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1.
Dark respiration causes an increase in leaf CO2 concentration (Ci), and the continuing increases in atmospheric [CO2] further increases Ci. Elevated leaf CO2 concentration causes stomatal pores to close. Here, we demonstrate that high intracellular CO2/HCO3 enhances currents mediated by the Arabidopsis thaliana guard cell S-type anion channel SLAC1 upon coexpression of any one of the Arabidopsis protein kinases OST1, CPK6, or CPK23 in Xenopus laevis oocytes. Split-ubiquitin screening identified the PIP2;1 aquaporin as an interactor of the βCA4 carbonic anhydrase, which was confirmed in split luciferase, bimolecular fluorescence complementation, and coimmunoprecipitation experiments. PIP2;1 exhibited CO2 permeability. Mutation of PIP2;1 in planta alone was insufficient to impair CO2- and abscisic acid-induced stomatal closing, likely due to redundancy. Interestingly, coexpression of βCA4 and PIP2;1 with OST1-SLAC1 or CPK6/23-SLAC1 in oocytes enabled extracellular CO2 enhancement of SLAC1 anion channel activity. An inactive PIP2;1 point mutation was identified that abrogated water and CO2 permeability and extracellular CO2 regulation of SLAC1 activity. These findings identify the CO2-permeable PIP2;1 as key interactor of βCA4 and demonstrate functional reconstitution of extracellular CO2 signaling to ion channel regulation upon coexpression of PIP2;1, βCA4, SLAC1, and protein kinases. These data further implicate SLAC1 as a bicarbonate-responsive protein contributing to CO2 regulation of S-type anion channels.  相似文献   

2.
Cryptococcus gattii is unique among human pathogenic fungi with specialized ecological niche on trees. Since leaves concentrate CO2, we investigated the role of this gaseous molecule in C. gattii biology and virulence. We focused on the genetic analyses of β-carbonic anhydrase (β-CA) encoded by C. gattii CAN1 and CAN2 as later is critical for CO2 sensing in a closely related pathogen C. neoformans. High CO2 conditions induced robust development of monokaryotic hyphae and spores in C. gattii. Conversely, high CO2 completely repressed hyphae development in sexual mating. Both CAN1 and CAN2 were dispensable for CO2 induced morphogenetic transitions. However, C. gattii CAN2 was essential for growth in ambient air similar to its reported role in C. neoformans. Both can1 and can2 mutants retained full pathogenic potential in vitro and in vivo. These results provide insight into C. gattii adaptation for arboreal growth and production of infectious propagules by β-CA independent mechanism(s).  相似文献   

3.
Significant differences in CO2 compensation concentration measured in the field among varieties of the species Zea mays L. are reported for the first time. CO2 compensation concentrations were significantly (P≤ 0.01) and negatively correlated with apparent photosynthesis at 300 μl CO2/liter air. The Michaelis constant (as defined) for a leaf was significantly (P≤ 0.01) and positively correlated with apparent photosynthesis among varieties. While the first correlation is similar to behavior of CO2 compensation among species of different photosynthetic efficiency, the latter correlation is the converse of the behavior of Km among species.  相似文献   

4.
Periostin (PN, gene name POSTN) is an extracellular matrix protein that is up-regulated in bronchial epithelial cells and lung fibroblasts by TH-2 cytokines. Its paralog, TGF-β-induced protein (βig-h3, gene name TGFBI), is also expressed in the lung and up-regulated in bronchial myofibroblasts by TGF-β. PN and βig-h3 contain fasciclin 1 modules that harbor putative recognition sequences for γ-glutamyl carboxylase and are annotated in UniProt as undergoing vitamin K-dependent γ-carboxylation of multiple glutamic acid residues. γ-carboxylation profoundly alters activities of other proteins subject to the modification, e.g., blood coagulation factors, and would be expected to alter the structure and function of PN and βig-h3. To analyze for the presence of γ-carboxylation, proteins extracted from fibrotic lung were reacted with monoclonal antibodies specific for PN, βig-h3, or modification with γ-carboxyglutamic acid (Gla). In Western blots of 1-dimensional gels, bands stained with anti-PN or -βig-h3 did not match those stained with anti-Gla. In 2-dimensional gels, anti-PN-positive spots had pIs of 7.0 to >8, as expected for the unmodified protein, and there was no overlap between anti-PN-positive and anti-Gla-positive spots. Recombinant PN and blood coagulation factor VII were produced in HEK293 cells that had been transfected with vitamin K 2, 3-epoxide reductase C1 to optimize γ-carboxylation. Recombinant PN secreted from these cells did not react with anti-Gla antibody and had pIs similar to that found in extracts of fibrotic lung whereas secreted factor VII reacted strongly with anti-Gla antibody. Over 67% coverage of recombinant PN was achieved by mass spectrometry, including peptides with 19 of the 24 glutamates considered targets of γ-carboxylation, but analysis revealed no modification. Over 86% sequence coverage and three modified glutamic acid residues were identified in recombinant fVII. These data indicate that PN and βig-h3 are not subject to vitamin K-dependent γ-carboxylation.  相似文献   

