pH regulation in apoplastic and cytoplasmic cell compartments of leaves

 The regulation of pH in the apoplast, cytosol and chloroplasts of intact leaves was studied by means of fluorescent pH indicators and as a response of photosynthesis to acid stress. The apoplastic pH increased under anaerobiosis. Aeration reversed this effect. Apoplastic responses to CO₂, HCl or NH₃ differed considerably. Whereas HCl and ammonia caused rapid acidification or alkalinization, the return to initial pH values was slow after cessation of fumigation. Addition of CO₂ either did not produce the acidification expected on the basis of known apoplastic buffering or even caused some alkalinization. Removal of CO₂ shifted the apoplastic pH into the alkaline range before the pH returned to initial steady-state levels. In the presence of vanadate, the alkaline shift was absent and the apoplastic pH returned slowly to the initial level when CO₂ was removed from the atmosphere. In contrast to the response of the apoplast, anaerobiosis acidified the cytosol or, in some species, had little effect on its pH. Acidification was rapidly reversed upon re-admission of oxygen. The CO₂-dependent pH changes were very fast in the cytosol. Considerable alkalinization was observed after removal of CO₂ under aerobic, but not under anaerobic conditions. Rates of the re-entry of protons into the cytosol during recovery from CO₂ stress increased in the presence of oxygen with the length of previous exposure to high CO₂. Effective pH regulation in the chloroplasts was indicated by the recovery of photosynthesis after the transient inhibition of photosynthetic electron flow when CO₂ was increased from 0.038% to 16% in air. As photosynthesis became inhibited under high CO₂, reduction of the electron transport chain increased transiently. The time required for recovery of photosynthesis from inhibition during persistent CO₂ stress was similar to the time required for establishing steady-state pH values in the cytosol under acid stress. The high capacity of leaf cells for the rapid re-attainment of pH homeostasis in the apoplast and the cytoplasm under acid or alkaline stress suggested the rapid activation or deactivation of membrane-localised proton-transporting enzymes and corresponding ion channel regulation for co-transport of anions or counter-transport of cations together with proton fluxes. Acidification of the cytoplasm appeared to activate energy-dependent proton export primarily into the vacuoles whereas apoplastic alkalinization resulted in the pumping of protons into the apoplast. Proton export rates from the cytosol into the apoplast after anaerobiosis were about 100 nmol (m² leaf area)⁻¹ s⁻¹ or less. Proton export under acid stress into the vacuole was about 1200 nmol m⁻² s⁻¹. The kinetics of pH responses to the addition or withdrawal of CO₂ indicated the presence of carbonic anhydrase in the cytosol, but not in the apoplast. Received: 19 July 1999 / Accepted: 29 December 1999     

Chloroplast pH values and buffer capacities in darkened leaves as revealed by CO2 solubilization in vivo

Leaves of the C₃ plants Brassica oleracea L., Datura suaveolens Humb. & Bonpl. ex Willd., Helianthus annuus L. and Nicotiana tabacum L. with open stomata were exposed in a leaf chamber in the dark to CO₂ concentrations varying from 1 to 20% in air. When they were transferred back into CO₂-free air, CO₂ was rapidly released. It originated from dissolved CO₂ and from the bicarbonate in the chloroplast stroma, since vacuoles are acidic and chloroplasts contain carbonic anhydrase which rapidly liberates CO₂ from bicarbonate. The data were fitted to a model which accounts for the CO₂/bicarbonate equilibrium in buffers with different CO₂ concentrations and initial pH values. From this, pH values and CO₂-dependent pH changes in the chloroplast stroma were calculated. The full range of external CO₂ concentration caused acidic shifts up to 1 pH unit. The best fits of the data points were obtained with stromal buffer concentrations ranging from 45 to 65 mM and stromal pH values at low CO₂ between 7.5 and 7.9. Calculated buffer capacities ranged from 23 to 31 mM H⁺ per pH unit. The work shows that measurements of solubilized CO₂ are useful to investigate proton buffering and pH regulation in the chloroplast stroma of intact leaves.Abbreviation Chl chlorophyll This work was supported by Sonderforschungsbereich 251 of the University of Würzburg and the Volkswagenstiftung grant I-67762. We are very grateful to Dr. V. Oja for helpful advice.     

