Photosynthetic Gas Exchange and Discrimination against 13CO2 and C18O16O in Tobacco Plants Modified by an Antisense Construct to Have Low Chloroplastic Carbonic Anhydrase

The physiological role of chloroplastic carbonic anhydrase (CA) was examined by antisense suppression of chloroplastic CA (on average 8% of wild type) in Nicotiana tabacum. Photosynthetic gas-exchange characteristics of low-CA and wild-type plants were measured concurrently with short-term, on-line stable isotope discrimination at varying vapor pressure deficit (VPD) and light intensity. Low-CA and wild-type plants were indistinguishable in the responses of assimilation, transpiration, stomatal conductance, and intercellular CO2 concentration to changing VPD or light intensity. At saturating light intensity, low-CA plants had lower discrimination against 13CO2 than wild-type plants by 1.2 to 1.8[per mille (thousand) sign]. Consequently, tissue of the low-CA plants was higher in 13C than the control plants. It was calculated that low-CA plants had chloroplast CO2 concentrations 13 to 22 [mu]mol mol-1 lower than wild-type plants. Discrimination against C18O16O in low-CA plants was 20% of that of the wild type, confirming a role of chloroplastic CA in the mechanism of discrimination against C18O16O ([delta]C18O16O). As VPD increased, stomatal closure caused a reduction in chloroplastic C02 concentration, and since VPD and chloroplastic CO2 concentration act in opposing directions on [delta]C18O16O, no effect of VPD was seen on [delta]C18O16O.     

Discrimination Processes and Shifts in Carboxylation during the Phases of Crassulacean Acid Metabolism

The magnitude and extent of Crassulacean acid metabolism (CAM) activity in two Clusia species was manipulated to investigate the regulation of the distinct CAM phases. First, in response to leaf-air vapor pressure deficit at night, changes in leaf conductance altered on-line carbon-isotope discrimination throughout the theoretical range for dark CO2 uptake during CAM. These ranged from the limit set by phosphoenolpyruvate carboxylase (PEPc) (-6[per mille (thousand) sign], [delta]13C equivalent of -2[per mille (thousand) sign]) to that imposed by diffusion limitation (+4[per mille (thousand) sign], [delta]13C equivalent of -12[per mille (thousand) sign]), but the lowest carbon-isotope discrimination occurred when P[square root]pa was only 0.7. Second, when the availability of external or internal sources of CO2 was reduced for both field- and greenhouse-grown plants, CO2 uptake by day via PEPc during phase II largely compensated. Third, by reducing the dark period, plants accumulated low levels of acidity, and CO2 uptake occurred throughout the subsequent light period. Discrimination switched from being dominated by PEPc (phase II) to ribulose 1,5-bisphosphate carboxylase/oxygenase (phase III), with both enzymes active during phase IV. Under natural conditions, photochemical stability is maintained by extended PEPc activity in phase II, which enhances acid accumulation and delays decarboxylation until temperature and light stress are maximal at midday.     

Carbon Isotopic Fractionation Does Not Occur during Dark Respiration in C3 and C4 Plants

The magnitude of possible carbon isotopic fractionation during dark respiration was investigated with isolated mesophyll cells from mature leaves of common bean (Phaseolus vulgaris L.), a C3 plant, and corn (Zea mays L.), a C4 plant. Mesophyll protoplasts were extracted from greenhouse-grown leaves and incubated in culture solutions containing different carbohydrate substrates (fructose, glucose, and sucrose) with known [delta]13C values. The CO2 produced by protoplasts after incubation in the dark was collected, purified, and analyzed for its carbon isotope ratio. From observations of the isotope ratios of the substrate and respired CO2, we calculated the carbon isotope discrimination associated with metabolism of each of these substrates. In eight of the 10 treatment combinations, the carbon isotope ratio discrimination was not significantly different from 0. In the remaining two treatment combinations, the carbon isotope ratio discrimination was 1[per mille (thousand) sign]. From these results, we conclude that there is no significant carbon isotopic discrimination during mitochondrial dark respiration when fructase, glucose, or sucrose are used as respiratory substrates.     

