Influence of Photorespiration on ATP/ADP Ratios in the Chloroplasts, Mitochondria, and Cytosol, Studied by Rapid Fractionation of Barley (Hordeum vulgare) Protoplasts

Using the principle described by R McC Lilley, M Stitt, G Mader, HW Heldt (1982 Plant Physiol 70: 965-970), an apparatus for rapid fractionation of barley leaf (Hordeum vulgare) protoplasts by membrane filtration was built. From studies of ATP/ADP ratios, it is concluded that the quenching of metabolic reactions is very fast, making it possible to use the method for studies on metabolic interactions between different compartments in plant cells. The fractionation method was used to study the influence of photorespiration on ATP/ADP ratios in the chloroplasts, mitochondria, and cytosol of barley leaf protoplasts. The cytosolic ATP/ADP ratio was higher under photorespiratory conditions than under nonphotorespiratory conditions. Aminoacetonitrile, an inhibitor of the photorespiratory conversion of glycine to serine, had a very small effect on the ATP/ADP ratios in the different subcellular compartments during photosynthesis in nonphotorespiratory conditions (saturating CO₂). In photorespiratory conditions (limiting CO₂), on the other hand, aminoacetonitrile increased the ATP/ADP ratio in the chloroplasts and decreased the ATP/ADP ratios in the mitochondria and the cytosol. These results are consistent with the hypothesis, that during photorespiration glycine oxidation is coupled to oxidative phosphorylation to provide ATP to the cytosol.     

The effects of oligomycin on content of adenylates in mesophyll protoplasts,chloroplasts and mitochondria from Pb<Superscript>2+</Superscript> treated pea and barley leaves

The effects of oligomycin on photosynthesis and respiration in relation to ATP production in chloroplasts and mitochondria were investigated in protoplasts isolated from the detached pea (Pisum sativum L cv. Iłowiecki.) and barley (Hordeum vulgare L. cv. Gunilla) leaves treated 5 mM Pb(NO₃)₂. The oligomycin (OM), an inhibitor of oxidative phosphorylation at 0.1 μM concentration caused the inhibition of photosynthesis rate in the protoplasts from both the control and the Pb-treated pea leaves. The respiration rate and ATP/ADP ratio in the protoplasts and the activity of ATPase in mitochondria, were also diminished in the control protoplasts. These effects were not observed in the protoplasts and mitochondria isolated from the Pb-treated leaves. Oligomycin, an inhibitor of photophosphorylation at 10 μM concentration decreased ATPase activity in chloroplasts from both the control and the Pb- treated leaves. Using the method of rapid fractionation of barley protoplasts it was shown that the ATP/ADP ratio in the mitochondria from Pb-treated leaves was largely suppressed (from 1.8 to 0.4) by OM under nonphotorespiratory conditions (high CO₂), whereas under photorespiratory conditions (low CO₂) this ratio was high (5.3) and under OM decreased less (to 3.1). Our results indicate that oligomycin, in organelle isolated from Pb-treated leaves, had no inhibitory effect on the mitochondrial ATPase, whereas it inhibited chloroplasts ATPase. We suggest that Pb ions affected the catalytic cycle and/or conformational changes of ATPase in pea chloroplasts differently than in mitochondria. The differences in Pb responses may reflect fine mechanisms for the regulation of ATP production in the plant cells under stress conditions.     

Relationship between Respiration and Photosynthesis in Guard Cell and Mesophyll Cell Protoplasts of Commelina communis L

A mass spectrometric method combining ¹⁶O/¹⁸O and ¹²C/¹³C isotopes was used to quantify the unidirectional fluxes of O₂ and CO₂ during a dark to light transition for guard cell protoplasts and mesophyll cell protoplasts of Commelina communis L. In darkness, O₂ uptake and CO₂ evolution were similar on a protein basis. Under light, guard cell protoplasts evolved O₂ (61 micromoles of O₂ per milligram of chlorophyll per hour) almost at the same rate as mesophyll cell protoplasts (73 micromoles of O₂ per milligram of chlorophyll per hour). However, carbon assimilation was totally different. In contrast with mesophyll cell protoplasts, guard cell protoplasts were able to fix CO₂ in darkness at a rate of 27 micromoles of CO₂ per milligram of chlorophyll per hour, which was increased by 50% in light. At the onset of light, a delay observed for guard cell protoplasts between O₂ evolution and CO₂ fixation and a time lag before the rate of saturation suggested a carbon metabolism based on phosphoenolpyruvate carboxylase activity. Under light, CO₂ evolution by guard cell protoplasts was sharply decreased (37%), while O₂ uptake was slowly inhibited (14%). A control of mitochondrial activity by guard cell chloroplasts under light via redox equivalents and ATP transfer in the cytosol is discussed. From this study on protoplasts, we conclude that the energy produced at the chloroplast level under light is not totally used for CO₂ assimilation and may be dissipated for other purposes such as ion uptake.     