A CO2-Flux Mechanism Operating via pH-Polarity in Hydrilla Verticillata Leaves With C3 and C4 Photosynthesis

The aquatic angiosperm Hydrilla verticillata lacks Kranz anatomy, but has an inducible, C₄-based, CO₂ concentrating mechanism (CCM) that concentrates CO₂ in the chloroplasts. Both C₃ and C₄ Hydrilla leaves showed light-dependent pH polarity that was suppressed by high dissolved inorganic carbon (DIC). At low DIC (0.25 mol m⁻³), pH values in the unstirred water layer on the abaxial and adaxial sides of the leaf were 4.2 and10.3, respectively. Abaxial apoplastic acidification served as a CO₂ flux mechanism (CFM), making HCO₃ ⁻ available for photosynthesis by conversion to CO₂. DIC at 10 mol m⁻³ completely suppressed acidification and alkalization. The data, along with previous results, indicated that inhibition was specific to DIC, and not a buffer effect. Acidification and alkalization did not necessarily show 1:1 stoichiometry; their kinetics for the apolar induction phase differed, and alkalization was less inhibited by 2.5 mol m⁻³ DIC. At low irradiance (50 μmol photons m⁻² s⁻¹), where CCM activity in C₄ leaves is minimized, both leaf types had similar DIC inhibition of pH polarity. However, as irradiance increased, DIC inhibition of C₃ leaves decreased. In C₄ leaves the CFM and CCM seemed to compete for photosynthetic ATP and/or reducing power. The CFM may require less, as at low irradiance it still operated maximally, if [DIC] was low. Iodoacetamide (IA), which inhibits CO₂ fixation in Hydrilla, also suppressed acidification and alkalization, especially in C₄ leaves. IA does not inhibit the C₄ CCM, which suggests that the CFM and CCM can operate independently. It has been hypothesized that irradiance and DIC regulate pH polarity by altering the chloroplastic [DIC], which effects the chloroplast redox state and subsequently redox regulation of a plasma-membrane H⁺-ATPase. The results lend partial support to a down-regulatory role for high chloroplastic [DIC], but do not exclude other sites of DIC action. IA inhibition of pH polarity seems inconsistent with the chloroplast NADPH/NADP⁺ ratio being the redox transducer. The possibility that malate and oxaloacetate shuttling plays a role in CFM regulation requires further investigation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effects of NH4 +, NO3 − and HCO3 − on apoplast pH in the outer cortex of root zones of maize, as measured by the fluorescence ratio of fluorescein boronic acid

A fluorimetric ratio technique was elaborated to measure apoplastic pH in the outer root cortex of maize (Zea mays L.) grown hydroponically. A newly synthesized fluorescent probe, fluorescein boronic acid (pK_a = 5.48), which covalently binds to the cell wall of the outer cell layers, was used. Under conditions of saturating ion concentrations the apoplastic pH was determined along the root axis ranging from 1 to 30 mm behind the root tip. Apoplastic pH was recorded for root segment areas (1 mm²), and pH values of high statistical significance were obtained. With an external solution of pH 5, the apoplastic pH was about pH 5.1 in the division zone, between pH 4.8 and 4.9 in the elongation region and about pH 4.9 in the root hair zone. At an external pH of 8.6, the difference between the external pH and the apoplastic pH was considerably more, with a pH of 5.2–5.3 in all root zones. Addition of 1 mM NH₄ ⁺ caused a small apoplastic pH decrease (0.05 of a pH unit) in all root zones. Apoplastic alkalization upon application of 6 mM NO₃ ⁻ was highest (0.3 of a pH unit) in the zone where root hairs emerge; in the division and early elongation zones, apoplastic pH increased only transiently. In the presence of 10 mM HCO₃ ⁻, NO₃ ⁻ elicited a higher and persistent alkalization (0.06–0.25 of a pH unit) in all root zones. Application of fusicoccin reduced apoplastic pH from 4.85 to 4.75 in the elongation zone, while inhibition of the H⁺-ATPase with vanadate alkalized the apoplast in the root hair zone from pH 5.4 to 5.6. The observed pH differences along the root axis upon differential N supply and application of HCO₃ ⁻ provide evidence that this new pH technique is a useful tool with which to measure apoplastic pH, and in future may permit measurements at microsites at the cell level by use of microscope imaging. Received: 26 August 1998 / Accepted: 4 May 1999     