5.
Phanerochaete chrysosporium metabolized the radiolabeled lignin model compounds [γ-14C]guaiacylglycerol-β-guaiacyl ether and [4-methoxy-14C]veratrylglycerol-β-guaiacyl ether (VI) to 14CO2 in stationary and in shaking cultures. 14CO2 evolution was greater in stationary culture. 14CO2 evolution from [γ-14C]guaiacyl-glycerol-β-guaiacyl ether and [4-methoxy-14C]veratrylglycerol-β-guaiacyl ether in stationary cultures was two- to threefold greater when 100% O2 rather than air (21% O2) was the gas phase above the cultures. 14CO2 evolution from the metabolism of the substrates occurred only as the culture entered the stationary phase of growth. The presence of substrate levels of nitrogen in the medium suppressed 14CO2 evolution from both substrates in stationary cultures. [14C]veratryl alcohol and 4-ethoxy-3-methoxybenzyl alcohol were formed as products of the metabolism of VI and 4-ethoxy-3-methoxyphenylglycerol-β-guaiacyl ether, respectively.  相似文献   

6.
Experiments to determine the early labeled photosynthetic products in Elodea canadensis show that after 2 seconds of exposure to NaH14CO3, 45% of the 14C incorporated is located in malate and aspartate. Phosphoglyceric acid and sugars account for 27% of the label during similar exposures. Equivalent amounts of organic acids and C3 cycle products are present after 8 seconds. Four-carbon acids remain relatively unchanged throughout the first 45 seconds of exposure, while sugars increase in a linear fashion. Enzyme assays indicate that ribulose diphosphate and phosphoenolpyruvate carboxylase enzymes are present in a ratio of approximately 2:1. It appears that E. canadensis is able to synthesize significant amounts of four-carbon acids via β-carboxylation and this may play a role in maintaining a pH favorable for carboxylation in aquatic plants.  相似文献   

7.
Global climate change is expected to affect how plants respond to their physical and biological environments. In this study, we examined the effects of elevated CO2 ([CO2]) and low soil moisture on the physiological responses of mountain maple (Acer spicatum L.) seedlings to light availability. The seedlings were grown at ambient (392 µmol mol−1) and elevated (784 µmol mol−1) [CO2], low and high soil moisture (M) regimes, at high light (100%) and low light (30%) in the greenhouse for one growing season. We measured net photosynthesis (A), stomatal conductance (g s), instantaneous water use efficiency (IWUE), maximum rate of carboxylation (V cmax), rate of photosynthetic electron transport (J), triose phosphate utilization (TPU)), leaf respiration (R d), light compensation point (LCP) and mid-day shoot water potential (Ψx). A and g s did not show significant responses to light treatment in seedlings grown at low soil moisture treatment, but the high light significantly decreased the C i/C a in those seedlings. IWUE was significantly higher in the elevated compared with the ambient [CO2], and the effect was greater at high than the low light treatment. LCP did not respond to the soil moisture treatments when seedlings were grown in high light under both [CO2]. The low soil moisture significantly reduced Ψx but had no significant effect on the responses of other physiological traits to light or [CO2]. These results suggest that as the atmospheric [CO2] rises, the physiological performance of mountain maple seedlings in high light environments may be enhanced, particularly when soil moisture conditions are favourable.  相似文献   

