Transient alkalinization in the leaf apoplast of Vicia faba L. depends on NaCl stress intensity: an in situ ratio imaging study

The apoplast is suggested to be involved not only in the response, but also in the perception and transduction of various environmental signals. In this context, apoplastic alkalinization has previously been discussed as a general stress factor caused by abiotic and biotic stress events. In this study, an ion-sensitive fluorescence probe in combination with inverted fluorescence microscopy has been used for in planta monitoring of apoplastic shoot pH during challenging of Vicia faba L. plants by NaCl stress encountered at the roots. We demonstrate that transient increases in leaf apoplastic pH are dependent on the NaCl stress intensity. Moreover, we have visualized spatial pH gradients within the leaf apoplast. Our results indicate that these pH responses are propagated from root to leaf and that this occurs along the apoplast.     

Apoplastic domains and sub-domains in the shoots of etiolated corn seedlings

Light Green, an apoplastic probe, was applied to the cut mesocotyl base or to the cut coleoptile apex of etiolated seedlings of Zea mays L. cv. Silver Queen. Probe transport was measured and its tissue distribution determined. In the mesocotyl, there is an apoplastic barrier between cortex and stele. This barrier creates two apoplastic domains which are non-communicating. A kinetic barrier exists between the apoplast of the mesocotyl stele and that of the coleoptile. This kinetic barrier is not absolute and there is limited communication between the apoplasts of the two regions. This kinetic barrier effectively creates two sub-domains. In the coleoptile, there is communication between the apoplast of the vascular strands and that of the surrounding cortical tissue. No apoplastic communication was observed between the coleoptile cortex and the mesocotyl cortex. Thus, the apoplastic space of the coleoptile cortex is a sub-domain of the integrated coleoptile domain and is separate from that of the apoplastic domain of the mesocotyl cortex.     

Apoplastic pH during low-oxygen stress in Barley

BACKGROUND AND AIMS: Anoxia leads to an energy crisis, tolerance of which varies from plant to plant. Although the apoplast represents an important storage and reaction space, and engages in the mediation of membrane transport, this extracellular compartment has not yet been granted a role during oxygen shortage. Here, an attempt is made to highlight the importance of the apoplast during oxygen stress and to test whether information about it is transferred systemically in Hordeum vulgare. METHODS: Non-invasive ion-selective microprobes were used which, after being inserted through open stomata, directly contact the apoplastic fluid and continuously measure the apoplastic pH and changes to it. KEY RESULTS: (a) Barley leaves respond to oxygen stress with apoplastic alkalinization and membrane depolarization. These responses are persistent under anoxia (N2; O2 < 3%) but transient under hypoxia. (b) Being applied to the root, the information 'anoxia' is signalled to the leaf as an increase in pH, whereas 'hypoxia' is not: flooding of the roots within the first 2 h has no effect on the leaf apoplastic pH, whereas anoxia (N2) or chemical anoxia (NaCN/salicylic hydroxamic acid) rapidly increase the leaf apoplastic pH. (c) Under anoxia, the proton motive force suffers a decrease by over 70 %, which impairs H(+) -driven transport. CONCLUSIONS: Although anoxia-induced apoplastic alkalinization is a general response to stress, its impact on the proton motive force (reduction) and thus on transport mediation of energy-rich compounds is evident. It is concluded that anoxia tolerance depends on how the plant is able to hold the proton motive force and H(+) turnover at a level that guarantees sufficient energy is harvested to overcome the crisis.     

Does high bicarbonate supply to roots change availability of iron in the leaf apoplast?

