The Metabolism of Carbon-14 Specifically Labelled Glucose in Leaves Showing Tobacco Mosaic Virus Induced Local Necrotic Lesions

Glucose metabolism of healthy and tobacco mosaic virus-infected leaf-discs of Nicotiana tabocum L. var. Xanthi showing local-necrotic lesions was investigated using glucose-¹⁴C. Local lesion formation following inoculation with tobacco mosaic virus resulted in enhanced glucose metabolism reflected by an increased rate of release of ¹⁴CO₂ from glucose-U-¹⁴C and greater incorporation of ¹⁴C into all cell fractions. When specifically labelled glucose was fed to healthy and tobacco mosaic virus infected leaves, the C₆/C₁ ratio (rate of release of ¹⁴CO₂ from glucose-6-¹⁴C/rate of release of ¹⁴CO₂ from glucose-l-¹⁴C) was similar for healthy and virus-infected leaves. The C₆/C₁ ratios recorded from 0.30 to 0.50 indicate that both the glycolytic and pentose phosphate pathways participate in glucose catobolism in healthy and virus-infected leaves. Although the C₆/C₁ ratio was the same as that of the healthy leaf the rate of release of ¹⁴CO₂ from glucose-6-¹⁴C and glucose-1-¹⁴C was greatly increased in the virus-infected leaf. The increased glucose catabolism occurs by both glycolytic and pentose phosphate pathways in the virus-infected leaf.     

Respiration Rate and Adenosine Triphosphate Formation in Tissues4

When leaves of Nicotiana tabacum L. var. Xanthi were inoculatedwith TMV, local lesion formation gave enhanced ATP concentrationaccompanied by increased respiration. Phosphorus metabolismin healthy and infected leaves was investigated either by floatingleaf tissue on ³²P₁-phosphate buffer or by allowing intact leavesto take up ³²P₁-phosphate buffer through the petioles. Bothmethods gave increased ³²P₁ incorporation into inorganic andorganic acid-soluble phosphorus fractions of the infected leaf.Comparison of the specific activity of ³²P₁-phosphate buffersupplied with the specific activity of the -phosphate groupof ATP demonstrated that 50 per cent of the ATP in the healthyleaf compared with over 78 per cent of the ATP in the infectedleaf was metabolically active in respiration. As the ATP concentrationin the infected leaf is much greater than that in the healthyleaf, this means that the amount of ATP being utilized and resynthesizedas a result of respiratory metabolism in the infected leaf ismore than twice that in the healthy leaf. Pulse-labelling experimentsdemonstrated that the rate of ATP turnover was very similarin healthy and infected leaves; therefore the increased respirationin the infected leaf results from the larger ATP pool with aturnover rate of ATP similar to that in the healthy leaf. Withtobacco callus tissue cultures infected by TMV, where infectiondoes not result in local-lesion formation, phosphorus metabolismwas unaltered in the infected tissue. It is concluded that necroticlocal-lesion formation results from increased availability ofATP at sites of ATP utilization, and this is aggravated by over-productionof ATP in the infected leaf.     

Regulation of mitochondrial energy production in cardiac cells of rainbow trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>)

In skinned rat cardiac fibres, mitochondrial affinity for endogenous ADP generated by creatine kinase and Ca²⁺-activated ATPases is higher than for exogenous ADP added to the surrounding medium, suggesting that mitochondria are functionally coupled to creatine kinase and ATPases. Such a coupling may be weaker or absent in ectothermic vertebrate cardiac cells, because they typically have less elaborate intracellular membrane structures, higher glycolytic capacity and lower working temperature. Therefore, we examined skinned cardiac fibres from rainbow trout at 10 °C. The apparent mitochondrial affinity for endogenous ADP was obtained by stimulation with ATP and recording of the release of ADP into the surrounding medium. The apparent affinity for endogenous ADP was much higher than for exogenous ADP suggesting a functional coupling between mitochondria and ATPases. The apparent affinity for exogenous ADP and ATP was increased by creatine or an increase in Ca²⁺-activity, which should increase intrafibrillar turnover of ATP to ADP. In conclusion, ADP seems to be channelled from creatine kinase and ATPases to mitochondria without being released to the surrounding medium. Thus, despite difference in structure, temperature and metabolic capacity, trout myocardium resembles that of rat with regard to the regulation of mitochondrial respiration.Abbreviations ACR acceptor control ratio - ANT adenine nucleotide translocase - K_{M ADP} apparent mitochondrial affinity for ADP - K_{M ATP} apparent mitochondrial affinity for ATP - LDH lactate dehydrogenase - V_ADP ADP-stimulated respiration rate - V_{ADP max} maximal ADP-stimulated respiration rate - V_ATP ATP-stimulated respiration rate - V_{ATP max} maximal ATP-stimulated respiration rate - V₀ basal respiration rate in the absence of ADPCommunicated by G. Heldmaier     