Correlations between the 13C Content of Primary and Secondary Plant Products in Different Cell Compartments and That in Decomposing Basidiomycetes

Relative carbon isotope ratio ([delta]13C values) of primary and secondary products from different compartments of annual plants, pine needles, wood, and decomposing Basidiomycetes have been determined. An enrichment in 13C was found for storage tissues of annual plants, because of the high level of the primary storage products sucrose and starch; however, the enrichment was even greater in leaf starch. All of these compounds had the same relative 13C enrichment in positions 3 and 4 of glucose. Secondary products in conifer needles (lignin, lipids) were depleted in 13C by 1 to 2 [per mille (thousand) sign] relative to carbohydrates from the same origin. Air pollution caused a small decrease in [delta]13C values; however, the relative content of plant products, especially of the soluble polar compounds, was also affected. Decomposing fungi showed a global accumulation of 13C by 4[per mille (thousand) sign] relative to their substrates in wood. Their chitin was enriched by 2[per mille (thousand) sign] relative to the cellulose of the wood. Hence, Basidiomycetes preferentially metabolize "light" molecules, whereas "heavy" molecules are preferentially polymerized. Our results are discussed on the basis of a kinetic isotope effect on the fructose-1,6-bisphosphate aldolase reaction and of metabolic branching on the level of the triose phosphates with varying substrate fluxes.     

Carbon-Isotope Composition of Biochemical Fractions and the Regulation of Carbon Balance in Leaves of the C3-Crassulacean Acid Metabolism Intermediate Clusia minor L. Growing in Trinidad

Carbon-isotope ratios ([delta]13Cs) were measured for various bio-chemical fractions quantitatively extracted from naturally exposed and shaded leaves of the C3-Crassulacean acid metabolism (CAM) intermediate Clusia minor, sampled at dawn and dusk on days during the wet and dry seasons in Trinidad. As the activity of CAM increased in response to decreased availability of water and higher photon flux density, organic acids and soluble sugars were enriched in 13C by approximately 3.5 to 4%[per mille (thousand) sign] compared to plants sampled during the wet season. The induction of CAM was accompanied by a doubling in size of the reserve carbohydrate pools. Moreover, stoichiometric measurements indicated that degradation of both chloroplastic reserves and soluble sugars were necessary to supply phosphoenolpyruvate for the synthesis of organic acids at night. Results also suggest that two pools of soluble sugars exist in leaves of C. minor that perform CAM, one a vacuolar pool enriched in 13C and the second a transport pool depleted in 13C. Estimates of carbon-isotope discrimination expressed during CAM, derived from the trafficking among inorganic carbon, organic acids, and carbohydrate pools overnight, ranged from 0.9 to 3.1%[per mille (thousand) sign]. The [delta]13C of structural material did not change significantly between wet and dry seasons, indicating that most of the carbon used in growth was derived from C3 carboxylation.     

Steady-state nitrogen isotope effects of N2 and N2O production in Paracoccus denitrificans

Nitrogen stable-isotope compositions (delta15N) can help track denitrification and N2O production in the environment, as can knowledge of the isotopic discrimination, or isotope effect, inherent to denitrification. However, the isotope effects associated with denitrification as a function of dissolved-oxygen concentration and their influence on the isotopic composition of N2O are not known. We developed a simple steady-state reactor to allow the measurement of denitrification isotope effects in Paracoccus denitrificans. With [dO2] between 0 and 1.2 microM, the N stable-isotope effects of NO3- and N2O reduction were constant at 28.6 per thousand +/- 1.9 per thousand and 12.9 per thousand +/- 2.6 per thousand, respectively (mean +/- standard error, n = 5). This estimate of the isotope effect of N2O reduction is the first in an axenic denitrifying culture and places the delta15N of denitrification-produced N2O midway between those of the nitrogenous oxide substrates and the product N2 in steady-state systems. Application of both isotope effects to N2O cycling studies is discussed.     

