Light-Induced Polar pH Changes in Leaves of Elodea canadensis: II. Effects of Ferricyanide: Evidence for Modulation by the Redox State of the Cytoplasm

The effect of an extracellular electron acceptor, ferricyanide, on the light-induced polar leaf pH changes of the submerged angiosperm Elodea canadensis in light and in darkness was determined. The rate of transmembrane ferricyanide reduction was stimulated by increased light intensity and was inhibited by inorganic carbon, indicating that changes in the redox state of the chloroplast were reflected at the plasma membrane. The addition of ferricyanide inhibited the light-induced polar leaf pH reaction. This effect could be balanced by increasing the light intensity. In the dark, the acidification induced by ferricyanide was not influenced by diethylstilbestrol at concentrations that completely inhibited the polar leaf pH changes. This indicates that the ferricyanide-induced H⁺ extrusion and the H⁺ transport during the polar reaction were mediated by different mechanisms.     

Light-induced pH and K+ changes in the apoplast of intact leaves

The K⁺-sensitive fluorescent dye benzofuran isophthalate (PBFI) and the pH-sensitive fluorescein isothiocyanate dextran (FITC-Dextran) were used to investigate the influence of light/dark transitions on apoplastic pH and K⁺ concentration in intact leaves of Vicia faba L. with fluorescence ratio imaging microscopy. Illumination by red light led to an acidification in the leaf apoplast due to light-induced H⁺ extrusion. Similar apoplastic pH responses were found on adaxial and abaxial sides of leaves after light/dark transition. Stomatal opening resulted only in a slight pH decrease (0.2 units) in the leaf apoplast. Gradients of apoplastic pH exist in the leaf apoplast, being about 0.5–1.0 units lower in the center of the xylem veins as compared with surrounding cells. The apoplastic K⁺ concentration in intact leaves declined during the light period. A steeper light-induced decrease in apoplastic K⁺, possibly caused by higher apoplastic K⁺, was found on the abaxial side of leaves concentration. Simultaneous measurements of apoplastic pH and K⁺ demonstrated that a light-induced decline in apoplastic K⁺ concentration indicative of net K⁺ uptake into leaf cells occurs independent of apoplastic pH changes. It is suggested that the driving force that is generated by H⁺ extrusion into the leaf apoplast due to H⁺-ATPase activity is sufficient for passive K⁺ influx into the leaf cells. Received: 7 March 2000 / Accepted: 12 May 2000     

Identification and Characterization of the Na+/H+ Antiporter Nhas3 from the Thylakoid Membrane of Synechocystis sp. PCC 6803