Resistance to freshwater exposure in White Sea Littorina spp. II: Acid-base regulation

Parameters of acid-base and energy status were studied by in vivo ³¹P-nuclear magnetic resonance spectroscopy in three White Sea Littorina spp. (L.littorea, L. saxatilis and L. obtusata) during prolonged anaerobiosis in freshwater. Intracellular pH decreased significantly, especially during the early period of anaerobiosis, but later the decrease in intracellular pH slowed down considerably, suggesting a capacity for intracellular pH regulation in all three species. There was a trend for intracellular pH to fall most rapidly in the least freshwater-resistant species, L. obtusata, as compared to the most resistant, L. littorea. Non-bicarbonate, non-phosphate buffer values estimated by the homogenate technique were similar in the three studied species (28–37 mmol pH⁻¹ kg⁻¹ wet weight) and did not change during freshwater exposure. The CaCO₃ buffer value of the foot tissues was considerably higher (171–218 mmol pH⁻¹ kg⁻¹ wet weight) and decreased significantly during freshwater exposure. The contribution of the multiple tissue buffering systems to intracellular pH regulation in Littorina spp. shifts between different stages of freshwater exposure. Initially, the non-bicarbonate, non-phosphate tissue buffering system seems to be of major importance for metabolic proton buffering at intracellular pH between 7.5 and 7.0. During later stages of anaerobiosis and at lower intracellular pH, the CaCO₃ buffer is involved in proton buffering. Decrease in the CaCO₃ buffer value during freshwater exposure was in quantitative agreement with the amount of metabolic protons buffered, thus suggesting that CaCO₃ tissue stores may serve as a major buffering system during prolonged anaerobiosis in Littorina spp. Accepted: 23 December 1999     

Leaf chlorosis in oilseed rape plants (Brassica napus) grown on cadmium-polluted soil: causes and consequences for photosynthesis and growth

Brassica napus L. (oilseed rape) was grown from seeds on a reconstituted soil contaminated with cadmium (100 mg Cd kg⁻¹ dry soil), resulting in a marked chlorosis of the leaves which was investigated using a combination of biochemical, biophysical and physiological methods. Spectroscopic and chromatographic analyses of the photosynthetic pigments indicated that chlorosis was not due to a direct interaction of Cd with the chlorophyll biosynthesis pathway. In addition, mineral deficiency and oxidative stress were apparently not involved in the pigment loss. Leaf chlorosis was attributable to a marked decrease in the chloroplast density caused by a reduction in the number of chloroplasts per cell and a change in cell size, suggesting that Cd interfered with chloroplast replication and cell division. Relatively little Cd was found in the chloroplasts and the properties of the photosynthetic apparatus (electron transport, protein composition, chlorophyll antenna size, chloroplast ultrastructure) were not affected appreciably in plants grown on Cd-polluted soil. Depth profiling of photosynthetic pigments by phase-resolved photoacoustic spectroscopy revealed that the Cd-induced decrease in pigment content was very pronounced at the leaf surface (stomatal guard cells) compared to the leaf interior (mesophyll). This observation was consistent with light transmission and fluorescence microscopy analyses, which revealed that stomata density in the epidermis was noticeably reduced in Cd-exposed leaves. Concomitantly, the stomatal conductance estimated from gas-exchange measurements was strongly reduced with Cd. When plants were grown in a high-CO₂ atmosphere (4,000 μl CO₂ l⁻¹), the inhibitory effect of Cd on growth was not cancelled, suggesting that the reduced availability of CO₂ at the chloroplast level associated with the low stomatal conductance was not the main component of Cd toxicity in oilseed rape. Received: 14 July 2000 / Accepted: 27 August 2000     

Assimilate export by leaves of Ricinus communis L. growing under normal and elevated carbon dioxide concentrations: the same rate during the day, a different rate at night