ATP sulfurylase from Penicillium chrysogenum. Molecular basis of the sigmoidal velocity curves induced by sulfhydryl group modification

ATP sulfurylase from Penicillium chrysogenum is a noncooperative homooligomer containing three free sulfhydryl groups per subunit. Under nondenaturing conditions, one SH group per subunit was modified by 5,5'-dithiobis-(2-nitrobenzoate), or N-ethylmaleimide. Modification had only a small effect on kcat, but markedly increased the [S]0.5 values for the substrates, MgATP and SO4(2-). MgATP and adenosine-5'-phosphosulfate protected against modification. The SH-modified enzyme displayed sigmoidal velocity curves for both substrates with Hill coefficients (nH) of 2. Fluorosulfonate (FSO3-) and other dead-end inhibitors competitive with SO4(2-) activated the SH-modified enzyme at low SO4(2-) concentration. In order to determine whether the sigmoidicity resulted from true cooperative binding (as opposed to a kinetically based mechanism), the shapes of the binding curves were established from the degree of protection provided by a ligand against phenylglyoxal-dependent irreversible inactivation under noncatalytic conditions. Under standard conditions (0.05 M Na-N-(2-hydroxyethyl)piperazine-N'-3-propanesulfonic acid buffer, pH 8, 30 degrees C, and 3mM phenylglyoxal) the native enzyme was inactivated with a k of 2.67 +/- 0.25 X 10-3 s-1, whereas k for the SH-modified enzyme was 5.44 +/- 0.27 X 10-3 s-1. The increased sensitivity of the modified enzyme resulted from increased reactivity of ligand-protectable groups. Both the native and the SH-modified enzyme displayed hyperbolic plots of delta k (i.e. protection) versus [MgATP], or [FSO3-], or [S2O3(2-]) in the absence of coligand (nH = 0.98 +/- 0.06). The plots of delta k versus [ligand] for the native enzyme were also hyperbolic in the presence of a fixed concentration of coligand. However, in the presence of a fixed [FSO3-] or [S2O3(2-]), the delta k versus [MgATP] plot for the SH-modified enzyme was sigmoidal, as was the plot of delta k versus [FSO3-] or [S2O3(2-]) in the presence of a fixed [MgATP]. The nH values were 1.92 +/- 0.09. The results indicate that substrates (or analogs) bind hyperbolically to unoccupied SH-modified subunits, but in a subunit-cooperative fashion to form a ternary complex.     

Stereochemical determination of carbon partitioning between photosynthesis and photorespiration in C3 plants: use of (3R)-D-[3-3H1, 3-14C]glyceric acid

When (3R)-D-[3-3H1,3-14C]glyceric acid is supplied in tracer amounts to illuminated tobacco leaf discs, the acid penetrates to the chloroplasts without loss of 3H, and is phosphorylated there. Subsequent metabolism associated with the reductive photosynthetic cycle fully conserves 3H. Oxidation of ribulose bisphosphate (RuBP) by RuBP carboxylase-oxygenase (EC 4.1.1.39) results in the formation of (2R)-[2-3H1, 14C]glycolic acid which, on oxidation by glycolate oxidase (EC 1.1.3.1), releases 3H to water. Loss of 3H from the combined photosynthetic and photorespiratory systems is, therefore, associated with the oxidative photorespiratory loop. Assuming steady-state conditions and a basic metabolic model, the fraction of RuBP oxidized and the photorespiratory carbon flux relative to gross or net CO2 fixation can be calculated from the fraction of supplied 3H retained in the triose phosphates exported from the chloroplasts. This retention can be determined from the 3H:14C ratio for glucose obtained from isolated sucrose. The dependence of 3H retention upon O2 and CO2 concentrations can be deduced by assuming simple competitive kinetics for RuBP carboxylase-oxygenase. The experimental results confirmed the stereochemical assumptions made. Under conditions of negligible photorespiration 3H retention was essentially complete. The change in 3H retention with O2 and CO2 concentrations were investigated. For leaf discs (upper surface up) in normal air, it was estimated that 39% of the RuBP was oxidized, 32% of the fixed CO2 was photorespired, and the photorespiration rate was 46% of the net photosynthetic CO2 fixation rate. These are minimal estimates, as it is assumed that the only source of photorespired CO2 is glycine decarboxylation.     