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.     

Carbohydrate Metabolism and Translocation in Healthy and Cocoa Swollen Shoot Virus-Infected Cocoa Plants

Analyses of cocoa swollen shoot virus-infected and healthy cocoa (Theobroma cacao L.) plant tissues were made to determine the effect of virus infection on the metabolism and transport of carbohydrates in affected plants. Starch, sucrose and reducing sugars were found to accumulate in infected tissues. Translocation of photosynthates (mainly as sucrose) to the stem and root system, as estimated by the overnight loss of carbohydrates from the leaves and by ¹⁴CO₂ tracer experiments, was as efficient in the infected plants as in the healthy. Infected plants showed a higher diurnal turnover of carbohydrates in their leaves and, on unit leaf area basis, higher levels of ¹⁴C-labelled assimilates suggesting that they have a greater photosynthetic capacity than the healthy plants. The rate of respiration, as determined by the proportions of organic acids, amino acids and other intermediary metabolites formed from translocated ¹⁴C-labelled sugars, was generally higher in infected than in healthy plants. It is concluded from available data showing the presence in infected tissues of mineral nutrients, protein N and amino acids at the same concentrations as in healthy plants, and from the relatively high rates of photosynthesis and respiration that a high rate of metabolic activity is maintained in the host-virus system. Some factors possibly contributing to the stunted growth of infected plants are discussed in the light of these findings.     

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.     

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

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

Effects of nutrient enrichment and leaf quality on the breakdown of leaves in a hardwater stream

1. The breakdown of leaf litter in streams is influenced strongly by leaf quality and the concentration of dissolved nutrients, primarily inorganic nitrogen (N) and phosphorus (P) in the water. We examined the effect of nutrient enrichment on the breakdown of three species of leaves in a hardwater, nutrient‐rich stream. The rate of microbial respiration was also measured on the decomposing leaves. 2. The breakdown rates of dogwood (Cornus stolonifera), aspen (Populus tremuloides) and birch (Betula occidentalis), k‐values of 0.0461, 0.0307 and 0.0186 day^–1, respectively, were unaffected by nutrient enrichment and generally faster than reported previously. Microbial respiration on the leaves was greater than reported previously for leaves of congeneric species. It appears that leaf breakdown in the study stream was not nutrient limited. 3. Nitrogen‐based measures of leaf quality, such as percentage N and carbon (C)/nitrogen ratio, did not correspond to measured breakdown rates among the three leaf types. The best predictors of relative breakdown rates were percentage lignin and the percentage of the total carbon that occurred as lignin. We suggest that, when leaf breakdown is not nutrient limited, measures of carbon quality (i.e. lignin‐based measures) are a better assessment of overall leaf quality than are N‐based measures. 4. Previous studies have indicated that the enzymes produced by aquatic hyphomycetes (microfungi) operate most efficiently at a basic pH and in the presence of calcium ions. The hardwater conditions (pH=8.6, total hardness > 300 mg CaCO₃ L^–1) and abundance of dissolved NO₃ and soluble reactive phosphorous (SRP) (approximately 50 μg L^–1, each) in the study stream appear to have provided conditions that resulted in a high respiration rate and rapid breakdown of leaf litter.     

