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The C3-CAM intermediate plant Clusia fluminensis under well-watered at low light conditions opens stomata during the light period. In leaf extracts of this plant we have found two copper-zinc superoxide dismutases (CuZnSODs) and two manganese SODs: MnSOD-like protein (MnSOD II) and MnSOD I. Daily rhythm of the MnSOD I shows maximum activity during the afternoon hours and it is accompanied by only a very small tendency to increase in catalase (CAT) activity and lowering of citrate level. [ABSTRACT FROM AUTHOR]

Differences in the activity and structure of the vacuolar H+-ATPase (V-ATPase, EC 3.6.1.3) were investigated in the C3/CAM intermediate plant Kalanchoë blossfeldiana Poellnitz cv. Tom Thumb, with lower or higher expression of CAM, and Hordeum vulgare cv. Carina, grown with or without 150 mM NaCl. In K. blossfeldiana ATP-hydrolysis and H+-transport activity were higher with higher expression of CAM than in plants with very weak CAM. This was mainly due to a larger amount of V-ATPase. Statistical analysis of the diameter of intramembrane particles (IMPs) on freeze-fractures of tonoplast vesicles showed that IMPs were larger in tonoplast vesicle preparations of K. blossfeldiana with strong CAM expression (9.1 nm) than in preparations of K. blossfeldiana with low CAM expression (7.3 nm). As there is evidence that the majority of IMPs on freeze-fractures of tonoplast vesicles corresponds to the V0 domain of V-ATPase, the higher activity of V-ATPase in K. blossfeldiana with stronger CAM could be a result of additional structural changes in its membrane-integral domain. The higher activity of V-ATPase in K. blossfeldiana with stronger CAM is discussed in relation to the requirement for a higher proton pumping capacity for nocturnal malate accumulation in the vacuole. The ATP-dependent H+-pumping activity in H. vulgare was higher under salt stress than in control plants, while the rates of ATP-hydrolysis and the size of IMPs were not affected by the salt treatment. The data presented here indicate that different mechanisms might increase the transport capacity of V-ATPase to meet the higher requirements of secondary active transport related to CAM expression and adaptation to salt stress. [ABSTRACT FROM AUTHOR]

Clusia hilariana Schltdl. is described in literature as an obligate Crassulacean acid metabolism (CAM) species. In the present study we assessed the effect of irradiance with low light (LL, 200μmolm(-2)s(-1)) and high light (HL, 650-740μmolm(-2)s(-1)), on the interdependency of citrate and malate diurnal fluctuations. In plants grown at HL CAM-type oscillations of concentration of citrate and malate were obvious. However, at LL daily courses of both acids do not seem to indicate efficient utilization of these compounds as CO2 and NADPH sources. One week after transferring plants from LL to HL decarboxylation of malate was accelerated. Thus, in the CAM plant C. hilariana two independent rhythms of accumulation and decarboxylation of malate and citrate take place, which appear to be related to photosynthesis and respiration, respectively. Non photochemical quenching (NPQ) of photosystem II, especially well expressed during the evening hours was enhanced. Exposure to HL for 7 d activated oxidative stress protection mechanisms such as the interconversion of violaxanthin (V), antheraxanthin (A) and zeaxanthin (Z) (epoxydation/de-epoxydation) measured as epoxydation state (EPS). This was accompanied by a slight increase in the total amount of these pigments. However, all these changes were not observed in plants exposed to HL for only 2 d. Besides violaxanthin cycle components also lutein, which shows a small, but not significant increase, may be involved in dissipating excess light energy in C. hilariana., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

The halophyte Mesembryanthemum crystallinum adapts to salt stress by salt uptake and switching from C3 photosynthesis to CAM (crassulacean acid metabolism). An important role in this process is played by transport proteins in the tonoplast of the central vacuole. In the present study we examine dynamic changes in the protein composition during salt-stress adaptation in microsomes from M. crystallinum leaves. Plants challenged with 400 mM NaCl accumulate salt by day 4 of treatment and malic acid only at day 12; a switching to CAM hence follows any initial steps of salt adaptation with a delay. Using a label-free and semiquantitative approach, we identified the most dramatic changes between the proteome of control plants and plants harvested after 12 days of the treatment; the abundance of 14 proteins was significantly affected. The proteomic data revealed that the majority of the subunits of V-ATPase (vacuolar H(+)-ATPase) holoenzyme. The salt treatment somewhat decreased the abundance of all subunits in the short term (4 days). Long-term adaptation, including the switching to CAM, goes together with a strong increase in the representation of all detectable subunits. Because this increase is subunit-specific, with the highest rise occurring for subunits E and c, the data suggest that long-term adaptation to salt stress correlates with a change in V-ATPase subunit stoichiometry and highlight the structural plasticity of this holoenzyme.

