Intramitochondrial localisation of glycine decarboxylase in spinach leaves

Intact spinach leaf mitochondria are capable of oxidising glycine with good respiratory control and the oxidation is coupled to 3 phosphorylation sites. The intramitochondrial localisation of glycine decarboxyllation has been studied and it is demonstrated that the enzyme system is associated with the inner membrane of spinach leaf mitochondria. Both glycine decarboxylation and glycine dependent O₂ uptake are stimulated by ADP and FCCP and are sensitive to electron transport inhibitors. Both processes showed no requirements for co-factors. We suggest that glycine decarboxylase is coupled to the electron transport chain $\text{via}$ an NAD⁺-linked system and that during rapid photorespiration glycine oxidation synthesises considerable amounts of ATP outside of the chloroplast.     

Progress No. 9 Cultivar of Pea Has Alternative Respiratory Capacity and Normal Glycine Metabolism

Mitochondria from the dwarf pea cultivar Progress No. 9, havebeen reported to lack alternative respiration. However, therates of respiration with succinate and malate by mitochondriaisolated from Progress No. 9, although approximately 30% lowerthan those from Alaska, had the same percentage distributionof respiratory capacity between the cytochrome pathway and thealternative pathway. Immunoblots showed both cultivars containpolypeptides identified as components of the alternative oxidase.Both cultivars formed identical products during photosynthetic¹⁴CO₂ fixation, and the percentage distribution of ¹⁴C intoglycine and serine were similar. In spite of large amounts ofglycine decarboxylase in the isolated leaf mitochondria, ratesof O₂ uptake with glycine were similar to rates with malateor succinate. It appears that glycine oxidation was coupledto the alternative oxidase to the same extent as malate andsuccinate oxidation, and the alternative oxidase was not specificallyutilized for glycine oxidation. (Received May 21, 1990; Accepted December 19, 1990)     

Glycine metabolism and oxalacetate transport by pea leaf mitochondria

Isolated pea leaf mitochondria oxidatively decarboxylate added glycine. This decarboxylation could be linked to the respiratory chain (in which case it was coupled to three phosphorylations) or to mitochondrial malate dehydrogenase when oxalacetate was supplied. Decarboxylation rates measured as O₂ uptake, or CO₂ and NH₃ release were adequate to account for whole leaf photorespiration. Oxalacetate-supported glycine decarboxylation, measured by linking malate efflux to added malic enzyme, yielded rates considerably less than the electron transport rates. Butylmalonate inhibited malate efflux but not oxalacetate entry; phthalonate inhibited oxalacetate entry but had little effect on malate or α-ketoglutarate oxidation. It is suggested that oxalacetate and malate transport are catalyzed by separate carrier systems of the mitochondrial membrane.     

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

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

Metabolic regulation of carbon flux during C4 photosynthesis

The potential for glycolate and glycine metabolism and the mechanism of refixation of photorespiratory CO₂ in leaves of C₄ plants were studied by parallel inhibitor experiments with thin leaf slices, different leaf cell types and isolated mitochondria of C₃ and C₄ Panicum species. CO₂ evolution by leaf slices of P. bisulcatum, a C₃ species, fed glycolate or glycine was light-independent and O₂-sensitive. The C₄ P. maximum and P. miliaceum leaf slices fed glycolate or glycine evolved CO₂ in the dark but not in the light. In C₄ species, dark CO₂ evolution was abolished by the addition of phosphoenolpyruvate (PEP)⁴. The addition of maleate, a PEP carboxylase inhibitor, resulted in photorespiratory CO₂ efflux by C₄ leaf slices in the light also. However, PEP and maleate had no effect on either glycolate-dependent O₂ uptake by the C₄ leaf slices or on glycolate and glycine metabolism in C₃ leaf slices. The rate of photorespiratory CO₂ evolution in the C₃ Panicum species was 3 times higher than that observed with the C₄ species. The ratio of glycolate-dependent CO₂ evolution to O₂ uptake in both groups was 1:2. Isolated C₄ mesophyll protoplasts or their mitochondria did not metabolize glycolate or glycine. However, both C₃ mesophyll protoplasts and C₄ bundle sheath strands readily metabolized glycolate and glycine in a light-independent, O₂-sensitive manner, and the addition of PEP or maleate had no effect. C₄ bundle sheath- and C₃-mitochondria were capable of oxidizing glycine. This oxidation was linked to the mitochondrial electron transport chain, was coupled to three phosphorylation sites and was sensitive to electron transport inhibitors. C₄ bundle sheath- and C₃-mitochondrial glycine decarboxylation was stimulated by oxaloacetate and NAD had no effect. In marked contrast, mitochondria isolated from C₄ mesophyll cells were incapable of oxidizing or decarboxylating added glycine. The results suggest that in leaves of C₄ plants bundle sheath cells are the primary site of O₂-sensitive photorespiratory CO₂ evolution and the PEP carboxylase present in the mesophyll cells has the Potential for efficiently refixing CO₂ before it escapes out of the leaf. The relative role of the PEP carboxylase mediated CO₂ pump and reassimilation of photorespiratory CO₂ are discussed in relation to the apparent lack of photorespiration in leaves of C₄ species.Abbreviations BSA bovine serum albumin - Chl chlorophyll - PEP phosphoenolpyruvate - Rbu-P ₂ ribulose 1,5-bisphosphate - Rib-5-P ribose-5-phosphate - Ru-5-P ribuluse-5-phosphate - FCCP carbonyl cyanide p-trifluoromethoxyphenylhydrazone Journal Series Paper, New Jersey Agricultural Experiment Station     