Castor bean (Ricinus communis L.) plants were grown for 5–7 weeks in a controlled environment at 350 μl l⁻¹ or 700 μl l⁻¹ CO₂. Carbon assimilation, assimilate deposition, dark respiration and assimilate mobilization were measured in leaves 2, 3 and 4 (counted from the base of the plant), and a balance sheet of carbon input and export was elaborated for both CO₂ concentrations. Carbon dioxide assimilation was nearly constant over the illumination period, with only a slight depression occurring at the end of the day in mature source leaves, not in young source leaves. Assimilation was ca. 40% higher at 700 μl l⁻¹ than at 350 μl l⁻¹ CO₂. The source leaves increased steadily in weight per unit area during the first 3 weeks, more at 700 μl l⁻¹ than at 350 μl l⁻¹ CO₂. On top of an irreversible weight increase, there was a large gain in dry weight during the day, which was reversed during the night. This reversible weight gain was constant over the life time of the leaf and ca. 80% higher at 700 μl l⁻¹ than at 350 μl l⁻¹. Most of it was due to carbohydrates. The carbon content (as a percentage) was not altered by the CO₂ treatment. Respiration was 25% higher in high-CO₂ plants when based on leaf area, but the same when based on dry weight. The rate of carbon export via the phloem was the same during the daytime in plants grown at 350 μl l⁻¹ and 700 μl l⁻¹ CO₂. During the night the low-CO₂ plants had only 50% of the daytime export rate, in contrast to the high-CO₂ plants which maintained the high export rate. It was concluded that the phloem loading system is saturated during the daytime in both CO₂ regimes, whereas during the night the assimilate supply is reduced in plants in the normal CO₂ concentration. Two-thirds of the carbon exported from the leaves was permanently incorporated as plant dry matter in the residual plant parts. This “assimilation efficiency” was the same for both CO₂ regimes. It is speculated that under 350 μl l⁻¹ CO₂ the growing Ricinus plant operates at sink limitation during the day and at source limitation during the night. Received: 2 February 1999 / Accepted: 19 April 1999     

The effect of elevated CO2 concentrations on K+ and anion channels of Vicia faba L. guard cells

The effects of elevated CO₂ concentrations on stomatal movement, anion- and K⁺-channel activities were examined in guard cells from epidermal strips of Vicia faba. Membrane voltage was measured using intracellular, double-barrelled microelectrodes and ion-channel currents were recorded under voltage clamp during exposure to media equilibrated with ambient (350 μl · l⁻¹), 1000 μl · l⁻¹ and 10 000 μl · l⁻¹ CO₂ in 20% O₂ and 80% N₂. The addition of 1000 μl · l⁻¹ CO₂ to the bathing solution caused stomata to close with a halftime of approx. 40 min, and with 10 000 μl · l⁻¹ CO₂ closure occurred with a similar time course. Under voltage clamp, exposure to 1000 μl · l⁻¹ and 10 000 μl · l⁻¹ CO₂ resulted in a rapid increase (mean, 1.5 ± 0.2-fold, n = 8; range 1.3- to 2.5-fold) in the magnitude of current carried by outward-rectifying K⁺ channels (I_K,out). The effect of CO₂ on I_K,out was essentially complete within 30 s and was independent of clamp voltage, but was associated with 25–40% (mean, 30 ± 4%) decrease in the halftime for current activation. Exposure to CO₂ also resulted in a four-fold increase in background current near the free-running membrane voltage, recorded as the instantaneous current at the start of depolarising and hyperpolarising voltage steps, and a decrease in the magnitude of current carried by inward-rectifying K⁺ channels (I_K,in). The effect of CO₂ on I_K,in was generally slower than on I_K,out; it was allied with a transient acceleration of its activation kinetics during the first 60–120 s of treatment; and it was associated with a negative shift in the voltage-sensitivity of gating over a period of 3–5 min. Measurements carried out to isolate the background currents attributable to anion channels (I_Cl), using tetraethylammonium chloride and CsCl, showed that CO₂ also stimulated I_Cl and dramatically altered its relaxation kinetics. Within the timeframe of CO₂ action at the membrane, no significant effect was observed on cytosolic pH, measured using the fluorescent dye 2′,7′-bis-(2-carboxyethyl)-5,6-carboxyflourescein (BCECF) and ratio fluorescence microphotometry. These results are broadly consistent with the pattern of guard-cell response to abscisic acid, and indicate that guard cells control both anion and K⁺ channels to achieve net solute loss in CO₂. By contrast with the effects of abscisic acid, however, the data indicate that CO₂ action is not mediated through changes in cytosolic pH and thereby implicate new and, as yet, unidentified pathway(s) for channel regulation in the guard cells. Received: 8 January 1997 / Accepted: 28 February 1997     