The Fabaceae tree Butea monosperma (TAUB.; syn. Erythrina monosperma (LAM.)) is widely distributed in Central and West-India. We studied it at three sites, i.e. at two locations with contrasting exposure (NE and SW, respectively) in a small mountain range with poor soil on highly drained rocky slopes and at a third location in a plane with deeper soils and better water supply. The two mountain range sites differed in the light climate where the NE-slope obtained more day-integrated irradiance. Chlorophyll fluorescence was measured with a portable fluorometer and leaf samples for stable isotope analyses (δ(13)C, δ(15)N, δ(18)O) were collected. No differences were seen in carbon and nitrogen contents of leaves at the three sites. N and O isotope signatures of the leaves were similar at the two rocky hill slope sites. More positive values for both signatures were obtained in the leaves in the plane. For all sites saturation of ETR was only achieved well above a PPFD of 1,000 μmol m(-2) s(-1) indicating that the leaves were sun-type leaves. The photosynthetic performance of Butea at the plane was very similar to that at the SW-slope of the mountain range and higher ETRs were obtained at the NE-slope. Ecophysiological flexibility allows Butea to perform well in a variety of habitats and yet gives it particular fitness at specific sites. The best performance was observed in the highly insolated steep rocky hill site (NE-slope) underlining the suitability of the tree for reforestation.

Salinity tolerance in plants involves controlled Na(+) transport at the site of Na(+) accumulation and intracellular Na(+) compartmentation. The focus of this study was the identification and analysis of the expression of Na(+)/H(+) antiporters in response to NaCl stress in one particular plant, the facultative halophyte Mesembryanthemum crystallinum Na(+)/H(+) antiporters of M. crystallinum were cloned by RACE-PCR from total mRNA of leaf mesophyll cells. Functional complementation of Saccharomyces cerevisiae and Escherichia coli mutants was performed. The kinetics of changes in the expression of antiporters were quantified by real-time PCR in leaves and roots. Five Na(+)/H(+) antiporters (McSOS1, McNhaD, McNHX1, McNHX2 and McNHX3) were cloned, representing the entire set of these transporters in M. crystallinum. The functionality of McSOS1, McHX1 and McNhaD was demonstrated in complementation experiments. Quantitative analysis revealed a temporal correlation between salt accumulation and expression levels of genes in leaves, but not in roots, which was most pronounced for McNhaD. Results suggest a physiological role of McSOS1, McNhaD and McNHX1 in Na(+) compartmentation during plant adaptation to high salinity. The study also provides evidence for salt-induced expression and function of the Na(+)/H(+) antiporter McNhaD in chloroplasts and demonstrates that the chloroplast is one of the compartments involved in the response of cells to salt stress.

Clusia minor L., a C3-CAM intermediate, and Clusia multiflora H. B. K., a C3 obligate, present two physiotypes of a similar morphotype occurring sympatrically in the field. Both species, exposed 2 days to high light, show similar responses to this kind of stress: (i) the level of xanthophyll pigments in tested plants during the daycourse adapts to stress, (ii) the levels of antheraxanthin and zeaxanthin clearly increase during the afternoon showing increased de-epoxidation, (iii) the changes in the xanthophyll cycle are similar. Exposure to high light increases the malate levels in C. minor during the afternoon while decreases the day/night changes of the malate levels, and hence the Crassulacean Acid Metabolism (CAM) expression. It can be concluded that strong light applied as a single stress factor to well-watered plants is not effective in strengthing the CAM metabolism in a C3-CAM intermediate plant but rather suppresses the CAM activity despite exposure to high light energy. It is suggested that, when water supply is not limiting and other stresses do not prevail, C3 allows to use up the citrate pool, especially in the afternoon and enables a superior daily photon utilization.