Temperature acclimation in brook trout muscle: Adenine nucleotide concentrations, phosphorylation state and adenylate energy charge

Summary Cold acclimation in fish is associated with an elevation in metabolic rate. The present study investigates the role of adenine nucleotides and related compounds in metabolic regulation following temperature acclimation. Brook trout (Salvelinus fontinalis) were acclimated for 10 weeks to either +4°C or +24°C. Both groups of fish were exercised at 2.5 body lengths s^–1 for 2 weeks prior to sacrifice in order to control for differences in spontaneous activity.Concentrations of ATP, ADP, AMP, P _i and PC were approximately 2-fold higher in white than red muscles. Temperature acclimation had little effect on total adenine nucleotide concentration in either muscle type. In white fibres acclimation to 4°C results in a 39% increase in [ADP] and [AMP], a 35% decrease in [PC] (phosphorylcreatine), and no significant change in [P _i ]. In contrast temperature has little effect on concentrations of these compounds in red muscle.Parameters of metabolic control — adenylate energy charge ([ATP]+0.5 [ADP]/[ATP]+[ADP]+[AMP]), phosphorylation state ([ATP]/[ADP]·[P _i ]), and the ratios [ATP][ADP] and [ATP][AMP] — were significantly lower in cold- than warm-acclimated white muscle. The observed changes in phosphorylation state and [ATP][AMP] are consistent with an increase in mitochondrial respiration and glycolysis, respectively.In conclusion, changes in metabolites may be an important factor in producing an enhanced metabolic rate in cold-acclimated fish.     

Gas Exchange and Emission of Chlorophyll Fluorescence during the Monocycle of Rust, Angular Leaf Spot and Anthracnose on Bean Leaves as a Function of their Trophic Characteristics

Measurements related to gas exchange and chlorophyll fluorescence emission were taken from healthy and diseased bean leaves with rust, angular leaf spot, and anthracnose during lesion development for each disease. The experiments were performed at different temperatures of plant incubation, and using two bean cultivars. The main effect of temperature of plant incubation was in disease development. There was no significant difference between cultivars in relation to disease development and in magnitude of physiological alterations when disease severity was the same for each cultivar. These diseases reduced the net photosynthetic rate and increased the dark respiration of infected leaves after the appearance of visible symptoms and the differences between healthy and diseased leaves increased with disease development. The transpiration rate and stomatal conductance were stable during the monocycle of rust, however, these two variables decreased in leaves with angular leaf spot and anthracnose beginning with symptom appearance and continuing until lesion development was complete. Carboxylation resistance was probably the main factor related to reduction of photosynthetic rate of the apparently healthy area of leaves with rust and angular leaf spot. Reduction of the intercellular concentration of CO₂, due to higher stomatal resistance, was probably the main factor for leaves with anthracnose. Chlorophyll fluorescence assessments suggested that there was no change in electron transport capacity and generation of ATP and NADPH in apparently healthy areas of diseased leaves, but decreases in chlorophyll fluorescence emission occurred on visibly lesioned areas for all diseases. Minimal fluorescence was remarkably reduced in leaves with angular leaf spot. Maximal fluorescence and optimal quantum yield of photosystem II of leaves were reduced for all three diseases. Bean rust, caused by a biotrophic pathogen, induced less damage to the regulation mechanisms of the physiological processes of the remaining green area of diseased leaves than did bean angular leaf spot or anthracnose, caused by hemibiotrophic pathogens. The magnitude of photosynthesis reduction can be related to the host–pathogen trophic relationships.     

Effects of Leaf Age, Inoculum Dose and Freezing on Development of Chocolate Spot (Botrytis fabae) Lesions on Field Bean (Vicia faba) Leaves

Botrytis fabae spore suspensions containing c. 1, 10, 10², 10³, 10⁴, 10⁵, or 10⁶ spores/ml were used to inoculate 5, 17 or 30-day-old field bean leaves. The percentages of the leaf areas covered by, chocolate spot lesions and the percentages of the leaf areas bearing conidiophores were assessed 1, 6, 12, 14, and 19 days after inoculation. The percentage of the area covered by lesions and the percentage of the area bearing conidiophores (logit-transformed) increased linearly with increasing spore concentration (log₁₀-transformed). The proportions of leaf areas covered by lesions and bearing conidiophores were both greater on 17 and 30-day-old leaves than on 5-day-old leaves. The rate of lesion growth increased with both increasing inoculum dose and increasing leaf age. Generally there was no interaction between the effects of leaf age and the effects of inoculum dose on either lesion growth or sporulation. Two days after inoculation with suspensions of either 10⁴ or 10⁶ spores/ml, 7-day-old leaves grown at 15°C were transferred to –16°C or 2.5°C or kept at 15°C for 4 days. Two days later more spores had been produced on leaves which had been frozen (–16°C) than on, leaves kept at 2.5°C.     