Discrimination in the dark. Resolving the interplay between metabolic and physical constraints to phosphoenolpyruvate carboxylase activity during the crassulacean acid metabolism cycle

A model defining carbon isotope discrimination (delta13C) for crassulacean acid metabolism (CAM) plants was experimentally validated using Kalanchoe daigremontiana. Simultaneous measurements of gas exchange and instantaneous CO2 discrimination (for 13C and 18O) were made from late photoperiod (phase IV of CAM), throughout the dark period (phase I), and into the light (phase II). Measurements of CO2 response curves throughout the dark period revealed changing phosphoenolpyruvate carboxylase (PEPC) capacity. These systematic changes in PEPC capacity were tracked by net CO2 uptake, stomatal conductance, and online delta13C signal; all declined at the start of the dark period, then increased to a maximum 2 h before dawn. Measurements of delta13C were higher than predicted from the ratio of intercellular to external CO2 (p(i)/p(a)) and fractionation associated with CO2 hydration and PEPC carboxylations alone, such that the dark period mesophyll conductance, g(i), was 0.044 mol m(-2) s(-1) bar(-1). A higher estimate of g(i) (0.085 mol m(-2) s(-1) bar(-1)) was needed to account for the modeled and measured delta18O discrimination throughout the dark period. The differences in estimates of g(i) from the two isotope measurements, and an offset of -5.5 per thousand between the 18O content of source and transpired water, suggest spatial variations in either CO2 diffusion path length and/or carbonic anhydrase activity, either within individual cells or across a succulent leaf. Our measurements support the model predictions to show that internal CO2 diffusion limitations within CAM leaves increase delta13C discrimination during nighttime CO2 fixation while reducing delta13C during phase IV. When evaluating the phylogenetic distribution of CAM, carbon isotope composition will reflect these diffusive limitations as well as relative contributions from C3 and C4 biochemistry.     

Natural abundance carbon isotope composition of isoprene reflects incomplete coupling between isoprene synthesis and photosynthetic carbon flow

Isoprene emission from leaves is dynamically coupled to photosynthesis through the use of primary and recent photosynthate in the chloroplast. However, natural abundance carbon isotope composition (delta(13)C) measurements in myrtle (Myrtus communis), buckthorn (Rhamnus alaternus), and velvet bean (Mucuna pruriens) showed that only 72% to 91% of the variations in the delta(13)C values of fixed carbon were reflected in the delta(13)C values of concurrently emitted isoprene. The results indicated that 9% to 28% carbon was contributed from alternative, slow turnover, carbon source(s). This contribution increased when photosynthesis was inhibited by CO(2)-free air. The observed variations in the delta(13)C of isoprene under ambient and CO(2)-free air were consistent with contributions to isoprene synthesis in the chloroplast from pyruvate associated with cytosolic Glc metabolism. Irrespective of alternative carbon source(s), isoprene was depleted in (13)C relative to mean photosynthetically fixed carbon by 4 per thousand to 11 per thousand. Variable (13)C discrimination, its increase by partially inhibiting isoprene synthesis with fosmidomicin, and the associated accumulation of pyruvate suggested that the main isotopic discrimination step was the deoxyxylulose-5-phosphate synthase reaction.     