The role of the leaf apoplast in iron (Fe) uptake into the leaf symplast is insufficiently understood, particularly in relation to the supposed inactivation of Fe in leaves caused by elevated bicarbonate in calcareous soils. It has been supposed that high bicarbonate supply to roots increases the pH of the leaf apoplast which decreases the physiological availability of Fe in leaf tissues. The study reported here has been carried out with sunflower plants grown in nutrient solution and with grapevine plants grown on calcareous soil under field conditions. The data obtained clearly show that the pH of the leaf apoplastic fluid was not affected by high bicarbonate supply in the root medium (nutrient solution and field experiments). The concentrations of total, symplastic and apoplastic Fe were decreased in chlorotic leaves of both sunflower (nutrient solution experiment) and grapevine plants in which leaf expansion was slightly inhibited (field experiment). However, in grapevine showing severe inhibition of leaf growth, total Fe concentration in chlorotic leaves was the same or even higher than in green ones, indicative to the so-called `chlorosis paradox'. The findings do not support the hypothesis of Fe inactivation in the leaf apoplast as the cause of Fe deficiency chlorosis since no increase was found in the relative amount of apoplastic Fe (% of total leaf Fe) either in the leaves of sunflower or grapevine plants. It is concluded that high bicarbonate concentration in the soil solution does not decrease Fe availability in the leaf apoplast.     

Apoplastic pH and Fe(3+) reduction in intact sunflower leaves

It has been hypothesized that under NO(3)(-) nutrition a high apoplastic pH in leaves depresses Fe(3+) reductase activity and thus the subsequent Fe(2+) 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 NO(3)(-) 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 NO(3)(-)/proton cotransport and are supposed to be related to growing cells of a young leaf; they were not found in the case of sole NH(4)(+) or NH(4)NO(3) nutrition. Complementary to pH measurements, the formation of Fe(2+)-ferrozine from Fe(3+)-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 Fe(3+) reduction increased with decreasing apoplastic pH, with the highest rates at around pH 5. 0. In analogy to the monitoring of Fe(3+) reduction in the leaf xylem, we suggest that under alkaline nutritional conditions at interveinal microsites of increased apoplastic pH, Fe(3+) 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.     

Early changes in apoplast composition associated with defence and disease in interactions between Phaseolus vulgaris and the halo blight pathogen Pseudomonas syringae Pv. phaseolicola

The apoplast is the arena in which endophytic pathogens such as Pseudomonas syringae grow and interact with plant cells. Using metabolomic and ion analysis techniques, this study shows how the composition of Phaseolus vulgaris leaf apoplastic fluid changes during the first six hours of compatible and incompatible interactions with two strains of P. syringae pv. phaseolicola (Pph) that differ in the presence of the genomic island PPHGI‐1. Leaf inoculation with the avirulent island‐carrying strain Pph 1302A elicited effector‐triggered immunity (ETI) and resulted in specific changes in apoplast composition, including increases in conductivity, pH, citrate, γ‐aminobutyrate (GABA) and K⁺, that are linked to the onset of plant defence responses. Other apoplastic changes, including increases in Ca²⁺, Fe^2/3+ Mg²⁺, sucrose, β‐cyanoalanine and several amino acids, occurred to a relatively similar extent in interactions with both Pph 1302A and the virulent, island‐less strain Pph RJ3. Metabolic footprinting experiments established that Pph preferentially metabolizes malate, glucose and glutamate, but excludes certain other abundant apoplastic metabolites, including citrate and GABA, until preferred metabolites are depleted. These results demonstrate that Pph is well‐adapted to the leaf apoplast metabolic environment and that loss of PPHGI‐1 enables Pph to avoid changes in apoplast composition linked to plant defences.     

Effect of aluminium on the activity of apoplastic acid phosphatase and the exudation of macromolecules by roots and suspension-culture cells ofZea mays L.

Aluminium accumulates predominantly in the root apoplast where it binds to the pectin matrix of the cell wall with its negative charges. In this study, we investigated whether short-term Al treatment (2 h) affects the activity of apoplastic acid phosphatase and the exudation of macromolecules by roots and suspension-culture cells of Zea mays L. The pectin content of the cell cultures was modified by long-term adaptation to NaCl stress or long-term adaptation to the cellulose-synthesis inhibitor 2,6-dichlorbenzonitrile (DCB), and by short-term treatment for up to 15 min with pectolyase. At pH 4.5, neither acid phosphatase activity of commercial enzyme preparations nor of exudates from root-tips and suspension-cells of Zea mays L. were affected directly by Al. However, the exudation and the activity of apoplastic acid phosphatase was reduced to a greater extent by Al cells with high pectin content than in cells with normal pectin content. The strongest reduction of acid phosphatase exudation was observed in pectolyase-treated cells with the lowest pectin content. Al reduced not only the release of acid phosphatase from the suspension cells, but also the release of total proteins and pectins. However, no relationship existed between the magnitude of Al-induced reduction of protein and pectin release and the cell pectin contents. These results support the assumption that Al modifies cell-wall and plasma-membrane transport-properties for macromolecules and the activity of apoplastic enzymes thus modifying Al sensitivity.     