Regulation of pyruvate oxidation in blowfly flight muscle mitochondria: Requirement for ADP

Blowfly (Phormia regina) flight muscle mitochondria oxidized pyruvate (+ proline) in the presence of either ADP (coupled respiration) or carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP-uncoupled respiration). There was an absolute requirement for ADP (K_m = 8.0 μm) when pyruvate oxidation was stimulated by FCCP in the presence of oligomycin. This requirement for ADP was limited to the oxidation of pyruvate; uncoupled α-glycerolphosphate oxidation proceeded maximally even in the absence of added ADP. Atractylate inhibited uncoupled pyruvate oxidation whether added before (>99%) or after (95%) initiation of respiration with FCCP. In the presence of FCCP, oligomycin, and limiting concentrations of ADP (less than 110 μm), there was a shutoff in the uptake of oxygen. This inhibition of respiration was completely reversed by the addition of more ADP. Plots of net oxygen uptake as a function of the limiting ADP concentration were linear; the observed ADP/O ratio was 0.22 ± 0.025. An ADP/O ratio of 0.2 was predicted if phosphorylation occurred only at the succinyl-CoA synthetase step of the tricarboxylate cycle. Experiments performed in the presence of limiting concentrations of ADP, and designed to monitor changes in the mitochondrial content of ADP and ATP, demonstrated that the shutoff in oxygen uptake was not due to the presence of a high intramitochondrial concentration of ATP. Indeed, ATP, added to the medium prior to the addition of FCCP, inhibited uncoupled pyruvate oxidation; the apparent K_I was 0.8 mm. These results are consistent with the hypothesis that it is the intramitochondrial ATP/ADP ratio that is one of the controlling factors in determining the rate of flux through the tricarboxylate cycle. Changes in the mitochondrial content of citrate, isocitrate, α-ketoglutarate, and malate during uncoupled pyruvate oxidation in the presence of a limiting concentration of ADP were consistent with the hypothesis that the mitochondrial NAD⁺-linked isocitric dehydrogenase is a major site for such control through the tricarboxylate cycle.     

Translocation of Photosynthate in Curly Top Virus-infected Tomatoes

Photosynthate translocation in single leaflets of healthy and curly top virus-infected tomatoes was investigated using ¹⁴C as a marker. The amount of radioactivity found in plant parts not exposed to ¹⁴CO₂ was substantially lower in diseased than in healthy plants. The time lag for the appearance of ¹⁴C in the petiole was considerably longer in the infected plants than in the healthy. The kinetics of disappearance of ¹⁴C from the lamina during the 24-hour period following labeling showed a strong retention of recent assimilates within the diseased leaf, not accompanied by increased immobilization into insoluble forms. Sucrose was the predominant compound participating in photosynthate transport in both healthy and diseased leaves. The amount of ¹⁴CO₂ fixed was approximately 40% lower in curly top virus-infected leaves than in healthy leaves.     

Rapid Metabolic Changes in the Wounding Response of Leaf Discs following Excision

The dark respiration rate of discs from fully expanded tobacco leaves (Nicotiana tabacum) increased linearly with decreasing diameter, the relative increase being independent of leaf age. The wound respiration responsible for this situation reached a plateau within 15 minutes of excision. Metabolite analysis gave evidence for two independent effects, also unrelated to age. The first was a forward crossover between phosphoenolpyruvate and pyruvate which was found as early as 1 minute after excision and persisted for up to 40 minutes. It was attributed to activation of pyruvate kinase by a changed ionic balance resulting from membrane damage, was accompanied by a reverse crossover between triose phosphates and 3-phosphoglycerate, and was localized in the outer region of the discs. The second effect was a rapid rise in hexose monophosphate and ATP levels throughout the discs. After 1 to 10 minutes the ATP/ADP ratio rose strongly for at least 3 hours; after 20 to 40 minutes there was net synthesis of adenine nucleotide as ATP. These results indicate that extrapolation from leaf discs to intact leaves is highly inadvisable.     