Na⁺/H⁺ antiporters influence proton or sodium motive force across the membrane. Synechocystis sp. PCC 6803 has six genes encoding Na⁺/H⁺ antiporters, nhaS1–5 and sll0556. In this study, the function of NhaS3 was examined. NhaS3 was essential for growth of Synechocystis, and loss of nhaS3 was not complemented by expression of the Escherichia coli Na⁺/H⁺ antiporter NhaA. Membrane fractionation followed by immunoblotting as well as immunogold labeling revealed that NhaS3 was localized in the thylakoid membrane of Synechocystis. NhaS3 was shown to be functional over a pH range from pH 6.5 to 9.0 when expressed in E. coli. A reduction in the copy number of nhaS3 in the Synechocystis genome rendered the cells more sensitive to high Na⁺ concentrations. NhaS3 had no K⁺/H⁺ exchange activity itself but enhanced K⁺ uptake from the medium when expressed in an E. coli potassium uptake mutant. Expression of nhaS3 increased after shifting from low CO₂ to high CO₂ conditions. Expression of nhaS3 was also found to be controlled by the circadian rhythm. Gene expression peaked at the beginning of subjective night. This coincided with the time of the lowest rate of CO₂ consumption caused by the ceasing of O₂-evolving photosynthesis. This is the first report of a Na⁺/H⁺ antiporter localized in thylakoid membrane. Our results suggested a role of NhaS3 in the maintenance of ion homeostasis of H⁺, Na⁺, and K⁺ in supporting the conversion of photosynthetic products and in the supply of energy in the dark.Na⁺/H⁺ antiporters are integral membrane proteins that transport Na⁺ and H⁺ in opposite directions across the membrane and that occur in virtually all cell types. These transporters play an important role in the regulation of cytosolic pH and Na⁺ concentrations and influence proton or sodium motive force across the membrane (1, 2). In Escherichia coli, three Na⁺/H⁺ antiporters (NhaA, NhaB, and ChaA) have been described in detail. Of these, NhaA is the functionally best characterized transporter. The crystal structure of NhaA has been resolved (3). In addition, mutants of nhaA, nhaB, and chaA as well as the triple mutant have been generated (4). The triple mutant was shown to be hypersensitive to extracellular Na⁺. The genome of the cyanobacterium Synechocystis sp. PCC 6803 contains six genes encoding Na⁺/H⁺ antiporters (NhaS1–5 and sll0556). NhaS1 (slr1727) has also been designated SynNhaP (5, 6). Null mutants of nhaS1, nhaS2, nhaS4, and nhaS5 have been generated; however, a null mutant of nhaS3 could not be obtained, indicating that it is an essential gene (6–8). By heterologous expression in E. coli, Na⁺/H⁺ exchange activities could be shown for NhaS1–5 (5, 6). Inactivation of nhaS1 and nhaS2 results in retardation of growth of Synechocystis (5, 6). It has been reported that in these mutants the concentration of Na⁺ in cytosol and intrathylakoid space (lumen) increases and impairs the photosynthetic and/or respiratory activity of the cell (9, 10). Therefore the Na⁺ extrusion by Synechocystis Na⁺/H⁺ antiporters similar to E. coli NhaA, NhaB, and ChaA is essential for the adaptation to salinity stress.In contrast to the case in E. coli, Na⁺ is an essential element for the growth of some cyanobacteria (11, 12). Interestingly, the Na⁺/H⁺ antiporter homolog NhaS4 was identified as an uptake system for Na⁺ from the medium during a screen for mutations in Synechocystis that result in lack of growth at low Na⁺ concentrations (7). The requirement of a Na⁺ uptake antiporter for cell growth is consistent with the physiology of Synechocystis. Specifically, photoautotrophic bacteria like cyanobacteria share some components (plastoquinone, cytochrome b₆f, and c₆) of the thylakoid membrane for electron transport for both photophosphorylation and respiratory oxidative phosphorylation. Na⁺/H⁺ antiporters therefore may coordinate both H⁺ and Na⁺ gradients across the plasma and thylakoid membranes to adapt to daily environmental changes (11). It remains to be determined whether the six Na⁺/H⁺ antiporters are localized to the plasma membrane or to the thylakoid membrane in Synechocystis. Information on the membrane localization will also provide information on the physiological role in Synechocystis. In this study, we explored the membrane localization of NhaS3, the role of specific amino acid residues for its function, and the effect of CO₂ concentration and circadian rhythms on the expression pattern of nhaS3 to gain insight into the physiological role of NhaS3 in Synechocystis.     

Assembly of D1/D2 complexes of photosystem II: Binding of pigments and a network of auxiliary proteins

