Effect of low growth temperature on coupling between electron transport and proton flux in Vicia faba thylakoids

Coupling between electron transport and proton flux has been compared in chloroplasts from Vicia faba (cv. Windsor) plants grown at 20 and 5°C. Proton uptake by warm-grown thylakoids was sensitive to external pH and stimulated by micromolar adenine nucleotide above pH 7.0. Electron transport was modulated by pH, adenine nucleotide and energy transfer inhibitors (triphenyltin and Hg²⁺). By contrast, proton uptake by cold-grown thylakoids was generally lower and was insensitive to micromolar ATP. The rate of non-phosphorylating electron flow in cold-grown thylakoids was relatively insensitive to pH and Hg²⁺ and was not modulated by adenine nucleotides or triphenyltin. Stimulation of electron transport by phosphorylating conditions in cold-grown thylakoids was generally lower and insensitive to pH. It is concluded that the control of proton efflux through CF₀-CF₁ differs in thylakoids of V. faba grown at warm and cold temperatures.     

Light perception in guard cells

Abstract. Guard cells perceive light via two photoreceptor systems: a blue-light-dependent photosystem and the guard cell chloroplast. Chloroplasts stimulate stomatal opening by transducing photosynthetic active radiation into proton pumping at the guard cell plasma membrane. In addition, guard cell chloroplasts fix CO₂ photosynthetically. Sugar from guard cell photosynthesis can contribute to the osmotic build-up required for opening. The blue-light-dependent photosystem activates proton pumping at the guard cell plasma membrane and stimulates starch hydrolysis. Available information on the photobiological properties of guard cells makes it possible to describe stomatal function in terms of the cellular components regulating stomatal movements. The blue light response is involved in stomatal opening in the early morning and stomatal responses to sunflecks. The guard cell chloroplast is likely to be involved in stomatal adaptations to sun, shade and to temperature. Interactions between these photosystems, a third photoreceptor in guard cells, phytochrome, and other mechanisms transducing stomatal responses such as VPD and carbon dioxide, provide the cellular basis for stomatal regulation.     

Asymmetric effects of divalent cations and protons on active Ca2+ efflux and Ca2+-ATPase in intact red blood cells

Summary The influence of the asymmetric addition of various divalent cations and protons on the properties of active Ca²⁺ transport have been examined in intact human red blood cells. Active Ca²⁺ efflux was determined from the initial rate of⁴⁵Ca²⁺ loss after CoCl₂ was added to block Ca²⁺ loading via the ionophore A23187. Ca²⁺-ATPase activity was measured as phosphate production over 5 min in cells equilibrated with EGTA-buffered free Ca²⁺ in the presence of A23187. The apparent Ca affinity of active Ca²⁺ efflux (K _0.5=30–40 mol/liter cells) was significantly lower than that measured by the Ca²⁺-ATPase assay (K _0.5=0.4 m). Possible reasons for this apparent difference are considered. Both active Ca²⁺ efflux and Ca²⁺-ATPase activity were reduced to less than 5% of maximal levels (20 mmol/liter cells · hr) in Mg²⁺-depleted cells, and completely restored by reintroduction of intracellular Mg²⁺. Active Ca²⁺ efflux was inhibited almost completely by raising external CaCl₂ (but not MgCl₂) to 20mm, probably by interaction of Ca²⁺ at the externally oriented E₂P conformation of the pump. Cd²⁺ was more potent than Ca²⁺ in this inhibition, while Mn²⁺ was less potent and 10mm Ba²⁺ was without effect. A Ca²⁺: proton exchange mechanism for active Ca²⁺ efflux was supported by the results, as external protons (pH 6–6.5) stimulated active Ca²⁺ efflux at least twofold above the efflux rate at pH 7.8 Ca²⁺ transport was not affected by decreasing the membrane potential across the red cell.     

