The rate of nitrite reduction in leaves as indicated by O₂ and CO₂ exchange during photosynthesis

Light response (at 300 ppm CO(2) and 10-50 ppm O(2) in N(2)) and CO(2) response curves [at absorbed photon fluence rate (PAD) of 550 μmol m(-2) s(-1)] of O(2) evolution and CO(2) uptake were measured in tobacco (Nicotiana tabacum L.) leaves grown on either NO(3)(-) or NH(4)(+) as N source and in potato (Solanum tuberosum L.), sorghum (Sorghum bicolor L. Moench), and amaranth (Amaranthus cruentus L.) leaves grown on NH(4)NO(3). Photosynthetic O(2) evolution in excess of CO(2) uptake was measured with a stabilized zirconia O(2) electrode and an infrared CO(2) analyser, respectively, and the difference assumed to represent the rate of electron flow to acceptors alternative to CO(2), mainly NO(2)(-), SO(4)(2-), and oxaloacetate. In NO(3)(-)-grown tobacco, as well as in sorghum, amaranth, and young potato, the photosynthetic O(2)-CO(2) flux difference rapidly increased to about 1 μmol m(-2) s(-1) at very low PADs and the process was saturated at 50 μmol quanta m(-2) s(-1). At higher PADs the O(2)-CO(2) flux difference continued to increase proportionally with the photosynthetic rate to a maximum of about 2 μmol m(-2) s(-1). In NH(4)(+)-grown tobacco, as well as in potato during tuber filling, the low-PAD component of surplus O(2) evolution was virtually absent. The low-PAD phase was ascribed to photoreduction of NO(2)(-) which successfully competes with CO(2) reduction and saturates at a rate of about 1 μmol O(2) m(-2) s(-1) (9% of the maximum O(2) evolution rate). The high-PAD component of about 1 μmol O(2) m(-2) s(-1), superimposed on NO(2)(-) reduction, may represent oxaloacetate reduction. The roles of NO(2)(-), oxaloacetate, and O(2) reduction in the regulation of ATP/NADPH balance are discussed.     

Structural and biochemical characterization of the C₃-C₄ intermediate Brassica gravinae and relatives, with particular reference to cellular distribution of Rubisco

On the basis of its CO(2) compensation concentration, Brassica gravinae Ten. has been reported to be a C(3)-C(4) intermediate. This study investigated the structural and biochemical features of photosynthetic metabolism in B. gravinae. The cellular distribution of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) was also examined in B. gravinae, B. napus L. (C(3)), Raphanus sativus L. (C(3)), and Diplotaxis tenuifolia (L.) DC. (C(3)-C(4)) by immunogold electron microscopy to elucidate Rubisco expression during the evolution from C(3) to C(3)-C(4) intermediate plants. The bundle sheath (BS) cells of B. gravinae contained centrifugally located chloroplasts as well as centripetally located chloroplasts and mitochondria. Glycine decarboxylase P-protein was localized in the BS mitochondria. Brassica gravinae had low C(4) enzyme activities and high activities of Rubisco and photorespiratory enzymes, suggesting that it reduces photorespiratory CO(2) loss by the glycine shuttle. In B. gravinae, the labelling density of Rubisco was higher in the mesophyll chloroplasts than in the BS chloroplasts. A similar cellular pattern was found in other Brassicaceae species. These data demonstrate that, during the evolution from C(3) to C(3)-C(4) intermediate plants, the intercellular pattern of Rubisco expression did not change greatly, although the amount of chloroplasts in the BS cells increased. It also appears that intracellular variation in Rubisco distribution may occur within the BS cells of B. gravinae.     

Activated carbons from sisal waste by chemical activation with K₂CO₃: kinetics of paracetamol and ibuprofen removal from aqueous solution

Sisal waste was used as precursor to prepare carbons by chemical activation. The influence of the K₂CO₃ amount and activation temperature on the materials textural properties were studied through N₂ and CO₂ adsorption assays. As the severity of the treatment increases there is a development of supermicropores, and the micropore size distribution changes from mono to bimodal. A carbon with an apparent surface area of 1038 m² g⁻¹ and pore volume of 0.49 cm³ g⁻¹ was obtained. TPD results showed the incidence in acidic type groups although the pH_PZC reveals an almost neutral character of the surface. Adsorption kinetic data of ibuprofen and paracetamol show that the processes obey to a pseudo-second order kinetic equation. Regarding the removal efficiency the prepared samples attained values comparable to a commercial carbon (>65%), revealing that chemical activation of sisal wastes with K₂CO₃ allows obtaining samples suitable for pharmaceutical compounds removal from liquid phase.     

