The hydrogen peroxide-sensitive proteome of the chloroplast in vitro and in vivo

Muthuramalingam M, Matros A, Scheibe R, Mock H-P, Dietz K-J. The hydrogen peroxide-sensitive proteome of the chloroplast in vitro and in vivo. Frontiers in Plant Science. 2013;4:54-1-54-14.Hydrogen peroxide (H2O2) evolves during cellular metabolism and accumulates under various stresses causing serious redox imbalances. Many proteomics studies aiming to identify proteins sensitive to H2O2 used concentrations that were above the physiological range. Here the chloroplast proteins were subjected to partial oxidation by exogenous addition of H2O2 equivalent to 10% of available protein thiols which allowed for the identification of the primary targets of oxidation. The chosen redox proteomic approach employed differential labeling of non-oxidized and oxidized thiols using sequential alkylation with N-ethylmaleimide and biotin maleimide. The in vitro identified proteins are involved in carbohydrate metabolism, photosynthesis, redox homeostasis, and nitrogen assimilation. By using methyl viologen that induces oxidative stress in vivo, mostly the same primary targets of oxidation were identified and several oxidation sites were annotated. Ribulose-1,5-bisphosphate (RubisCO) was a primary oxidation target. Due to its high abundance, RubisCO is suggested to act as a chloroplast redox buffer to maintain a suitable redox state, even in the presence of increased reactive oxygen species release. 2-cysteine peroxiredoxins (2-Cys Prx) undergo redox-dependent modifications and play important roles in antioxidant defense and signaling. The identification of 2-Cys Prx was expected based on its high affinity to H2O2 and is considered as a proof of concept for the approach. Targets of Trx, such as phosphoribulokinase, glyceraldehyde-3-phosphate dehydrogenase, transketolase, and sedoheptulose-1,7-bisphosphatase have at least one regulatory disulfide bridge which supports the conclusion that the identified proteins undergo reversible thiol oxidation. In conclusion, the presented approach enabled the identification of early targets of H2O2 oxidation within the cellular proteome under physiological experimental conditions

Accumulation and nuclear import of HIF1 alpha during high and low oxygen concentration in skeletal muscle cells in primary culture

AbstractThe hypoxia-inducible-factor-1 (HIF1) mediates the transcriptional upregulation of several target genes during hypoxia. HIF1 itself is known to be regulated essentially by ubiquitinylation and proteolytic degradation of the subunit HIF1α of the dimeric transcription factor HIF1. In contrast to other tissues, skeletal muscle expresses high amounts of HIF1α in normoxia as well as in hypoxia. In view of this, we aimed to investigate HIF1α accumulation and subcellular localization as well as the transcriptional activity of the HIF1α-regulated gene of glyceraldehyde dehydrogenase (GAPDH) in skeletal muscle cells exposed to low oxygen concentration (3% O2), normoxia (20% O2) or high oxygen concentration (42% O2). Immunofluorescence analysis reveals that under normoxic and high oxygen conditions, significant amounts of HIF1α can be found exclusively in the cytoplasm of the myotubes. Muscle cells treated with CoCl2, a known inhibitor of HIF1α degradation, show even higher levels of HIF1α, again exclusively in the cytoplasm. Under conditions of low oxygen, HIF1α in controls as well as in CoCl2-treated cells is found in the nuclei. CdCl2 inhibits nuclear import of HIF1α at low oxygen concentration and leads to a transcriptional downregulation of the marker enzyme of anaerobic glycolysis GAPDH. Immunoprecipitation with anti-HIF1α antibody co-precipitates HSP90 in an oxygen-dependent manner, more at high pO2 than at low pO2. Cadmium-treated samples also show high amounts of co-immunoprecipitated HSP90, independent of oxygen concentration. We conclude that in skeletal muscle cells, HIF1α, in contrast to other tissues, may, in addition to its regulation by degradation, also be regulated by binding to HSP90 and subsequent inhibition of its import into the nuclei

