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The implantations were performed in 4H silicon carbide homoepitaxial layers deposited on (00.1) substrates with 8° offcut, and reference 4H-SiC substrates. The 2 MeV Se+ ions and 1 MeV Al+ ions were implanted with four fluences subsequently increased by the factor of 4–5×. The samples were studied by means of X-ray diffraction topography, high-resolution diffractometry, specular X-ray reflectometry, and Rutherford backscattering spectrometry\channeling method. The dislocation density in the samples evaluated from the diffraction topographs did not exceed 5 × 103 cm−2. The representative roughness values evaluated from the reflectometric measurements was 2.3 ± 0.1 nm for the substrates and less than 1.4 ± 0.1 nm for the epitaxial layers. A significantly higher damage level in the case of 2 MeV Se+ ions in comparison with 1 MeV Al+ ion and a linear increase of the strain with the fluence was indicated, but the highest doses of selenium ions caused the amorphization of the implanted layer. It was also possible to obtain a good fitting of the theoretical and experimental diffraction curves approximating the strain profiles by the distribution of the point defects calculated with the SRIM 2008 code. It was confirmed that the maximum coming from surface damages observed in channeling spectra of the virgin substrate wafers was significantly higher than in the case of epitaxial layers. Copyright © 2015 John Wiley & Sons, Ltd.

The occurrence of oxidative stress and oxidative damage in the halophyte Mesembryanthemum crystallinum during C 3 /crassulacean acid metabolism (CAM) transition evoked by salinity and high irradiance was investigated. The hypothesis tested was that CAM induction will effectively protect M. crystallinum against oxidative damage even under additionally applied oxidative stress via ozone (150 p.p.b.) exposure. Salinity (0.4 M NaCl), high irradiance and ozone induced the cytosolic CuZn-superoxide dismu- tase (SOD; EC 1.15.1.1) transcript abundance and activity indicating oxidative stress. However, ozone did not induce CAM. Contrary to the hypothesis, that ethylene may be involved in CAM induction, gassing with ethylene (200 p.p.b.) did not lead to CAM, but revealed that in M. crystallinum ethylene biosynthesis is induced by autostim- ulation. A putative fragment of the hitherto unknown nu- cleotide sequence of 1-aminocyclopropane-1-carboxylate oxidase (ACC; EC 4.4.1.14) was isolated and used as a specific probe against the ACC oxidase transcript to moni- tor oxidative damage. Ozone exposure increased the ACC oxidase expression only in plants with C 3 -photosynthesis which also showed severe necrosis and reduced photosyn- thetic activity, whereas plants in CAM mode (salt induced) remained unaffected. Natural stressors such as salinity and high irradiance induce CAM and up-regulate the ARS component CuZn-SOD very effectively to protect M. crys- tallinum from oxidative damage even when additional oxi- dative stress such as ozone is present.

There are two H+-pumping enzymes at the tonoplast membrane of plant vacuoles, the V-ATPase and the V-PPase. One attempt to explain the enigma of “two H+ pumps, one membrane” was the suggestion that the V-PPase has special functions in young developing and growing tissues in utilization of pyrophosphate produced in particularly active metabolism and in pumping of K+ for vacuolization. This should lead to reciprocal expression of both enzymes with time during development. Here we used stimulation of Kalanchoe blossfeldiana Poellnitz cv. Tom Thumb plants by short-day treatments to induce crassulacean acid metabolism and flowering and of Ricinus communis L. stem tissue by infection with Agrobacterium tumefaciens strain C58 to induce vigorous growth of tumours, and we compared these stimulated tissues with leaves of non-stimulated long-day controls and non-infected stem tissue, respectively. Activities and protein levels of both enzymes increased (K. blossfeldiana) or remained high (R. communis) in the stimulated tissues and decreased in the non-stimulated tissues with time. Time-dependent patterns of the two enzymes were concordant in all of the four cases and not inverse, i.e. two plants with two different conditions each, leading to very different developmental situations.

Microsomal membranes of Chlamydomonas reinhardtii possess PPase and V-ATPase activities. By immunogold labelling we have shown that H+-pyrophosphatase (PPase) is localized to membranes of lytic and contractile vacuoles of Chlamydomonas, in which the density of antigen in the latter is much higher. In addition, PPase is conspicuously present in trans cisternae and transpole elements of the Colgi apparatus. Such a distribution for PPase has hitherto not been reported. A positive in situ identification for PPase at the plasma membrane, including the flagellar membrane, was also made, and has also been confirmed by Western blotting and activity measurements on isolated plasma membranes. V-ATPase antisera which cross react with polypeptides of this transport complex from maize roots failed to recognize anything in Western blots of Chlamydomonas microsomal membranes. Thus immunogold labelling for V-ATPase was not possible with Chlamydomonas. On the other hand, surfaces of contractile vacuole membranes as revealed by deepetching were covered by conspicuous 9 − 11.5 nm diameter smooth particles which had a central hole. These were very similar to those previously identified by Heuser et al., (1993) as the V,-head of V-ATPase in Dictyostelium contractile vacuoles. Another type of membrane image, designated “intermediate-sized vesicle”, was found associated with the contractile vacuole. It was characterized by densely-packed 6 − 7.5nm diameter polygonal particles, which upon rotation analysis showed both 5- and 6-fold symmetries, also with a central hole. These particles are interpreted as representing either PPase complexes or the V0 body of the V-ATPase in etched fractured membrane surfaces. We have incorporated these findings into a model of contractile vacuole function.

