Dark Respiration during Photosynthesis in Wheat Leaf Slices

The metabolism of [¹⁴C]succinate and acetate was examined in leaf slices of winter wheat (Triticum aestivum L. cv Frederick) in the dark and in the light (1000 micromoles per second per square meter photosynthetically active radiation). In the dark [1,4-¹⁴C]succinate was rapidly taken up and metabolized into other organic acids, amino acids, and CO₂. An accumulation of radioactivity in the tricarboxylic acid cycle intermediates after ¹⁴CO₂ production became constant indicates that organic acid pools outside of the mitochondria were involved in the buildup of radioactivity. The continuous production of ¹⁴CO₂ over 2 hours indicates that, in the dark, the tricarboxylic acid cycle was the major route for succinate metabolism with CO₂ as the chief end product. In the light, under conditions that supported photorespiration, succinate uptake was 80% of the dark rate and large amounts of the label entered the organic and amino acids. While carbon dioxide contained much less radioactivity than in the dark, other products such as sugars, starch, glycerate, glycine, and serine were much more heavily labeled than in darkness. The fact that the same tricarboxylic acid cycle intermediates became labeled in the light in addition to other products which can acquire label by carboxylation reactions indicates that the tricarboxylic acid cycle operated in the light and that CO₂ was being released from the mitochondria and efficiently refixed. The amount of radioactivity accumulating in carboxylation products in the light was about 80% of the ¹⁴CO₂ release in the dark. This indicates that under these conditions, the tricarboxylic acid cycle in wheat leaf slices operates in the light at 80% of the rate occurring in the dark.     

Hydrogen evolution as a consumption mode of reducing equivalents in green algal fermentation

Dark anaerobic fermentation in the green algae Chlamydomonas MGA 161, Chlamydomonas reinhardtii, Chlorella pyrenoidosa, and Chlorococcum minutum was studied. Our isolate, Chlamydomonas MGA 161, was unusual in having high H₂ but almost no formate. The fermentation pattern in Chlamydomonas MGA 161 was altered by changes in the NaCl or NH₄Cl concentration. Glycerol formation increased at low (0.1%) and high (7%) NaCl concentrations; starch degradation, and formation of ethanol, H₂, and CO₂ increased with the addition of NH₄Cl to above 5 millimolar in N-deficient cells. C. reinhardtii and C. pyrenoidosa exhibited a very similar anaerobic metabolism, forming formate, acetate and ethanol in a ratio of about 2:2:1. C. minutum was also unusual in forming acetate, glycerol, and CO₂ as its main products, with H₂, formate, and ethanol being formed in negligible amounts. In the presence of CO, ethanol formation increased twofold in Chlamydomonas MGA 161 and C. reinhardtii, but the fermentation pattern in C. minutum did not change. An experiment with hypophosphite addition showed that dark H₂ evolution of the Escherichia coli type could be ruled out in Chlamydomonas MGA 161 and C. reinhardtii. Among the green algae investigated, three fermentation types were identified by the distribution pattern of the end products, which reflected the consumption mode of reducing equivalents in the cells.     

Zur Analyse des CO2-Austausches von Bryophyllum

Summary Starch consumption during the dark period in detached phyllodia of Bryophyllum tubiflorum is inhibited, when the phyllodia are held in an atmosphere free from carbon dioxide during the night. This is true also in other succulent plants with Crassulacean acid metabolism=CAM (examined were Bryophyllum calycinum and Sedum morganianum). This effect seems to indicate that the role of starch in CAM is production of CO₂ acceptors rather than production of carbon dioxide by respiration. If the CO₂ acceptors are not used, starch consumption comes to an end.This hypothesis could also explain results of experiments in which phyllodia were held at different temperatures during the dark period, and net CO₂ fixation, starch loss and malate gain were determined. At 10° CO₂ uptake was at a maximum (the necessary supply of CO₂ acceptors must have therefore been at a maximum, too). Under these conditions there was the greatest amount of starch consumption. At 23° C, CO₂ uptake was clearly lowered, and this was also true for starch consumption. At 35° C net CO₂ uptake was balanced by net CO₂, output (no CO₂ acceptors were needed in CO₂ dark fixation). At this temperature no starch loss could be measured.     