18O pattern and biosynthesis of natural plant products

Oxygen atoms in plant products originate from CO(2), H(2)O and O(2), precursors with quite different delta18O values. Furthermore their incorporation by different reactions implies isotope effects. On this base the resulting non-statistical 18O distributions in natural compounds are discussed. The delta18O value of cellulose is correlated to that of the leaf water, and the observed 18O enrichment (approximately +27 per thousand) is generally attributed to an equilibrium isotope effect between carbonyl groups and water. However, as soluble and heterotrophically synthesised carbohydrates show other correlations, a non-statistical 18O distribution - originating from individual biosynthetic reactions - is postulated for carbohydrates. Similarly, the delta18O values of organic acids, carbonyl compounds, alcohols and esters indicate water-correlated, but individual 18O abundances (e.g. O from acyl groups approximately +19% above water), depending upon origin and biosyntheses. Alcoholic groups introduced by monooxygenase reactions, e.g. in sterols and phenols, show delta18O values near +5 per thousand, in agreement with an assumed isotope fractionation factor of approximately 1.02 on the reaction with atmospheric oxygen (delta18O=+23.5 per thousand). Correspondingly, a "thermodynamically ordered isotope distribution" is only observed for oxygen in some functional groups correlated to an origin from CO(2) and H(2)O, not from O(2). The individual isotopic increments of functional groups permit the prediction of global delta18O values of natural compounds on the basis of their biosynthesis.     

Temporal variation in δ<Superscript>13</Superscript>C of ecosystem respiration in the Pacific Northwest: links to moisture stress

We measured seasonal and interannual variations in delta(13)C values within the carbon reservoirs (leaves and soil) and CO(2) fluxes (soil and ecosystem respired CO(2)) of an old growth coniferous forest in the Pacific Northwest USA with relation to local meteorological conditions. There were significant intra-annual and interannual differences in the carbon isotope ratios of CO(2) respired at both the ecosystem (delta(13)C(R)) and the soil levels (delta(13)C(R-soil)), but only limited variations in the carbon isotope ratios of carbon stocks. The delta(13)C(R) values varied by as much as 4.4 per thousand over a growing season, while delta(13)C(R-soil )values changed as much as 6.2 per thousand. The delta(13)C of soil organic carbon (delta(13)C(SOC)) and needle organic carbon (delta(13)C(P)) exhibited little or no significant changes over the course of this study. Carbon isotope discrimination within leaves (Delta(p)) showed systematic decreases with increased canopy height, but remained fairly constant throughout the year (Delta(p)=17.9 per thousand -19.2 per thousand at the top of the canopy, Delta(p)=19.6 per thousand -20.9 per thousand at mid-canopy, Delta(p)=23.3 per thousand -25.1 per thousand at the canopy base). The temporal variation in the delta(13)C of soil and ecosystem respired CO(2) was correlated ( r=0.93, P<0.001) with soil moisture levels, with dry summer months having the most (13)C-enriched values. The dynamic seasonal changes in delta(13)C of respired CO(2) are hypothesized to be the result of fast cycling of recently fixed carbon back to the atmosphere. One scaling consequence of the seasonal and interannual variations in delta(13)C(R) is that inversion-based carbon-cycle models dependent on observed atmospheric CO(2) concentration and isotope values may be improved by incorporating dynamic delta(13)C(R) values to interpret regional carbon sink strength.     

Simultaneous kinetic analysis of ribulose 1,5-bisphosphate carboxylase/oxygenase activities

An assay was developed for simultaneous kinetic analysis of the activities of the bifunctional plant enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase [EC 4.1.1.39]. [1-¹⁴C,5-³H]Ribulose 1,5-bisphosphate (RuBP) was used as the labeled substrate. Tritium enrichment of the doubly labeled 3-phosphoglycerate (3-PGA) product, common to both enzyme activities, may be used to calculate V_c/V_o ratios from the expression A/(B-A) where A and B represent the ³H/¹⁴C isotope ratios of doubly labeled RuBP and 3-PGA, and V_c and V_o represent the activities of carboxylase and oxygenase, respectively. Doubly labeled substrate was synthesized from [2-¹⁴C]glucose and [6-³H]glucose using the enzymes of the pentose phosphate pathway coupled with phosphoribulokinase.     