BSA, bovine serum albumin CAM, Crassulacean acid metabolism DTT, dithiothreitol EDTA, ethylenediaminetetraacetic acid FPLCfast protein liquid chromatography HEPES, N-(2-hydroxyethyl)piperazine-Ń-(ethanesulphonic acid) ME, β-mercaptoethanol NBT, nitro blue tetrazolium PAGE, polyacrylamide gel electrophoresis SDS, sodium dodecyl sulphate SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis Rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) SOD, superoxide dismutase (EC 1.15.1.1) TEMED, N,N,Ń,Ń-tetramethylethylenediamine Tris, Tris (hydroxymethyl) aminomethane Tricine, N-Tris(hydroxymethyl)methylglycine Treatment of Mesembryanthemum crystallinum for several days with 0·4 kmol m–3 NaCl in the root medium, in parallel to an increase of the cell sap osmolarity enhances activity of important antioxidative enzymes, such as superoxide dismutases (SODs). M. crystallinum is equipped with three SOD isoforms. These isoforms were identified as Mn-, Fe-, and Cu/Zn-SODs, respectively. Mn-SOD was found in the mitochondrial fraction, Fe-SOD in the chloroplast fraction, and Cu/Zn-SOD is probably localized in the cytosol. The Fe-SOD found in M. crystallinum is the first iron-containing SOD enzyme to be characterized in the plant family Aizoaceae. Salt treatment increases the activity of this isoform earlier than the other SODs. Molecular masses of SOD isoforms were determined as 82, 48 and 34 kDa for Mn-, Fe-, Cu/Zn-SODs, respectively. Native Mn-SOD seems to be a tetramer, while Fe-SOD and Cu/Zn-SOD are dimers. All SOD isoforms show high thermal stability. Mn-SOD is active even after short heating at 90 °C and Fe-SOD at 70 °C. Moreover, high concentrations of β-mercaptoethanol used as a reducing agent did not destroy the function of all isoforms. With the salinity treatment in M. crystallinum, Crassulacean acid metabolism (CAM) is induced. Build-up of large stationary O2 concentrations in the leaf air spaces is associated with the photosynthetic CO2 reduction behind closed stomata in phase III of CAM. This illustrates why M. crystallinum may require higher antioxidative activities under NaCl stress and also explains earlier findings that CAM plants are more resistant than C3 plants to environmental stress as imposed by, for example, SO2 and O3.

The effects of solubilization with Triton X-100 or Brij 58 on the polypeptide composition and the substrate affinity of the tonoplast H+-ATPase of plants of Mesembryanthemum crystallinum performing C3 photosynthesis or crassulacean acid metabolism (CAM) have been compared. Although all known subunits of the tonoplast H+-ATPase were present in the fraction of solubilized proteins after treatment with Brij 58 or Triton X-100, with Triton X-100 the apparent KM value for ATP hydrolysis was increased by a factor of 1.8 and 1.5 in preparations from C3 and CAM plants, respectively, even at low concentrations in contrast to treatment with Brij 58. This is explained by structural changes of the tonoplast H+-ATPase due to the Triton X-100 treatment. After solubilization with Brij 58 the tonoplast H+-ATPase was partially purified by Superose-6 size-exclusion FPLC. When Brij 58 was present, addition of lipids to the chromatography buffer was not necessary to conserve enzyme activity in contrast to previously described purification methods using Triton X-100. The substrate affinity of the partial purified H+-ATPase was similar to the substrate affinity obtained for ATP-hydrolysis of native tonoplast vesicles, indicating that the enzyme structure during partial purification was conserved by using Brij 58. The results underline that the lipid environment of the tonoplast H+-ATPase is important for enzyme structure and function.

Summary IA detergent removal technique was used to reconstitute solubilized tonoplast proteins of mesophyll cells of the CAM plant Kalanchoe daigremontiana into phosphatidylcholine liposomes. The proteoliposomes were able to hydrolyse ATP and to pump protons across the vesicle membrane. Both activities were inhibited by nitrate, an inhibitor of V-type ATPases. Freeze-fracture micrographs confirmed the incorporation of membrane proteins into liposomes. Increase of specific ATP-hydrolysis activity compared to solubilized tonoplast proteins and SDS-PAGE analysis of reconstituted proteins in comparison with the polypeptide pattern of the purified tonoplast H+-ATPase from the same plant source indicated a highly selective reconstitution of the tonoplast H+-ATPase.