated barley protoplasts were similar. The above results show that there was a fully reversible light-activation of the carboxylase in isolated barley protoplasts at physiologically significant CO₂ levels.     

Effects of glyoxylate on photosynthesis by intact chloroplasts

Because glyoxylate inhibits CO₂ fixation by intact chloroplasts and purified ribulose bisphosphate carboxylase/oxygenase, glyoxylate might be expected to exert some regulatory effect on photosynthesis. However, ribulose bisphosphate carboxylase activity and activation in intact chloroplasts from Spinacia oleracea L. leaves were not substantially inhibited by 10 millimolar glyoxylate. In the light, the ribulose bisphosphate pool decreased to half when 10 millimolar glyoxylate was present, whereas this pool doubled in the control. When 10 millimolar glyoxylate or formate was present during photosynthesis, the fructose bisphosphate pool in the chloroplasts doubled. Thus, glyoxylate appeared to inhibit the regeneration of ribulose bisphosphate, but not its utilization.

The fixation of CO₂ by intact chloroplasts was inhibited by salts of several weak acids, and the inhibition was more severe at pH 6.0 than at pH 8.0. At pH 6.0, glyoxylate inhibited CO₂ fixation by 50% at 50 micromolar, and glycolate caused 50% inhibition at 150 micromolar. This inhibition of CO₂ fixation seems to be a general effect of salts of weak acids.

Radioactive glyoxylate was reduced to glycolate by chloroplasts more rapidly in the light than in the dark. Glyoxylate reductase (NADP⁺) from intact chloroplast preparations had an apparent K_m (glyoxylate) of 140 micromolar and a V_max of 3 micromoles per minute per milligram chlorophyll.

     

Veränderliche Markierungsmuster bei 14CO2-Fütterung von Bryophyllum tubiflorum zu verschiedenen Zeitpunkten der Hell-Dunkelperiode

Summary The distribution of radioactivity between the products of ¹⁴CO₂ light fixation in phyllodia of Bryophyllum tubiflorum could be influenced experimentally by manipulating the malic acid content of the cells. Accelerating the deacidification of the tissue during the light period by application of higher light intensities accelerated the increase of malate labelling and the decrease of the sucrose labelling after ¹⁴CO₂ light fixation under our standard conditions (10 min preillumination, 15 min ¹⁴CO₂ light fixation, 8000 lux).In other experiments different malate contents of the tissues were induced by treating the phyllodia with different temperatures during the night period. In the morning, phyllodia with low malate content transferred most of the label into malate, and phyllodia with high malate content incorporated most of the ¹⁴C radioactivity into sugars. However, this was true only after preillumination of 1 hour. When the phyllodia fixed ¹⁴CO₂ without preillumination, no differences in the labelling patterns between acidified and non-acidified phyllodia could be observed.In experiments using leaf tissue slices of Bryophyllum daigremontianum we could again observe that malate was labelled more heavily in the deacidified tissue than in the acidified controls, with less radioactivity being transferred into phosphate esters and sugars. The rates of ¹⁴CO₂ light fixation were identical in tissue slices with high and low malate content. However, the rates of CO₂ dark fixation in the acidified samples were clearly lower than those in the deacidified ones. The low rate of CO₂ dark fixation in acidified samples could not be inhibited by an inhibitor of PEP-carboxylase as the high CO₂ dark fixation rate of the deacidified tissue could be inhibited.The results are discussed in relation to the feed back inhibition of PEP-carboxylase in vivo by malate. Compartmentation also seemed to be involved in controlling the flow of carbon during CO₂ light fixation in succulent tissue.     

Effect of Oxygen on Photosynthesis, Photorespiration and Respiration in Detached Leaves. II. Corn and other Monocotyledons

The effect of O₂ on the CO₂ exchange of detached leaves of corn (Zea mays), wheat (Triticum vulgare), oats (Avena sativa), barley (Hordeum vulgare), timothy (Phleum pratense) and cat-tail (Typha angustifolia) was measured with a Clark oxygen electrode and infrared carbon dioxide analysers in both open and closed systems.