Glyoxylate decarboxylation during photorespiration

At 25° C under aerobic conditions with or without gluamate 10% of the [1-¹⁴C]glycollate oxidised in spinach leaf peroxisomes was released as ¹⁴CO₂. Without glutamate only 5% of the glycollate was converted to glycine, but with it over 80% of the glycollate was metabolised to glycine. CO₂ release was probably not due to glycine breakdown in these preparations since glycine decarboxylase activity was not detected. Addition of either unlabelled glycine or isonicotinyl hydrazide (INH) did not reduce ¹⁴CO₂ release from either [1-¹⁴C]glycollate or [1-¹⁴C]glyoxylate. Furthermore, the amount of available H₂O₂ (Grodzinski and Butt, 1976) was sufficient to account for all of the CO₂ release by breakdown of glyoxylate. Peroxisomal glycollate metabolism was unaffected by light and isolated leaf chloroplasts alone did not metabolise glycollate. However, in a mixture of peroxisomes and illuminated chloroplasts the rate of glycollate decarboxylation increased three fold while glycine synthesis was reduced by 40%. Although it was not possible to measure available H₂O₂ directly, the data are best explained by glyoxylate decarboxylation. Catalase reduced CO₂ release and enhanced glycine synthesis. In addition, when a model system in which an active preparation of purified glucose oxidase generating H₂O₂ at a known rate was used to replace the chloroplasts, similar rates of ¹⁴CO₂ release and [¹⁴C]glycine synthesis from [1-¹⁴C]glycollate were measured. It is argued that in vivo glyoxylate metabolism in leaf peroxisomes is a key branch point of the glycollate pathway and that a portion of the photorespired CO₂ arises during glyoxylate decarboxylation under the action of H₂O₂. The possibility that peroxisomal catalase exerts a peroxidative function during this process is discussed.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid - INH isonicotinylhydrazide - PHMS pyridyl-2-yl--hydroxymethane sulphonic acid     

Mechanism of decarboxylation of glycine and glycolate by isolated soybean cells

Isolated soybean leaf mesophyll cells decarboxylated exogenously added [1-¹⁴C]glycolate and [1-¹⁴C]glycine in the dark. The rate of CO₂ release from glycine was inhibited over 90% by isonicotinic acid hydrazide and about 80% by KCN, two inhibitors of the glycine to serine plus CO₂ reaction. The release of CO₂ from glycolate was inhibited by less than 50% under the same conditions. This indicates that about 50% of the CO₂ released from glycolate occurred at a site other than the glycine to serine reaction. The sensitivity of this alternative site of CO₂ release to an inhibitor of glycolate oxidase (methyl-2-hydroxy-3-butynoate) but not an inhibitor of the glutamate:glyoxylate aminotransferase (2,3-epoxypropionate) indicates that this alternative (isonicotinic acid hydrazide insensitive) site of CO₂ release involved glyoxylate. Catalase inhibited this CO₂ release. Under the conditions used it is suggested that about half of the CO₂ released from glycolate occurred at the conversion of glycine to serine plus CO₂ while the remaining half of the CO₂ loss resulted from the direct oxidation of glyoxylate by H₂O₂.     