Light-enhanced dark respiration in leaves and mesophyll protoplasts of pea in relation to photorespiration,respiration and some metabolites content

The respiration rate of leaves and mesophyll protoplasts of pea (Pisum sativum L.), from plants which were previously kept in darkness for 24 h was doubled following a period of photosynthesis at ambient level of O₂ (21 %), whereas the low level of O₂ (1 % and 4 % for leaves and protoplasts, respectively) reduced this light-enhanced dark respiration (LEDR) to the rate as noted before the illumination. Similarly to respiration rate, the oxygen at used concentrations had no effect on the ATP/ADP ratio in the dark-treated leaves. However, the ATP/ADP ratio in leaves photosynthesizing at 21 % O₂ was higher (up to 40 %, dependence on CO₂ concentration in the range 40–1600 1 dm⁻³) than in those photosynthesizing at 1 % O₂ or darkened at air (21 % O₂). Also, at 1 % O₂ the accumulation of malate was suppressed (by about 40 %), to a value noted for leaves darkened at 21 % O₂. The dark-treatment of leaves reduced the ability of isolated mitochondria to oxidize glycine (by about twofold) and succinate, but not malate. Mitochondria from both the light- and dark-treated leaves did not differ in qualitative composition of free amino acids, however, there were significant quantitative differences especially with respect to aspartate, alanine, glutamate and major intermediates of the photorespiratory pathway (glycine, serine). Our results suggest that accumulation of photorespiratory and respiratory metabolites in pea leaves during photosynthesis at 1 % O₂ is reduced, hence the suppression of postillumination respiration rate.     

Photosynthesis by isolated protoplasts, protoplast extracts, and chloroplasts of wheat: influence of orthophosphate, pyrophosphate, and adenylates

Protoplasts, protoplast extracts (intact chloroplasts plus extrachloroplastic material), and chloroplasts isolated from protoplasts of wheat (Triticum aestivum) have rates of photosynthesis as measured by light-dependent O₂ evolution of about 100 to 150 micromoles of O₂ per milligram of chlorophyll per hour at 20 C and saturating bicarbonate. The assay conditions sufficient for this activity were 0.4 molar sorbitol, 50 millimolar N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid KOH (pH 7.6), and 10 millimolar NaHCO₃ with protoplast, plus a requirement of 1 to 10 millimolar ethylenediaminetetraacetate (EDTA) and 0.2 to 0.5 millimolar inorganic orthophosphate (Pi) with protoplast extracts and chloroplasts. Protoplast extracts evolved approximately 6 micromoles of O₂ per milligram of chlorophyll before photosynthesis became largely dependent on exogenous Pi while photosynthesis by chloroplasts had a much stronger dependence on exogenous Pi from the outset.

Photosynthesis by chloroplasts from 6-day-old wheat plants under optimum levels of Pi was similar to that with the addition of 5 millimolar inorganic pyrophosphate (PPi) plus 0.2 millimolar adenosine-5′-diphosphate (ADP). Either PPi or ADP added separately inhibited photosynthesis. When chloroplasts were incubated in the dark for 2 to 6 minutes, photosynthesis was strongly inhibited by 5 millimolar PPi and this inhibiting was relieved by including adenosine-5′-triphosphate (ATP) or ADP (0.2 to 0.6 millimolar). Chloroplasts from 9-day-old wheat leaves were slightly less sensitive to inhibition by PPi and showed little or no inhibition by ADP.

Chloroplasts isolated from protoplasts and assayed with 0.3 millimolar Pi added before illumination have an induction time from less than 1 minute up to 16 minutes depending on the time of the assay after isolation and the components of the medium. In order to obtain maximum rates of photosynthesis and minimum induction time, NaHCO₃ and chelating agents, EDTA or PPi (+ATP), are required in the chloroplast isolation, resuspension and assay medium. With these inclusions in the isolation and resuspension medium the induction time decreased rapidly during the first 20 to 30 minutes storage of chloroplasts on ice. Requirements for isolating intact and photosynthetically functional chloroplasts from wheat protoplasts are discussed.