Photosystem II (PSII) is the multi-subunit light-driven oxidoreductase that drives photosynthetic electron transport using electrons extracted from water. To investigate the initial steps of PSII assembly, we used strains of the cyanobacterium Synechocystis sp. PCC 6803 arrested at early stages of PSII biogenesis and expressing affinity-tagged PSII subunits to isolate PSII reaction center assembly (RCII) complexes and their precursor D1 and D2 modules (D1_mod and D2_mod). RCII preparations isolated using either a His-tagged D2 or a FLAG-tagged PsbI subunit contained the previously described RCIIa and RCII* complexes that differ with respect to the presence of the Ycf39 assembly factor and high light-inducible proteins (Hlips) and a larger complex consisting of RCIIa bound to monomeric PSI. All RCII complexes contained the PSII subunits D1, D2, PsbI, PsbE, and PsbF and the assembly factors rubredoxin A and Ycf48, but we also detected PsbN, Slr1470, and the Slr0575 proteins, which all have plant homologs. The RCII preparations also contained prohibitins/stomatins (Phbs) of unknown function and FtsH protease subunits. RCII complexes were active in light-induced primary charge separation and bound chlorophylls (Chls), pheophytins, beta-carotenes, and heme. The isolated D1_mod consisted of D1/PsbI/Ycf48 with some Ycf39 and Phb3, while D2_mod contained D2/cytochrome b₅₅₉ with co-purifying PsbY, Phb1, Phb3, FtsH2/FtsH3, CyanoP, and Slr1470. As stably bound, Chl was detected in D1_mod but not D2_mod, formation of RCII appears to be important for stable binding of most of the Chls and both pheophytins. We suggest that Chl can be delivered to RCII from either monomeric Photosystem I or Ycf39/Hlips complexes.

Analysis of isolated assembly complexes provides new insights into the early stages of photosystem II biogenesis.     

Effect of Exogenous Putrescine, Spermidine, and Spermine on K Uptake and H Extrusion through Plasmamembrane in Maize Root Segments

The action of exogenous polyamines (putrescine, spermidine, and spermine) on `washing' and fusicoccin-stimulated K⁺ uptake and H⁺ extrusion through the plasmamembrane in maize (Zea mays L., hybrid line Plenus S 516) root apical segments was studied. The results showed that polyamines inhibit the washing-stimulated K⁺ influx and H⁺ extrusion without interfering with K⁺ uptake and H⁺ extrusion stimulated by fusicoccin. Spermidine appeared to be the most effective in inhibiting K⁺ uptake and H⁺ extrusion while putrescine showed a smaller inhibiting action with respect to the others. The analysis of kinetic constants indicated that the polyamines behave as competitive inhibitors with respect to K⁺.     

Discovery of a Chlorophyll Binding Protein Complex Involved in the Early Steps of Photosystem II Assembly in Synechocystis

Efficient assembly and repair of the oxygen-evolving photosystem II (PSII) complex is vital for maintaining photosynthetic activity in plants, algae, and cyanobacteria. How chlorophyll is delivered to PSII during assembly and how vulnerable assembly complexes are protected from photodamage are unknown. Here, we identify a chlorophyll and β-carotene binding protein complex in the cyanobacterium Synechocystis PCC 6803 important for formation of the D1/D2 reaction center assembly complex. It is composed of putative short-chain dehydrogenase/reductase Ycf39, encoded by the slr0399 gene, and two members of the high-light-inducible protein (Hlip) family, HliC and HliD, which are small membrane proteins related to the light-harvesting chlorophyll binding complexes found in plants. Perturbed chlorophyll recycling in a Ycf39-null mutant and copurification of chlorophyll synthase and unassembled D1 with the Ycf39-Hlip complex indicate a role in the delivery of chlorophyll to newly synthesized D1. Sequence similarities suggest the presence of a related complex in chloroplasts.     