Corn leaves did not produce CO₂ in the light at any O₂ concentration, as was shown by the zero CO₂ compensation point and the absence of a CO₂ burst in the first minute of darkness. The rate of photosynthesis was inhibited by O₂ and the inhibition was not completely reversible. On the other hand, the steady rate of respiration after a few minutes in the dark was not affected by O₂.

These results were interpreted as indicating the absence of any measurable respiration during photosynthesis. Twelve different varieties of corn studied all responded to O₂ in the same way.

The other 5 monocotyledons studied did produce CO₂ in the light. Moreover, the CO₂ compensation point increased linearly with O₂ indicating a stimulation of photorespiration.

The implications of the lack of photorespiration in studies of primary productivity are discussed.

     

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.

     

Enhanced Dark CO(2) Fixation by Preilluminated Chlorella pyrenoidosa and Anacystis nidulans

The products of short time photosynthesis and of enhanced dark ¹⁴CO₂ fixation (illumination in helium prior to addition of ¹⁴CO₂ in dark) by Chlorella pyrenoidosa and Anacystis nidulans were compared. Glycerate 3-phosphate, phosphoenolpyruvate, alanine, and aspartate accounted for the bulk of the ¹⁴C assimilated during enhanced dark fixation while hexose and pentose phosphates accounted for the largest fraction of isotope assimilated during photosynthesis. During the enhanced dark fixation period, glycerate 3-phosphate is carboxyl labeled and glucose 6-phosphate is predominantly labeled in carbon atom 4 with lesser amounts in the upper half of the C₆ chain and traces in carbon atoms 5 and 6. Tracer spread throughout all the carbon atoms of photosynthetically synthesized glycerate 3-phosphate and glucose 6-phosphate. During the enhanced dark fixation period, there was a slow formation of sugar phosphates which subsequently continued at 5 times the initial rate long after the cessation of ¹⁴CO₂ uptake. To explain the kinetics of changes in the labelling patterns and in the limited formation of the sugar phosphates during enhanced dark CO₂ fixation, the suggestion is made that most of the reductant mediating these effects did not have its origin in the preillumination phase.

It is concluded that a complete photosynthetic carbon reduction cycle operates to a limited extent, if at all, in the dark period subsequent to preillumination.

     

Photorespiration and Glycolate Metabolism: A Re-examination and Correlation of Some Previous Studies

Some previous studies of photorespiration and glycolate oxidation were re-examined and correlated by infra-red CO₂ analysis. Data about rate of photosynthesis and oxygen sensitivity indicated that complete inhibition of photosynthesis with 3-(3,4-dichlorophenyl)-1,1 dimethyl urea (DCMU) allowed dark respiration to continue in the light. Photorespiration was also inhibited. The oxygen sensitivity of glycolate-stimulated CO₂ production was found to be compatible with the proposal that glycolate is a substrate of photorespiration. Both `in vivo' and `in vitro' studies of the alga Nitella flexilis have revealed a pathway of glycolate oxidation similar to that of higher plants. DCMU inhibition of photosynthesis by Nitella gave results similar to those for the monocotyledons tested. Under very low light intensity, carbon dioxide compensation in corn was measurable but was not sensitive to high oxygen concentration. It appears that the lack of photorespiration in this plant is not the end result of efficient internal recycling of CO₂ to photosynthesis.     