     

Limitation of Photosynthesis by Carbon Metabolism : I. Evidence for Excess Electron Transport Capacity in Leaves Carrying Out Photosynthesis in Saturating Light and CO(2)

It has been investigated how far electron transport or carbon metabolism limit the maximal rates of photosynthesis achieved by spinach leaves in saturating light and CO₂. Leaf discs were illuminated with high light until a steady state rate of O₂ evolution was attained, and then subjected to a 30 second interruption in low light, to generate an increased demand for the products of electron transport. Upon returning to high light there is a temporary enhancement of photosynthesis which lasts 15 to 30 seconds, and can be up to 50% above the steady state rate of O₂ evolution. This temporary enhancement is only found when saturating light intensities are used for the steady state illumination, is increased when low light rather than darkness is used during the interruption, and is maximal following a 30 to 60 seconds interruption in low light. Decreasing the temperature over the 10 to 30°C range led to the transient enhancement becoming larger. The temporary enhancement is associated with an increased ATP/ADP ratio, a decreased level of 3-phosphoglycerate, and increased levels of triose phosphate and ribulose 1,5-bisphosphate. Since electron transport can occur at higher rates than in steady state conditions, and generate a higher energy status, it is concluded that leaves have a surplus electron transport capacity in saturating light and CO₂. From the alterations of metabolites, it can be calculated that the enhanced O₂ evolution must be accompanied by an increased rate of ribulose 1,5-bisphosphate regeneration and carboxylation. It is suggested that the capacity for sucrose synthesis ultimately limits the maximal rates of photosynthesis, by restricting the rate at which inorganic phosphate can be recycled to support electron transport and carbon fixation in the chloroplast.     

Photosynthetic response of Chlamydomonas reinhardtii to short-term storage on ice in darkness. Dependence of the response on growth conditions

The effect of storage of the unicellular green alga Chlamydomonas reinhardtii (strain 137+) in the pelleted state in darkness on ice (0.2–0.5°C) (further simply “SPDI-treatment”) on its photosynthetic and respiratory activities was studied. To this end, the steady-state rates of O₂ exchange in darkness (dark respiration) and under saturating light (apparent photosynthesis) as well as the induction periods (IP) of apparent photosynthesis were measured at 25°C in the SPDI-untreated and SPDI-treated for the period from ～0.5 to ～30 h algal cells. In contrast to expectations, the SPDI-treatment consistently affected the rate and IP of photosynthesis depending on the physiological state of C. reinhardtii. Dark respiration was affected by the SPDI-treatment as well. However, in absolute values the respiratory changes were much less than the photosynthetic ones, and they were insufficiently reproducible. The SPDI-treatment affected photosynthesis most significantly in high-CO₂-grown cells (cells grown at 5% CO₂ in white light). The rate of photosynthesis in these cells declined quasi-exponentially as a function of time during the SPDI-treatment with a t _1/2 ～1.5 h and finally became by about 60% lower than that before the SPDI-treatment. This decline of photosynthesis was accompanied by continuous and essential increase in the photosynthetic IP. The SPDI-induced photosynthetic changes in high-CO₂-grown cells resulted from the firm disfunction of the photosynthetic apparatus. After switch from growth at 5% CO₂ in white light to growth at ～0.03% CO₂ (air) in white, blue, or red light, the alga gradually transited to a physiological state, in which the negative effects of the SPDI-treatment on the rate and IP of photosynthesis became weak and absent, respectively. Remarkably, this transition was faster in blue (≤5 h) than in white and red light (>10 h). Similar changes in the response of the alga to the SPDI-treatment occurred when high-CO₂-grown cells (5% CO₂, white light, 26°C) were incubated in darkness (air, 24–26°C) for 20–25 h. The results of study were analyzed in the light of literature data relating to the effects of CO₂ concentration, darkness, and light quality on carbohydrates in plant organisms. The analysis led to suggestion that there is connection between the negative effect of the SPDI-treatment on C. reinhardtii and nonstructural carbohydrates presented in the alga: the more carbohydrates contain the alga, the more extensive inactivation of the photosynthetic apparatus occurs in it during its storage in the dense (pelleted) state in darkness on ice.     