SpAHA1 and SpSOS1 Coordinate in Transgenic Yeast to Improve Salt Tolerance

In plant cells, the plasma membrane Na⁺/H⁺ antiporter SOS1 (salt overly sensitive 1) mediates Na⁺ extrusion using the proton gradient generated by plasma membrane H⁺-ATPases, and these two proteins are key plant halotolerance factors. In the present study, two genes from Sesuvium portulacastrum, encoding plasma membrane Na⁺/H⁺ antiporter (SpSOS1) and H⁺-ATPase (SpAHA1), were cloned. Localization of each protein was studied in tobacco cells, and their functions were analyzed in yeast cells. Both SpSOS1 and SpAHA1 are plasma membrane-bound proteins. Real-time polymerase chain reaction (PCR) analyses showed that SpSOS1 and SpAHA1 were induced by salinity, and their expression patterns in roots under salinity were similar. Compared with untransformed yeast cells, SpSOS1 increased the salt tolerance of transgenic yeast by decreasing the Na⁺ content. The Na⁺/H⁺ exchange activity at plasma membrane vesicles was higher in SpSOS1-transgenic yeast than in the untransformed strain. No change was observed in the salt tolerance of yeast cells expressing SpAHA1 alone; however, in yeast transformed with both SpSOS1 and SpAHA1, SpAHA1 generated an increased proton gradient that stimulated the Na⁺/H⁺ exchange activity of SpSOS1. In this scenario, more Na⁺ ions were transported out of cells, and the yeast cells co-expressing SpSOS1 and SpAHA1 grew better than the cells transformed with only SpSOS1 or SpAHA1. These findings demonstrate that the plasma membrane Na⁺/H⁺ antiporter SpSOS1 and H⁺-ATPase SpAHA1 can function in coordination. These results provide a reference for developing more salt-tolerant crops via co-transformation with the plasma membrane Na⁺/H⁺ antiporter and H⁺-ATPase.     

Comparative Analysis of kdp and ktr Mutants Reveals Distinct Roles of the Potassium Transporters in the Model Cyanobacterium Synechocystis sp. Strain PCC 6803

Photoautotrophic bacteria have developed mechanisms to maintain K⁺ homeostasis under conditions of changing ionic concentrations in the environment. Synechocystis sp. strain PCC 6803 contains genes encoding a well-characterized Ktr-type K⁺ uptake transporter (Ktr) and a putative ATP-dependent transporter specific for K⁺ (Kdp). The contributions of each of these K⁺ transport systems to cellular K⁺ homeostasis have not yet been defined conclusively. To verify the functionality of Kdp, kdp genes were expressed in Escherichia coli, where Kdp conferred K⁺ uptake, albeit with lower rates than were conferred by Ktr. An on-chip microfluidic device enabled monitoring of the biphasic initial volume recovery of single Synechocystis cells after hyperosmotic shock. Here, Ktr functioned as the primary K⁺ uptake system during the first recovery phase, whereas Kdp did not contribute significantly. The expression of the kdp operon in Synechocystis was induced by extracellular K⁺ depletion. Correspondingly, Kdp-mediated K⁺ uptake supported Synechocystis cell growth with trace amounts of external potassium. This induction of kdp expression depended on two adjacent genes, hik20 and rre19, encoding a putative two-component system. The circadian expression of kdp and ktr peaked at subjective dawn, which may support the acquisition of K⁺ required for the regular diurnal photosynthetic metabolism. These results indicate that Kdp contributes to the maintenance of a basal intracellular K⁺ concentration under conditions of limited K⁺ in natural environments, whereas Ktr mediates fast potassium movements in the presence of greater K⁺ availability. Through their distinct activities, both Ktr and Kdp coordinate the responses of Synechocystis to changes in K⁺ levels under fluctuating environmental conditions.     

Genetic Analysis of the Hox Hydrogenase in the Cyanobacterium Synechocystis sp. PCC 6803 Reveals Subunit Roles in Association,Assembly, Maturation,and Function

