Carbon dioxide diffusion across stomata and mesophyll and photo-biochemical processes as affected by growth CO2 and phosphorus nutrition in cotton

Nutrients such as phosphorus may exert a major control over plant response to rising atmospheric carbon dioxide concentration (CO₂), which is projected to double by the end of the 21st century. Elevated CO₂ may overcome the diffusional limitations to photosynthesis posed by stomata and mesophyll and alter the photo-biochemical limitations resulting from phosphorus deficiency. To evaluate these ideas, cotton (Gossypium hirsutum) was grown in controlled environment growth chambers with three levels of phosphate (Pi) supply (0.2, 0.05 and 0.01 mM) and two levels of CO₂ concentration (ambient 400 and elevated 800 μmol mol⁻¹) under optimum temperature and irrigation. Phosphate deficiency drastically inhibited photosynthetic characteristics and decreased cotton growth for both CO₂ treatments. Under Pi stress, an apparent limitation to the photosynthetic potential was evident by CO₂ diffusion through stomata and mesophyll, impairment of photosystem functioning and inhibition of biochemical process including the carboxylation efficiency of ribulose-1,5-bisphosphate carboxylase/oxyganase and the rate of ribulose-1,5-bisphosphate regeneration. The diffusional limitation posed by mesophyll was up to 58% greater than the limitation due to stomatal conductance (g_s) under Pi stress. As expected, elevated CO₂ reduced these diffusional limitations to photosynthesis across Pi levels; however, it failed to reduce the photo-biochemical limitations to photosynthesis in phosphorus deficient plants. Acclimation/down regulation of photosynthetic capacity was evident under elevated CO₂ across Pi treatments. Despite a decrease in phosphorus, nitrogen and chlorophyll concentrations in leaf tissue and reduced stomatal conductance at elevated CO₂, the rate of photosynthesis per unit leaf area when measured at the growth CO₂ concentration tended to be higher for all except the lowest Pi treatment. Nevertheless, plant biomass increased at elevated CO₂ across Pi nutrition with taller plants, increased leaf number and larger leaf area.     

Cytochrome c oxidase loses catalytic activity and structural integrity during the aging process in Drosophila melanogaster

The hypothesis, that structural deterioration of cytochrome c oxidase (CcO) is a causal factor in the age-related decline in mitochondrial respiratory activity and an increase in H₂O₂ generation, was tested in Drosophila melanogaster. CcO activity and the levels of seven different nuclear DNA-encoded CcO subunits were determined at three different stages of adult life, namely, young-, middle-, and old-age. CcO activity declined progressively with age by 33%. Western blot analysis, using antibodies specific to Drosophila CcO subunits IV, Va, Vb, VIb, VIc, VIIc, and VIII, indicated that the abundance these polypeptides decreased, ranging from 11% to 40%, during aging. These and previous results suggest that CcO is a specific intra-mitochondrial site of age-related deterioration, which may have a broad impact on mitochondrial physiology.     

The naturally split Npu DnaE intein exhibits an extraordinarily high rate in the protein trans-splicing reaction

We have studied the naturally split α subunit of the DNA polymerase III (DnaE) intein from Nostoc punctiforme PCC73102 (Npu) using purified proteins and determined an apparent first-order rate constant of (1.1±0.2)×10^-2 s⁻¹ at 37 °C. This represents the highest rate reported for the protein trans-splicing reaction so far (t_1/2 of ∼ 60 s). Furthermore, the reaction was very robust and high-yielding with respect to different extein sequences, temperatures from 6 to 37 °C, and the presence of up to 6 M urea. Given these outstanding properties, the Npu DnaE intein appears to be the intein of choice for many applications in protein and cellular chemistry.     