Markedly low requirement of added CO2 for photosynthesis by mesophyll protoplasts of pea (Pisum sativum): possible roles of photorespiratory CO2 and carbonic anhydrase

Mesophyll protoplasts of pea required only 74.1 μM CO₂ for maximal photosynthesis, unlike chloroplasts, which required up to 588 μM CO₂. Such a markedly low requirement for CO₂ could be because of an internal carbon source and/or a CO₂ concentrating mechanism in mesophyll protoplasts. Ethoxyzolamide (EZA), an inhibitor of internal carbonic anhydrase (CA) suppressed photosynthesis by mesophyll protoplasts at low CO₂ (7.41 μM) but had no significant effect at high CO₂ (741 μM). However, acetazolamide, another inhibitor of CA, did not exert as much dramatic effect as EZA. Three photorespiratory inhibitors, aminoacetonitrile or glycine hydroxamate (GHA) or aminooxyacetate inhibited markedly photosynthesis at low CO₂ but not at high CO₂. Inhibitors of glycolysis or tricarboxylic acid cycle (NaF, sodium malonate) or phosphoenolpyruvate carboxylase (3,3‐dichloro‐2‐dihydroxy phosphinoyl‐methyl‐2‐propenoate) had no significant effect on photosynthesis. The CO₂ requirement of protoplast photosynthesis and the sensitivity of photosynthesis to EZA were much higher at low oxygen (65 nmol ml^?1) than that at normal oxygen (212 nmol ml^?1). In contrast, the inhibitory effect of photorespiratory inhibitors on protoplast photosynthesis was similar in both normal and low oxygen medium. The marked elevation of glycine/serine ratio at low O₂ or in presence of GHA confirmed the suppression of photorespiratory decarboxylation by GHA. While demonstrating interesting difference between the response of protoplasts and chloroplasts to CO₂, we suggest that photorespiration could be a significant source of CO₂ for photosynthesis in mesophyll protoplasts at limiting CO₂ and at atmospheric levels of oxygen. Obviously, carbonic anhydrase is essential to concentrate or retain CO₂ in mesophyll cells.     

Oscillations in photosynthesis are initiated and supported by imbalances in the supply of ATP and NADPH to the Calvin cycle

Oscillations in the rate of photosynthesis of sunflower (Helianthus annuus L.) leaves were induced by subjecting leaves, whose photosynthetic apparatus had been activated, to a sudden transition from darkness or low light to high-intensity illumination, or by transfering them in the light from air to an atmosphere containing saturating CO₂. It was found that at the first maximum, light-and CO₂-saturated photosynthesis can be much faster than steady-state photosynthesis. Both Q_A in the reaction center of PS II and P₇₀₀ in the reaction center of PS I of the chloroplast electron-transport chain were more oxidized during the maxima of photosynthesis than during the minima. Maxima of P₇₀₀ oxidation slightly preceded maxima in photosynthesis. During a transition from low to high irradiance, the assimilatory force F_A, which was calculated from ratios of dihydroxyacetone phosphate to phosphoglycerate under the assumption that the reactions catalyzed by NADP-dependent glyceraldehydephosphate dehydrogenase, phosphoglycerate kinase and triosephosphate isomerase are close to equilibrium, oscillated in parallel with photosynthesis. However, only one of its components, the calculated phosphorylation potential (ATP)/(ADP)(P_i), paralleled photosynthesis, whereas calculated NADPH/NADP ratios exhibited antiparallel behaviour. When photosynthetic oscillations were initiated by a transition from low to high CO₂, the assimilatory force F_A declined, was very low at the first minimum of photosynthesis and increased as photosynthesis rose to its second maximum. The observations indicate that the minima in photosynthesis are caused by lack of ATP. This leads to overreduction of the electron-transport chain which is indicated by the reduction of P₇₀₀. During photosynthetic oscillations the chloroplast thylakoid system is unable to adjust the supply of ATP and NADPH rapidly to demand at the stoichiometric relationship required by the carbonreduction cycle.Abbreviations PGA 3-phosphoglycerate - DHAP dihydroxyacetone phosphate - P₇₀₀ electron-donor pigment in the reaction enter of PS I - Q_A quinone acceptor in the reaction center of PS II This work received support from the Estonian Academy of Sciences, the Bavarian Ministry of Science and Art and the Sonderforschungsbereich 251 of the University of Würzburg. We are grateful for criticism by D.A. Walker, Robert Hill Institute, University of Sheffield, U.K. and by Mark Stitt, Institute of Botany, University of Heidelberg, FRG.     