Relationship between iron chlorosis and alkalinity in Zea mays

Mengel, K. and Geurtzen, G. 1988. Relationship between iron chlorosis and alkalinity in Zea mays . - Physiol. Plant. 72: 460–465.
Maize ( Zea mays L. cv. Anjou 21) grown in nutrient solution with Fe-EDTA and with nitrate as the sole nitrogen source showed typical Fe-chlorosis symptoms after a growth period of 14–21 days. Alkalinity in roots, stems and leaves of the chlorotic plants was high. Transferring the chlorotic plants from the nitrate-containing nutrient solution to a solution of (NH₄)₂SO₄ resulted in a regreening of leaves within 2–3 days which was associated with a decrease in solution pH, a decrease in alkalinity of plant parts, a translocation of Fe from roots to tops and a release of Fe into the outer solution. Similar effects were obtained when Fe chlorotic plants were transferred to a dilute HO solution with pH 3.5.
Spraying chlorotic leaves with indoleacetic acid or with fusicoccin led also to a regreening of leaves without having a major effect on leaf alkalinity.
Interpretation of the experimental results is based on the assumption that nitrate as sole N source leads to a high pH level in the apoplast resulting in the precipitation of Fe compounds, probably Fe oxide hydrate. Ammonium nutrition has the reverse effect since it lowers the apoplast pH and this can result in the dissolution of Fe compounds. Application of indoleacetic acid as well as fusicoccin supposedly stimulates the proton pumps in the plasmalemma of the leaf tissue. The resulting decrease in apoplast leaf pH in the microenvironment also leads to a dissolution of Fe compounds in the apoplast and thus promotes the uptake of Fe by the symplasm.     

Extreme pKa displacements at the active sites of FMN-dependent alpha-hydroxy acid-oxidizing enzymes.

Flavocytochrome b2 (or L-lactate dehydrogenase) from baker's yeast is thought to operate by the initial formation of a carbanion, as do the evolutionarily related alpha-hydroxy acid-oxidizing FMN-dependent oxidases. Previous work has shown that, in the active site of the unligated reduced flavocytochrome b2, the group that has captured the substrate alpha-proton has a high pKapp, calculated to lie around 15 through the use of Eigen's equation. A detailed inspection of the now known three-dimensional structure of the enzyme leads to the conclusion that the high pKa belongs to His 373, an active site group that plays the role of general base in the forward reaction and of general acid in the reverse direction. Moreover, consideration of the kinetics of proton transfer during the catalytic cycle suggests that the pKa of the reduced FMN N5 position should be lowered by several pH units compared to its pKa of 20 or more when free. The features of the three-dimensional structure possibly responsible for these pK shifts are analyzed; they are proposed to consist of a network of hydrogen bonds with the solvent and of a mutual electrostatic stabilization of anionic reduced flavin and the imidazolium ion. Finally, it is suggested that similar pK shifts affect the active sites of the alpha-hydroxy acid-oxidizing flavooxidases, which are homologous to flavocytochrome b2. The functional significance of these pK shifts in terms of catalysis and semiquinone stabilization is discussed.     

ATP-driven proton pumping in two species of Chara differing in salt tolerance

Chara corallina is an obligate freshwater alga, while C. buckellii can be grown in salt and freshwater culture. When grown in fresh water, C. buckellii has electrophysiological properties similar to C. corallina, but when cultured in salt water, it has a less negative membrane potential and has a higher conductance. We show in internally perfused, tonoplast-free cells that the ATP-dependence of the two species cultured in fresh water is similar, although C. buckellii hyperpolarizes at lower ATP concentrations. We determined the pump parameters in perfused and intact cells. Using both techniques, C. corallina and C. buckellii cultured in fresh water show similar values of E_p, G_p and I_p. However, there is a significant difference between the two techniques: E_p is more negative (–400 to –700 mV) in perfused cells than in intact cells (–220 to –260mV); G_p is lower (0·1–0·2 versus 0·3–0·9 S m^?2); and I_p is higher (40–60 versus 10–18 mA m^?2). Salt-cultured C. buckellii was compared with freshwater C. buckellii using intact cells; G_p and E_p were similar, but I_p was much higher in salt-cultured cells (60 versus 15mA m^?2). This higher pump rate is due to the depolarization of the membrane of salt-cultured algae, which is caused by a higher passive conductance. The significance of the less negative membrane potential and the higher rate of proton pumping is discussed with respect to the banding pattern and salt stress.     