Evidence for active Phloem loading in the minor veins of sugar beet

Phloem loading in source leaves of sugar beet (Beta vulgaris, L.) was studied to determine the extent of dependence on energy metabolism and the involvement of a carrier system. Dinitrophenol at a concentration of 4 mm uncoupled respiration, lowered source leaf ATP to approximately 40% of the level in the control leaf and inhibited translocation of exogenously supplied ¹⁴C-sucrose to approximately 20% of the control. Dinitrophenol at a concentration of 8 mm inhibited rather than promoted CO₂ production, indicating a mechanism of inhibition other than uncoupling of respiration. The 8 mm dinitrophenol also reduced ATP to approximately 40% of the level in the control source leaf and reduced translocation of exogenous sucrose to approximately 10% of the control. Application of 4 mm ATP to an untreated source leaf promoted the translocation rate by approximately 80% over the control, while in leaves treated with 4 mm dinitrophenol, 4 mm ATP restored translocation to the control level. No recovery of translocation was observed when ATP was applied to leaves treated with 8 mm dinitrophenol. The results indicate an energy-requiring process for both phloem loading and translocation in the source leaf.     

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).     

On the Role of Mitochondrial Oxidative Phosphorylation in Photosynthesis Metabolism as Studied by the Effect of Oligomycin on Photosynthesis in Protoplasts and Leaves of Barley (Hordeum vulgare)

Low concentrations of oligomycin, which strongly inhibit mitochondrial oxidative phosphorylation but do not affect chloroplast photophosphorylation, caused an inhibition of photosynthesis by 30 to 40% in barley (Hordeum vulgare L.) leaf protoplasts. This inhibition is reversed and the full rate of photosynthesis is regained when the protoplasts are ruptured so as to leave the chloroplasts intact. Oligomycin fed into barley leaves by the transpiration stream inhibited photosynthesis in these leaves by up to 60%. The measurement of metabolites in protoplast and leaf extracts showed that oligomycin caused a decrease in the ATP/ADP ratio and an increase in the content of glucose- and fructose 6-phosphate. Subcellular analysis of protoplasts revealed that the decrease in ATP/ADP ratio in the cytosol was larger than in the stroma and that the increase in hexose monophosphates was restricted to the cytosol, whereas the stromal hexosemonophosphates decreased upon the addition of oligomycin. Moreover, oligomycin caused an increase in the triosephosphate-3-phosphoglycerate ratio. It is concluded from these results that during photosynthesis of a plant leaf cell mitochondrial oxidative phosphorylation contributes to the ATP supply of the cell and prevents overreduction of the chloroplast redox carriers by oxidizing reductive equivalents generated by photosynthetic electron transport.     

Mitochondria contribute to increased photosynthetic capacity of leaves of winter rye (Secale cereale L.) following cold-hardening

Cold-hardening of winter rye (Secale cereale L. cv. Musketeer) increased dark respiration from ?2.2 to ?3.9 μmol O₂ m^?2s^?1 and doubled light-and CO₂-saturated photosynthesis at 20°C from 18.1 to 37.0μmol O₂ m^?2 s^?1 We added oligomycin at a concentration that specifically inhibits oxidative phosphorylation to see whether the observed increase in dark respiration reflected an increase in respiration in the light, and whether this contributed to the enhanced photosynthesis of cold-hardened leaves. Oligomycin inhibited light- and CO₂-saturated rates of photosynthesis in non-hardened and cold-hardened leaves by 14 and 25%, respectively, and decreased photochemical quenching of chlorophyll a fluorescence to a greater degree in cold-hardened than in non-hardened leaves. These data indicate an increase both in the rate of respiration in the light, and in the importance of respiration to photosynthesis following cold-hardening. Analysis of metabolite pools indicated that oligomycin inhibited photosynthesis by limiting regeneration of ribulose-1,5-bisphosphate. This limitation was particularly severe in cold-hardened leaves, and the resulting low 3-phospho-glycerate pools led to a feed-forward inhibition of sucrose-phosphate synthase activity. Thus, it does not appear that oxidative phosphorylation supports the increase in photo-synthetic O₂ evolution following cold-hardening by increasing the availability of cytosolic ATP. The data instead support the hypothesis that the mitochondria function in the light by using the reducing equivalents generated by non-cyclic photosynthetic electron transport.     