Hydrogenases are metalloenzymes that catalyze 2H⁺ + 2e⁻ ↔ H₂. A multisubunit, bidirectional [NiFe]-hydrogenase has been identified and characterized in a number of bacteria, including cyanobacteria, where it is hypothesized to function as an electron valve, balancing reductant in the cell. In cyanobacteria, this Hox hydrogenase consists of five proteins in two functional moieties: a hydrogenase moiety (HoxYH) with homology to heterodimeric [NiFe]-hydrogenases and a diaphorase moiety (HoxEFU) with homology to NuoEFG of respiratory Complex I, linking NAD(P)H ↔ NAD(P)⁺ as a source/sink for electrons. Here, we present an extensive study of Hox hydrogenase in the cyanobacterium Synechocystis sp. PCC 6803. We identify the presence of HoxEFUYH, HoxFUYH, HoxEFU, HoxFU, and HoxYH subcomplexes as well as association of the immature, unprocessed large subunit (HoxH) with other Hox subunits and unidentified factors, providing a basis for understanding Hox maturation and assembly. The analysis of mutants containing individual and combined hox gene deletions in a common parental strain reveals apparent alterations in subunit abundance and highlights an essential role for HoxF and HoxU in complex/subcomplex association. In addition, analysis of individual and combined hox mutant phenotypes in a single strain background provides a clear view of the function of each subunit in hydrogenase activity and presents evidence that its physiological function is more complicated than previously reported, with no outward defects apparent in growth or photosynthesis under various growth conditions.     

Plasma-membrane H+-ATPases are expressed in pitchers of the carnivorous plant Nepenthes alata Blanco

Nepenthes is a unique genus of carnivorous plants that can capture insects in trapping organs called pitchers and digest them in pitcher fluid. The pitcher fluid includes digestive enzymes and is strongly acidic. We found that the fluid pH decreased when prey accumulates in the pitcher fluid of Nepenthes alata. The pH decrease may be important for prey digestion and the absorption of prey-derived nutrients. To identify the proton pump involved in the acidification of pitcher fluid, plant proton-pump homologs were cloned and their expressions were examined. In the lower part of pitchers with natural prey, expression of one putative plasma-membrane (PM) H⁺-ATPase gene, NaPHA3, was considerably higher than that of the putative vacuolar H⁺-ATPase (subunit A) gene, NaVHA1, or the putative vacuolar H⁺-pyrophosphatase gene, NaVHP1. Expression of one PM H⁺-ATPase gene, NaPHA1, was detected in the head cells of digestive glands in the lower part of pitchers, where proton extrusion may occur. Involvement of the PM H⁺-ATPase in the acidification of pitcher fluid was also supported by experiments with proton-pump modulators; vanadate inhibited proton extrusion from the inner surface of pitchers, whereas bafilomycin A₁ did not, and fusicoccin induced proton extrusion. These results strongly suggest that the PM H⁺-ATPase is responsible for acidification of the pitcher fluid of Nepenthes. Received: 8 June 2000 / Accepted: 8 August 2000     

Photosynthetic Electron Transport Involved in PxcA-Dependent Proton Extrusion in Synechocystis sp. Strain PCC6803: Effect of pxcA Inactivation on CO2, HCO3−, and NO3− Uptake

The product of pxcA (formerly known as cotA) is involved in light-induced Na⁺-dependent proton extrusion. In the presence of 2,5-dimethyl-p-benzoquinone, net proton extrusion by Synechocystis sp. strain PCC6803 ceased after 1 min of illumination and a postillumination influx of protons was observed, suggesting that the PxcA-dependent, light-dependent proton extrusion equilibrates with a light-independent influx of protons. A photosystem I (PS I) deletion mutant extruded a large number of protons in the light. Thus, PS II-dependent electron transfer and proton translocation are major factors in light-driven proton extrusion, presumably mediated by ATP synthesis. Inhibition of CO₂ fixation by glyceraldehyde in a cytochrome c oxidase (COX) deletion mutant strongly inhibited the proton extrusion. Leakage of PS II-generated electrons to oxygen via COX appears to be required for proton extrusion when CO₂ fixation is inhibited. At pH 8.0, NO₃⁻ uptake activity was very low in the pxcA mutant at low [Na⁺] (~100 μM). At pH 6.5, the pxcA strain did not take up CO₂ or NO₃⁻ at low [Na⁺] and showed very low CO₂ uptake activity even at 15 mM Na⁺. A possible role of PxcA-dependent proton exchange in charge and pH homeostasis during uptake of CO₂, HCO₃⁻, and NO₃⁻ is discussed.     