Isolation and characterization of metabolically competent mitochondria from spinach leaf protoplasts

Intact mitochondria were prepared from spinach (Spinacia oleracea L. var. Kyoho) leaf protoplasts and purified by Percoll discontinuous gradient centrifugation. Assays of several marker enzymes showed that the final mitochondrial preparations obtained are nearly free from other contaminating organelles, e.g. chloroplasts, peroxisomes, and endoplasmic reticulum. These mitochondria oxidized malate, glycine, succinate, and NADH, tightly coupled to oxidative phosphorylation with high values of ADP to O ratio as well as respiratory control ratio. The rate of NADH oxidation was 331 nmoles O₂ per milligram mitochondrial protein per minute, which is comparable to that obtained by highly purified potato or mung bean mitochondria. However, the activity of glutamine synthetase was barely detectable in the isolated mitochondrial fraction. This finding rules out a hypothetical scheme (Jackson, Dench, Morris, Lui, Hall, Moore 1971 Biochem Soc Trans 7: 1122) dealing with the role of the mitochondrial glutamine synthetase in the reassimilation of NH₃, which is released during the step of photorespiratory glycine decarboxylation in green leaf tissues, but it is consistent with the photosynthetic nitrogen cycle (Keys, Bird, Cornelius, Lea, Wallsgrove, Miflin 1978 Nature (Lond) 275: 741), in which NH₃ reassimilation occurs outside the mitochondria.     

Effects of pH, NADH, succinate and malate on the oxidation of glycine in spinach leaf mitochondria

The effect of external pH on several reactions catalyzed by glycine decarboxylase in spinach leaf mitochondria was investigated. Glycine-dependent oxygen consumption showed a pH optimum at 7.6, whereas the release of CO₂ and NH3 from glycine in the presence of oxaloacetate both showed pH maxima at 8.1. Glycine-dependent reduction of 2,6-dichlorophenolindophenol. on the other hand showed a pH optimum at 8.4. It is concluded that these three reactions have different rate-limiting steps. The rate of the glycine-bicarbonate exchange reaction catalyzed by glycine decarboxylase showed no optimum in the pH range investigated, pH 7–9, but increased with decreasing pH. This suggests that CO₂ may be the true substrate in this reaction.
The oxidation of glycine inhibited the oxidation of both malate, succinate and external NADH since the addition of malate, succinate or NADH to mitochondria oxidizing glycine in state 3 resulted in a rate of oxygen consumption which was lower than the sum of the rates when the substrates were oxidized individually. The addition of malate, succinate or NADH did not, however, decrease the rate of CO₂ or NH, release from glycine. It is suggested that the preferred oxidation of glycine by-spinach leaf mitochondria may constitute an important regulatory mechanism for the function of leaf mitochondria during photosynthesis.     

Regulation of Respiration in the Leaves and Roots of Two Lolium perenne Populations with Contrasting Mature Leaf Respiration Rates and Crop Yields

Measurements of O₂ uptake were made on leaves and roots of two populations of Lolium perenne L. cv S23 (GL66 and GL72), previously shown to have contrasting rates of CO₂ evolution and yields of dry matter. O₂ uptake was faster in the mature leaves of GL66 than those of GL72, but no difference was observed in the respiratory rates of meristematic leaf bases or mature roots. The growth rate of GL72 was faster than that of GL66. Cyanide resistance was substantial in mature leaves but the alternative path did not contribute to O₂ uptake in the dark. In both populations, adding malate and glycine stimulated O₂ uptake, but exogenous sucrose only stimulated when uncoupler was also present. The difference between the respiratory rates of the two populations was maintained under all investigated conditions. We conclude that the rate of mature leaf respiration in the dark in L. perenne is limited by adenylate control of glycolysis. The difference between the fast (GL66) and slow (GL72) respiring populations reflected a greater respiratory capacity and higher turnover of ATP in GL66. Alternative path capacity was also high in the roots of both and contributed substantially to O₂ uptake, as indicated by inhibition by salicylhydroxamic acid in the absence of KCN. The alternative path capacity of meristematic leaf bases was considerably less than that in mature leaves.