Vacuolar H+-translocating ATPases from plants: Structure,function, and isoforms

The vacuolar H⁺-translocating ATPase (V-type ATPase) plays a central role in the growth and development of plant cells. In a mature cell, the vacuole is the largest intracellular compartment, occupying about 90% of the cell volume. The proton electrochemical gradient (acid inside) formed by the vacuolar ATPase provides the primary driving force for the transport of numerous ions and metabolites against their electrochemical gradients. The uptake and release of solutes across the vacuolar membrane is fundamental to many cellular processes, such as osmoregulation, signal transduction, and metabolic regulation. Vacuolar ATPases may also reside on endomembranes, such as Golgi and coated vesicles, and thus may participate in intracellular membrane traffic, sorting, and secretion.Plant vacuolar ATPases are large complexes (400–650 kDa) composed of 7–10 different subunits. The peripheral sector of 5–6 subunits includes the nucleotide-binding catalytic and regulatory subunits of 70 and 60 kDa, respectively. Six copies of the 16-kDa proteolipid together with 1–3 other subunits make up the integral sector that forms the H⁺ conducting pathway. Isoforms of plant vacuolar ATPases are suggested by the variations in subunit composition observed among and within plant species, and by the presence of a small multigene family encoding the 16-kDa and 70-kDa subunits. Multiple genes may encode isoforms with specific properties required to serve the diverse functions of vacuoles and endomembrane compartments.Abbreviations DCCD N,N-dicyclohexylcarbodiimide - CAM Crassulacean acid metabolism - Nbd-Cl 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole - Bz-ATP 3-O-(4-benzoyl)benzolyadenosine 5-triphosphate - DIDS 4,4-diisothiocyanostilbene-2,2-disulfonic acid - NEM N-ethylmaleimide - IP₃ inositol-1,4,5-triphosphate - H⁺-PPase H⁺-translocating pyrophosphatase - V-type vacuolar-type - P-type phosphorylated intermediate- or plasma membrane-type - F-type F₁F_o-type - V-ATPase vacuolar-type H⁺-ATPase     

Cytochrome bc 1 and b 6 f complexes of photosynthetic membranes

All photosynthetic membranes contain a cytochrome bc ₁ or b ₆ f complex that catalyzes the oxidation of quinols and the reduction of a high-potential electron carrier, such as cytochrome c ₂ or plastocyanin. The cytochrome complex also functions in the translocation of protons across the membrane and as a consequence, establishes the proton motive force that is used for the synthesis of ATP. The structure and function of the cytochrome complexes are first reviewed in this chapter. Amino acid sequence information for almost all of the protein subunits of these complexes is now available, and these allow for a detailed consideration of functional domains in the protein subunits and for a further discussion of the evolution of the cytochrome complex in photosynthetic organisms.     

类囊体膜的垛叠、松散与它的功能关系

菠菜完整叶绿体置于4mM MgCl_2或20 mM KCl低浓度介质中低渗10秒钟后,得到由Mg~(++)或K~+离子诱导的类囊体垛叠膜和松散膜。它们在功能上表现出明显的差异。垛叠膜有较高的毫秒级延迟光发射(ms-DLE),松散膜显著降低DLE的快相,垛叠膜比松散膜的9-AA荧光猝灭快,并保持稳定;而松散膜有H~+渗漏。在非循环或Fd催化的循环光合磷酸化中,垛叠膜比松散膜活力高。但是,若在同样的低渗介质中低渗1分钟以上,Mg~(++)离子诱导的垛叠膜,在显微结构上不同于低渗过10秒钟的垛叠膜,它垛叠较松,而且在磷酸化活力上也与松散膜差别不大。揭示了H~+传递速度受二个光系统、电子载体间的距离及偶联程度的限制。新鲜制备的垛叠或松散膜,在NADP~+还原系统中,具有相同的电子传递放O_2速度,说明电子传递速度在一定范围内不受膜间的距离和偶联程度的影响。但是松散膜不稳定,随着膜的老化而解联,牛血清白蛋白(BSA)能稳定松散膜的电子传递。