Stable carbon isotope fractionation by sulfate-reducing bacteria

Biogeochemical transformations occurring in the anoxic zones of stratified sedimentary microbial communities can profoundly influence the isotopic and organic signatures preserved in the fossil record. Accordingly, we have determined carbon isotope discrimination that is associated with both heterotrophic and lithotrophic growth of pure cultures of sulfate-reducing bacteria (SRB). For heterotrophic-growth experiments, substrate consumption was monitored to completion. Sealed vessels containing SRB cultures were harvested at different time intervals, and delta(13)C values were determined for gaseous CO(2), organic substrates, and products such as biomass. For three of the four SRB, carbon isotope effects between the substrates, acetate or lactate and CO(2), and the cell biomass were small, ranging from 0 to 2 per thousand. However, for Desulfotomaculum acetoxidans, the carbon incorporated into biomass was isotopically heavier than the available substrates by 8 to 9 per thousand. SRB grown lithoautotrophically consumed less than 3% of the available CO(2) and exhibited substantial discrimination (calculated as isotope fractionation factors [alpha]), as follows: for Desulfobacterium autotrophicum, alpha values ranged from 1.0100 to 1.0123; for Desulfobacter hydrogenophilus, the alpha value was 0.0138, and for Desulfotomaculum acetoxidans, the alpha value was 1.0310. Mixotrophic growth of Desulfovibrio desulfuricans on acetate and CO(2) resulted in biomass with a delta(13)C composition intermediate to that of the substrates. The extent of fractionation depended on which enzymatic pathways were used, the direction in which the pathways operated, and the growth rate, but fractionation was not dependent on the growth phase. To the extent that environmental conditions affect the availability of organic substrates (e.g., acetate) and reducing power (e.g., H(2)), ecological forces can also influence carbon isotope discrimination by SRB.     

Partial reduction in ribulose 1,5-bisphosphate carboxylase/oxygenase activity by carboxypeptidase A

Treatment with carboxypeptidase A of ribulose bisphosphate carboxylase/oxygenase (rubisco) from spinach and Chlamydomonas, but not tobacco, reduced activity by 60-70%. Further studies with the spinach enzyme indicated that only one amino acid from each of the large (valine) and small (tyrosine) subunits was removed and the loss of activity was correlated with modification of the large subunit. The modified enzyme also had a two-fold greater Km for RuBP but CO2/O2 specificity was only 5% lower and may not be significantly different. The relative rates of release of valine and tyrosine also depended on the presence or absence of RuBP or CO2 plus Mg during treatment. The results indicate that the C-terminal amino acid in the large subunit of spinach, which is not located near the active site region, plays a previously unrecognized role in determining the catalytic activity of the enzyme.     

Wildtype and engineered monomeric triosephosphate isomerase from Trypanosoma brucei: partitioning of reaction intermediates in D2O and activation by phosphite dianion