Transverse and cross-sections were made of mature leaves of both populations to study anatomical features which might explain the differences in ATP turnover, suggested by the biochemical experiments. Leaves of GL72 were thicker but did not show a different anatomy when compared with GL66. The increased thickness was not due to more or larger cells but entirely to a larger intercellular volume.

     

Effect of oxygen on photosynthesis by spinach leaf protoplasts

The photosynthetic CO₂ fixation by spinach leaf (Spinacia oleracea L. var. Kyoho) protoplasts was inhibited by substituting an atmosphere of N₂ with one of either air (21% O₂) or 100% O₂. The inhibitory effect of 100% O₂ was greater than that of air. The mode of inhibition by 100% O₂ and air was competitive with respect to CO₂; Ki(O₂) value was 0.32 mM at pH 7 and 0.28 mM at pH 8.5 The labeling patterns of compounds in protoplasts exposed to ¹⁴CO₂ in light after transferring them from N₂ to O₂ atmospheres were examined. There was no detectable ¹⁴CO₂ incorporation into glycolate under anaerobic and O₂ atmospheres; a more marked labeling of glycine occurred under an oxidative environment compared to that under the anaerobic condition, presumably because of a rapid transformation of glycolate to glycine in the protoplasts.     

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

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

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

     

Modification of Carbon Partitioning, Photosynthetic Capacity, and O2 Sensitivity in Arabidopsis Plants with Low ADP-Glucose Pyrophosphorylase Activity

Wild-type Arabidopsis plants, the starch-deficient mutant TL46, and the near-starchless mutant TL25 were evaluated by noninvasive in situ methods for their capacity for net CO₂ assimilation, true rates of photosynthetic O₂ evolution (determined from chlorophyll fluorescence measurements of photosystem II), partitioning of photosynthate into sucrose and starch, and plant growth. Compared with wild-type plants, the starch mutants showed reduced photosynthetic capacity, with the largest reduction occurring in mutant TL25 subjected to high light and increased CO₂ partial pressure. The extent of stimulation of CO₂ assimilation by increasing CO₂ or by reducing O₂ partial pressure was significantly less for the starch mutants than for wild-type plants. Under high light and moderate to high levels of CO₂, the rates of CO₂ assimilation and O₂ evolution and the percentage inhibition of photosynthesis by low O₂ were higher for the wild type than for the mutants. The relative rates of ¹⁴CO₂ incorporation into starch under high light and high CO₂ followed the patterns of photosynthetic capacity, with TL46 showing 31% to 40% of the starch-labeling rates of the wild type and TL25 showing less than 14% incorporation. Overall, there were significant correlations between the rates of starch synthesis and CO₂ assimilation and between the rates of starch synthesis and cumulative leaf area. These results indicate that leaf starch plays an important role as a transient reserve, the synthesis of which can ameliorate any potential reduction in photosynthesis caused by feedback regulation.     

Direct and indirect effects of elevated CO2 on leaf respiration in a forest ecosystem

We measured the short‐term direct and long‐term indirect effects of elevated CO₂ on leaf dark respiration of loblolly pine (Pinus taeda) and sweetgum (Liquidambar styraciflua) in an intact forest ecosystem. Trees were exposed to ambient or ambient + 200 µmol mol^?1 atmospheric CO₂ using free‐air carbon dioxide enrichment (FACE) technology. After correcting for measurement artefacts, a short‐term 200 µmol mol^?1 increase in CO₂ reduced leaf respiration by 7–14% for sweetgum and had essentially no effect on loblolly pine. This direct suppression of respiration was independent of the CO₂ concentration under which the trees were grown. Growth under elevated CO₂ did not appear to have any long‐term indirect effects on leaf maintenance respiration rates or the response of respiration to changes in temperature (Q₁₀, R₀). Also, we found no relationship between mass‐based respiration rates and leaf total nitrogen concentrations. Leaf construction costs were unaffected by growth CO₂ concentration, although leaf construction respiration decreased at elevated CO₂ in both species for leaves at the top of the canopy. We conclude that elevated CO₂ has little effect on leaf tissue respiration, and that the influence of elevated CO₂ on plant respiratory carbon flux is primarily through increased biomass.     