8.
Assimilatory nitrate reductase activity (NRA) in crude spinach leaf (Spinacia oleracea) extracts undergoes rapid changes following fluctuations in photosynthesis brought about by changes in external CO2 or by water stress (WM Kaiser, E Brendle-Behnisch [1991] Plant Physiol 96:363-367). A modulation of NRA sharing several characteristics (stability, response to Mg2+ or Ca2+, kinetic constants) with the in vivo modulation was obtained in vitro by preincubating desalted leaf extracts with physiological concentrations of Mg2+ and ATP (deactivating) or AMP (activating). When nitrate reductase (NR) was inactivated in vivo by illuminating leaves at the CO2 compensation point, it could be reactivated in vitro by incubating leaf extracts with AMP. For the in vitro inactivation, ATP could be replaced by GTP or UTP. Nonhydrolyzable ATP analogs (β, γ-imido ATP, β, γ-methyl-ATP) had no effect on NR, whereas γ-S-ATP caused an irreversible inactivation. This suggests that NR modulation involves ATP hydrolysis. In contrast to NR in crude leaf extracts, partially purified NR did not respond to ATP or AMP. ATP and AMP levels in whole leaf extracts changed in the way predicted by the modulation of NRA when leaves were transferred from photosynthesizing (low ATP/AMP) to photorespiratory (high ATP/AMP) conditions. Adenine nucleotide levels in leaves could be effectively manipulated by feeding mannose through the leaf petiole. NRA followed these changes as expected from the in vitro results. This suggests that cytosolic ATP/AMP levels are indeed the central link between NRA in the cytosol and photosynthesis in the chloroplast. Phosphorylation/dephosphorylation of NR or of NR-regulating protein factors is discussed as a mechanism for a reversible modulation of NR by ATP and AMP.  相似文献   

9.
Methionine residues in α/β-type small, acid-soluble spore proteins (SASP) of Bacillus species were readily oxidized to methionine sulfoxide in vitro by t-butyl hydroperoxide (tBHP) or hydrogen peroxide (H2O2). These oxidized α/β-type SASP no longer bound to DNA effectively, but DNA binding protected α/β-type SASP against methionine oxidation by peroxides in vitro. Incubation of an oxidized α/β-type SASP with peptidyl methionine sulfoxide reductase (MsrA), which can reduce methionine sulfoxide residues back to methionine, restored the α/β-type SASP’s ability to bind to DNA. Both tBHP and H2O2 caused some oxidation of the two methionine residues of an α/β-type SASP (SspC) in spores of Bacillus subtilis, although one methionine which is highly conserved in α/β-type SASP was only oxidized to a small degree. However, much more methionine sulfoxide was generated by peroxide treatment of spores carrying a mutant form of SspC which has a lower affinity for DNA. MsrA activity was present in wild-type B. subtilis spores. However, msrA mutant spores were no more sensitive to H2O2 than were wild-type spores. The major mechanism operating for dealing with oxidative damage to α/β-type SASP in spores is DNA binding, which protects the protein’s methionine residues from oxidation both in vitro and in vivo. This may be important in vivo since α/β-type SASP containing oxidized methionine residues no longer bind DNA well and α/β-type SASP-DNA binding is essential for long-term spore survival.  相似文献   

10.
Mesophyll cells were rapidly isolated from soybean (Glycine max [L.]) leaves using a combined Macerase enzyme-stirring technique. About 50% to 70% of the leaf cells on a chlorophyll basis from 3 grams of leaves could be isolated in 15 minutes. The cells obtained by this method were capable of high rates of photosynthesis even after storage in the dark for periods of up to 9 hours. The CO2-saturated rate of photosynthesis increased from 5 μm CO2/mg Chl·hour at 5 C to 170 μm CO2/mg Chl·hour at 40 C. At atmospheric CO2 concentration, the rate varied from 5 to 55 μm CO2/mg Chl·hour over this temperature range. The reduced temperature response of photosynthesis at low CO2 concentration was due to an increased Km(CO2) of the cells with increasing temperature. The products of photosynthesis in the isolated cells were similar to the products of leaf photosynthesis.  相似文献   

11.