Light Stimulation of Ethylene Release from Leaves of Gomphrena globosa L

The effect of light and CO₂ on both the endogenous and 1-aminocyclopropane-1-carboxylic acid (ACC)-dependent ethylene evolution from metabolically active detached leaves and leaf discs of Gomphrena globosa L. is reported. Treatment with varying concentrations of ACC did not appear to inhibit photosynthesis, respiration, or stomatal behavior. In all treatments, more ethylene was released into a closed flask from ACC-treated tissue, but the pattern of ethylene release with respect to light/dark/CO₂ treatments was the same.

Leaf tissue in the light with a source of CO₂ sufficient to maintain photosynthesis always generates 3 to 4 times more ethylene than tissue in the dark. Conversely, the lowest rate of ethylene release occurs when leaf tissue is illuminated and photosynthetic activity depletes the CO₂ to the compensation point. Ethylene release in the dark is also stimulated by CO₂ either added to the flask as bicarbonate or generated by dark respiration. Ethylene release increases dramatically and in parallel with photosynthesis at increasing light intensities in this C₄ plant. Ethylene release appears dependent on CO₂ both in the light and in the dark. Therefore, it is suggested that the important factor regulating the evolution of ethylene gas from leaves of Gomphrena may be CO₂ metabolism rather than light per se.

     

Photochemical disproportionation of sulfur into sulfide and sulfate byChlorobium limicola formathiosulfatophilum

The anaerobic bacteriumChlorobium assimilates carbon dioxide in the light with various sulfur compounds as electron donors. The well-known metabolic pathway proceeds from the oxidation of sulfide via sulfur to sulfate. In the dark the reaction is partially reversed when sulfur is reduced to hydrogen sulfide. The fermenting cells thereby release an excess of reductant. We have now found a hydrogen sulfide production from sulfur, which is light-dependent. It is more than ten times faster than the dark reaction. This appears in experiments where the cell suspension is illuminated in absence of CO₂ and flushed continuously with H₂ or Ar. The H₂S is trapped with ZnCl₂ and the S^2- titrated with iodine. The total amount of H₂S evolved in the light increases proportionally with the amount of sulfur added, and about one-half of the added sulfur is converted to H₂S. Another part of the metabolized sulfur appears at the same time as sulfate, but all the sulfur oxidized to sulfate does not account for the larger amount of sulfur reduced to hydrogen sulfide. Very likely other unanalyzed oxidized sulfur compounds must also have been produced. Use of H₂ instead of Ar as the anaerobic gas phase does not increase the amount of H₂S produced, nor does the addition of thiosulfate; sulfur itself is the preferred electron donor for the sulfur reduction. Up to a light intensity of 10000 ergs cm^-2sec^-1 CO₂ does not affect H₂S production. Without CO₂, saturation of the light-dependent evolution of H₂S is reached at about 40000 ergs cm^-2sec^-1. In contrast, presence of CO₂ at this light intensity makes the sulfide production disappear completely. On application of mass spectrometry to the gas exchange upon illumination, at high light intensity a H₂S gush is found during the first 3 min. This is followed by CO₂ fixation, while simultaneously the reductant H₂S is now taken up. WithRhodospirillum rubrum, the addition of sulfur leads to a moderate evolution of H₂S. In contrast toChlorobium this reaction inR. rubrum is not light-sensitive, nor does it produce detectable amounts of sulfate. After addition of malate the rate of H₂S evolution does increase in the light, since the cells use malate as an electron donor during their photochemical metabolism.     

Photosynthetic Production of Hydrogen Peroxide by Anacystis nidulans

A sensitive assay based upon fluorescence of scopoletin allowed continuous recording of H₂O₂ production in illuminated intact cells of Anacytis nidulans. Onset of illumination was followed by a 5 to 10 second lag, a burst of very rapid production continuing for up to 5 minutes, and finally a slow and continuing steady rate of H₂O₂ production. Size of the H₂O₂ burst was decreased by 3-(3,4-dichlorophenyl)-1, 1-dimethylurea, by low O₂, and by certain Calvin cycle intermediates; it was increased by high light intensity, CO₂ depletion, Calvin cycle inhibitors (as iodoacetamide), cold shock, carbonyl cyanide m-chlorophenylhydrazone, and certain organic acids as glycolate). The H₂O₂ burst was explained by the following hypothesis: a low potential reductant is produced more rapidly than it can be used in the normal pathway to CO₂ reduction and, instead, reacts with oxygen. H₂O₂ production is regarded as a metabolic defect observable in Anacystis most dramatically during the transition from a very low rate of oxidative dark metabolism to a high rate of photosynthetic metabolism.     