Involvement of respiratory processes in the transient knockout of net CO2 uptake in Mimosa pudica upon heat stimulation

Leaf photosynthesis of the sensitive plant Mimosa pudica displays a transient knockout in response to electrical signals induced by heat stimulation. This study aims at clarifying the underlying mechanisms, in particular, the involvement of respiration. To this end, leaf gas exchange and light reactions of photosynthesis were assessed under atmospheric conditions largely eliminating photorespiration by either elevated atmospheric CO₂ or lowered O₂ concentration (i.e. 2000 μmol mol^?1 or 1%, respectively). In addition, leaf gas exchange was studied in the absence of light. Under darkness, heat stimulation caused a transient increase of respiratory CO₂ release simultaneously with stomatal opening, hence reflecting direct involvement of respiratory stimulation in the drop of the net CO₂ uptake rate. However, persistence of the transient decline in net CO₂ uptake rate under illumination and elevated CO₂ or 1% O₂ makes it unlikely that photorespiration is the metabolic origin of the respiratory CO₂ release. In conclusion, the transient knockout of net CO₂ uptake is at least partially attributed to an increased CO₂ release through mitochondrial respiration as stimulated by electrical signals. Putative CO₂ limitation of Rubisco due to decreased activity of carbonic anhydrase was ruled out as the photosynthesis effect was not prevented by elevated CO₂.     

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.     

Metabolism of <Superscript>14</Superscript>C-glycine as a substrate for photorespiration of the leaf at different developmental stages of ephemeroides

The metabolism of ¹⁴C-glycine (a substrate for photorespiration) was studied in the light and in darkness under natural CO₂ concentration (0.03%) in the leaves of ephemeroides Scilla sibirica Haw. and Ficaria verna Huds. at different developmental stages. Using one and the same sample, potential photosynthesis (at 1% CO₂), true photosynthesis (at 0.03% CO₂), and leaf respiratory capacity were measured by the radiometric and manometric methods, respectively. All measurements were performed at 15°C, an average temperature during ephemer growth. It was found that, in the white zone of the Scilla leaf, the rate of CO₂ evolution resulting from metabolization of exogenous ¹⁴C-glycine was similar in the light and in darkness. In the green zone of the Scilla leaf and in the green leaf of Ficaria, both ¹⁴C-glycine absorption and ¹⁴CO₂ evolution were lower in the light as compared with darkness, which is explained by CO₂ reassimilation. In all treatments of both plant species, a specific inhibitor of glycine decarboxylase complex (GDC), aminoacetonitrile (5 mM) suppressed CO₂ evolution by 20–40%. It was concluded that in ephemeroides mitochondrial GDC, responsible for CO₂ evolution in photorespiration, is formed at the earliest stage of leaf development. This indicates that photorespiration can occur simultaneously with the development of the leaf photosynthetic activity. On the basis of the assumption that carbon losses in the form of CO₂ evolved during photorespiration comprise 25% of true photosynthesis, it was calculated that, in ephemer leaves, the highest rates of photorespiration and photosynthesis were attained during flowering when the leaf area was the largest and the rate of dark respiration was reduced by 1.5–2.0 times. The highest rates of dark respiration were observed in the beginning of growth. In senescing leaves by the end of the plant vegetation, potential photosynthesis and true photosynthesis were reduced, whereas dark respiration remained essentially unchanged. It is concluded that the high rates of potential and true photosynthesis are characteristic of ephemeroides when they complete their short developmental program in early spring (at 15°C); theoretically, photorespiration also occurs at a high rate during this period, when this process provides for a defense against the threat of photoinhibition at low temperature and high insolation.     

Acclimation of snow gum (Eucalyptus pauciflora) leaf respiration to seasonal and diurnal variations in temperature: the importance of changes in the capacity and temperature sensitivity of respiration

