Rafael Ratajczak

Early Salt Stress Effects on the Differential Expression of Vacuolar H+-ATPase Genes in Roots and Leaves of Mesembryanthemum crystallinum

In Mesembryanthemum crystallinum, the salt stress-induced metabolic switch from C3 photosynthesis to Crassulacean acid metabolism is accompanied by major changes in gene expression. However, early effects of salt exposure (i.e. prior to Crassulacean acid metabolism induction) on genes coding for vacuolar transport functions have not yet been studied. Therefore, the expression of vacuolar H+-ATPase genes was analyzed in different organs of 4-week-old plants stressed with 400 mM NaCl for 3, 8, or 24 h. Partial cDNAs for the subunits A, B, and c were cloned and used as homologous probes for northern blot analysis. In control plants, the mRNA levels for the different subunits showed organ-specific differences. In fully expanded leaves, subunit c mRNA was very low but increased transiently during the light period. Plant organs also differed in their salt-stress response. In roots and young leaves, mRNA levels for all three subunits increased about 2-fold compared to control plants, whereas in fully expanded leaves only subunit c mRNA responded to salt. The results indicate that the expression of vacuolar H+-ATPase genes does not always involve a fixed stoichiometry of mRNAs for the different subunits and that the mRNA level for subunit c is particularly sensitive to developmental and environmental changes.

Specific regulation of SOD isoforms by NaCl and osmotic stress in leaves of the C3 halophyte Suaeda salsa L.

The halophyte Suaeda salsa L., exposed to different NaCl concentrations (100 and 400 mmol/L) and polyethylene glycol (isoosomotic to 100 mmol/L NaCl) containing nutrient solutions under normal or K+-deficient conditions for 7 days, was used to study effects of NaCl salinity and osmotic stress on chlorophyll content, chlorophyll fluorescence characteristics, malonedialdehyde (MDA) content, and superoxide dismutase (SOD) isoform activities. Photosynthetic capacity was not decreased by NaCl treatment, indicating that S. salsa possesses an effective antioxidative response system for avoiding oxidative damage. Seven SOD activity bands were detected in S. salsa leaf extracts, including an Mn-SOD and several isoforms of Fe-SOD and CuZn-SOD. It turned out that NaCl salinity and osmotic stress lead to a differential regulation of distinct SOD isoenzymes. This differential regulation is suggested to play a major role in stress tolerance of S. salsa.

Influence of light intensity and salt-treatment on mode of photosynthesis and enzymes of the antioxidative response system of Mesembryanthemum crystallinum

The metabolic switch from C3-photosynthesis to crassulacean acid metabolism (CAM),and the antioxidative response of Mesembryanthemum crystallinum L. plants cultured under severe salt stress and high light intensities, and a combination of both stress conditions, were studied. High light conditions led to a more rapid CAM induction than salinity. The induction time was still shortened when both stress factors were combined. A main pattern observed in CAM plants was a decrease in mitochondrial Mn-superoxide dismutase (SOD) activity during the day. The activities of the chloroplastic Fe-SOD and cytosolic CuZn-SOD were increased due to salt treatment after a lag phase, while catalase activity was decreased. Combination of salt and light stress did not lead to a higher SOD activity as found after application of one stress factor alone, indicating that there is a threshold level of the oxidative stress response. The fact that salt-stressed plants grown under high light conditions showed permanent photoinhibition and lost the ability for nocturnal malate storage after 9 d of treatment indicate serious malfunction of metabolism, leading to accelerated senescence. Comparison of CuZn-SOD activity with CuZn-SOD protein amount, which was determined immunologically, indicates that the activity of the enzyme is at least partially post-translationally regulated.

The Physiology, Biochemistry and Molecular Biology of the Plant Vacuolar ATPase

Publisher Summary This chapter discusses the physiology, biochemistry, and molecular biology of the plant vacuolar ATPase. The transmembrane movement of solute particles by direct consumption of energy, available from dissociation of covalent phosphoric acid–ester bonds, is limited to a few simple cations such as H + and Ca 2+ and possibly Na + and Cl – , with the H + - and Ca 2+ -transporting ATPases at the plasma membrane. H + –ATPases in general are located in different membrane systems. They are important for cellular metabolism in different ways. The V–ATPase can be visualized in the electron microscope by preparation of negatively stained membrane vesicles or of replicas of freeze fracture surfaces of vesicle membranes. By pumping protons into the vacuole, the V–ATPase removes H + ions from the cytosol and thus, it is part of a cytoplasmic pH–stat mechanism. It also energizes the tonoplast by establishing an electrochemical proton gradient across this membrane. Both of these functions are basic physiological requirements in plant cell biology.

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.

Three-dimensional map of a plant V-ATPase based on electron microscopy.

V-ATPases pump protons into the interior of various subcellular compartments at the expense of ATP. Previous studies have shown that these pumps comprise a membrane-integrated, proton-translocating (V0), and a soluble catalytic (V1) subcomplex connected to one another by a thin stalk region. We present two three-dimensional maps derived from electron microscopic images of the complete V-ATPase complex from the plantKalanchoë daigremontiana at a resolution of 2.2 nm. In the presence of a non-hydrolyzable ATP analogue, the details of the stalk region between V0 and V1 were revealed for the first time in their three-dimensional organization. A central stalk was surrounded by three peripheral stalks of different sizes and shapes. In the absence of the ATP analogue, the tilt of V0changed with respect to V1, and the stalk region was less clearly defined, perhaps due to increased flexibility and partial detachment of some of the peripheral stalks. These structural changes corresponded to decreased stability of the complex and might be the initial step in a controlled disassembly.