NADPH fluorescence in the cyanobacterium Synechocystis sp. PCC 6803: A versatile probe for in vivo measurements of rates,yields and pools

We measured the kinetics of light-induced NADPH formation and subsequent dark consumption by monitoring in vivo its fluorescence in the cyanobacterium Synechocystis PCC 6803. Spectral data allowed the signal changes to be attributed to NAD(P)H and signal linearity vs the chlorophyll concentration was shown to be recoverable after appropriate correction. Parameters associated to reduction of NADP⁺ to NADPH by ferredoxin–NADP⁺-oxidoreductase were determined: After single excitation of photosystem I, half of the signal rise is observed in 8 ms; Evidence for a kinetic limitation which is attributed to an enzyme bottleneck is provided; After two closely separated saturating flashes eliciting two photosystem I turnovers in less than 2 ms, more than 50% of the cytoplasmic photoreductants (reduced ferredoxin and photosystem I acceptors) are diverted from NADPH formation by competing processes. Signal quantitation in absolute NADPH concentrations was performed by adding exogenous NADPH to the cell suspensions and by estimating the enhancement factor of in vivo fluorescence (between 2 and 4). The size of the visible (light-dependent) NADP (NADP⁺ + NADPH) pool was measured to be between 1.4 and 4 times the photosystem I concentration. A quantitative discrepancy is found between net oxygen evolution and NADPH consumption by the light-activated Calvin–Benson cycle. The present study shows that NADPH fluorescence is an efficient probe for studying in vivo the energetic metabolism of cyanobacteria which can be used for assessing multiple phenomena occurring over different time scales.     

Membrane Potential Changes Related to Active Transport of Glycine in Lemna gibba G1

Accumulation of ¹⁴C-labeled glycine and microelectrode techniques were employed to study glycine transport and the effect of glycine on the membrane potential (Δψ) in Lemna gibba G1. Evidence is presented that two processes, a passive uptake by diffusion and a carrier-mediated uptake, are involved in glycine transport into Lemna cells. At the onset of active glycine uptake the component of Δψ which depended on metabolism was decreased. The depolarized membrane repolarized in the presence of glycine. This glycine-induced depolarization followed a saturation curve with increasing glycine concentration which corresponded to carrier-mediated glycine influx kinetics. The transport of glycine was correlated with the metabolically dependent component of Δψ. It is suggested (a) that the transient change in Δψ reflects the operation of an H⁺-glycine cotransport system driven by an electrochemical H⁺ gradient; and (b) that this system is energized by an active H⁺ extrusion. Therefore the maximum depolarization of the membrane consequently depended on both the rate of glycine uptake and the activity of the proton extrusion pump.     

The enthalpy changes upon hydrolysis of guanosine triphosphate anhydride and ester bonds

The effects of N,N′-dicyclohexylcarbodiimide (DCCD), triphenyltin chloride (TPT), and 3,5-di-tert-butyl-4-hydroxybenzylidenemalonomtrile (SP6847) were tested on the light-dependent activities of Halobacterium halobium R₁mR which contains a new retinal protein pigment designated as halorhodopsin but no bacteriorhodospin. DCCD inhibited ATP synthesis either in the light- or in the dark-aerobic conditions without affecting the light-induced proton uptake (ΔH⁺). Although DCCD lowered the membrane potential under dark-anaerobic conditions, the potential increased in the light as high as the control (the light-dependent membrane potential increment Δψ became apparently larger in the presence of DCCD). TPT had negligible effect on ATP synthesis both in the dark or in the light but inhibited markedly ΔH⁺ and partly Δψ. After R₁mR was treated with DCCD, TPT abolished ΔH⁺ almost completely but Δψ only partly. The remaining Δψ was collapsed by SF6847 with a concomitant proton incorporation (pH increase). These results led to the following postulations: (i) In R₁mR, ATP is synthesized by a H⁺-ATPase coupled either to respiration and/or light energization by halorhodopsin; (ii) the majority of protons are incorporated in the light by a mechanism which differs from H⁺-ATPase but is driven by the Δψ generated by halorhodopsin; (iii) TPT acts in this system as a chloride/hydroxide exchanger; (iv) the uncoupler SF6847 carries protons into cells in response to Δψ.     