We investigated the relationship between daily and seasonal temperature variation and dark respiratory CO₂ release by leaves of snow gum (Eucalyptus pauciflora Sieb. ex Spreng) that were grown in their natural habitat or under controlled‐environment conditions. The open grassland field site in SE Australia was characterized by large seasonal and diurnal changes in air temperature. On each measurement day, leaf respiration rates in darkness were measured in situ at 2–3 h intervals over a 24 h period, with measurements being conducted at the ambient leaf temperature. The rate of respiration at a set measuring temperature (i.e. apparent ‘respiratory capacity’) was greater in seedlings grown under low average daily temperatures (i.e. acclimation occurred), both in the field and under controlled‐environment conditions. The sensitivity of leaf respiration to diurnal changes in temperature (i.e. the Q₁₀ of leaf respiration) exhibited little seasonal variation over much of the year. However, Q₁₀ values were significantly greater on cold winter days (i.e. when daily average and minimum air temperatures were below 6° and –1 °C, respectively). These differences in Q₁₀ values were not due to bias arizing from the contrasting daily temperature amplitudes in winter and summer, as the Q₁₀ of leaf respiration was constant over a wide temperature range in short‐term experiments. Due to the higher Q₁₀ values in winter, there was less difference between winter and summer leaf respiration rates measured at 5 °C than at 25 °C. The net result of these changes was that there was relatively little difference in total daily leaf respiratory CO₂ release per unit leaf dry mass in winter and summer. Under controlled‐environment conditions, acclimation of respiration to growth temperature occurred in as little as 1–3 d. Acclimation was associated with a change in the concentration of soluble sugars under controlled conditions, but not in the field. Our data suggest that acclimation in the field may be associated with the onset of cold‐induced photo‐inhibition. We conclude that cold‐acclimation of dark respiration in snow gum leaves is characterized by changes in both the temperature sensitivity and apparent ‘capacity’ of the respiratory apparatus, and that such changes will have an important impact on the carbon economy of snow gum plants.     

Role of enzymatic and non-enzymatic processes in H2O2 removal by rat liver and heart mitochondria

We compared the capacity of rat liver and heart mitochondria to remove exogenously produced H₂O₂, determining their ability to decrease fluorescence generated by H₂O₂ detector system. In the absence of substrates, liver and heart mitochondria removed H₂O₂ at similar rates. Respiratory substrate addition increased removal rates, indicating a respiration-dependent process. Moreover, the rates were higher with pyruvate/malate than with succinate and in heart than in liver mitochondria. Generally, the changes in H₂O₂ removal rates mirrored those of H₂O₂ release rates excluding the possibility that endogenous and exogenous H₂O₂ competed for the removing system. This idea was supported by the observation that the heaviest of three liver mitochondrial fractions exhibited the highest rates of both H₂O₂ release and removal. Pharmacological inhibition showed tissue-linked differences in antioxidant enzyme contribution to H₂O₂ removal which were consistent with the differences in antioxidant system activities. The enzymatic processes accounted only in part for net H₂O₂ removal and the non-enzymatic ones participated to H₂O₂ scavenging to a degree that was higher for heart than for liver mitochondria. The idea that non-enzymatic scavenging was due in great part to hemoproteins action was consistent with observation that the concentration of cytochromes, in particular cytochrome c, was higher in heart mitochondria. Indirect support was also obtained by a technique of enhanced luminescence, utilizing the capacity of cytochrome c/H₂O₂ to catalyze the luminol oxidation, which showed that luminescence response to an oxidative challenge was higher in heart mitochondria.     

Effects of glycine hydroxamate, carbon dioxide, and oxygen on photorespiratory carbon and nitrogen metabolism in spinach mesophyll cells

The effects of added glycine hydroxamate on the photosynthetic incorporation of ¹⁴CO₂ into metabolites by isolated mesophyll cells of spinach (Spinacia oleracea L.) was investigated under conditions favorable to photorespiratory (PR) metabolism (0.04% CO₂ and 20% O₂) and under conditions leading to nonphotorespiratory (NPR) metabolism (0.2% CO₂ and 2.7% O₂). Glycine hydroxamate (GH) is a competitive inhibitor of the photorespiratory conversion of glycine to serine, CO₂ and NH₄⁺. During PR fixation, addition of the inhibitor increased glycine and decreased glutamine labeling. In contrast, labeling of glycine decreased under NPR conditions. This suggests that when the rate of glycolate synthesis is slow, the primary route of glycine synthesis is through serine rather than from glycolate. GH addition increased serine labeling under PR conditions but not under NPR conditions. This increase in serine labeling at a time when glycine to serine conversion is partially blocked by the inhibitor may be due to serine accumulation via the “reverse” flow of photorespiration from 3-P-glycerate to hydroxypyruvate when glycine levels are high. GH increased glyoxylate and decreased glycolate labeling. These observations are discussed with respect to possible glyoxylate feedback inhibition of photorespiration.