An analytical model of non-photorespiratory CO₂release in the light and dark in leaves of C₃species based on stoichiometric flux balance

Leaf respiration continues in the light but at a reduced rate. This inhibition is highly variable, and the mechanisms are poorly known, partly due to the lack of a formal model that can generate testable hypotheses. We derived an analytical model for non‐photorespiratory CO₂ release by solving steady‐state supply/demand equations for ATP, NADH and NADPH, coupled to a widely used photosynthesis model. We used this model to evaluate causes for suppression of respiration by light. The model agrees with many observations, including highly variable suppression at saturating light, greater suppression in mature leaves, reduced assimilatory quotient (ratio of net CO₂ and O₂ exchange) concurrent with nitrate reduction and a Kok effect (discrete change in quantum yield at low light). The model predicts engagement of non‐phosphorylating pathways at moderate to high light, or concurrent with processes that yield ATP and NADH, such as fatty acid or terpenoid synthesis. Suppression of respiration is governed largely by photosynthetic adenylate balance, although photorespiratory NADH may contribute at sub‐saturating light. Key questions include the precise diel variation of anabolism and the ATP : 2e^‐ ratio for photophosphorylation. Our model can focus experimental research and is a step towards a fully process‐based model of CO₂ exchange.     

Effects of low atmospheric CO2 and elevated temperature during growth on the gas exchange responses of C3, C3–C4 intermediate, and C4 species from three evolutionary lineages of C4 photosynthesis

This study evaluates acclimation of photosynthesis and stomatal conductance in three evolutionary lineages of C(3), C(3)-C(4) intermediate, and C(4) species grown in the low CO(2) and hot conditions proposed to favo r the evolution of C(4) photosynthesis. Closely related C(3), C(3)-C(4), and C(4) species in the genera Flaveria, Heliotropium, and Alternanthera were grown near 380 and 180 μmol CO(2) mol(-1) air and day/night temperatures of 37/29°C. Growth CO(2) had no effect on photosynthetic capacity or nitrogen allocation to Rubisco and electron transport in any of the species. There was also no effect of growth CO(2) on photosynthetic and stomatal responses to intercellular CO(2) concentration. These results demonstrate little ability to acclimate to low CO(2) growth conditions in closely related C(3) and C(3)-C(4) species, indicating that, during past episodes of low CO(2), individual C(3) plants had little ability to adjust their photosynthetic physiology to compensate for carbon starvation. This deficiency could have favored selection for more efficient modes of carbon assimilation, such as C(3)-C(4) intermediacy. The C(3)-C(4) species had approximately 50% greater rates of net CO(2) assimilation than the C(3) species when measured at the growth conditions of 180 μmol mol(-1) and 37°C, demonstrating the superiority of the C(3)-C(4) pathway in low atmospheric CO(2) and hot climates of recent geological time.     

Hydrogen bonds determine the structures of the ternary heterocyclic complexes C₂H₄O···2HF, C₂H₅N···2HF and C₂H₄S···2HF: density functional theory and topological calculations

A theoretical study of structural, electronic, topological and vibrational parameters of the ternary hydrogen-bonded complexes C₂H₄O···2HF, C₂H₅N···2HF and C₂H₄S···2HF is presented here. Different from binary systems with a single proton donor, the tricomplexes have the property of forming multiple hydrogen bonds, which are analyzed from a structural and vibrational point of view, but verified only by means of the quantum theory of atoms in molecules (QTAIM). As traditionally done in the hydrogen bond theory, the charge transfer between proton donors and acceptors was computed using the CHELPG calculations, which also revealed agreement with dipole moment variation and a cooperative effect on the tricomplexes. Furthermore, redshift events on proton donor bonds were satisfactorily identified, although, in this case, an absence of experimental data led to the use of a theoretical argument to interpret these spectroscopic shifts. It was therefore the use of the QTAIM parameters that enabled all intermolecular vibrational modes to be validated. The most stable tricomplex in terms of energy was identified via the strength of the hydrogen bonds, which were modeled as directional and bifurcated.     

The kinetics of interconversion of intermediates of the reaction of pig muscle lactate dehydrogenase with oxidized nicotinamide–adenine dinucleotide and lactate

Oxamate competes with pyruvate for the substrate binding site on the E(NADH) complex of pig skeletal muscle lactate dehydrogenase. When this enzyme was mixed with saturating concentrations of NAD(+) and lactate in a stopped-flow rapid-reaction spectrophotometer there was no transient accumulation of enzyme complexes with the reduced nucleotide. The steady-state rate of formation of free NADH was reached within the dead-time of the instrument (3ms). When oxamate was added to inhibit the steady state and to uncouple the equilibration: [Formula: see text] through the rapid formation of E(NADH) (Oxamate), the rate of formation of E(NADH) could be measured by observation of the first turnover. This pH-dependent transient is controlled by the rate of dissociation of pyruvate and the fraction of the enzyme in the form E(NADH) (Pyruvate).     