Background

Members of the canonical Transient Receptor Potential (TRPC) class of cationic channels function downstream of Gαq and PLCβ in Drosophila photoreceptors for transducing visual stimuli. Gαq has recently been implicated in olfactory sensing of carbon dioxide (CO2) and other odorants. Here we investigated the role of PLCβ and TRPC channels for sensing CO2 in Drosophila.

Methodology/Principal Findings

Through behavioral assays it was demonstrated that Drosophila mutants for plc21c, trp and trpl have a reduced sensitivity for CO2. Immuno-histochemical staining for TRP, TRPL and TRPγ indicates that all three channels are expressed in Drosophila antennae including the sensory neurons that express CO2 receptors. Electrophysiological recordings obtained from the antennae of protein null alleles of TRP (trp343) and TRPL (trpl302), showed that the sensory response to multiple concentrations of CO2 was reduced. However, trpl302; trp343 double mutants still have a residual response to CO2. Down-regulation of TRPC channels specifically in CO2 sensing olfactory neurons reduced the response to CO2 and this reduction was obtained even upon down-regulation of the TRPCs in adult olfactory sensory neurons. Thus the reduced response to CO2 obtained from the antennae of TRPC RNAi strains is not due to a developmental defect.

Conclusion

These observations show that reduction in TRPC channel function significantly reduces the sensitivity of the olfactory response to CO2 concentrations of 5% or less in adult Drosophila. It is possible that the CO2 receptors Gr63a and Gr21a activate the TRPC channels through Gαq and PLC21C.  相似文献   

12.
G protein β subunits (Gβ) play essential roles in phototransduction as part of G protein βγ (Gβγ) and regulator of G protein signaling 9 (RGS9)-Gβ5 heterodimers. Both are obligate dimers that rely on the cytosolic chaperone CCT and its co-chaperone PhLP1 to form complexes from their nascent polypeptides. The importance of PhLP1 in the assembly process was recently demonstrated in vivo in a retinal rod-specific deletion of the Phlp1 gene. To test whether this is a general mechanism that also applies to other cell types, we disrupted the Phlp1 gene specifically in mouse cones and measured the effects on G protein expression and cone visual signal transduction. In PhLP1-deficient cones, expression of cone transducin (Gt2) and RGS9-Gβ5 subunits was dramatically reduced, resulting in a 27-fold decrease in sensitivity and a 38-fold delay in cone photoresponse recovery. These results demonstrate the essential role of PhLP1 in cone G protein complex formation. Our findings reveal a common mechanism of Gβγ and RGS9-Gβ5 assembly in rods and cones, highlighting the importance of PhLP1 and CCT-mediated Gβ complex formation in G protein signaling.  相似文献   

13.
Photosynthesis: action spectra for leaves in normal and low oxygen   总被引:1,自引:1,他引:0       下载免费PDF全文
The action spectrum of apparent photosynthesis for attached radish (Raphanus sativus L. var. Early Scarlet Globe) and corn (Zea mays L. var. Pride V.) leaves was measured at 300 μl/l CO2 and both 21% and 2% O2. The spectra were measured at light intensities where apparent photosynthesis was proportional to intensity. For radish, a high compensation point plant, oxygen had an inhibiting effect on photosynthesis at all wavelengths from 402 to 694 mμ. If a constant rate of photosynthesis at 21% O2 for the different wavelengths was chosen, then the percent increase in net CO2 fixation at 2% O2 was constant. For corn, a low compensation point plant, no inhibitory effect of oxygen concentration from 2% to 21% O2 was found over the visible spectrum. The CO2 compensation point for light intensities greater than the light compensation point was found to be constant and independent of wavelength for both radish and corn leaves. For radish, the lowering of the oxygen concentration from 21% to 2% at these intensities was found to reduce the CO2 compensation point by the same amount for the wavelengths studied.  相似文献   