Ribulose-1,5-bisphosphate carboxylase-oxygenase,other Calvin-cycle enzymes,and chlorophyll decrease when glucose is supplied to mature spinach leaves via the transpiration stream

The inhibition of photosynthesis after supplying glucose to detached leaves of spinach (Spinacia oleracea L.) was used as a model system to search for mechanisms which potentially contribute to the sink regulation of photosynthesis. Detached leaves were supplied with 50 mM glucose or water for 7 d through the transpiration stream, holding the leaves in low irradiance (16 mol photons · m^–2 · s^–1) and a cycle of 9 h light/15 h darkness to prevent any endogenous accumulation of carbohydrate. Leaves supplied with water only showed marginal changes of photosynthesis, respiration, enzyme levels or metabolites. When leaves were supplied with 50 mM glucose, photosynthesis was gradually inhibited over several days. The inhibition was most marked when photosynthesis was measured in saturating irradiance and ambient CO₂, less marked in saturating irradiance and saturating CO₂, and least marked in limiting irradiance. There was a gradual loss of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) protein, fructose-1,6-bisphosphatase, NADP-glyceraldehyde-3-phosphate dehydrogenase and chlorophyll. The inhibition of photosynthesis was accompanied by a large decrease of glycerate-3-phosphate, an increase of triose-phosphates and fructose-1,6-bisphospate, and a small decrease of ribulose-1,5-bisphosphate. The stromal NADPH/NADP ratio increased (as indicated by increased activation of NADP-malate dehydrogenase), and the ATP/ADP ratio increased. Chlorophyll-fluorescence analysis indicated that thylakoid energisation was increased, and that the acceptor side of photosystem II was more reduced. Similar results were obtained when glucose was supplied by floating leaf discs in low irradiance on glucose solution, and when detached spinach leaves were held in high light to produce an endogenous accumulation of carbohydrate. Feeding glucose also led to an increased rate of respiration. This was not accompanied by any changes of pyruvate kinase, phosphofructokinase, or pyrophosphate: fructose-6-phosphate phosphotransferase activity. There was a decrease of phosphoenolpyruvate, glycerate-3-phosphate and glycerate-2-phosphate, an increase of pyruvate and triose-phosphates, and an increased ATP/ADP ratio. These results show (i) that accumulation of carbohydrate can inhibit photosynthesis via a long-term mechanism involving a decrease of Rubisco and other Calvin-cycle enzymes and (ii) that respiration is stimulated due to an unknown mechanism, which increases the utilisation of phosphoenolpyruvate.Abbreviations and Symbols C_i CO₂ concentration in the air space within the leaf - F_m fluorescence yield with a saturating pulse in dark-adapted material - F_o ground level of fluorescence using a weak non-actinic modulated beam in the dark - Fru1,6bisP fructose-1,6-bisphosphate - Fru1,6Pase fructose-1,6-bisphosphatase - Fru2,6bisP fructose-2,6-bisphosphate - IRGA infrared gas analyser - NAD-MDH NAD-dependent malate dehydrogenase - NADP-MDH NADP-dependent malate dehydrogenase - NADP-GAPDH NADP-dependent glyceraldehyde-3-phosphate dehydrogenase - PEP phosphoenolpyruvate - PFK phospho-fructokinase - PFP pyrophospate: fructose-6-phosphate-phosphotransferase - 3-PGA glycerate-3-phospate - P_i inorganic phosphate - Ru1,5bisP ribulose 1,5-bisphosphate - Rubisco ribulose-1,5-bisphosphate carboxylase-oxygenase - triose-phosphates sum of glyceraldehyde-3-phosphate and dihydroxyacetone phosphate This research was supported by the Deutsche Forschungsgemeinschaft (SFB 137).     