The effect of Cd2+ on photosynthetic reactions of mesophyll protoplasts

Photosynthetic CO₂-fixation of mesophyll protoplasts of lambs lettuce [Valerianella locusta (L.) Betcke] was inhibited by short time exposure to Cd⁺. Inhibition was due to uptake of the metal ion into the protoplasts and increased with increasing Cd²⁺ concentrations and the time of preincubation. A 10 min pretreatment at 2 mM Cd²⁺ reduced CO₂-fixation by 40–60%. Inhibition of photosynthesis was independent of the light intensity to which the protoplasts were exposed. Measurement of the lightinduced electrochromic pigment absorption change at 518nm and chlorophyll fluorescence studies revealed that primary photochemical reactions associated with the thylakoid membranes were not affected by the metal ion. Also, light activation of the ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) was not inhibited by Cd²⁺. Under rate-limiting CO₂ concentrations, inhibition of CO₂-fixation was smaller than at V_max of CO₂ reduction indicating that the carboxylation reaction of the Calvin cycle is not susceptible to Cd²⁺. Cd²⁺ treatment of protoplasts significantly extended the lagphase of CO₂-supported O₂-evolution and partly inhibited light activation of the glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13) and the ribulose-5-phosphate kinase (EC 2.7.1.19). Measurement of relative concentrations of [¹⁴C]-labeled Calvin cycle intermediates showed that Cd²⁺ caused a decrease in the 3-phosphoglycerate/triose phosphate ratio and an increase in the triose phosphate/ribulose-1,5-bisphosphate ratio. It is concluded that in protoplasts Cd²⁺ affects photosynthesis mainly at the level of dark reactions and that the site of inhibition may be localized in the regenerative phase of the Calvin cycle.     

Photosynthesis, Respiration and Post-illumination Fixation of CO2 by Corn Leaves as Influenced by Light and Oxygen Concentration

The effect of oxygen concentration on the rate of CO₂-uptake in continuous and intermittent light was studied as well as the CO₂-fixation during a short dark period after light was turned off. In addition the dark respiration and the CO₂-compensation point of attached and detached corn leaves were determined. Leaves of 4 to 22-day old plants were used as experimental material. A closed circuit system of an infrared carbon dioxide analyzer was employed to measure the rate of CO₂-exchange. It was found that in an atmosphere consisting of 100 % oxygen, there was about 50 per cent inhibition of the rate of CO₂-uptake in continuous and intermittent light compared to that in an atmosphere consisting of 21% oxygen. The same was true of the rate of CO₂-fixation in darkness during a short period after the light was turned off. Since the response to oxygen concentration of the CO₂-uptake in light and of the CO₂-fixation in darkness after the light was turned off were similar, it is concluded that the fixation of CO₂ in the short dark period represents an over- shoot of photosynthesis. The rate of dark respiration was little affected by the oxygen concentration in the ranges used in the experiments. The carbon dioxide compensation point which has been observed in leaves of 4 to 14-day old plants was not influenced by either oxygen concentration or light intensity. Since the changes in the rate of CO₂-uptake due to changes in the concentration of oxygen and light intensity had no effect on the CO₂-compensation point, it is concluded that a reabsorption of respiratory CO₂ by photosynthesis could not account for the low value of this point. These results are interpreted as a further corroboration of the statement that the leaves of corn lack the process of photorespiration and that dark respiration is inhibited in light. It was observed that the rate of the CO₂-uptake gradually increased in plants which were from 4 to 22-days old. The inhibitory effect of high concentration of oxygen on the rate of CO₂-uptake was relatively higher in old plants than in young ones.     

Reduction of Adenosine Triphosphate Levels in Susceptible Maize Mesophyll Protoplasts by Helminthosporium maydis Race T Toxin