Rapid apoplastic pH measurement in Arabidopsis leaves using a fluorescent dye

In plants, the extracellular space (apoplast) is one of the main places where exchange of molecules occurs between cells. Not only is this compartment involved in the storage of multiple metabolites and ions, including calcium and protons, but it also plays a role in the transmission of signaling molecules for cell-to-cell communication. It has recently been shown multiple times that these two aspects are linked and can influence each other. In particular, apoplast pH was shown as a primary regulator of auxin (IAA) transport in Arabidopsis thaliana. To prove the role of apoplastic pH, we have developed a protocol for apoplastic fluid extraction from Arabidopsis leaves, followed by pH determination using the 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) fluorescent dye. This technique successfully allows one to monitor apoplastic pH variations among different plant lines and to link changes in apoplastic pH to cellular responses in the plant.     

Solute accumulation differs in the vacuoles and apoplast of ripening grape berries

Phloem unloading is thought to switch from a symplastic route to an apoplastic route at the beginning of ripening in grape berries and some other fleshy fruits. However, it is unclear whether different solutes accumulate in both the mesocarp vacuoles and the apoplast. We modified a method developed for tomato fruit to extract apoplastic sap from grape berries and measured the changes in apoplastic and vacuolar pH, soluble sugars, organic acids, and potassium in ripening berries of Vitis vinifera ‘Merlot’ and V. labruscana ‘Concord’. Solute accumulation varied by genotype, compartment, and chemical species. The apoplast pH was substantially higher than the vacuolar pH, especially in Merlot (approximately two units). However, the vacuole–apoplast proton gradient declined during ripening and in Merlot, but not in Concord, collapsed entirely at maturity. Hexoses accumulated in both the vacuoles and apoplast but at different rates. Organic acids, especially malate, declined much more in the vacuoles than in the apoplast. Potassium accumulated in the vacuoles and apoplast of Merlot. In Concord, by contrast, potassium increased in the vacuoles but decreased in the apoplast. These results suggest that solutes in the fruit apoplast are tightly regulated and under developmental control.     

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     

Apoplastic pH and FeIII reduction in young sunflower (Helianthus annuus) roots

The relationship between the apoplastic pH in young sunflower roots ( Helianthus annuus L.) and the plasmalemma ferric chelate reductase (FC-R; EC 1.16.1.7) activity in roots was investigated. The hypothesis was tested that a high apoplastic pH depresses FC-R activity, thereby restricting the uptake of Fe²⁺ into the cytosol. Until recently, little has been known about this relationship, because pH and redox reaction measurements are difficult to perform within the confines of the root apoplast. We recorded the apoplastic pH by means of the fluorescence ratio in conjunction with video microscopy by covalently tagging fluorescein boronic acid to OH groups of the root cell wall. Fe^III reduction was measured using a similar approach by tagging ferrozine diboronic acid with OH groups of the cell wall. Ferrozine forms an Fe²⁺ complex, thus indicating the reduction of ferric iron. In roots bathing in buffered outer solutions of different pH, a high pH sensitivity of apoplastic Fe^III reduction was found, with the highest ferric iron reduction rates at an apoplastic pH of 4.9; above an apoplastic pH of 5.3, no reduction was observed. Nitrate in the bathing solution increased the apoplastic pH and hence depressed the Fe^III reduction; ammonium had the reverse effect. Nitrate together with HCO₃^–, a combination which is typical of calcareous soils, had the strongest depressing effect. From the results, it can be concluded that the main reason for the frequently occurring iron deficiency chlorosis of plants grown on calcareous soils is the inhibition of Fe^III reduction in the apoplast, and hence Fe²⁺ uptake into the cytosol.     