14.
Stable Carbon Isotope Fractionation by Sulfate-Reducing Bacteria   总被引:5,自引:1,他引:4       下载免费PDF全文
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 δ13C values were determined for gaseous CO2, organic substrates, and products such as biomass. For three of the four SRB, carbon isotope effects between the substrates, acetate or lactate and CO2, and the cell biomass were small, ranging from 0 to 2‰. However, for Desulfotomaculum acetoxidans, the carbon incorporated into biomass was isotopically heavier than the available substrates by 8 to 9‰. SRB grown lithoautotrophically consumed less than 3% of the available CO2 and exhibited substantial discrimination (calculated as isotope fractionation factors [α]), as follows: for Desulfobacterium autotrophicum, α values ranged from 1.0100 to 1.0123; for Desulfobacter hydrogenophilus, the α value was 0.0138, and for Desulfotomaculum acetoxidans, the α value was 1.0310. Mixotrophic growth of Desulfovibrio desulfuricans on acetate and CO2 resulted in biomass with a δ13C 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., H2), ecological forces can also influence carbon isotope discrimination by SRB.  相似文献   

15.
We here report a study on the activation of the ι-class bacterial CA from Burkholderia territorii (BteCAι). This protein was recently characterised as a zinc-dependent enzyme that shows a significant catalytic activity (kcat 3.0 × 105 s−1) for the physiological reaction of CO2 hydration to bicarbonate and protons. Some amino acids and amines, among which some proteinogenic derivatives as well as histamine, dopamine and serotonin, showed efficient activating properties towards BteCAι, with activation constants in the range 3.9–13.3 µM. L-Phe, L-Asn, L-Glu, and some pyridyl-alkylamines, showed a weaker activating effect towards BteCAι, with KA values ranging between 18.4 µM and 45.6 µM. Nowadays, no information is available on active site architecture, metal ion coordination and catalytic mechanism of members of the ι-group of CAs, and this study represents another contribution towards a better understanding of this still uncharacterised class of enzymes.  相似文献   

16.
Understanding the influences of climatic changes on water use efficiency (WUE) of Tibetan alpine meadows is important for predicting their long-term net primary productivity (NPP) because they are considered very sensitive to climate change. Here, we collected wool materials produced from 1962 to 2010 and investigated the long-term WUE of an alpine meadow in Tibet on basis of the carbon isotope values of vegetation (δ 13Cveg). The values of δ 13Cveg decreased by 1.34‰ during 1962–2010, similar to changes in δ 13C values of atmospheric CO2. Carbon isotope discrimination was highly variable and no trend was apparent in the past half century. Intrinsic water use efficiency (W i) increased by 18 μmol·mol–1 (approximately 23.5%) during 1962–2010 because the increase in the intercellular CO2 concentration (46 μmol·mol–1) was less than that in the atmospheric CO2 concentration (C a, 73 μmol·mol–1). In addition, W i increased significantly with increasing growing season temperature and C a. However, effective water use efficiency (W e) remained relatively stable, because of increasing vapor pressure deficit. C a, precipitation, and growing season temperature collectively explained 45% of the variation of W e. Our findings indicate that the W e of alpine meadows in the Tibetan Plateau remained relatively stable by physiological adjustment to elevated C a and growing season temperature. These findings improve our understanding and the capacity to predict NPP of these ecosystems under global change scenarios.  相似文献   

17.
18.

Background

The monocled cobra (Naja kaouthia) is responsible for snakebite fatality in Indian subcontinent and in south-western China. Phospholipase A2 (PLA2; EC 3.1.1.4) is one of the toxic components of snake venom. The present study explores the mechanism and rationale(s) for the differences in anticoagulant potency of two acidic PLA2 isoenzymes, Nk-PLA2α (13463.91 Da) and Nk-PLA2β (13282.38 Da) purified from the venom of N. kaouthia.