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.     

The role of Hox hydrogenase in the H2 metabolism of Thiocapsa roseopersicina

The purple sulfur phototrophic bacterium Thiocapsa roseopersicina BBS synthesizes at least three NiFe hydrogenases (Hox, Hup, Hyn). We characterized the physiological H₂ consumption/evolution reactions in mutants having deletions of the structural genes of two hydrogenases in various combinations. This made possible the separation of the functionally distinct roles of the three hydrogenases. Data showed that Hox hydrogenase (unlike the Hup and Hyn hydrogenases) catalyzed the dark fermentative H₂ evolution and the light-dependent H₂ production in the presence of thiosulfate. Both Hox⁺ and Hup⁺ mutants demonstrated light-dependent H₂ uptake stimulated by CO₂ but only the Hup⁺ mutant was able to mediate O₂-dependent H₂ consumption in the dark. The ability of the Hox⁺ mutant to evolve or consume hydrogen was found to depend on a number of interplaying factors including both growth and reaction conditions (availability of glucose, sulfur compounds, CO₂, H₂, light). The study of the redox properties of Hox hydrogenase supported the reversibility of its action. Based on the results a scheme is suggested to describe the role of Hox hydrogenase in light-dependent and dark hydrogen metabolism in T. roseopersicina BBS.     

Relationships between Photosynthetically Active Radiation, Nocturnal Acid Accumulation, and CO(2) Uptake for a Crassulacean Acid Metabolism Plant, Opuntia ficus-indica

The influences of photosynthetically active radiation (PAR) and water status on nocturnal Crassulacean acid metabolism (CAM) were quantitatively examined for a widely cultivated cactus, Opuntia ficus-indica (L.) Miller. When the total daily PAR was maintained at 10 moles photons per square meter per day but the instantaneous PAR level varied, the rate of nocturnal H⁺ accumulation (tissue acidification) became 90% saturated near 700 micromoles per square meter per second, a PAR level typical for similar light saturation of C₃ photosynthesis. The total nocturnal H⁺ accumulation and CO₂ uptake reached 90% of maximum for a total daily PAR of about 22 moles per square meter per day. Light compensation occurred near 0 moles per square meter per day for nocturnal H⁺ accumulation and 4 moles per square meter per day for CO₂ uptake. Above a total daily PAR of 36 moles per square meter per day or for an instantaneous PAR of 1150 micromoles per square meter per second for more than 6 hours, the nocturnal H⁺ accumulation actually decreased. This inhibition, which occurred at PAR levels just above those occurring in the field, was accompanied by a substantial decrease in chlorophyll content over a 1-week period.

A minimum ratio of H⁺ accumulated to CO₂ taken up of 2.5 averaged over the night occurred for a total daily PAR of 31 moles per square meter per day under wet conditions. About 2 to 6 hours into the night under such conditions, a minimum H⁺-to-CO₂ ratio of 2.0 was observed. Under progressively drier conditions, both nocturnal H⁺ accumulation and CO₂ uptake decreased, but the H⁺-to-CO₂ ratio increased. A ratio of two H⁺ per CO₂ is consistent with the H⁺ production accompanying the conversion of starch to malic acid, and it apparently occurs for O. ficus-indica when CAM CO₂ uptake is strongly favored over respiratory activity.

     

Effect of Osmotic Stress on Carbon Metabolism in Chlamydomonas reinhardtii: Accumulation of Glycerol as an Osmoregulatory Solute

NaCl, KCl, and sucrose at equiosmolar concentrations had similar inhibitory effects on photosynthetic carbon metabolism by the freshwater green alga, Chlamydomonas reinhardtii. Inhibitory concentrations of these solutes altered the products of photosynthetic ¹⁴CO₂ incorporation, resulting in reduced incorporation into starch, sugar phosphates, lactate, and glycolate, but caused an accumulation of glycerol both intracellularly and in the medium.     