Physiological and anatomical differentiation of two sympatric weed populations

Neuffer B, Schorsch M, Hameister S, Knuesting J, Selinski J, Scheibe R. Physiological and anatomical differentiation of two sympatric weed populations. PEERJ. 2020;8: e9226.In the vineyards of Rhineland-Palatinate (Germany), two different types of Shepherd's Purse (Capsella bursa-pastoris) coexist: (1) the common type called 'wild type', and (2) the decandric type called Capsella apetala or 'Spe' with four stamens in place of the four petals. In this study, we compare the anatomical and physiological characters of rosette leaves of the respective types. Progeny of individual plants was cultivated in growth chambers under low- and high-light conditions. Under low-light conditions, the stomata densities of the adaxial and abaxial epidermis did not differ between the two types. When grown under high-light conditions, wild type and Spe, both exhibited increased stomata densities compared to low-light conditions, but Spe to a lesser extent than the wild type. The maximal photosynthetic capacity of Spe was lower in both, low-light and high-light conditions compared to wild-type plants. Under all CO2 concentrations, Spe seemed to be less productive. The less effective CO2 assimilation of the Spe mutant C. apetala was accompanied by later flowering. This fact prolonged the vegetative phase of Spe by about two weeks and was sufficient for the maintenance of both populations stably over years

Regulation of gene expression by photosynthetic signals triggered through modified CO(2 )availability

BACKGROUND: To coordinate metabolite fluxes and energy availability, plants adjust metabolism and gene expression to environmental changes through employment of interacting signalling pathways. RESULTS: Comparing the response of Arabidopsis wild-type plants with that of the mutants adg1, pgr1 and vtc1 upon altered CO(2)-availability, the regulatory role of the cellular energy status, photosynthetic electron transport, the redox state and concentration of ascorbate and glutathione and the assimilatory force was analyzed in relation to the transcript abundance of stress-responsive nuclear encoded genes and psaA and psbA encoding the reaction centre proteins of photosystem I and II, respectively. Transcript abundance of Bap1, Stp1, psaA and psaB was coupled with seven metabolic parameters. Especially for psaA and psaB, the complex analysis demonstrated that the assumed PQ-dependent redox control is subordinate to signals linked to the relative availability of 3-PGA and DHAP, which define the assimilatory force. For the transcripts of sAPx and Csd2 high correlations with the calculated redox state of NADPH were observed in pgr1, but not in wild-type, suggesting that in wild-type plants signals depending on thylakoid acidification overlay a predominant redox-signal. Strongest correlation with the redox state of ascorbate was observed for 2CPA, whose transcript abundance regulation however was almost insensitive to the ascorbate content demonstrating dominance of redox regulation over metabolite sensing. CONCLUSION: In the mutants, signalling pathways are partially uncoupled, demonstrating dominance of metabolic control of photoreaction centre expression over sensing the redox state of the PQ-pool. The balance between the cellular redox poise and the energy signature regulates sAPx and Csd2 transcript abundance, while 2CPA expression is primarily redox-controlled

Expression of the minor isoform pea ferredoxin in tobacco alters photosynthetic electron partitioning and enhances cyclic electron flow

Ferredoxins (Fds) are ferrosulfoproteins that function as low-potential electron carriers in plants. The Fd family is composed of several isoforms that share high sequence homology but differ in functional characteristics. In leaves, at least two isoforms conduct linear and cyclic photosynthetic electron transport around photosystem I, and mounting evidence suggests the existence of at least partial division of duties between these isoforms. To evaluate the contribution of different kinds of Fds to the control of electron fluxes along the photosynthetic electron transport chain, we overexpressed a minor pea (Pisum sativum) Fd isoform (PsFd1) in tobacco (Nicotiana tabacum) plants. The transplastomic OeFd1 plants exhibited variegated leaves and retarded growth and developmental rates. Photosynthetic studies of these plants indicated a reduction in carbon dioxide assimilation rates, photosystem II photochemistry, and linear electron flow. However, the plants showed an increase in nonphotochemical quenching, better control of excitation pressure at photosystem II, and no evidence of photoinhibition, implying a better dynamic regulation to remove excess energy from the photosynthetic electron transport chain. Finally, analysis of P700 redox status during illumination confirmed that the minor pea Fd isoform promotes enhanced cyclic flow around photosystem I. The two novel features of this work are: (1) that Fd levels achieved in transplastomic plants promote an alternative electron partitioning even under greenhouse light growth conditions, a situation that is exacerbated at higher light intensity measurements; and (2) that an alternative, minor Fd isoform has been overexpressed in plants, giving new evidence of labor division among Fd isoforms.Fil: Blanco, Nicolás Ernesto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; Argentina. Universidad de Umea; SueciaFil: Ceccoli, Romina Denis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Dalla Via, Maria Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; ArgentinaFil: Voss, Ingo. Universität Osnabrück; AlemaniaFil: Segretin, Maria Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones En Ingeniería Genética y Biología Molecular; ArgentinaFil: Bravo Almonacid, Fernando Felix. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones En Ingeniería Genética y Biología Molecular; ArgentinaFil: Melzer, Michael. Leibniz-Institut für Pflanzengenetik und Kulturpflanzenforschung, Physiologie und Zellbiologie; AlemaniaFil: Hajirezaei, Mohammad Reza. Leibniz-Institut für Pflanzengenetik und Kulturpflanzenforschung, Physiologie und Zellbiologie; ArgentinaFil: Scheibe, Renate. Universität Osnabrück; AlemaniaFil: Hanke, Guy T.. Universität Osnabrück; Alemani