Glucose Respiration in the Intact Chloroplast of Chlamydomonas reinhardtii

Chloroplastic respiration was monitored by measuring ¹⁴CO₂ from ¹⁴C glucose in the darkened Chlamydomonas reinhardtii F-60 chloroplast. The patterns of ¹⁴CO₂ evolution from labeled glucose in the absence and presence of the inhibitors iodoacetamide, glycolate-2-phosphate, and phosphoenolpyruvate were those expected from the oxidative pentose phosphate cycle and glycolysis. The K_m for glucose was 56 micromolar and for MgATP was 200 micromolar. Release of ¹⁴CO₂ was inhibited by phloretin and inorganic phosphate. Comparing the inhibition of CO₂ evolution generated by pH 7.5 with respect to pH 8.2 (optimum) in chloroplasts given C-1, C-2, and C-6 labeled glucose indicated that a suboptimum pH affects the recycling of the pentose phosphate intermediates to a greater extent than CO₂ evolution from C-1 of glucose. Respiratory inhibition by pH 7.5 in the darkened chloroplast was alleviated by NH₄Cl and KCl (stromal alkalating agents), iodoacetamide (an inhibitor of glyceraldehyde 3-phosphate dehydrogenase), or phosphoenolpyruvate (an inhibitor of phosphofructokinase). It is concluded that the site which primarily mediates respiration in the darkened Chlamydomonas chloroplast is the fructose-1,6-bisphosphatase/phosphofructokinase junction. The respiratory pathways described here can account for the total oxidation of a hexose to CO₂ and for interactions between carbohydrate metabolism and the oxyhydrogen reaction in algal cells adapted to a hydrogen metabolism.     

Light-induced alkalization of the chloroplast stroma in vivo as estimated from the CO2 capacity of intact sunflower leaves

Rapid CO₂ gas exchange by Helianthus leaves was analysed kinetically using a computer model which distinguished different components of the gas exchange by different time constants. A rapid phase of CO₂ uptake was ascribed to the solubilization of CO₂ in all leaf compartments and to the conversion of the dissolved CO₂ to HCO₃⁻ in the chloroplast stroma which contains carbonic anhydrase. From stromal $\text{HCO}{}_3{}^{\mathrm{-}}\text{CO}{}_2$ ratios the stroma pH of darkened leaves was estimated to be close to 7.5. Occasionally, values as high as 8 or as low as 7 were also obtained. If fast HCO₃⁻ formation also occurs in the cytosol, pH values may be lower by about 0.3 pH units than those calculated under the assumption that carbonic anhydrase is localized in chloroplasts only. Illumination with a light intensity close to saturation of photosynthesis caused an increase in CO₂ solubilization which indicated the alkalization of the chloroplast stroma by about 0.6 pH units. This is an underestimation, if the pH of cytosol decreases in the light liberating CO₂ by the action of carbon anhydrase. An alkalization of the stroma by 0.6 pH units indicates the export of about 450 nmol H⁺/mg chlorophyll from the stroma. This forms the basis of a large transthylakoid pH gradient which drives light-dependent ATP synthesis. A pH gradient between stroma and cytosol is capable of supporting secondary gradients between these compartments in the light, such as a gradient in the $\text{ATP}\text{ADP}$ ratio. On darkening, the stroma alkalization was reversed. The rate of stroma acidification was much higher in the presence of CO₂ than in its absence.     

Solute accumulation and decreased photosynthesis in leaves of potato plants expressing yeast-derived invertase either in the apoplast, vacuole or cytosol