Diurnal starch accumulation and utilization in phosphorus-deficient soybean plants

The effects of phosphorus deficiency on carbohydrate accumulation and utilization in 34-day-old soybean (Glycine max L. Merr.) plants were characterized over a diurnal cycle to evaluate the mechanisms by which phosphorus deficiency restricts plant growth. Phosphorus deficiency decreased the net CO₂ exchange rate throughout the light period. The decrease in the CO₂ exhange rate was associated with a decrease in stomatal conductance and an increase in the internal CO₂ concentration. These observations indicate that phosphorus deficiency increased mesophyll resistance. Assimilate export rate from the youngest fully expanded leaves was decreased by phosphorus deficiency, whereas starch concentrations in these leaves were increased. Higher starch concentrations in phosphorus-deficient youngest fully expanded leaves resulted from a longer period of net starch accumulation and a shorter period of net starch degradation relative to those for phosphorus-sufficient controls. Phosphorus deficiency decreased sucrose-P synthase activity by 27% (averaged over the diurnal cycle), and essentially eliminated diurnal variation in sucrose-P-synthase activity. Diurnal variations in nonstructural carbohydrate concentrations in leaves and stems were also less pronounced in phosphorus-deficient plants than in controls. In phosphorus-deficient plants, only 30% of the whole plant starch present at the end of a light phase was utilized during the subsequent 12-hour dark phase as compared with 68% for phosphorus-sufficient controls. Although phosphorus deficiency decreased the CO₂ exchange rate and whole plant leaf area, accumulation of high starch concentrations in leaves and stems and restricted starch utilization in the dark indicate that growth processes (i.e. sink activities) were restricted to a greater extent than photosynthetic capacity. Further experimentation is required to determine whether decreased starch utilization in phosphorus-deficient plants is the cause or the result of restricted growth.     

Hydrogen metabolism by filamentous cyanobacteria

Apparent discrepancies in the literature concerning the amounts of H₂ produced by strains of Anabaena cylindrica are explained. These are not due to differences in strains used by different workers nor to differences in growth conditions, but rather appear to be due to the fact that cultures show an increasing dependence with age on CO₂ for sustained H₂ production. Two distinct hydrogenase activities were measured and characterized, both in vivo and in vitro in A. cylindrica B629; these were H₂ uptake activity and H₂ evolution from reduced methyl viologen. Gentle cell disruption techniques were used to gain further evidence that the latter activity was soluble. H₂ uptake was strongly inhibited by acetylene in vivo in the light or in the dark with phenazine methosulfate added, but only after a prolonged lag period. In extracts this lag did not occur. A detailed study of the nitrogenase and hydrogen uptake activities and their interrelationship both in the light and in the dark in A. cylindrica B629 showed that only in the dark in the presence of O₂ did H₂ uptake support C₂H₂ reduction significantly. Under several conditions in which nitrogenase activity was inhibited H₂ uptake was unaffected. H₂ metabolism was tested in three nonheterocystous filamentous cyanobacteria under different growth and incubation conditions. These were Plectonema boryanum, Schizothrix calcicola, and Oscillatoria brevis. Myxosarcina chroococcoides and Fischerella muscicola were also investigated. Cyanobacterial species vary markedly in their hydrogen metabolism and in the composition of the three H₂ metabolizing enzymes.     

Enhancement of Ethylene Release from Leaf Tissue during Glycolate Decarboxylation : A Possible Role for Photorespiration

When leaf discs of Xanthium strumarium L. and Salvia splendens L. are incubated in sealed flasks in the light, more C₂H₄ gas is released in the presence of added CO₂ (30-200 millimolar NaHCO₃) than without CO₂. In Salvia, the maximum rate of C₂H₄ release occurs when sufficient CO₂ (above 125 millimolar NaHCO₃) is added to saturate photosynthesis confirming previous studies. The maximum rate of C₂H₄ release from illuminated discs is similar to the rate in the dark with or without CO₂ in both species. Glycolate enhances a CO₂-dependent C₂H₄ evolution from illuminated leaf discs. However, the maximum rate of C₂H₄ release with glycolate is the same as that observed with saturating CO₂. When photosynthesis is inhibited by darkness or by 3-(3,4-dichlorophenyl)-1,1-dimethylurea, glycolate has no effect.