Principal Findings

By LC-MS/MS analysis, these PLA2s showed highest similarity (23.5% sequence coverage) with PLA2 III isolated from monocled cobra venom. The catalytic activity of Nk-PLA2β exceeds that of Nk-PLA2α. Heparin differentially regulated the catalytic and anticoagulant activities of these Nk-PLA2 isoenzymes. The anticoagulant potency of Nk-PLA2α was comparable to commercial anticoagulants warfarin, and heparin/antithrombin-III albeit Nk-PLA2β demonstrated highest anticoagulant activity. The anticoagulant action of these PLA2s was partially contributed by a small but specific hydrolysis of plasma phospholipids. The strong anticoagulant effect of Nk-PLA2α and Nk-PLA2β was achieved via preferential, non-enzymatic inhibition of FXa (Ki = 43 nM) and thrombin (Ki = 8.3 nM), respectively. Kinetics study suggests that the Nk-PLA2 isoenzymes inhibit their “pharmacological target(s)” by uncompetitive mechanism without the requirement of phospholipids/Ca2+. The anticoagulant potency of Nk-PLA2β which is higher than that of Nk-PLA2α is corroborated by its superior catalytic activity, its higher capacity for binding to phosphatidylcholine, and its greater strength of thrombin inhibition. These PLA2 isoenzymes thus have evolved to affect haemostasis by different mechanisms. The Nk-PLA2β partially inhibited the thrombin-induced aggregation of mammalian platelets suggesting its therapeutic application in the prevention of unwanted clot formation.

Conclusion/Significance

In order to develop peptide-based superior anticoagulant therapeutics, future application of Nk-PLA2α and Nk-PLA2β for the treatment and/or prevention of cardiovascular disorders are proposed.  相似文献   

19.
Integrins are important mammalian receptors involved in normal cellular functions and the pathogenesis of inflammation and disease. Entamoeba histolytica is a protozoan parasite that colonizes the gut, and in 10% of infected individuals, causes amebic colitis and liver abscess resulting in 105 deaths/year. E. histolytica-induced host inflammatory responses are critical in the pathogenesis of the disease, yet the host and parasite factors involved in disease are poorly defined. Here we show that pro-mature cysteine proteinase 5 (PCP5), a major virulent factor that is abundantly secreted and/or present on the surface of ameba, binds via its RGD motif to αVβ3 integrin on Caco-2 colonic cells and stimulates NFκB-mediated pro-inflammatory responses. PCP5 RGD binding to αVβ3 integrin triggered integrin-linked kinase(ILK)-mediated phosphorylation of Akt-473 that bound and induced the ubiquitination of NF-κB essential modulator (NEMO). As NEMO is required for activation of the IKKα-IKKβ complex and NFκB signaling, these events markedly up-regulated pro-inflammatory mediator expressions in vitro in Caco-2 cells and in vivo in colonic loop studies in wild-type and Muc2−/− mice lacking an intact protective mucus barrier. These results have revealed that EhPCP5 RGD motif is a ligand for αVβ3 integrin-mediated adhesion on colonic cells and represents a novel mechanism that E. histolytica trophozoites use to trigger an inflammatory response in the pathogenesis of intestinal amebiasis.  相似文献   

20.
Patients with chronic obstructive pulmonary disease, acute lung injury, and critical care illness may develop hypercapnia. Many of these patients often have muscle dysfunction which increases morbidity and impairs their quality of life. Here, we investigated whether hypercapnia leads to skeletal muscle atrophy. Mice exposed to high CO2 had decreased skeletal muscle wet weight, fiber diameter, and strength. Cultured myotubes exposed to high CO2 had reduced fiber diameter, protein/DNA ratios, and anabolic capacity. High CO2 induced the expression of MuRF1 in vivo and in vitro, whereas MuRF1−/− mice exposed to high CO2 did not develop muscle atrophy. AMP-activated kinase (AMPK), a metabolic sensor, was activated in myotubes exposed to high CO2, and loss-of-function studies showed that the AMPKα2 isoform is necessary for muscle-specific ring finger protein 1 (MuRF1) up-regulation and myofiber size reduction. High CO2 induced AMPKα2 activation, triggering the phosphorylation and nuclear translocation of FoxO3a, and leading to an increase in MuRF1 expression and myotube atrophy. Accordingly, we provide evidence that high CO2 activates skeletal muscle atrophy via AMPKα2-FoxO3a-MuRF1, which is of biological and potentially clinical significance in patients with lung diseases and hypercapnia.  相似文献   

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