The influence of light quality on C4 photosynthesis under steady-state conditions in Zea mays and Miscanthus×giganteus: changes in rates of photosynthesis but not the efficiency of the CO2 concentrating mechanism

Differences in light quality penetration within a leaf and absorption by the photosystems alter rates of CO₂ assimilation in C₃ plants. It is also expected that light quality will have a profound impact on C₄ photosynthesis due to disrupted coordination of the C₄ and C₃ cycles. To test this hypothesis, we measured leaf gas exchange, ¹³CO₂ discrimination (Δ¹³C), photosynthetic metabolite pools and Rubisco activation state in Zea mays and Miscanthus × giganteus under steady‐state red, green, blue and white light. Photosynthetic rates, quantum yield of CO₂ assimilation, and maximum phosphoenolpyruvate carboxylase activity were significantly lower under blue light than white, red and green light in both species. However, similar leakiness under all light treatments suggests the C₄ and C₃ cycles were coordinated to maintain the photosynthetic efficiency. Measurements of photosynthetic metabolite pools also suggest coordination of C₄ and C₃ cycles across light treatments. The energy limitation under blue light affected both C₄ and C₃ cycles, as we observed a reduction in C₄ pumping of CO₂ into bundle‐sheath cells and a limitation in the conversion of C₃ metabolite phosphoglycerate to triose phosphate. Overall, light quality affects rates of CO₂ assimilation, but not the efficiency of CO₂ concentrating mechanism.     

The effect of potato plant transformation with the gene encoding Δ12-acyl-lipid desaturase on the CO2 exchange and activities of antioxidant enzymes under hypothermia

The effects of potato (Solanum tuberosum L., cv. Desnitsa) plant transformation with the desA gene encoding Δ12-acyl-lipid desaturase from Synechocystis sp. PCC 6803 on the regulation of free-radical processes in relation to plant tolerance to hypothermia are considered. It was shown that the content of polyunsaturated fatty acids (PUFA) in transformed plants was higher than in wild-type ones. In particular, the content of linoleic acid in transformants was higher by 35% and the content of linolenic acid was by 41% higher than in untransformed plants. In addition, transformation induced an increase in the absolute content of C₁₆-PUFA and on the whole resulted in a marked accumulation of membrane lipids. As judged from the values of the damage index and the ratio of photosynthesis to respiration in wild-type and transformed plants under cold treatment, these changes in lipid metabolism favored the protection of coupling membranes, thus preventing plants against free-radical oxidation under low-temperature stress. As a result, the intensity of oxidative stress in transformed plants was much lower than in wild-type ones, whereas antioxidant enzymes (superoxide dismutase, catalase, peroxidase) were not substantially activated under hypothermia.     

The intramolecular ¹³C-distribution in ethanol reveals the influence of the CO₂ -fixation pathway and environmental conditions on the site-specific ¹³C variation in glucose

Efforts to understand the cause of ¹²C versus ¹³C isotope fractionation in plants during photosynthesis and post‐photosynthetic metabolism are frustrated by the lack of data on the intramolecular ¹³C‐distribution in metabolites and its variation with environmental conditions. We have exploited isotopic carbon‐13 nuclear magnetic resonance (¹³C NMR) spectrometry to measure the positional isotope composition (δ¹³C_i, ‰) in ethanol samples from different origins: European wines, liquors and sugars from C₃, C₄ and crassulacean acid metabolism (CAM) plants. In C₃‐ethanol samples, the methylene group was always ¹³C‐enriched (～2‰) relative to the methyl group. In wines, this pattern was correlated with both air temperature and δ¹⁸O of wine water, indicating that water vapour deficit may be a critical defining factor. Furthermore, in C₄‐ethanol, the reverse relationship was observed (methylene‐C relatively ¹³C‐depleted), supporting the concept that photorespiration is the key metabolic process leading to the ¹³C distribution in C₃‐ethanol. By contrast, in CAM‐ethanol, the isotopic pattern was similar to but stronger than C₃‐ethanol, with a relative ¹³C‐enrichment in the methylene‐C of up to 13‰. Plausible causes of this ¹³C‐pattern are briefly discussed. As the intramolecular δ¹³C_i‐values in ethanol reflect that in source glucose, our data point out the crucial impact on the ratio of metabolic pathways sustaining glucose synthesis.     