Studies with [2,3-¹⁴C]-1-aminocyclopropane-1-carboxylic acid (ACC) show that the pattern of C₂H₄ release and the specific activity of the ¹⁴C₂H₄ in the presence and absence of glycolate is similar to that described above, indicating that glycolate does not alter uptake of the exogenously supplied precursor (ACC) or stimulate C₂H₄ release from an endogenous source at appreciable rates. Glycolate oxidase in vitro generates H₂O₂ which stimulates a slow breakdown of ACC to C₂H₄, but since exogenous glycolate is oxidized to CO₂ in both the light and the dark it is argued that the glycolate-dependent increase in C₂H₄ release from illuminated leaf discs is not mediated directly by the action of enzymes of glycolate catabolism. The effects of glycolate and CO₂ are not easily explained by changes in stomatal resistance. The data support the view that glycolate decarboxylation at subsaturating levels of CO₂ in the light stimulates C₂H₄ release by raising the CO₂ level in the tissue.

     

Carbon dioxide fixation in isolated kalanchoe chloroplasts

Chloroplasts isolated from Kalanchoe diagremontiana leaves were capable of photosynthesizing at a rate of 5.4 μmoles of CO₂ per milligram of chlorophyll per hour. The dark rate of fixation was about 1% of the light rate. A high photosynthetic rate was associated with low starch content of the leaves. Ribose 5-phosphate, fructose 1,6-diphosphate, and dithiothreitol stimulated fixation, whereas phosphoenolpyruvate and azide were inhibitors. The products of CO₂ fixation were primarily those of the photosynthetic carbon reduction cycle.     

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.     

The CO2-concentrating mechanism in a starchless mutant of the green unicellular alga Chlorella pyrenoidosa

The CO₂-concentrating mechanism (CCM) was induced in the green unicellular alga Chlorella when cells were transferred from high (5% CO₂) to low (0.03%) CO₂ concentrations. The induction of the CCM correlated with the formation of a starch sheath specifically around the pyrenoid in the chloroplast. With the aim of clarifying whether the starch sheath was involved in the operation of the CCM, we isolated and physiologically characterized a starchless mutant of Chlorella pyrenoidosa, designated as IAA-36. The mutant strain grew as vigorously as the wild type under high and low CO₂ concentrations, continuous light and a 12 h light/12 h dark photoperiod. The CO₂ requirement for half-maximal rates of photosynthesis [K_0.5(CO₂)] decreased from 40 μM to 2–3 μM of CO₂ when both wild type and mutant were switched from high to low CO₂. The high affinity for inorganic carbon indicates that the IAA-36 mutant is able to induce a fully active CCM. Since the mutant does not have the pyrenoid starch sheath, we conclude that the sheath is not involved in the operation of the CCM in Chlorella cells.     

Rewiring cyanobacterial photosynthesis by the implementation of an oxygen-tolerant hydrogenase

Molecular hydrogen (H₂) is considered as an ideal energy carrier to replace fossil fuels in future. Biotechnological H₂ production driven by oxygenic photosynthesis appears highly promising, as biocatalyst and H₂ syntheses rely mainly on light, water, and CO₂ and not on rare metals. This biological process requires coupling of the photosynthetic water oxidizing apparatus to a H₂-producing hydrogenase. However, this strategy is impeded by the simultaneous release of oxygen (O₂) which is a strong inhibitor of most hydrogenases. Here, we addressed this challenge, by the introduction of an O₂-tolerant hydrogenase into phototrophic bacteria, namely the cyanobacterial model strain Synechocystis sp. PCC 6803. To this end, the gene cluster encoding the soluble, O₂-tolerant, and NAD(H)-dependent hydrogenase from Ralstonia eutropha (ReSH) was functionally transferred to a Synechocystis strain featuring a knockout of the native O₂ sensitive hydrogenase. Intriguingly, photosynthetically active cells produced the O₂ tolerant ReSH, and activity was confirmed in vitro and in vivo. Further, ReSH enabled the constructed strain Syn_ReSH⁺ to utilize H₂ as sole electron source to fix CO₂. Syn_ReSH⁺ also was able to produce H₂ under dark fermentative conditions as well as in presence of light, under conditions fostering intracellular NADH excess. These findings highlight a high level of interconnection between ReSH and cyanobacterial redox metabolism. This study lays a foundation for further engineering, e.g., of electron transfer to ReSH via NADPH or ferredoxin, to finally enable photosynthesis-driven H₂ production.     