Pre-anthesis high-temperature acclimation alleviates damage to the flag leaf caused by post-anthesis heat stress in wheat

The objective of this study was to investigate the effect of pre-anthesis high-temperature acclimation on leaf physiology of winter wheat in response to post-anthesis heat stress. The results showed that both pre- and post-anthesis heat stresses significantly depressed flag leaf photosynthesis and enhanced cell membrane peroxidation, as exemplified by increased O₂⁻ production rate and reduction in activities of antioxiditave enzymes. However, under post-anthesis heat stress, plants with pre-anthesis high-temperature acclimation (HH) showed much higher photosynthetic rates than those without pre-anthesis high-temperature acclimation (CH). Leaves of HH plants exhibited a higher Chl a/b ratio and lower chlorophyll/carotenoid ratio and superoxide anion radical release rate compared with those of the CH plants. In addition, antioxidant enzyme activities in HH plants were significantly higher than in CH. Coincidently, expressions of photosythesis-responsive gene encoding Rubisco activase B (RcaB) and antioxidant enzyme-related genes encoding mitochondrial manganese superoxide dismutase (Mn-SOD), chloroplastic Cu/Zn superoxide dismutase (Cu/Zn-SOD), catalase (CAT) and cytosolic glutathione reductase (GR) were all up-regulated under HH, whereas a gene encoding a major chlorophyll a/b-binding protein (Cab) was up-regulated by post-anthesis heat stress at 10 DAA, but was down-regulated at 13 DAA. The changes in the expression levels of the HH plants were more pronounced than those for the CH. Collectively, the results indicated that pre-anthesis high-temperature acclimation could effectively alleviate the photosynthetic and oxidative damage caused by post-anthesis heat stress in wheat flag leaves, which was partially attributable to modifications in the expression of the photosythesis-responsive and antioxidant enzymes-related genes.     

Genetic diversity of microsatellite loci in hierarchically structured populations

Microsatellite loci are widely used for investigating patterns of genetic variation within and among populations. Those patterns are in turn determined by population sizes, migration rates, and mutation rates. We provide exact expressions for the first two moments of the allele frequency distribution in a stochastic model appropriate for studying microsatellite evolution with migration, mutation, and drift under the assumption that the range of allele sizes is bounded. Using these results, we study the behavior of several measures related to Wright’s F_ST, including Slatkin’s R_ST. Our analytical approximations for F_ST and R_ST show that familiar relationships between N_em and F_ST or R_ST hold when the migration and mutation rates are small. Using the exact expressions for F_ST and R_ST, our numerical results show that, when the migration and mutation rates are large, these relationships no longer hold. Our numerical results also show that the diversity measures most closely related to F_ST depend on mutation rates, mutational models (stepwise versus two-phase), migration rates, and population sizes. Surprisingly, R_ST is relatively insensitive to the mutation rates and mutational models. The differing behaviors of R_ST and F_ST suggest that properties of the among-population distribution of allele frequencies may allow the roles of mutation and migration in producing patterns of diversity to be distinguished, a topic of continuing investigation.     

Modelling ¹⁸O₂ and ¹⁶O₂ unidirectional fluxes in plants: I. regulation of pre-industrial atmosphere

In closed systems, the O₂ compensation point (Γ_O) was previously defined as the upper limit of O₂ level, at a given CO₂ level, above which plants cannot have positive carbon balance and survive. Studies with ¹⁸O₂ measure the actual O₂ uptake by photorespiration due to the dual function of Rubisco, the enzyme that fixes CO₂ and takes O₂ as an alternative substrate. One-step modelling of CO₂ and O₂ uptakes allows calculating a plant specificity factor (Sp) as the sum of the biochemical specificity of Rubisco and a biophysical specificity, function of the resistance to CO₂ transfer from the atmosphere to Rubisco. The crossing points (Cx, Ox) are defined as CO₂ and O₂ concentrations for which O₂ and CO₂ uptakes are equal. It is observed that: (1) under the preindustrial atmosphere, photorespiration of C3 plants uses as much photochemical energy as photosynthesis, i.e. the Cx and Ox are equal or near the CO₂ and O₂ concentrations of that epoch; (2) contrarily to Γ_C, a Γ_O does not practically limit the plant growth, i.e. the plant net CO₂ balance is positive up to very high O₂ levels; (3) however, in a closed biosystem, Γ_O exists; it is not the limit of plant growth, but the equilibrium point between photosynthesis and the opposite respiratory processes; (4) a reciprocal relationship exists between Γ_O and Γ_C, as unique functions of the respective CO₂ and O₂ concentrations and of Sp, this invalidates some results showing two different functions for Γ_O and Γ_C, and, consequently, the associated analyses related to greenhouse effects in the past; (5) the pre-industrial atmosphere levels of O₂ and CO₂ are the Γ_O and Γ_C of the global bio-system. They are equal to or near the values of Cx and Ox of C3 plants, the majority of land plants in preindustrial period. We assume that the crossing points represent favourable feedback conditions for the biosphere-atmosphere equilibrium and could result from co-evolution of plants-atmosphere-climate. We suggest that the evolution of Rubisco and associated pathways is directed by an optimisation between photosynthesis and photorespiration.     