Potato (Solanum tuberosum cv. Désirée) plants expressing yeast invertase directed either to the apoplast, vacuole or cytosol were biochemically and physiologically characterised. All lines of transgenic plants showed similarities to plants growing under water stress. Transformants were retarded in growth, and accumulated hexoses and amino acids, especially proline, to levels up to 40-fold higher than those of the wild types. In all transformants rates of CO₂ assimilation and leaf conductance were reduced. From the unchanged intercellular partial pressure of CO₂ and apoplastic cis-abscisic acid (ABA) content of transformed leaves it was concluded that the reduced rate of CO₂ assimilation was not caused by a limitation in the availability of CO₂ for␣the ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco). In the transformants the amount of Rubisco protein was not reduced, but both activation state and carboxylation efficiency of photosynthesis were lowered. In vacuolar and cytosolic transformants this inhibition of Rubisco might be caused by a changed ratio of organic bound and inorganic phosphate, as indicated by a doubling of phosphorylated intermediates. But in apoplastic transformants the pattern of phosphorylated intermediates resembled that of leaves of water-stressed potato plants, although the cause of inhibition of photosynthesis was not identical. Whereas in water-stressed plants increased contents of the phytohormone ABA are supposed to mediate the adaptation to water stress, no contribution of ABA to reduction of photosynthesis could be detected in invertase transformants. Received: 29 May 1996 / Accepted: 30 December 1996     

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     

Discrepancy between nitrate reduction rates in intact leaves and nitrate reductase activity in leaf extracts: What limits nitrate reduction in situ?

Nitrate reductase (NR) activity in spinach leaf extracts prepared in the presence of a protein phosphatase inhibitor (50 μM cantharidine) was measured in the presence of Mg²⁺ (NRact) or EDTA (NRmax), under substrate saturation. These in-vitro activities were compared with nitrate reduction rates in leaves from nitrate-sufficient plants. Spinach leaves containing up to 60 μmol nitrate per g fresh weight were illuminated in air with their petiole in water. Their nitrate content decreased with time, permitting an estimation of nitrate reduction in situ. The initial rates (1–2 h) of nitrate consumption were usually lower than NRact, and with longer illumination time (4 h) the discrepancy grew even larger. When leaves were fed through their petiole with 30 mM nitrate, initial in-situ reduction rates calculated from nitrate uptake and consumption were still lower than NRact. However, nitrate feeding through the petiole maintained the in situ-nitrate reduction rate for a longer time. Initial rates of nitrate reduction in situ only matched NRact when leaves were illuminated in 5% CO₂. In CO₂-free air or in the dark, both NRact and in-situ nitrate reduction decreased, but NRact still exceeded in-situ reduction. More extremely, under anoxia or after feeding 5-amino-4-imidazole carboxyamide ribonucleoside in the dark, NR was activated to the high light level; yet in spite of that, nitrate reduction in the leaf remained very low. It was examined whether the standard assay for NRact would overestimate the in-situ rates due to a dissociation of the inactive phospho-NR-14-3-3 complex after extraction and dilution, but no evidence for that was found. In-situ NR obviously operates below substrate saturation, except in the light at high ambient CO₂. It is suggested that in the short term (2 h), nitrate reduction in situ is mainly limited by cytosolic NADH, and cytosolic nitrate becomes limiting only after the vacuolar nitrate pool has been partially emptied. Received: 19 June 1999 / Accepted: 12 October 1999     

The size of the lumenal proton pool in leaves during induction and steady-state photosynthesis

This report describes a new method to measure the chloroplastic lumenal proton pool in leaves (tobacco and sunflower). The method is based on measurement of CO₂ outbursts from leaves caused by the shift in the CO₂ + H₂O ↔ HCO₃ ⁻ + H⁺ equilibrium in the chloroplast stroma as protons return from the lumen after darkening. Protons did not accumulate in the lumen to a significant extent when photosynthesis was light-limited, but a large pool of >100 μmol H⁺ m⁻² accumulated in the lumen as photosynthesis became light-saturated. During thylakoid energization in the light, large amounts of protons are moved from binding sites in the stroma to binding sites in the lumen. The transthylakoidal difference in the chemical potential of free protons (ΔpH) is largely based on the difference in the chemical potential of bound protons in the lumenal and stromal compartments (pK). Over the course of the dark-light induction of photosynthesis protons accumulate in the lumen during reduction of 3-phosphoglycerate. The accumulation of electrons in reduced compounds of the stroma and cytosol is the natural cause for accumulation of a stoichiometric pool of lumenal protons during this transient event.     