Product yields for the reactions of (R)-glyceraldehyde 3-phosphate (GAP) in D2O at pD 7.9 catalyzed by wildtype triosephosphate isomerase from Trypanosoma brucei brucei (Tbb TIM) and a monomeric variant (monoTIM) of this wildtype enzyme were determined by (1)H NMR spectroscopy and were compared with the yields determined in earlier work for the reactions catalyzed by TIM from rabbit and chicken muscle [O'Donoghue, A. C., Amyes, T. L., and Richard, J. P. (2005), Biochemistry 44, 2610 - 2621]. Three products were observed from the reactions catalyzed by TIM: dihydroxyacetone phosphate (DHAP) from isomerization with intramolecular transfer of hydrogen, d-DHAP from isomerization with incorporation of deuterium from D2O into C-1 of DHAP, and d-GAP from incorporation of deuterium from D2O into C-2 of GAP. The yield of DHAP formed by intramolecular transfer of hydrogen decreases from 49% for the muscle enzymes to 40% for wildtype Tbb TIM to 34% for monoTIM. There is no significant difference in the ratio of the yields of d-DHAP and d-GAP for wildtype TIM from muscle sources and Trypanosoma brucei brucei, but partitioning of the enediolate intermediate of the monoTIM reaction to form d-DHAP is less favorable ((k(C1))(D)/(k(C2))(D) = 1.1) than for the wildtype enzyme ((k(C1))(D)/(k(C2))(D) = 1.7). Product yields for the wildtype Tbb TIM and monoTIM-catalyzed reactions of glycolaldehyde labeled with carbon-13 at the carbonyl carbon ([1-(13)C]-GA) at pD 7.0 in the presence of phosphite dianion and in its absence were determined by (1)H NMR spectroscopy [Go, M. K., Amyes, T. L., and Richard, J. P. (2009) Biochemistry 48, 5769-5778]. There is no detectable difference in the yields of the products of wildtype muscle and Tbb TIM-catalyzed reactions of [1-(13)C]-GA in D2O. The kinetic parameters for phosphite dianion activation of the reactions of [1-(13)C]-GA catalyzed by wildtype Tbb TIM are similar to those reported for the enzyme from rabbit muscle [Amyes, T. L. and Richard, J. P. (2007) Biochemistry 46, 5841-5854], but there is no detectable dianion activation of the reaction catalyzed by monoTIM. The engineered disruption of subunit contacts at monoTIM causes movement of the essential side chains of Lys-13 and His-95 away from the catalytic active positions. We suggest that this places an increased demand that the intrinsic binding energy of phosphite dianion be utilized to drive the change in the conformation of monoTIM back to the active structure for wildtype TIM.     

Oxidative decarboxylation of glycollate and glyoxylate by leaf peroxisomes

1. The conditions under which peroxisomal preparations from leaves of spinach beet and spinach catalyse the release of (14)CO(2) from [1-(14)C]glycollate and [1-(14)C]glyoxylate were investigated. 2. At pH8, (14)CO(2) production from [1-(14)C]glyoxylate was accompanied by equivalent quantities of formate. The accumulation of oxalate and the effects of various reagents, especially catalase inhibitors, show that glyoxylate is non-enzymically oxidized by H(2)O(2), which is generated by the oxidation of glyoxylate to oxalate by the action of glycollate oxidase. 3. (14)CO(2) is shown to be generated from [1-(14)C]glycollate at pH8 by a similar reaction, but the H(2)O(2) is generated mainly by the oxidation of glycollate to glyoxylate. 4. The physiological significance of these reactions is discussed, with special reference to their role in photorespiration.     

Annual rainfall does not directly determine the carbon isotope ratio of leaves of Eucalyptus species