Respiration of barley protoplasts before and after illumination

Respiratory O₂ consumption was investigated in dark-adapted barley (Hordeum vulgare L. cv. Gunilla) protoplasts and after illumination for 10 min at high and very low CO₂ in the presence of respiratory and photorespiratory inhibitors. In dark-adapted protoplasts no difference was observed between inhibitor treatments in high and very low CO₂. The respiratory rate increased somewhat after illumination and a difference in responce to inhibitors was in some cases observed between high and very low CO₂. Thus, the operation of the mitochondrial electron transport chain is affected following a period of active photosynthesis. In all situations tested, oligomycin inhibited respiratiory O₂ uptake indicating that respiration of mitochondria in protoplasts is not strictly ADP limited. Antimycin A inhibited respiration more in dark-adapted protoplasts than after illumination whereas SHAM gave the opposite response. Rotenone inhibited respiration both in dark-adapted protoplasts (about 30%) and after illumination where the inhibition was much greater in very low CO₂ (50%) than in high CO₂ (10%). After illumination in very low CO₂. SHAM + rotenone inhibited respiration almost completely (70%). Photorespiratory inhibitors had very small effect on O₂ consumption in darkness. After illumination the effect of aminoacetonitrile (AAN) was also very low whereas α-hydroxypyridine-2-methane sulphonate (HPMS) in photorespiratory conditions inhibited O₂ uptake much stronger (35%). The addition of glyoxylate enhanced respiration in the presence of HPMS up to the control level suggesting that alternative pathways of glyoxylate conversion might be operating. The differences in inhibitor responses may reflect fine mechanisms for the regulation of energetic balance in the plant cell which consists of switching from electron transport coupled to ATP production to non-coupled transport. Photorespiratory flux is also very flexible, and the suppression of glycine decarboxylation can induce bypass reactions of glyoxylate metabolism.     

14CO2 fixation and glycolate metabolism in the dark in isolated maize (Zea mays L.) bundle sheath strands

Bundle sheath strands capable of assimilating up to 68 μmoles CO₂ per mg chlorophyll per hr in the dark have been isolated from fully expanded leaves of Zea mays L. This dark CO₂-fixing system is dependent on exogenous ribose-5-phosphate, ADP or ATP, and Mg²⁺ for maximum activity. The principal product of dark fixation in this system is 3-phosphoglycerate, indicating that the CO₂-fixing reaction is mediated by ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39). The rate of dark CO₂ uptake in the strands in the presence of saturating levels of ribose-5-phosphate plus ADP is inhibited by oxygen. The inhibitory effect of oxygen is rapidly and completely reversible, and is relieved by increased levels of CO₂. Glycolate is synthesized in this dark system in the presence of [U-¹⁴C]ribose-5-phosphate, ADP, oxygen, and an inhibitor of glycolate oxidase (EC 1.1.3.1). Glycolate formation is completely abolished by heating the strands, and the rate of glycolate synthesis is markedly reduced by either lowering the oxygen tension or increasing the level of CO₂.These results, obtained with intact cells in the absence of light, indicate that the direct inhibitory effect of oxygen on photosynthesis is associated with photosynthetic carbon metabolism, probably at the level of ribulose-1,5-bisphosphate carboxylase, and not with photophosphorylation or photosynthetic electron transport. Furthermore, the findings indicate that the synthesis of glycolate from exogenous substrate can readily occur in the absence of photosynthetic electron transport, an observation consistent with the ribulose-1, 5-bisphosphate “oxygenase” scheme for glycolate formation during photosynthesis.     

Evidence for Mitochondrial Regulation of Photosynthesis by a Starchless Mutant of Nicotiana sylvestris

Mutant NS458 of Nicotiana sylvestris (Speg. et Comes) contains a defective plastid phosphoglucomutase and accumulates only trace amounts of starch. Determinations of carbon partitioning using tracer d-[3-¹⁴C]glyceric acid showed that the maximal CO₂ assimilation by mature leaves of the mutant at saturating [CO₂] and light and low [O₂] was close to the flux for sucrose formation in the wild type. The mutant is characterized by exceptionally slow oscillations in maximal CO₂ assimilation. The postulate that these slow oscillations follow changes in the cytosolic rate of sucrose phosphate synthesis has been investigated. Studies with wild-type and mutant leaf discs subjected to various treatments failed to indicate that any significant activation-inactivation cycle in sucrose-P synthase activity can occur. The rate of sucrose phosphate synthesis, however, might be altered by variations in the supply of uridine UDP-glucose which is controlled by the rate of ATP regeneration (via UTP regeneration). Treating mutant leaf protoplasts and young leaves with oligomycin, an inhibitor of mitochondrial ATP regeneration, reduced photosynthesis by as much as 25 and 40%, respectively. The wild type failed to show inhibition by oligomycin, i.e. its effect is masked when starch and sucrose synthesis can interact. It is concluded that maximal CO₂ assimilation in the mutant is fine tuned by mitochondrial metabolism such that interactions between sucrose synthesis and mitochondrial processes may generate the observed oscillations.     