Effects of substrate limitation on product distribution and H2/CO2 ratio inKlebsiella pneumoniae during anaerobic fermentation of glycerol

Product formation during anaerobic degradation of glycerol byKlebsiella pneumoniae DSM 2026, under glycerol limitation and glycerol excess in continugius cultures, has been investigated. Major and minor products and by-products as well as gaseous products were measured. The results indicated a positive correlation between specific glycerol uptake and most product formation rates under glycerol limitation. The production of 1,3-propanediol, lactate, formate, acetate, succinate and the by-products of anaerobic glycerol degradation byK. pneumoniae, acetoin and 2,3-butanediol, was favoured by glycerol excess, while hydrogen generation and ethanol formation were best under glycerol limitation. It was also found that under glycerol limitation the rate of hydrogen evolution was generally higher than the CO₂ production rate while under excess glycerol the reverse was true. Hence, on the basis of the ratio of the specific rates of evolution of H₂ and CO₂ (q _{H 2}/q _{CO 2}), it is possible to infer the existence of glycerol limitation. On the basis of the carbon and available electron balances, which are independent of metabolic pathways, the data are consistent. The NADH₂ balance, which took into consideration the pathways of product formation, was also tested to check the validity of the assumed pathways and to check critically the consistency of the data. Good balances were also obtained.[     

CO2 and malate metabolism in starch-containing and starch-lacking guard-cell protoplasts

Isolated, purified mesophyll and guard-cell protoplasts of Vicia faba L. and Allium cepa L. were exposed to ¹⁴CO₂ in the light and in the dark. The guard-cell protoplasts of Vicia and Allium did not show any labeling in phosphorylated products of the Calvin cycle, thus appearing to lack the ability to reduce CO₂ photosynthetically. In Vicia, high amounts of radioactivity (35%) appeared in starch after 60-s pulses of ¹⁴CO₂ both in the light and in the dark. Presumably, the ¹⁴CO₂ is fixed into the malate via PEP carboxylase and then metabolized into starch as the final product of gluconeogenesis. This is supported by the fact that guard-cell protoplasts exposed to malic acid uniformly labeled with ¹⁴CO₂ showed high amounts of labeled starch after the incubation, whereas cells labeled with [4-¹⁴C]malate had minimal amounts of labeled starch (1/120).In contrast, the starch-deficient Allium, guard-cell protoplasts did not show any significant ¹⁴CO₂ fixation. However, adding PEP to an homogenate stimulated ¹⁴CO₂ uptake, thus supporting the interpretation that the presence of starch as a source of PEP is necessary for incorporating CO₂ and delivering malate. With starch-containing Vicia guard-cell protoplasts, the correlation between changes in volume and the interconversion of malate and starch was demonstrated. It was shown that the rapid gluconeogenic conversion of malate into starch prevents an increase of the volume of the protoplasts, whereas the degradation of starch to malate is accompanied by a swelling of the protoplasts.Abbreviations GCPs guard-cell protoplasts - MCPs mesophyll cell protoplasts - PEP phosphoenolpyruvate - DTT dithiothreitol - 3-PGA 3-phosphoglyceric acid - RiBP ribulose 1,5 bisphosphate - MDH malate dehydrogenase - MES 2-(N-morpholino)ethane sulfonic acid - CAM crassulacean acid metabolism     

Experimental determination of the respiration associated with soybean/rhizobium nitrogenase function, nodule maintenance, and total nodule nitrogen fixation

The total metabolic cost of soybean (Glycine max L. Mer Clark) nodule nitrogen fixation was empirically separated into respiration associated with electron flow through nitrogenase and respiration associated with maintenance of nodule function.

Rates of CO₂ evolution and H₂ evolution from intact, nodulated root systems under Ar:O₂ atmospheres decreased in parallel when plants were maintained in an extended dark period. While H₂ evolution approached zero after 36 hours of darkness at 22°C, CO₂ evolution rate remained at 38° of the rate measured in light. Of the remaining CO₂ evolution, 62% was estimated to originate from the nodules and represents a measure of nodule maintenance respiration. The nodule maintenance requirement was temperature dependent and was estimated at 79 and 137 micromoles CO₂ (per gram dry weight nodule) per hour at 22°C and 30°C, respectively.

The cost of N₂ fixation in terms of CO₂ evolved per electron pair utilized by nitrogenase was estimated from the slope of H₂ evolution rate versus CO₂ evolution rate. The cost was 2 moles CO₂ evolved per mole H₂ evolved and was independent of temperature.

In this symbiosis, nodule maintenance consumed 22% of total respiratory energy while the functioning of nitrogenase consumed a further 52%. The remaining respiratory energy was calculated to be associated with ammonia assimilation, transport of reduced N, and H₂ evolution.