The diurnal course of leaf gas exchange of the C4 species Amaranthus retroflexus under field conditions in a ‘cool’ climate: Comparison with the C3 species Glycine max and Chenopodium album

Summary The gas exchange of leaves of Amaranthus retroflexus (C₄) measured under fluctuating environmental conditions in an experimental garden in Würzburg was compared with that of Glycine max and Chenopodium album (C₃). Consistent with previous observations, Amaranthus had higher leaf diffusion resistance than the C₃ species and low internal air space carbon dioxide concentration. Due to high photosynthetic capacity, Amaranthus fixed as much carbon during the light as the C₃ species, even at low temperatures and low light intensities. Low rates of dark respiration of leaves potentially enhances the ability of Amaranthus to grow rapidly after establishment in a disturbed habitat. The data suggest that some populations of Amaranthus retroflexus are adapted to cool climate conditions but are also able to exploit high temperature situations.     

Seasonal dynamics in the stable carbon isotope composition δ¹³C from non-leafy branch, trunk and coarse root CO₂ efflux of adult deciduous (Fagus sylvatica) and evergreen (Picea abies) trees

Respiration is a substantial driver of carbon (C) flux in forest ecosystems and stable C isotopes provide an excellent tool for its investigation. We studied seasonal dynamics in δ¹³C of CO₂ efflux (δ¹³C_E) from non‐leafy branches, upper and lower trunks and coarse roots of adult trees, comparing deciduous Fagus sylvatica (European beech) with evergreen Picea abies (Norway spruce). In both species, we observed strong and similar seasonal dynamics in the δ¹³C_E of above‐ground plant components, whereas δ¹³C_E of coarse roots was rather stable. During summer, δ¹³C_E of trunks was about ?28.2‰ (Beech) and ?26.8‰ (Spruce). During winter dormancy, δ¹³C_E increased by 5.6–9.1‰. The observed dynamics are likely related to a switch from growth to starch accumulation during fall and remobilization of starch, low TCA cycle activity and accumulation of malate by PEPc during winter. The seasonal δ¹³C_E pattern of branches of Beech and upper trunks of Spruce was less variable, probably because these organs were additionally supplied by winter photosynthesis. In view of our results and pervious studies, we conclude that the pronounced increases in δ¹³C_E of trunks during the winter results from interrupted access to recent photosynthates.     

Secondary structure of β-hydroxydecanoyl thiol ester dehydrase, a 39-kDa protein, derived from Hα, Cα, Cβ and CO signal assignments and the Chemical Shift Index: Comparison with the crystal structure

Summary Nearly complete backbone ¹H, ¹⁵N and ¹³C signal assignments are reported for -hydroxydecanoyl thiol ester dehydrase, a 39-kDa homodimer containing 342 amino acids. Although ¹⁵N relaxation data show that the protein has a rotational correlation time of 18 ns, assignments were derived from triple-resonance experiments recorded at 500 MHz and pH 6.8, without deuteration. The Chemical Shift Index, CSI, identified two long helices and numerous -strands in dehydrase. The CSI predictions are in close agreement with the secondary structure identified in the recently derived crystal structure, particularly when one takes account of the numerous bulges in the -strands. The assignment of dehydrase and a large deuterated protein [Yamazaki et al. (1994) J. Am. Chem. Soc., 116, 11655–11666] suggest that assignment of 40–60 kDa proteins is feasible. Hence, further progress in understanding the chemical shift/structure relationship could open the way to determine the structures of such large proteins. Supplementary Material is available on request, comprising Table S1 listing the spectral parameters; Table S2 listing the assignments; Fig. S1 showing the 2D ¹H–¹⁵N HSQC spectrum; Fig. S2 showing sequential NOEs, secondary shifts, H-exchange and ³J_HN data; and Fig. S3 showing plots of the H, C, CO and C Chemical Shift Indexes.To whom correspondence should be addressed.     

The membrane topology of the Rhizobium meliloti C4-dicarboxylate permease (DctA) as derived from protein fusions with Escherichia coli K12 alkaline phosphatase (PhoA) and β-galactosidase (LacZ)

The Rhizobium meliloti dctA gene encodes the C₄-dicarboxylate permease which mediates uptake of C₄-dicarboxylates, both in free-living and symbiotic cells. Based on the hydrophobicity of the DctA protein, 12 putative membrane spanning regions were predicted. The membrane topology was further analysed by isolating in vivo fusions of DctA to Escherichia coli alkaline phosphatase (PhoA) and E. coli β-galactosidase (LacZ). Of 10 different fusions 7 indicated a periplasmic and 3 a cytoplasmic location of the corresponding region of the DctA protein. From these data a two-dimensional model of DctA was constructed which comprised twelve transmembrane α-helices with the amino-terminus and the carboxy-terminus located in the cytoplasm. In addition, four conserved amino acid motifs present in many eukaryotic and prokaryotic transport proteins were observed.