Relationship between proton motive force and potassium ion transport in Halobacterium halobium envelope vesicles.

Membrane vesicles prepared from Halobacterium halobium extrude protons during illumination, and a pH difference (inside alkaline) and an electrical potential (inside negative) develop. The sizes of these gradients and their relative magnitudes are dependent on a complex interaction among the proton-pumping activity of bacteriorhodopsin, Na⁺ extrusion through an antiport system, and the ability of K⁺ and Cl^? to act as counterions to the electrogenic movement of H⁺. The net result of these variable effects is that the electrical potential is relatively independent of external pH, whereas the pH difference tends toward zero when the pH is increased to 7.5–8. Although the light-induced pH difference is greater in KCl than in NaCl, and the electrical potential smaller, this is not caused by a high permeability of the vesicle membranes to K⁺. The vesicle membrane is poorly permeable to K⁺, as shown by: lack of a K⁺ diffusion potential in the absence of valinomycin, light-induced electrical potentials which are in excess of the chemical potential difference for K⁺, and direct measurements of the slow rate of K⁺ influx during illumination. The finding that the rate of K⁺ uptake is a linear function of external K⁺ concentration between 0 and 1 m is inconsistent with the existence of a specific K⁺ permeation mechanism in these vesicles. Since at external K⁺ concentrations < 1.4 m the extrusion of Na⁺ during illumination proceeds much more rapidly than K⁺ influx, it must be concluded that the vesicles also lose Cl^? and water. Measurements of light-scattering changes confirm that under these conditions the vesicles collapse. The light-induced collapse is diminished only when the inward movement of K⁺ is increased, either by increasing the external K⁺ concentration or by adding valinomycin.     

Ion Fluxes and Phytochrome Protons in Mung Bean Hypocotyl Segments: II. Fluxes of Chloride, Protons, and Orthophosphate in Apical and Subhook Segments

The active form of phytochrome (Pfr) decreased CI⁻ uptake by subhypocotyl hook segments of Phaseolus aureus Roxb. and increased uptake by apical segments. Pfr had similar effects on Pi [³²Pi] uptake. Modulations of Pi [³²Pi] uptake were detectable 10 minutes following photoconversion. Pfr may modulate Pi influx across the plasmalemma. Pfr inhibited H⁺ extrusion by subhook segments and enhanced extrusion by apical hook segments. No rapid effects on H⁺ extrusion were found. Phytochrome may regulate a K⁺ -H⁺ exchange process. The differential responses of the two regions of the hypocotyl are discussed with respect to Pfr-mediated changes in growth and development.     

Light-induced h secretion and the relation to senescence of oat leaves

When abraded oat (Avena sativa L. cv Victory) leaf segments are floated on KCl solution, white light causes acidification of the solution external to leaf tissue. The presence of mannitol amplifies the light-induced proton secretion. Mature leaves as well as young ones acidify the medium in light, while senescing leaves (after 3 to 4 days incubated in water in the dark) lose the ability to produce this response to light. The decrease in H⁺ secretion is already measureable after as little as 30 minutes in darkness, while the increase in proteolysis rate was detected only after 6 hours in dark. The decrease in capacity to secrete protons is one of the symptoms of leaf senescence. Moreover, fusicoccin mimics light in stimulating H⁺ pumping and delaying the senescence in the dark. On the other hand vanadate, an apparent inhibitor of plasma membrane H⁺ ATPase, blocks the acidification and promotes the chlorophyll and protein degradation in leaf segments during the 2-day period of incubation. These results, which show a parallel between cessation of H⁺ secretion and acceleration of senescence, may suggest a regulatory role for H⁺ secretion in leaf senescence.     