The effects of different external nitrate concentrations on growth of Phaseolus vulgaris cv. Seafarer at chilling temperatures

The effects of different applied nitrate concentrations (1 to 50 mol m³) on growth of Phaseolus vulgaris cv. Seafarer at temperatures around 15°C was examined. Total plant dry weight and carbon content decreased sharply with increased applied nitrate 1 to 10 mol m^-3 then decreased slightly with further increases in applied N. Total plant reduced -N content increased sharply with increased applied nitrate concentration from 1 to 5 mol m^-3, changed little with increased applied nitrate from 5 to 25 mol m^-3, then increased when applied nitrate was increased from 25 to 50 mol m^-3. Nitrate concentration in all tissues increased sharply with applied nitrate increased from 1 to 10 mol m³ and showed a further increase at 50 mol m³ applied nitrate. Fresh weight to dry weight ratio for all leaves and specific leaf area for all secondary leaves increased sharply with applied nitrate concentration from 1 to 5 mol m^-3 then decreased with applied nitrate 25 to 50 mol m³ Secondary leaf chlorophyll concentration decreased sharply when applied nitrate increased from 1 to 5 mol m^-3 but increased with applied nitrate from 25 to 50 mol m^-3. Initially, the rate of leaf extension was greater at 20 mol m^-3 applied nitrate than 1 mol m^-3 applied nitrate. It is proposed that decreased growth with increased applied nitrate in the range 1 to 10 mol m^-3 is due to increased leaf damage caused by a greater rate of leaf expansion.     

Response of total night-time respiration to differences in total daily photosynthesis for leaves in a Quercus rubra L. canopy: implications for modelling canopy CO2 exchange

Measurements of photosynthesis and respiration were made on leaves in summer in a Quercus rubra L. canopy at approximately hourly intervals throughout 5 days and nights. Leaves were selected in the upper canopy in fully sunlit conditions (upper) and in the lower canopy (lower). In addition, leaves in the upper canopy were shaded (upper shaded) to decrease photosynthesis rates. The data were used to test the hypothesis that total night‐time respiration is dependent on total photosynthesis during the previous day and that the response is mediated through changes in storage in carbohydrate pools. Measurements were made on clear sunny days with similar solar irradiance and air temperature, except for the last day when temperature, especially at night, was lower than that for the previous days. Maximum rates of photosynthesis in the upper leaves (18.7 μmol m^?2 s^?1) were approximately four times higher than those in the lower leaves (4.3 μmol m^?2 s^?1) and maximum photosynthesis rates in the upper shaded leaves (8.0 μmol m^?2 s^?1) were about half those in the upper leaves. There was a strong linear relationship between total night‐time respiration and total photosynthesis during the previous day when rates of respiration were normalized to a fixed temperature of 20°C, removing the effects of temperature from this relationship. Measurements of specific leaf area, nitrogen and chlorophyll concentration and calculations of the maximum rate of carboxylation activity, V_cmax, were not significantly different between upper and upper shaded leaves 5 days after the shading treatment was started. There were small, but significant decreases in the rate of apparent maximum electron transport at saturating irradiance, J_max (P>0.05), and light use efficiency, ? (P<0.05), for upper shaded leaves compared with those for upper leaves. This suggests that the duration of shading in the experiment was sufficient to initiate changes in the electron transport, but not the carboxylation processes of photosynthesis. Support for the hypothesis was provided from analysis of soluble sugar and starch concentrations in leaves. Respiration rates in the upper shaded leaves were lower than those expected from a relationship between respiration and soluble sugar concentration for fully exposed upper and lower leaves. However, there was no similar difference in starch concentrations. This suggests that shading for the duration of several days did not affect sugar concentrations but reduced starch concentrations in leaves, leading to lower rates of respiration at night. A model was used to quantify the significance of the findings on estimated canopy CO₂ exchange for the full growing season. Introducing respiration as a function of total photosynthesis on the previous day resulted in a decrease in growing season night‐time respiration by 23% compared with the value when respiration was held constant. This highlights the need for a process‐based approach linking respiration to photosynthesis when modelling long‐term carbon exchange in forest ecosystems.