Regulation of photosynthesis in nitrogen deficient wheat seedlings

Nitrogen effects on the regulation of photosynthesis in wheat (Triticum aestivum L., cv Remia) seedlings were examined. Ribulose 1,5-bisphosphate carboxylase/oxygenase was rapidly extracted and tested for initial activity and for activity after incubation in presence of CO₂ and Mg²⁺. Freeze clamped leaf segments were extracted for determinations of foliar steady state levels of ribulose 1,5-bisphosphate, triose phosphate, 3-phosphoglycerate, ATP, and ADP. Nitrogen deficient leaves showed increased ATP/ADP and triose phosphate/3-phosphoglycerate ratios suggesting increased assimilatory power. Ribulose 1,5-bisphosphate levels were decreased due to reduced pentose phosphate reductive cycle activity. Nevertheless, photosynthesis appeared to be limited by ribulose 1,5-bisphosphate carboxylase/oxygenase, independent of nitrogen nutrition. Its degree of activation was increased in nitrogen deficient plants and provided for maximum photosynthesis at decreased enzyme protein levels. It is suggested that ribulose 1,5-bisphosphate carboxylase/oxygenase activity is regulated according to the amount of assimilatory power.     

Importance of oxidative electron transport over oxidative phosphorylation in optimizing photosynthesis in mesophyll protoplasts of pea (Pisum sativum L.)

The role of mitochondrial respiration in optimizing photosynthesis was assessed in mesophyll protoplasts of pea ( Pisum sativum L., cv. Arkel) by using low concentrations of oligomycin (an inhibitor of oxidative phosphorylation), antimycin A (inhibits cytochrome pathway of electron transport) and salicylhydroxamic acid (SHAM, an inhibitor of alternative oxidase). All three compounds decreased the rate of photosynthetic O₂ evolution in mesophyll protoplasts, but did not affect chloroplast photosynthesis. The inhibition of photosynthesis by these mitochondrial inhibitors was stronger at optimal CO₂ (1.0 m M NaHCO₃) than that at limiting CO₂ (0.1 m M NaHCO₃). We conclude that mitochondrial metabolism through both cytochrome and alternative pathways is essential for optimizing photosynthesis at limiting as well as at optimal CO₂. The ratios of ATP to ADP in whole protoplast extracts were hardly affected, despite the marked decrease in their photosynthetic rates by SHAM. Similarly, the decrease in the ATP/ADP ratio by oligomycin or antimycin A was more pronounced at limiting CO₂ than at optimal CO₂. The mitochondrial oxidative electron transport, through both cytochrome and alternative pathways, therefore akppears to be more important than oxidative phosphorylation in optimizing photosynthesis, particularly at limiting CO₂ (when ATP demand is expected to be low). Our results also confirm that the alternative pathway has a significant role in contributing to the cellular ATP, when the cytochrome pathway is limited.     

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.     

Temperature Dependence of Photosynthesis in Agropyron smithii Rydb. : III. Responses of Protoplasts and Intact Chloroplasts

Protoplasts and intact chloroplasts isolated from Agropyron smithii Rybd. were utilized in an effort to determine the limiting factor(s) for photosynthesis at supraoptimal temperatures. Saturated CO₂-dependent O₂ evolution had a temperature optimum of 35°C for both protoplasts and intact chloroplasts. A sharp decline in activity was observed as assay temperature was increased above 35°C, and at 45°C only 20% of the maximal rate remained. The temperature optimum for 3-phosphoglycerate reduction by intact chloroplasts was 35°C. Above this temperature, 3-phosphoglycerate reduction was more stable than CO₂-dependent O₂ evolution. Reduction of nitrite in coupled intact chloroplasts had a temperature optimum of 40°C with only slight variation in activity between 35°C and 45°C. Reduction of nitrite in uncoupled chloroplasts had a temperature optimum of 40°C, but increasing the assay temperature to 45°C resulted in a complete loss of activity. Reduction of p-benzoquinone by protoplasts and intact chloroplasts had a temperature optimum of 32°C when measured in the presence of dibromothymoquinone. This photosystem II activity exhibited a strong inhibition of O₂ evolution as assay temperature increased above the optimum. It is concluded that, below the temperature optimum, ATP and reductant were not limiting photosynthesis in these systems or intact leaves. Above the temperature optimum, photosynthesis in these systems is limited in part by the phosphorylation potential of the stromal compartment and not by the available reductant.     