In the Crassulacean acid metabolism (CAM) plants Clusia alata Triana and Planch., decarboxylation of citrate during phase III of CAM took place later than malate decarboxylation. The interdependence of these two CO(2) and NADPH sources is discussed. High light accelerated malate decarboxylation during the day and lowered citrate levels. Strong light stress also activated mechanisms that can protect the plant against oxidative stress. Upon transfer from low light (200micromol m(-2)s(-1)) to high light (650-740micromol m(-2)s(-1)), after 2 days, there was a transient increase of non-photochemical quenching (NPQ) of fluorescence of chlorophyll a of photosystem II. This indicated acute photoinhibition, which declined again after 7 days of exposure. Conversely, after 1 week exposure to high light, the mechanisms of interconversion of violaxanthin (V), antheraxanthin (A), zeaxanthin (Z) (epoxydation/de-epoxydation) were activated. This was accompanied by an increase in pigment levels at dawn and dusk.

The genome of Arabidopsis thaliana contains unusual members of the glutathione S-transferase (GST) superfamily with a cysteine in place of a serine at the active site. Four of these genes (at-dhar 1-4) have an appreciable homology to intracellular Cl- channels (CLICs) from vertebrates and invertebrates. Transient expression of AtDHAR1 as wild type protein or as a chimera with GFP in mammalian HEK293 or Chinese hamster ovary cells generated a distinct inward rectifying conductance with a characteristic biphasic kinetics but no apparent ion selectivity. Analysis of the subcellular localization of AtDHRA1::GFP showed that the bulk of the protein was located as soluble form in the cytoplasm; however, an appreciable fraction of it could also be found in association with the non-soluble microsomal fraction. These data suggest that plant members of the GST superfamily have similar to those from animals multiple functions. The increase of ion conductance by AtDHAR1 is better explained by a CLIC-like channel activity than by a modification of endogenous channel proteins.

Crassulacean acid metabolism (CAM) was induced in Mesembryanthemum crystallinum L. by either NaCl- or high light (HL)- stress. This generated in mesophyll cells predominantly of NaCl-stressed plants two different types of vacuoles: the generic acidic vacuoles for malic acid accumulation and additionally less acidic ("neutral") vacuoles for NaCl sequestration. To examine differences in the tonoplast properties of the two types of vacuoles, we separated microsomal membranes of HL- and NaCl-stressed M. crystallinum plants by centrifugation in sucrose density gradients. Positive immunoreactions of a set of antibodies directed against tonoplast specific proteins and tonoplast specific ATP- and PPi-hydrolytic activity were used as markers for vacuolar membranes. With these criteria tonoplast membranes were detected in both HL- and NaCl-stressed plants in association with the characteristic low sucrose density but also at an unusual high sucrose density. In HL-stressed plants most of the ATP- and PPi-hydrolytic activity and cross reactivity with antibodies including that directed against the Na+/H+-antiporter from Arabidopsis thaliana was detected with light sucrose density. This relationship was inverted in NaCl-stressed plants; they exhibited most pump activity and immunoreactivity in the heavy fraction. The relative abundance of the heavy membrane fraction reflects the relative occurrence of "neutral" vacuoles in either HL- or NaCl-stressed plants. This suggests that tonoplasts of the "neutral" vacuoles sediment at high sucrose densities. This is consistent with the view that this type of vacuoles serves for Na+ sequestration and is accordingly equipped with a high capacity of proton pumping and Na+ uptake via the Na+/H+-antiporter.

It is a common belief that plant mesophyll cells are occupied up to 95% by a single multipurpose vacuole. The common ice plant, Mesembryanthemum crystallinum L., however, requires two contrasting functions of the vacuole under salt stress. Large amounts of NaCl have to be sequestered permanently for osmotic purpose and for protecting the cytoplasm from NaCl toxicity. A dynamic exchange with the cytoplasm is required because photosynthesis proceeds under these conditions via the metabolic cycle of crassulacean acid metabolism (CAM). Nocturnally acquired CO2 must be kept as malate in the vacuole and re-mobilized in the daytime. Here, we show that two large independent types of vacuoles with different transport properties meet the requirements for the contrasting functions within the same cell.