Effects of agronomic application of olive mill wastewater in a field of olive trees on carbohydrate profiles, chlorophyll a fluorescence and mineral nutrient content

Olive mill wastewater (OMW) management is a serious environmental issue for the Mediterranean area where there is the most production of olive oil. OMW contains a high organic load, substantial amounts of plant nutrients but also several compounds with recognized toxicity towards living organisms. Moreover, OMW may represent a low cost source of water. We studied the influence of irrigation with OMW (amounts applied: 30, 60, 100 and 150 m³ h⁻¹) in a field of olive trees on root colonization, photosynthesis, chlorophyll fluorescence, leaf nutrient concentration and soluble carbohydrate. The soil fatty acid methyl ester (FAME) 16:1ω5 was used to quantify biomass of arbuscular mycorrhizal (AM) fungi and the root FAME 16:1ω5 analysis was used as index for the development of colonization in the roots. Agronomic application of OMW decreased significantly the abundance of the soil FAME 16:1ω5 and the root FAME 16:1ω5 in the soil amended with 60, 100 and 150 m³ ha⁻¹ OMW. Decreased root FAME 16:1ω5 due to OMW amendment was associated with a significant reduction of tissue nutrient concentrations in the olive trees. The highest application of OMW to the soil reduced significantly the olive trees uptake of N, P, K, Ca, Mg, Fe, Cu, Mn and Zn. Land spreading of OMW increased concentration of soluble carbohydrate in the olive leaves, mostly due to decreased sink demand for carbon by the root. In the olive trees amended with 150 m³ ha⁻¹ OMW, net CO₂ uptake rate (A), quantum yield of photosystem II electron transport (Φ_PSII), maximal photochemical efficiency of photosystem II (Fv/Fm), photochemical quenching (q_p) and the electron transport rate (ETR) were significantly depressed, whereas non-photochemical quenching (NPQ) was found to increase. Taken with data from experiments in field conditions, our results suggest that agronomic application of OMW alters the functioning of arbuscular mycorrhizas and can even disrupt the relationship between AM fungi and olive trees.     

Elevated Proton Leak of the Intermediate OH in Cytochrome c Oxidase

The kinetics of the formation and relaxation of transmembrane electric potential (Δψ) during the complete single turnover of CcO was studied in the bovine heart mitochondrial and the aa₃-type Paracoccus denitrificans enzymes incorporated into proteoliposome membrane. The real-time Δψ kinetics was followed by the direct electrometry technique. The prompt oxidation of CcO and formation of the activated, oxidized (O_H) state of the enzyme leaves the enzyme trapped in the open state that provides an internal leak for protons and thus facilitates dissipation of Δψ (τ^app ≤ 0.5-0.8 s). By contrast, when the enzyme in the O_H state is rapidly re-reduced by sequential electron delivery, Δψ dissipates much slower (τ^app > 3 s). In P. denitrificans CcO proteoliposomes the accelerated Δψ dissipation is slowed down by a mutational block of the proton conductance through the D-, but not K-channel. We concluded that in contrast to the other intermediates the O_H state of CcO is vulnerable to the elevated internal proton leak that proceeds via the D-channel.     