Leaf carbon isotope discrimination (delta13C) was widely considered to directly reflect the rainfall environment in which the leaf developed, but recent observations have queried this. The relationship between delta13C and rainfall was explored in Eucalyptus species growing along a rainfall gradient in Australia. The leaves of 43 species of Eucalyptus and the closely related Corymbia species produced in 2003 were sampled in September 2004 at 50 sites and grouped into 15 locations along a rainfall gradient in southwest Western Australia. At 24 sites, the same species and same trees were sampled as in a study in September 2003 when leaves produced in 2002 were sampled. The rainfall in 2004 was on average 190 mm (range 135-270 mm) higher at all locations than in 2003. In the leaves sampled in 2004, the mean carbon isotope discrimination (delta13C) across the 15 locations decreased 2.9 per thousand per 1000 mm of rainfall, the specific leaf area (SLA) increased by 2.9 m2 kg(-1) per 1000 mm of rainfall and the nitrogen (N) content decreased by 1.56 g m(-2) per 1000 mm of rainfall. In contrast, a comparison between the leaves produced in the drier 2002 year compared with the wetter 2003 year showed that there was a strong correlation (r2= 0.85) between the SLA values between years and a trend for higher values with increasing SLA, but the values of delta(13)C were on average only 0.38 per thousand lower (more negative) at all locations in the wetter year, equivalent to a decrease of 2.0 per thousand per 1000 mm of rainfall. The results suggest that while there may be constitutive differences in leaf morphology, SLA and N content per unit area, increasing rainfall or cloudiness associated with higher rainfall increases SLA and decreases N content per unit area. We conclude that rainfall does not directly influence delta13C, but induces leaf morphological and physiological changes that affect the resultant delta13C.     

Carbon isotope fractionation during photorespiration and carboxylation in Senecio

The magnitude of fractionation during photorespiration and the effect on net photosynthetic (13)C discrimination (Delta) were investigated for three Senecio species, S. squalidus, S. cineraria, and S. greyii. We determined the contributions of different processes during photosynthesis to Delta by comparing observations (Delta(obs)) with discrimination predicted from gas-exchange measurements (Delta(pred)). Photorespiration rates were manipulated by altering the O(2) partial pressure (pO(2)) in the air surrounding the leaves. Contributions from (13)C-depleted photorespiratory CO(2) were largest at high pO(2). The parameters for photorespiratory fractionation (f), net fractionation during carboxylation by Rubisco and phosphoenolpyruvate carboxylase (b), and mesophyll conductance (g(i)) were determined simultaneously for all measurements. Instead of using Delta(obs) data to obtain g(i) and f successively, which requires that b is known, we treated b, f, and g(i) as unknowns. We propose this as an alternative approach to analyze measurements under field conditions when b and g(i) are not known or cannot be determined in separate experiments. Good agreement between modeled and observed Delta was achieved with f = 11.6 per thousand +/- 1.5 per thousand, b = 26.0 per thousand +/- 0.3 per thousand, and g(i) of 0.27 +/- 0.01, 0.25 +/- 0.01, and 0.22 +/- 0.01 mol m(-2) s(-1) for S. squalidus, S. cineraria, and S. greyii, respectively. We estimate that photorespiratory fractionation decreases Delta by about 1.2 per thousand on average under field conditions. In addition, diurnal changes in Delta are likely to reflect variations in photorespiration even at the canopy level. Our results emphasize that the effects of photorespiration must be taken into account when partitioning net CO(2) exchange of ecosystems into gross fluxes of photosynthesis and respiration.     

Partition kinetics of ribulose-1,5-bisphosphate carboxylase from Rhodospirillum rubrum

When the enzymatically generated intermediate 2-carboxy-3-keto-D-arabinitol-1,5-bisphosphate (II) was used as a substrate with fresh enzyme, 70% reacted to produce 3-phosphoglycerate (3PGA). When a reaction mixture of enzyme plus [1-32P]ribulose 1,5-bisphosphate (RuBP) was quenched in the steady state with the tightly bound inhibitor 2-carboxyarabinitol-1,5-bisphosphate, 30% of the enzyme-bound species was released as 3PGA and 70% as RuBP. The major source for this partition was the ternary substrates Michaelis complex. The level of carboxylated intermediate in the steady state was determined to be 8% of active sites under the conditions of substrate saturation. No burst was seen in the appearance of product when 6.5 eq of [1-32P]RuBP was mixed with enzyme plus saturating CO2 and the reaction followed in the steady state. From these data plus the steady-state Vmax and Km of RuBP it is possible to derive the five bulk rate constants represented in the scheme ECO2 + RuBP in equilibrium ERuBPCO2 in equilibrium E X II----E + 2(3PGA).