Elke Fischer-Schliebs

Immunolocalization of plasma-membrane H+-ATPase and tonoplast-type pyrophosphatase in the plasma membrane of the sieve element-companion cell complex in the stem of Ricinus communis L.

Abstract. 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.

Evidence for two different nitrate-reducing activities at the plasma membrane in roots of Zea mays L.

Plasma-membrane (PM) vesicles isolated from 6-d-old corn roots by sucrose gradient centrifugation or two-phase partitioning showed an NADH-dependent nitrate reductase (NR) activity averaging at 40 nmol per milligram PM protein per hour. This membrane-associated NR activity could not be removed from two-phase-partitioned PM vesicles by salt washing, osmotic shock treatment, sonication, or freeze-thawing to reverse vesicle sidedness. Therefore, it could not be attributed to contamination of membrane vesicles by the soluble, cytosolic NR. Plasma-membrane vesicles reduced NO3- in the presence of the electron donors NADH or NADPH at an activity ratio of 2.2. The NADH- and NADPH-dependent NR activities of outside-out oriented PM vesicles differed in their sensitivity toward the detergent Brij 58, leading to a latency of 65% or 29% using NADH or NADPH as electron donor, respectively. The activities of NO3- reduction in the presence of saturating concentrations of NADH and NADPH were additive. Furthermore, both activities were characterized by a different pH dependence with a pH optimum of 7.5 for the NADH-dependent activity and of 6.8 for the NADPH-dependent activity. The membrane-associated NAD(P)H-dependent NR activities responded to different nitrogen nutrition of plants in a manner different from the soluble forms of the enzyme. The data confirm the existence of a corn PM NR and suggest that there may be two different NO3--reducing enzymes located at the PM of corn roots.

Na+/H+ antiporters are differentially regulated in response to NaCl stress in leaves and roots of Mesembryanthemum crystallinum.

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.

Differential Immunological Cross-Reactions with Antisera against the V-ATPase of Kalanchoë daigremontiana Reveal Structural Differences of V-ATPase Subunits of Different Plant Species

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.

A Plant Homolog of Animal Chloride Intracellular Channels (CLICs) Generates an Ion Conductance in Heterologous Systems

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.

Dynamics of activity and structure of the tonoplast vacuolar-type H+-ATPase in plants with differing CAM expression and in a C3 plant under salt stress

SummaryDifferences in the activity and structure of the vacuolar H+-ATPase (V-ATPase, EC 3.6.1.3) were investigated in the C3/CAM intermediate plantKalanchoë blossfeldiana Poellnitz cv. Tom Thumb, with lower or higher expression of CAM, andHordeum vulgare cv. Carina, grown with or without 150 mM NaCl. InK. 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 ofK. blossfeldiana with strong CAM expression (9.1 nm) than in preparations ofK. 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 inK. blossfeldiana with stronger CAM could be a result of additional structural changes in its membrane-integral domain. The higher activity of V-ATPase inK. 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 inH. 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.

Adaptation of the obligate CAM plant Clusia alata to light stress: Metabolic responses

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.

Stimulation of H+transport activity of vacuolar H+ATPase by activation of H+PPase in Kalanchoë blossfeldiana

In Kalanchoë blossfeldiana cv. Tom Thumb the initial rate of ATP-dependent H+-transport into tonoplast vesicles was stimulated up to three times if the H+-ATPase (EC 3.6.1.3) was energized a few minutes after pre-energization of the H+-PPase (EC 3.6.1.1). H+-PPase-activated ATP-dependent H+-transport was observed in plants of K. blossfeldiana cultivated in short day (SD) or long day (LD) conditions expressing different degrees of crassulacean acid metabolism (CAM). However, based on the higher activity and protein amount of H+-PPase and H+-ATPase present in the vacuolar membrane of SD plants the maximum H+-transport activity in the stimulated mode of the H+-ATPase was significantly higher in tonoplast vesicles of SD plants than of LD plants. Hence, a co-ordinated action of the H+-PPase and H+-ATPase at the tonoplast of Kalanchoë could allow a higher transport capacity at the vacuolar membrane when plants perform high CAM. Immunoprecipitation experiments with an antiserum raised against the A-subunit of the vacuolar H+-ATPase of Mesembryanthemum crystallinum L. showed that in SD and LD plants of K. blossfeldiana the H+-PPase was co-precipitated with the vacuolar H+-ATPase holoenzyme. The co-percipitation of the two transport proteins indicates a close structural localization of the H+-PPase and the A-subunit of the vacuolar H+-ATPase.

Proteomic analysis of Mesembryanthemum crystallinum leaf microsomal fractions finds an imbalance in V-ATPase stoichiometry during the salt-induced transition from C3 to CAM

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.

Na+/H+-transporter, H+-pumps and an aquaporin in light and heavy tonoplast membranes from organic acid and NaCl accumulating vacuoles of the annual facultative CAM plant and halophyte Mesembryanthemum crystallinum L.

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 Arabidopsisthaliana 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.