Field-acclimated Gossypium hirsutum cultivars exhibit genotypic and seasonal differences in photosystem II thermostability

Previous investigations have demonstrated that photosystem II (PSII) thermostability acclimates to prior exposure to heat and drought, but contrasting results have been reported for cotton (Gossypium hirsutum). We hypothesized that PSII thermotolerance in G. hirsutum would acclimate to environmental conditions during the growing season and that there would be differences in PSII thermotolerance between commercially-available U.S. cultivars. To this end, three cotton cultivars were grown under dryland conditions in Tifton Georgia, and two under irrigated conditions in Marianna Arkansas. At Tifton, measurements included PSII thermotolerance (T₁₅, the temperature causing a 15% decline in maximum quantum yield), leaf temperatures, air temperatures, midday (1200 to 1400 h) leaf water potentials (Ψ_MD), leaf-air vapor pressure deficit (VPD), actual quantum yield (Φ_PSII) and electron transport rate through PSII (ETR) on three sample dates. At Marianna, T₁₅ was measured on two sample dates. Optimal air and leaf temperatures were observed on all sample dates in Tifton, but PSII thermotolerance increased with water deficit conditions (Ψ_MD = −3.1 MPa), and ETR was either unaffected or increased under water-stress. Additionally, T₁₅ for PHY 499 was ∼5 °C higher than for the other cultivars examined (DP 0912 and DP 1050). The Marianna site experienced more extreme high temperature conditions (20–30 days T_max ≥ 35 °C), and showed an increase in T₁₅ with higher average T_max. When average T₁₅ values for each location and sample date were plotted versus average daily T_max, strong, positive relationships (r² from .954 to .714) were observed between T_max and T₁₅. For all locations T₁₅ was substantially higher than actual field temperature conditions. We conclude that PSII thermostability in G. hirsutum acclimates to pre-existing environmental conditions; PSII is extremely tolerant to high temperature and water-deficit stress; and differences in PSII thermotolerance exist between commercially-available cultivars.     

Population differentiation and migration: coalescence times in a two-sex island model for autosomal and X-linked loci

Evolutionists have debated whether population-genetic parameters, such as effective population size and migration rate, differ between males and females. In humans, most analyses of this problem have focused on the Y chromosome and the mitochondrial genome, while the X chromosome has largely been omitted from the discussion. Past studies have compared F_ST values for the Y chromosome and mitochondrion under a model with migration rates that differ between the sexes but with equal male and female population sizes. In this study we investigate rates of coalescence for X-linked and autosomal lineages in an island model with different population sizes and migration rates for males and females, obtaining the mean time to coalescence for pairs of lineages from the same deme and for pairs of lineages from different demes. We apply our results to microsatellite data from the Human Genome Diversity Panel, and we examine the male and female migration rates implied by observed F_ST values.     

The effect of CO2 enrichment on leaf photosynthetic rates and instantaneous water use efficiency of Andropogon gerardii in the tallgrass prairie

Open-top chambers were used to study the effects of CO₂ enrichment on leaf-level photosynthetic rates of the C₄ grass Andropogon gerardii in the native tallgrass prairie ecosystem near Manhattan, Kansas. Measurements were made during a year with abundant rainfall (1993) and a year with below-normal rainfall (1994). Treatments included: No chamber, ambient CO₂ (A); chamber with ambient CO₂ (CA); and chamber with twice-ambient CO₂ (CE). Measurements of photosynthesis were made at 2-hour intervals, or at midday, on cloudless days throughout the growing season using an open-flow gas-exchange system. No significant differences in midday rates of photosynthesis or in daily carbon accumulation as a result of CO₂ enrichment were found in the year with abundant precipitation. In the dry year, midday rates of photosynthesis were significantly higher in the CE treatment than in the CA or A treatments throughout the season. Estimates of daily carbon accumulation also indicated that CO₂ enrichment allowed plants to maximize carbon acquisition on a diurnal basis. The increased carbon accumulation was accounted for by greater rates of photosynthesis in the CE plots during midday. During the wet year, CO₂ enrichment decreased stomatal conductance, which allowed plants to decrease transpiration while still photosynthesizing at rates similar to plants in ambient conditions. During the dry year, CO₂ enrichment allowed plants to maintain photosynthetic rates even though stomatal conductance and transpiration had been reduced in all treatments due to stress. Estimates of instantaneous water-use efficiency were reduced under CO₂ enrichment for both years. This revised version was published online in June 2006 with corrections to the Cover Date.     