The lutein-epoxide cycle (Lx cycle) is an auxiliary xanthophyll cycle known to operate only in some higher-plant species. It occurs in parallel with the common violaxanthin cycle (V cycle) and involves the same epoxidation and de-epoxidation reactions as in the V cycle. In this study, the occurrence of the Lx cycle was investigated in the two major families of mistletoe, the Loranthaceae and the Viscaceae. In an attempt to find the limiting factor(s) for the occurrence of the Lx cycle, pigment profiles of mistletoes with and without the Lx cycle were compared. The availability of lutein as a substrate for the zeaxanthin epoxidase appeared not to be critical. This was supported by the absence of the Lx cycle in the transgenic Arabidopsis plant lutOE, in which synthesis of lutein was increased at the expense of V by overexpression of epsilon-cyclase, a key enzyme for lutein synthesis. Furthermore, analysis of pigment distribution within the mistletoe thylakoids excluded the possibility of different localizations for the Lx- and V-cycle pigments. From these findings, together with previous reports on the substrate specificity of the two enzymes in the V cycle, we propose that mutation to zeaxanthin epoxidase could have resulted in altered regulation and/or substrate specificity of the enzyme that gave rise to the parallel operation of two xanthophyll cycles in some plants. The distribution pattern of Lx in the mistletoe phylogeny inferred from 18S rRNA gene sequences also suggested that the occurrence of the Lx cycle is determined genetically. Possible molecular evolutionary processes that may have led to the operation of the Lx cycle in some mistletoes are discussed.

The model plant tobacco (Nicotiana tabacum L.) was chosen for a survey of the subunit composition of the V-ATPase at the protein level. V-ATPase was purified from tobacco leaf cell tonoplasts by solubilization with the nonionic detergent Triton X-100 and immunoprecipitation. In the purified fraction 12 proteins were present. By matrix-assisted laser-desorption ionization mass spectrometry (MALDI-MS) and amino acid sequencing 11 of these polypeptides could be identified as subunits A, B, C, D, F, G, c, d and three different isoforms of subunit E. The polypeptide which could not be identified by MALDI analysis might represent subunit H. The data presented here, for the first time, enable an unequivocal identification of V-ATPase subunits after gel electrophoresis and open the possibility to assign changes in polypeptide composition to variations in respective V-ATPase subunits occurring as a response to environmental conditions or during plant development.

Plasma-membrane-located primary pumps were investigated in the sieve element (SE)-companion cell complex in the transport phloem of 2-week-old stems of Ricinus communis L. and, for comparison, in stems of Cucurbita pepo L. and in the secondary phloem of Agrobacterium tumefaciens-induced crown galls as a typical sink tissue. The plasma-membrane (PM) H+-ATPase and the tonoplast-type pyrophosphatase (PPase) were immunolocalized by epifluorescence and confocal laser scanning microscopy (CLSM) upon single or double labeling with specific monoclonal and polyclonal antibodies. Quantitative fluorescence evaluation by CLSM revealed both pumps in one membrane, the sieve-element PM. Different PM H+-ATPase antibody clones, raised against the PM H+-ATPase of Zea mays coleoptiles, induced in mouse and produced in mouse hybridoma cells, discriminated between different phloem cell types. Clones 30D5C4 and 44B8A1 labeled sieve elements and clone 46E5B11D5 labeled companion cells, indicating the existence of different phloem PM H+-ATPase isoforms. The results are discussed in terms of energization of SE transporters for retrieval of leaking sucrose, K+ and amino acids, as one of the unknown roles of ATP found in SEs. The function of the PPase could be related to phloem sucrose metabolism in support of ATP-requiring processes.

Two antisera (ATP88 and ATP95) raised against the V-ATPase holoenzyme of Kalanchoë daigremontiana were tested for their cross-reactivity with subunits of V-ATPases from other plant species. V-ATPases from Kalanchoë blossfeldiana, Mesembryanthemum crystallinum, Nicotiana tabacum, Lycopersicon esculentum, Citrus limon, Lemna gibba, Hordeum vulgare and Zea mays were immunoprecipitated with an antiserum against the catalytic V-ATPase subunit A of M. crystallinum. As shown by silver staining and Western blot analysis with ATP88, subunits A, B, C, D and c were present in all immunoprecipitated V-ATPases. In contrast, ATP95 recognized the whole set of subunits only in K. blossfeldiana, M. crystallinum, H. vulgare and Z. mays. This differential cross reactivity of ATP95 indicates the presence of structural differences of certain V-ATPase subunits. Based on the Bafilomycin A1-sensitive ATPase activity of tonoplast enriched vesicles, and on the amount of V-ATPase solubilized and immunoprecipitated, the specific ATP-hydrolysis activity of the V-ATPases under test was determined. The structural differences correlate with the ability of V-ATPases from different species to hydrolyze ATP at one given assay condition for ATP-hydrolysis measurements. Interestingly V-ATPases showing cross-reactivity of subunits A, B, C, D and c with ATP95 showed higher rates of specific ATP hydrolysis compared to V-ATPases containing subunits which were not labeled by ATP95. Thus, V-ATPases with high turnover rates in our assay conditions may show common structural characteristics which separate them from ATPases with low turnover rates.