Inhibition of chloroplastic respiration by osmotic dehydration

The respiratory capacity of isolated spinach (Spinacia oleracea L.) chloroplasts, measured as the rate of ¹⁴CO₂ evolved from the oxidative pentose phosphate cycle in darkened chloroplasts exogenously supplied with [¹⁴C]glucose, was progressively diminished by escalating osmotic dehydration with betaine or sorbitol. Comparing the inhibitions of CO₂ evolution generated by osmotic dehydration in chloroplasts given C-1 and C-6 labeled glucose, 54% and 84% respectively, indicates that osmotic dehydration effects to a greater extent the recycling of the oxidative pentose phosphate intermediates, fructose-6P and glyceraldehyde-3P. Respiratory inhibition in the darkened chloroplast could be alleviated by addition of NH₄Cl (a stromal alkylating agent), iodoacetamide) an inhibitor of glyceraldehyde-3P dehydrogenase), or glycolate-2P (an inhibitor of phosphofructokinase). It is concluded that the site which primarily mediates respiratory inhibition in the darkened chloroplast occurs at the fructose 1,6-bisphosphatase/phosphofructokinase junction.     

Effects of elevated CO<Subscript>2</Subscript> applied to potato roots on the anatomy and ultrastructure of leaves

The root system of potato (Solanum tuberosum L. cv. Favorita) plants was treated with different O₂ and CO₂ concentrations for 35 d in aeroponic culture. Under 21 or 5 % O₂ in the root zones, the thickness of leaves and palisade parenchyma significantly increased at 3 600 μmol(CO₂) mol⁻¹ in the root zone, compared with CO₂ concentration 380 μmol mol⁻¹ or low CO₂ concentration (100 μmol mol⁻¹). In addition, smaller cells of palisade tissue, more intercellular air spaces and partially two layers of palisade cells were observed in the leaves with root-zone CO₂ enrichment. Furthermore, there was a significant increase in the size of chloroplasts and starch grains, and the number of starch grains per chloroplast due to elevated CO₂ only under 21 % O₂. In addition, a significant decline in the thickness of grana and the number of lamellas, but no significant differences in the number of grana per chloroplast were observed under elevated CO₂ concentration. The accumulation of starch grains in the chloroplast under elevated CO₂ concentration could change the arrangement of grana thylakoids and consequently inhibited the absorption of sun radiation and photosynthesis of potato plants.     

Regulation of the photosynthetic electron transport chain

The regulation of electron transport between photosystems II and I was investigated in the plant Silene dioica L. by means of measurement of the kinetics of reduction of P₇₀₀ following a light-to-dark transition. It was found that, in this species, the rate constant for P₇₀₀ reduction is sensitive to light intensity and to the availability of CO₂. The results indicated that at 25 °C the rate of electron transport is down-regulated by approximately 40–50% relative to the maximum rate achievable in saturating CO₂ and that this down-regulation can be explained by regulation of the electron transport chain itself. Measurements of the temperature sensitivity of this rate constant indicated that there is a switch in the rate-limiting step that controls electron transport at around 20 °C: at higher temperatures, CO₂ availability is limiting; at lower temperatures some other process regulates electron transport, possibly a diffusion step within the electron transport chain itself. Regulation of electron transport also occurred in response to drought stress and sucrose feeding. Measurements of non-photochemical quenching of chlorophyll fluorescence did not support the idea that electron transport is regulated by the pH gradient across the thylakoid membrane, and the possibility is discussed that the redox potential of a stromal component may regulate electron transport. Received: 4 March 1999 / Accepted: 25 May 1999     

Photosynthetic carbon metabolism in leaves of transgenic tobacco (Nicotiana tabacum L.) containing decreased amounts of fructose 2,6-bisphosphate