Helminthosporium maydis race T (HMT) toxin caused a reduction in the steady-state ATP levels when leaf mesophyll protoplasts isolated from maize containing Texas male-sterile (T) but not male-fertile (N) cytoplasm were incubated in the dark. At a toxin concentration 10 times the mean effectived dose for inhibition of root growth, the ATP levels began to fall in 30 to 90 seconds, fell by 50% in about 4 minutes, and reached 23% of the original levels in 100 minutes. This is faster than any previously observed response of whole cells or tissues to HMT toxin. In protoplasts incubated in the light, ATP levels were 25% higher than in the dark and were either unaffected or only slightly diminished by toxin. 3-(3,4-Dichlorophenyl)-1, 1-dimethylurea (DCMU), an inhibitor of photosynthetic electron transport, overcame the effect of light on both toxin-treated and control protoplasts. Oligomycin, an inhibitor of mitochondrial ATP synthesis, mimicked the effects of toxin in the dark, in the light, and in the light plus DCMU, but it was not specific for T cytoplasm. During the first 24 hours of culture, ATP levels in control protoplasts increased in both the light and dark. In the dark, ATP was not detectable after 24-hour incubation in the presence of toxin, whereas in the light a substantial amount of ATP remained. Our results are compatible with the hypothesis that mitochondria in vivo are inhibited by HMT toxin. Other responses of cells and tissues to toxin can be explained in terms of reduced ATP levels.     

Light control of the respiration of exogenous glycerol in the red macroalga Grateloupia doryphora

Heterotrophic activity in macroalgae has been little studied, but the red macroalga Grateloupia doryphora is known to grow in light at a higher rate in a glycerol-containing medium than in seawater. The effects of 0·1 M exogenous glycerol in seawater (SW90-gly) on the respiration rate of G. doryphora and the role played by light were investigated. The algae pretreated for 2 h in the light and in SW90-gly evolved oxygen and fixed carbon dioxide (H¹⁴CO₃ ^?), but also evolved radioactive ¹⁴CO₂ from [¹⁴C]glycerol. The rate of oxygen evolution was lower than that of samples in seawater, due to a high respiration rate and/or a partial inhibition of photosynthesis induced by glycerol. In contrast, the rate of inorganic carbon fixation was higher in SW90-gly than in control samples in seawater, suggesting that non-photosynthetic patterns were operating. In darkness, after pretreatment in the light in SW90-gly, samples showed a high oxygen uptake rate just after the light was turned off. Twenty minutes of darkness were enough to decrease this high respiration rate to that of samples in seawater. The oxygen uptake observed in all experiments with glycerol was mitochondrial as it was inhibited by potassium cyanide and salicylhydroxamic acid (SHAM). Pretreatment of samples in the light in SW90-gly with the photosynthetic inhibitor DCMU did not inhibit ensuing dark respiration, thus providing evidence for a non-photosynthetic effect of the light. The highest dark respiration rate was observed after the samples were pretreated in monochromatic blue light in glycerol-containing media.     

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.     

Demonstration of Both a Photosynthetic and a Nonphotosynthetic CO(2) Requirement for NH(4) Assimilation in the Green Alga Selenastrum minutum

Nitrogen-limited and nitrogen-sufficient cell cultures of Selenastrum minutum (Naeg.) Collins (Chlorophyta) were used to investigate the dependence of NH₄⁺ assimilation on exogenous CO₂. N-sufficient cells were only able to assimilate NH₄⁺ maximally in the presence of CO₂ and light. Inhibition of photosynthesis with 3-(3,4-dichlorophenyl)-1,1-dimethylurea, diuron also inhibited NH₄⁺ assimilation. These results indicate that NH₄⁺ assimilation by N-sufficient cells exhibited a strict requirement for photosynthetic CO₂ fixation. N-limited cells assimilated NH₄⁺ both in the dark and in the light in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, diuron, indicating that photosynthetic CO₂ fixation was not required for NH₄⁺ assimilation. Using CO₂ removal techniques reported previously in the literature, we were unable to demonstrate CO₂-dependent NH₄⁺ assimilation in N-limited cells. However, employing more stringent CO₂ removal techniques we were able to show a CO₂ dependence of NH₄⁺ assimilation in both the light and dark, which was independent of photosynthesis. The results indicate two independent CO₂ requirements for NH₄⁺ assimilation. The first is as a substrate for photosynthetic CO₂ fixation, whereas the second is a nonphoto-synthetic requirement, presumably as a substrate for the anaplerotic reaction catalyzed by phosphoenolpyruvate carboxylase.