T Tubules and Surface Membranes Provide Equally Effective Pathways of Carbonic Anhydrase-Facilitated Lactic Acid Transport in Skeletal Muscle

We have studied lactic acid transport in the fast mouse extensor digitorum longus muscles (EDL) by intracellular and cell surface pH microelectrodes. The role of membrane-bound carbonic anhydrases (CA) of EDL in lactic acid transport was investigated by measuring lactate flux in muscles from wildtype, CAIV-, CAIX- and CAXIV-single ko, CAIV-CAXIV double ko and CAIV–CAIX–CAXIV-triple ko mice. This was complemented by immunocytochemical studies of the subcellular localization of CAIV, CAIX and CAXIV in mouse EDL. We find that CAXIV and CAIX single ko EDL exhibit markedly but not maximally reduced lactate fluxes, whereas triple ko and double ko EDL show maximal or near-maximal inhibition of CA-dependent lactate flux. Interpretation of the flux measurements in the light of the immunocytochemical results leads to the following conclusions. CAXIV, which is homogeneously distributed across the surface membrane of EDL fibers, facilitates lactic acid transport across this membrane. CAIX, which is associated only with T tubular membranes, facilitates lactic acid transport across the T tubule membrane. The removal of lactic acid from the lumen of T tubuli towards the interstitial space involves a CO2-HCO3- diffusional shuttle that is maintained cooperatively by CAIX within the T tubule and, besides CAXIV, by the CAIV, which is strategically located at the opening of the T tubules. The data suggest that about half the CA-dependent muscular lactate flux occurs across the surface membrane, while the other half occurs across the membranes of the T tubuli.Public Library of Scienc

Multiple strategies to prevent oxidative stress in Arabidopsis plants lacking the malate valve enzyme NADP-malate dehydrogenase

The nuclear-encoded chloroplast NADP-dependent malate dehydrogenase (NADP-MDH) is a key enzyme controlling the malate valve, to allow the indirect export of reducing equivalents. Arabidopsis thaliana (L.) Heynh. T-DNA insertion mutants of NADP-MDH were used to assess the role of the light-activated NADP-MDH in a typical C3 plant. Surprisingly, even when exposed to high-light conditions in short days, nadp-mdh knockout mutants were phenotypically indistinguishable from the wild type. The photosynthetic performance and typical antioxidative systems, such as the Beck–Halliwell–Asada pathway, were barely affected in the mutants in response to high-light treatment. The reactive oxygen species levels remained low, indicating the apparent absence of oxidative stress, in the mutants. Further analysis revealed a novel combination of compensatory mechanisms in order to maintain redox homeostasis in the nadp-mdh plants under high-light conditions, particularly an increase in the NTRC/2-Cys peroxiredoxin (Prx) system in chloroplasts. There were indications of adjustments in extra-chloroplastic components of photorespiration and proline levels, which all could dissipate excess reducing equivalents, sustain photosynthesis, and prevent photoinhibition in nadp-mdh knockout plants. Such metabolic flexibility suggests that the malate valve acts in concert with other NADPH-consuming reactions to maintain a balanced redox state during photosynthesis under high-light stress in wild-type plants