Effects of Light on the Membrane Potential of Protoplasts from Samanea saman Pulvini : Involvement of K Channels and the H -ATPase

Rhythmic light-sensitive movements of the leaflets of Samanea saman depend upon ion fluxes across the plasma membrane of extensor and flexor cells in opposing regions of the leaf-movement organ (pulvinus). We have isolated protoplasts from the extensor and flexor regions of S. saman pulvini and have examined the effects of brief 30-second exposures to white, blue, or red light on the relative membrane potential using the fluorescent dye, 3,3′-dipropylthiadicarbocyanine iodide. White and blue light induced transient membrane hyperpolarization of both extensor and flexor protoplasts; red light had no effect. Following white or blue light-induced hyperpolarization, the addition of 200 millimolar K⁺ resulted in a rapid depolarization of extensor, but not of flexor protoplasts. In contrast, addition of K⁺ following red light or in darkness resulted in a rapid depolarization of flexor, but not of extensor protoplasts. In both flexor and extensor protoplasts, depolarization was completely inhibited by tetraethylammonium, implicating channel-mediated movement of K⁺ ions. These results suggest that K⁺ channels are closed in extensor plasma membranes and open in flexor plasma membranes in darkness and that white and blue light, but not red light, close the channels in flexor plasma membranes and open them in extensor plasma membranes. Vanadate treatment inhibited hyperpolarization in response to blue or white light, but did not affect K⁺ -induced depolarization. This suggests that white or blue light-induced hyperpolarization results from activation of the H⁺ -ATPase, but this hyperpolarization is not the sole factor controlling the opening of K⁺ channels.     

Recent advances in understanding the assembly and repair of photosystem II

Background

Photosystem II (PSII) is the light-driven water:plastoquinone oxidoreductase of oxygenic photosynthesis and is found in the thylakoid membrane of chloroplasts and cyanobacteria. Considerable attention is focused on how PSII is assembled in vivo and how it is repaired following irreversible damage by visible light (so-called photoinhibition). Understanding these processes might lead to the development of plants with improved growth characteristics especially under conditions of abiotic stress.

Scope

Here we summarize recent results on the assembly and repair of PSII in cyanobacteria, which are excellent model organisms to study higher plant photosynthesis.

Conclusions

Assembly of PSII is highly co-ordinated and proceeds through a number of distinct assembly intermediates. Associated with these assembly complexes are proteins that are not found in the final functional PSII complex. Structural information and possible functions are beginning to emerge for several of these ‘assembly’ factors, notably Ycf48/Hcf136, Psb27 and Psb28. A number of other auxiliary proteins have been identified that appear to have evolved since the divergence of chloroplasts and cyanobacteria. The repair of PSII involves partial disassembly of the damaged complex, the selective replacement of the damaged sub-unit (predominantly the D1 sub-unit) by a newly synthesized copy, and reassembly. It is likely that chlorophyll released during the repair process is temporarily stored by small CAB-like proteins (SCPs). A model is proposed in which damaged D1 is removed in Synechocystis sp. PCC 6803 by a hetero-oligomeric complex composed of two different types of FtsH sub-unit (FtsH2 and FtsH3), with degradation proceeding from the N-terminus of D1 in a highly processive reaction. It is postulated that a similar mechanism of D1 degradation also operates in chloroplasts. Deg proteases are not required for D1 degradation in Synechocystis 6803 but members of this protease family might play a supplementary role in D1 degradation in chloroplasts under extreme conditions.