E versus Z geometry in β-d-arabino-hexopyranosidulose oximes

Koenigs-Knorr-type glycosidations of peracylated 2Z-benzoyloxyimino-glycopyranosyl bromides invariably proceed with retention of the Z-geometry. Accordingly, the many β-d-hexosidulose oximes in literature which were prepared in this way and for which the oxime geometry has not been addressed explicitly, are the Z-oximes throughout. By contrast, oximation of β-d-hexopyranosid-2-uloses leads to mixtures of E and Z oximes readily separable and structurally verifiable by ¹H and ¹³C NMR. Configurational assignments rested on comparative evaluation of NMR data of E and Z isomers, and, most notably on an X-ray structural analysis of the pivaloylated isopropyl 2E-benzoyloxyimino-2-deoxy-β-d-arabino-hexopyranoside revealing the unusual ¹S₅?^1,4B conformation for the pyranoid ring.     

Water deficit in field-grown Gossypium hirsutum primarily limits net photosynthesis by decreasing stomatal conductance,increasing photorespiration,and increasing the ratio of dark respiration to gross photosynthesis

Much effort has been expended to improve irrigation efficiency and drought tolerance of agronomic crops; however, a clear understanding of the physiological mechanisms that interact to decrease source strength and drive yield loss has not been attained. To elucidate the underlying mechanisms contributing to inhibition of net carbon assimilation under drought stress, three cultivars of Gossypium hirsutum were grown in the field under contrasting irrigation regimes during the 2012 and 2013 growing season near Camilla, Georgia, USA. Physiological measurements were conducted on three sample dates during each growing season (providing a broad range of plant water status) and included, predawn and midday leaf water potential (Ψ_PD and Ψ_MD), gross and net photosynthesis, dark respiration, photorespiration, and chlorophyll a fluorescence. End-of-season lint yield was also determined. Ψ_PD ranged from −0.31 to −0.95 MPa, and Ψ_MD ranged from −1.02 to −2.67 MPa, depending upon irrigation regime and sample date. G. hirsutum responded to water deficit by decreasing stomatal conductance, increasing photorespiration, and increasing the ratio of dark respiration to gross photosynthesis, thereby limiting P_N and decreasing lint yield (lint yield declines observed during the 2012 growing season only). Conversely, even extreme water deficit, causing a 54% decline in P_N, did not negatively affect actual quantum yield, maximum quantum yield, or photosynthetic electron transport. It is concluded that P_N is primarily limited in drought-stressed G. hirsutum by decreased stomatal conductance, along with increases in respiratory and photorespiratory carbon losses, not inhibition or down-regulation of electron transport through photosystem II. It is further concluded that Ψ_PD is a reliable indicator of drought stress and the need for irrigation in field-grown cotton.     

Transmission dynamics of Toxoplasma gondii along an urban-rural gradient

Recently, several authors have proposed that the availability of intermediate hosts (IHs) for definitive hosts (DHs) may contribute to determining the dynamics and evolutionary ecology of parasites with facultative complex life cycles. The protozoa Toxoplasma gondii may be transmitted to DHs either via predation of infected IHs through a complex life cycle (CLC) or directly from a contaminated environment through a simple life cycle (SLC). This parasite is also present in contrasting host density environments. We tested the hypothesis that the relative contributions of the CLC and SLC along an urban-rural gradient depend on the IH supply. We built and analysed a deterministic model of the T. gondii transmission cycle. The SLC relative contribution is important only in urban-type environments, i.e., with low predation rate on IHs. In contrast, the parasite is predominantly transmitted through a CLC in suburban and rural environments. The association of the two cycles enables the parasite to spread in situations of low IH availability and low DH population size for which each cycle alone is insufficient.     