Redox control of oxidative stress responses in the C3-CAM intermediate plant Mesembryanthemum crystallinum

Abstract Crassulacean acid metabolism (CAM) is named after the Crassulaceae family of succulent plants, in which this type of metabolism was first discovered at the beginning of the 19th century. In recent years, Mesembryanthemum crystallinum, a facultative halophyte and C3–CAM intermediate plant, has become a favoured plant for studying stress response mechanisms during C3–CAM shifts. Recent studies in this and related areas can provide a new model of how such mechanisms could operate for acclimation to high salinity or excess excitation energy. These include roles for photosynthetic electron transport chain components and reactive oxygen species. The diurnal rhythms of catalase, superoxide dismutase and some CAM-related enzyme activities are discussed in relation to the protective role of photorespiration during C3–CAM transition. The role of excess excitation energy and redox events in the proximity of photosystem II (PSII) in regulation of ascorbate peroxidase (APX), superoxide dismutase (SOD): copper/zinc SOD (Cu/ZnSOD), iron SOD (FeSOD), and NAD(P)-malic enzyme gene expression are also discussed. We suggest a model in which the chloroplast plays a major role in regulation of acclimation to high salinity and/or excess exitation energy.

Changed Densities and Diameters of Intra-Membrane Tonoplast Particles of Mesembryanthemum crystallinum in Correlation with NaCl-Induced CAM

Summary Plants of Mesembryanthemum crystallinum L., a facultative halophyte and CAM plant, were treated with four different levels of salinity in the root medium, i.e. 0, 100, 200 and 400 mM NaCl. The degree of expression of CAM, as given by day/night changes of malate levels, and the densities and diameters of intramembraneous tonoplast particles were followed over a time course of 23 days after the beginning of the salt treatment. Particle densities and diameters were counted and measured, respectively, on electron micrographs of freeze-fracture replicas of purified tonoplast vesicles. Changes in the density and an increase of the diameters of these particles, which largely although perhaps not exclusively belong to the H + -transporting tonoplast ATPase, were observed in relation to the progressing salt treatment and ageing of the plants. Particle densities and diameters were strongly correlated with the actual degree of CAM-expression. Sodium-dodecylsulfate gel electrophoresis showed that the intramembraneous H + -channel subunit peptide (c-subunit) of the tonoplast ATPase increased in its staining density with the salt treatment and ageing of plants. It might be speculated that this is related to the observed changes in particle densities and diameters.

Localization of pyrophosphatase in membranes of cauliflower inflorescence cells

Abstract. Using a polyclonal antiserum specific for the tonoplastic H+-pyrophosphatase (tPPase), significant amounts of antigenic polypeptides of the correct molecular mass were detected in Western blots of plasma membrane isolated from cauliflower (Brassica oleracea L.) inflorescence by phase-partitioning and subsequent sucrose density centrifugation. Potassium iodide-stripped plasma membranes continued to give a strong positive signal, indicating that the PPase antigen detected was not a result of contamination through soluble PPase released during homogenisation. The same preparation contained negligible vacuolar (v)H+-ATPase activity and the A subunit of the vATPase could not be detected by immunoblotting. Plasma membrane fractions exhibited a proton-pumping activity with ATP as substrate, but such an activity was not measurable with pyrophosphate, although the hydrolysis of this substrate was recorded. By contrast, pyrophosphate supported proton pumping in tonoplast-containing fractions. Immunogold electron microscopy confirmed the presence of PPase at the plasma membrane as well as at the tonoplast, trans Golgi network, and multivesicular bodies. The density of immunogold label was higher at the plasma membrane than at the tonoplast, except for membrane fragments occurring in the lumen of the vacuoles which stained very conspicuously.

Transport of organic molecules across the tonoplast

Publisher Summary This chapter discusses transport of organic molecules across the tonoplast. The vacuole is the largest compartment of a mature plant cell and may occupy more than 80% of the total cell volume. In such mature cells, the cytosol is visible only as a thin layer which is separated from the cell wall by the plasmalemma and from the vacuolar sap (cell sap) by the vacuolar membrane (tonoplast). The constituents of the cell sap are mainly inorganic salts and water. Compartmentation experiments showed that besides inorganic salts and potentially toxic compounds, products of the primary metabolism such as sugars, amino acids, and organic acids are stored within the vacuole. In photosynthesizing protoplasts from barley leaves, the carbon fluxes to the vacuole can be studied using a very fast vacuole isolation procedure. The bulk of newly fixed carbon is converted into sucrose. Compartmentation analysis showed that, after a short lag phase, sucrose is transferred to the vacuole at rates comparable to those of its synthesis. Transport of organic acids, such as malate or citrate, is even faster while only a minor fraction of amino acids are transferred to the vacuole within the time span tested. Starch and sucrose are the main storage products of primary metabolism. Both are transitorily accumulated in leaves during the photosynthetic period, when the loading capacity of the phloem is limiting, and starch is degraded and sucrose exported during the night.