The aim of this work was to examine the role of fructose 2,6-bisphosphate (Fru-2,6-P₂) in photosynthetic carbon partitioning. The amount of Fru-2,6-P₂ in leaves of tobacco (Nicotiana tabacum L. cv. Samsun) was reduced by introduction of a modified mammalian gene encoding a functional fructose-2,6-bisphosphatase (EC 3.1.3.46). Expression of this gene in transgenic plants reduced the Fru-2,6-P₂ content of darkened leaves to between 54% and 80% of that in untransformed plants. During the first 30 min of photosynthesis sucrose accumulated more rapidly in the transgenic lines than in the untransformed plants, whereas starch production was slower in the transgenic plants. On illumination, the proportion of ¹⁴CO₂ converted to sucrose was greater in leaf disks of transgenic lines possessing reduced amounts of Fru-2,6-P₂ than in those of the control plants, and there was a corresponding decrease in the proportion of carbon assimilated to starch in the transgenic lines. Furthermore, plants with smaller amounts of Fru-2,6-P₂ had lower rates of net CO₂ assimilation. In illuminated leaves, decreasing the amount of Fru-2,6-P₂ resulted in greater amounts of hexose phosphates, but smaller amounts of 3-phosphoglycerate and dihydroxyacetone phosphate. These differences are interpreted in terms of decreased inhibition of cytosolic fructose-1,6-bisphosphatase resulting from the lowered Fru-2,6-P₂ content. The data provide direct evidence for the importance of Fru-2,6-P₂ in co-ordinating chloroplastic and cytosolic carbohydrate metabolism in leaves in the light. Received: 8 February 2000 / Accepted: 25 April 2000     

Apoplastic pH and Fe3+ Reduction in Intact Sunflower Leaves

It has been hypothesized that under NO₃⁻ nutrition a high apoplastic pH in leaves depresses Fe³⁺ reductase activity and thus the subsequent Fe²⁺ transport across the plasmalemma, inducing Fe chlorosis. The apoplastic pH in young green leaves of sunflower (Helianthus annuus L.) was measured by fluorescence ratio after xylem sap infiltration. It was shown that NO₃⁻ nutrition significantly increased apoplastic pH at distinct interveinal sites (pH ≥ 6.3) and was confined to about 10% of the whole interveinal leaf apoplast. These apoplastic pH increases presumably derive from NO₃⁻/proton cotransport and are supposed to be related to growing cells of a young leaf; they were not found in the case of sole NH₄⁺ or NH₄NO₃ nutrition. Complementary to pH measurements, the formation of Fe²⁺-ferrozine from Fe³⁺-citrate was monitored in the xylem apoplast of intact leaves in the presence of buffers at different xylem apoplastic pH by means of image analysis. This analysis revealed that Fe³⁺ reduction increased with decreasing apoplastic pH, with the highest rates at around pH 5.0. In analogy to the monitoring of Fe³⁺ reduction in the leaf xylem, we suggest that under alkaline nutritional conditions at interveinal microsites of increased apoplastic pH, Fe³⁺ reduction is depressed, inducing leaf chlorosis. The apoplastic pH in the xylem vessels remained low in the still-green veins of leaves with intercostal chlorosis.     

Flexible coupling between light-dependent electron and vectorial proton transport in illuminated leaves of C3 plants. Role of photosystem I-dependent proton pumping

The role of cyclic electron transport has been re-examined in leaves of C₃ plants because the bioenergetics of chloroplasts (H⁺/e = 3 in the presence of a Q-cycle; H⁺/ATP = 4 of ATP synthesis) had suggested that cyclic electron flow has no function in C₃ photosynthesis. After light activation of pea leaves, the dark reduction of P700 (the donor pigment of PSI) following far-red oxidation was much accelerated. This corresponded to loss of sensitivity of P700 to oxidation by far-red light and a large increase in the number of electrons available to reduce P700⁺ in the dark. At low CO₂ and O₂ molar ratios, far-red light was capable of decreasing the activity of photosystem II (measured as the ratio of variable to maximal chlorophyll fluorescence, F_v/F_m) and of increasing light scattering at 535 nm and zeaxanthin synthesis, indicating formation of a transthylakoid pH gradient. Both the light-induced increase in the number of electrons capable of reducing far-red-oxidised P700 and the decline in F_v/F_m brought about by far-red in leaves were prevented by methyl viologen. Antimycin A inhibited CO₂-dependent O₂ evolution of pea leaves at saturating but not under limiting light; in its presence, far-red light failed to decrease F_v/F_m. The results indicate that cyclic electron flow regulates the quantum yield of photosystem II by decreasing the intrathylakoid pH when there is a reduction in the availability of electron acceptors at the PSI level (e.g. during drought or cold stresses). It also provides ATP for the carbon-reduction cycle under high light. Under these conditions, the Q-cycle is not able to maintain a H⁺/e ratio of 3 for ATP synthesis: we suggest that the ratio is flexible, not obligatory. Received: 23 February 1999 / Accepted: 19 August 1999