Internal charge transfer in cytochrome c oxidase at a limited proton supply: Proton pumping ceases at high pH

Background

In the membrane-bound enzyme cytochrome c oxidase, electron transfer from cytochrome c to O₂ is linked to proton uptake from solution to form H₂O, resulting in a charge separation across the membrane. In addition, the reaction drives pumping of protons across the membrane.

Methods

In this study we have measured voltage changes as a function of pH during reaction of the four-electron reduced cytochrome c oxidase from Rhodobacter sphaeroides with O₂. These electrogenic events were measured across membranes containing purified enzyme reconstituted into lipid vesicles.

Results

The results show that the pH dependence of voltage changes (primarily associated with proton transfer) during O₂ reduction does not match that of the previously studied absorbance changes (primarily associated with electron transfer). Furthermore, the voltage changes decrease with increasing pH.

Conclusions

The data indicate that cytochrome c oxidase does not pump protons at high pH (10.5) (or protons are taken from the “wrong” side of the membrane) and that at this pH the net proton-uptake stoichiometry is ∼ 1/2 of that at pH 8. Furthermore, the results provide a basis for interpretation of results from studies of mutant forms of the enzyme.

General significance

These results provide new insights into the function of cytochrome c oxidase.     

Metabolic and energetic aspects of biohydrogen production of Clostridium tyrobutyricum: The effects of hydraulic retention time and peptone addition

This study evaluates the microbial metabolism and energy demand in fermentative biohydrogen production using Clostridium tyrobutyricum FYa102 at different hydraulic retention times (HRT) over a period of 1-18 h. The hydrogen yield shows a positive correlation with the butyrate yield, the B/A ratio, and the Y_H2/2(Y_HAc+Y_HBu) ratio, but a negative correlation with the lactate yield. A decrease in HRT, which is accompanied by an increased biomass growth, tends to decrease the B/A ratio, due presumably to a higher energy demand for microbial growth. The production of lactate at a low HRT, however, may involve an unfavorable change in e⁻ equiv distribution to result in a reduced hydrogen production. Finally, the relatively high hydrogen yields observed in the bioreactor with the peptone addition may be ascribed to the utilization of peptone as an additional energy and/or amino-acid source, thus reducing the glucose demand for biomass growth during the hydrogen production process.     

Modelling O2 and O2 unidirectional fluxes in plants. III: Fitting of experimental data by a simple model

Photosynthetic assimilation of CO₂ in plants results in the balance between the photochemical energy developed by light in chloroplasts, and the consumption of that energy by the oxygenation processes, mainly the photorespiration in C₃ plants. The analysis of classical biological models shows the difficulties to bring to fore the oxygenation rate due to the photorespiration pathway. As for other parameters, the most important key point is the estimation of the electron transport rate (ETR or J), i.e. the flux of biochemical energy, which is shared between the reductive and oxidative cycles of carbon. The only reliable method to quantify the linear electron flux responsible for the production of reductive energy is to directly measure the O₂ evolution by ¹⁸O₂ labelling and mass spectrometry. The hypothesis that the respective rates of reductive and oxidative cycles of carbon are only determined by the kinetic parameters of Rubisco, the respective concentrations of CO₂ and O₂ at the Rubisco site and the available electron transport rate, ultimately leads to propose new expressions of biochemical model equations. The modelling of ¹⁸O₂ and ¹⁶O₂ unidirectional fluxes in plants shows that a simple model can fit the photosynthetic and photorespiration exchanges for a wide range of environmental conditions. Its originality is to express the carboxylation and the oxygenation as a function of external gas concentrations, by the definition of a plant specificity factor Sp that mimics the internal reactions of Rubisco in plants. The difference between the specificity factors of plant (Sp) and of Rubisco (Sr) is directly related to the conductance values to CO₂ transfer between the atmosphere and the Rubisco site. This clearly illustrates that the values and the variation of conductance are much more important, in higher C₃ plants, than the small variations of the Rubisco specificity factor. The simple model systematically expresses the reciprocal variations of carboxylation and oxygenation exchanges illustrated by a “mirror effect”. It explains the protective sink effect of photorespiration, e.g. during water stress. The importance of the CO₂ compensation point, in classical models, is reduced at the benefit of the crossing points Cx and Ox, concentration values where carboxylation and oxygenation are equal or where the gross O₂ uptake is half of the gross O₂ evolution. This concept is useful to illustrate the feedback effects of photorespiration in the atmosphere regulation. The constancy of Sp and of Cx for a great variation of P under several irradiance levels shows that the regulation of the conductance maintains constant the internal CO₂ and the ratio of photorespiration to photosynthesis (PR/P). The maintenance of the ratio PR/P, in conditions of which PR could be reduced and the carboxylation increased, reinforces the hypothesis of a positive role of photorespiration and its involvement in the plant-atmosphere co-evolution.     