Stomatal Responses to CO(2) in Paphiopedilum and Phragmipedium: Role of the Guard Cell Chloroplast

A role of the guard cell chloroplasts in the CO₂ response of stomata was investigated through a comparison of the leaf gas exchange characteristics of two closely related orchids: Paphiopedilum harrisianum, which lacks guard cell chloroplasts and Phragmipedium longifolium, which has chlorophyllous guard cells. Leaves of both species had an apparent quantum yield for assimilation of about 0.05, with photosynthesis saturating at 0.300 to 0.400 millimoles per square meter per second. CO₂ curves were obtained by measuring steady-state assimilation and stomatal conductance under 0.180 or 0.053 millimoles per square meter per second white light, or darkness, at 0 to 400 microliters per liter ambient CO₂. The response of assimilation to changes in CO₂ was similar in the two species, but the response of conductance was consistently weaker in Paphiopedilum than in Phragmipedium. The data suggest involvement of guard cell chloroplasts in the stomatal response to CO₂ and in the coupling of assimilation and conductance in the intact leaf.     

Influence of Etherel and Gibberellic Acid on Carbon Metabolism,Growth, and Essential Oil Accumulation in Spearmint (Mentha Spicata)

Controlled environment chamber and glasshouse studies were conducted on six herbaceous annual species grown at 350 (AC) and 700 (EC) mol(CO₂) mol^-1 to determine whether growth at EC resulted in acclimation of the apparent quantum yield of photosynthesis (QY) measured at limiting photosynthetic photon flux density (PPFD), or in acclimation of net photosynthetic rate (P _N) measured at saturating PPFD. It was also determined whether acclimation in P _N at limiting PPFD was correlated with acclimation of carboxylation efficiency or ribulose-1,5-bisphosphate (RuBP) regeneration rate measured at saturating PPFD. Growth at EC reduced both the QY and P _N at limiting PPFD in three of the six species. The occurrence of photosynthetic acclimation measured at a rate limiting PPFD was independent of whether photosynthetic acclimation was apparent at saturating measurement PPFD. At saturating measurement PPFD, acclimation to EC in the apparent carboxylation efficiency and RuBP regeneration capacity also occurred independently. Thus at least three components of the photosynthetic system may adjust independently when leaves are grown at EC. Estimates of photosynthetic acclimation at both high and low PPFD are necessary to accurately predict photosynthesis at the whole plant or canopy level as [CO₂] increases.     

Response of photosynthetic carbon assimilation in mesophyll protoplasts to restriction on mitochondrial oxidative metabolism: Metabolites related to the redox status and sucrose biosynthesis

The patterns of cellular metabolites related to redox status and sucrose biosynthesis in mesophyll protoplasts of pea (Pisum sativum L.) were examined in the absence or presence of oligomycin (inhibitor of oxidative phosphorylation) or antimycin A (inhibitor of cytochrome pathway) or salicylhydroxamic acid (SHAM) (inhibitor of alternative pathway). The increase on illumination in the rate of photosynthesis or cellular metabolites was more at optimal CO₂ (1.0 mM NaHCO₃) compared to that at limiting CO₂ (0.1 mM NaHCO₃). Furthermore, the inhibition of photosynthesis in presence of mitochondrial inhibitors was more pronounced at optimal CO₂ than that at limiting CO₂. There was a marked increase in steady-state levels of triose-P/PGA (phosphoglyceric acid) and glucose-6-phosphate (Glc-6-P) in the presence of oligomycin and antimycin A. In contrast, SHAM caused a marked increase in malate/OAA (oxaloacetate). We suggest that dissipation of excess redox equivalents generated in photosynthesis occurs through both cytochrome and alternative pathways, while sucrose biosynthesis is backed up by cytochrome pathway alone. Thus, mitochondrial respiration (through both cytochrome and alternative pathways of mitochondrial electron transport) optimizes chloroplast photosynthesis by modulating cellular metabolites related to both intracellular redox state and sucrose biosynthesis.