Modelling ¹⁸O₂ and ¹⁶O₂ unidirectional fluxes in plants: II. analysis of rubisco evolution

The studies of Rubisco characteristics observed during plant evolution show that the variation of the Rubisco specificity factor only improved by two times from cyanobacteria to modern C3 plants. However we note important variations of the ratio between the maximum rates of oxygenation and carboxylation (V_O/V_C). Modelling in vivo¹⁸O₂ data in plant gas exchange shows that the oxygenation reaction of Rubisco plays a regulating role when the photochemical energy exceeds the carboxylation capacity. A protective index ‘oxygenation capacity’ is postulated, related to the ratio V_O/V_C of Rubisco, and hence to the sink energy effect of photorespiration. Analysing the trends of Rubisco parameters along the evolutionary scale, we show: (1) the increase of both V_C and V_O; (2) the enhancement of CO₂ affinity; and (3) the rise in oxygenation capacity at the expense of the CO₂ specificity. Hence, the factors of evolutionary pressure have not only directed the enzyme towards a more efficient utilisation of CO₂, but mainly to positively use the unavoidable great loss of energy and assimilated carbon in the process of photorespiration. These observations reinforce the hypothesis of plant-atmosphere co-evolution and of the complex role of Rubisco, which seems to be selected to develop both better CO₂ affinity and oxygenation capacity. The latter increases the capacity of sink of photorespiration, in particular, during water stress or under high irradiance, the two conditions experienced by plants in terrestrial environments. These observations help to explain some handicaps of C4 plants, and the supremacy of CAM and C3 perennial higher plants in arid environments.     

Structural and functional studies of Leishmania braziliensis Hsp90

The ubiquitous Hsp90 is critical for protein homeostasis in the cells, stabilizing “client” proteins in a functional state. Hsp90 activity depends on its ability to bind and hydrolyze ATP, involving various conformational changes that are regulated by co-chaperones, posttranslational modifications and small molecules. Compounds like geldanamycin (GA) and radicicol inhibit the Hsp90 ATPase activity by occupying the ATP binding site, which can lead client protein to degradation and also inhibit cell growth and differentiation in protozoan parasites. Our goal was to produce the recombinant Hsp90 of Leishmania braziliensis (LbHsp90) and construct of its N-terminal (LbHsp90N) and N-domain and middle-domain (LbHsp90NM), which lacks the C-terminal dimerization domain, in order to understand how Hsp90 works in protozoa. The recombinant proteins were produced folded as attested by spectroscopy experiments. Hydrodynamic experiments revealed that LbHsp90N and LbHsp90NM behaved as elongated monomers while LbHsp90 is an elongated dimer. All proteins prevented the in vitro citrate synthase and malate dehydrogenase aggregation, attesting that they have chaperone activity, and interacted with adenosine ligands with similar dissociation constants. The LbHsp90 has low ATPase activity (k_cat = 0.320 min^− 1) in agreement with Hsp90 orthologs, whereas the LbHsp90NM has negligible activity, suggesting the importance of the dimeric protein for this activity. The GA interacts with LbHsp90 and with its domain constructions with different affinities and also inhibits the LbHsp90 ATPase activity with an IC₅₀ of 0.7 μM. All these results shed light on the LbHsp90 activity and are the first step to understanding the Hsp90 molecular chaperone system in L. braziliensis.