Priming xylem for stress recovery depends on coordinated activity of sugar metabolic pathways and changes in xylem sap pH

Some plant species are capable of significant reduction of xylem embolism during recovery from drought despite stem water potential remains negative. However, the functional biology underlying this process is elusive. We subjected poplar trees to drought stress followed by a period of recovery. Water potential, hydraulic conductivity, gas exchange, xylem sap pH, and carbohydrate content in sap and woody stems were monitored in combination with an analysis of carbohydrate metabolism, enzyme activity, and expression of genes involved in sugar metabolic and transport pathways. Drought resulted in an alteration of differential partitioning between starch and soluble sugars. Upon stress, an increase in the starch degradation rate and the overexpression of sugar symporter genes promoted the efflux of disaccharides (mostly maltose and sucrose) to the apoplast. In turn, the efflux activity of the sugar‐proton cotransporters caused a drop in xylem pH. The newly acidic environment induced the activity of apoplastic invertases leading to the accumulation of monosaccharides in the apoplast, thus providing the main osmoticum necessary for recovery. During drought and recovery, a complex network of coordinated molecular and biochemical signals was activated at the interface between xylem and parenchyma cells that appeared to prime the xylem for hydraulic recovery.     

The apoplastic pH of the substomatal cavity of Vicia faba leaves and its regulation responding to different stress factors.

The apoplastic pH of the substomatal cavity is an essential determinant of stomatal movement. In detached leaves of Vicia faba substomatal apoplastic pH and its dependence on external (stress) factors was investigated using a non-invasive approach: pH-microsensors were inserted into open stomata, and upon contact with the apoplastic fluid, pH was measured continuously, as apoplastic pH was challenged by changed conditions of light, atmosphere (NH(3), CO(2)), and xylem sap (abscisic acid, cyanide, fusicoccin, pH, inorganic salts). Apoplastic pH proved extremely sensitive to infiltration and local flooding, which rapidly increased the apoplastic pH by more than 1.5 pH units. Recovery from infiltration took several hours, during which light effects on the apoplastic pH were strongly impeded. This indicates that pH tests carried out under such conditions may not be representative of the undisturbed leaf. NH(3), flushed across the stomata, yielded a rapid apoplastic alkalinization from which an apoplastic buffer capacity of 2-3 mM per pH unit was calculated. Fusicoccin, fed into the xylem sap acidified the apoplast, whereas cyanide alkalized it, thus underscoring the importance of the plasma membrane H(+) pump for apoplastic pH regulation. To address the question to what extent pH was a drought signal, the effect of iso-osmotic pH changes, fed into the xylem through the petiole were tested. It is demonstrated that the apoplastic response remained below 0.1 pH per pH unit imposed, regardless of the buffer capacity. An increase in the osmolarity of the bath solution (harbouring the cut petiole) using KCl, NaCl, CaCl(2) or sorbitol alkalized the substomatal apoplast. It is suggested that pH may only act as drought signal when accompanied by elevated osmolarity.     

Nitrate does not result in iron inactivation in the apoplast of sunflower leaves

It has been hypothesized that nitrate (NO(3)(-)) nutrition might induce iron (Fe) deficiency chlorosis by inactivation of Fe in the leaf apoplast (H.U. Kosegarten, B. Hoffmann, K. Mengel [1999] Plant Physiol 121: 1069-1079). To test this hypothesis, sunflower (Helianthus annuus L. cv Farnkasol) plants were grown in nutrient solutions supplied with various nitrogen (N) forms (NO(3)(-), NH(4)(+) and NH(4)NO(3)), with or without pH control by using pH buffers [2-(N-morpholino)ethanesulfonic acid or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid]. It was shown that high pH in the nutrient solution restricted uptake and shoot translocation of Fe independently of N form and, therefore, induced Fe deficiency chlorosis at low Fe supply [1 micro M ferric ethylenediaminedi(O-hydroxyphenylacetic acid)]. Root NO(3)(-) supply (up to 40 mM) did not affect the relative distribution of Fe between leaf apoplast and symplast at constant low external pH of the root medium. Although perfusion of high pH-buffered solution (7.0) into the leaf apoplast restricted (59)Fe uptake rate as compared with low apoplastic solution pH (5.0 and 6.0, respectively), loading of NO(3)(-) (6 mM) showed no effect on (59)Fe uptake by the symplast of leaf cells. However, high light intensity strongly increased (59)Fe uptake, independently of apoplastic pH or of the presence of NO(3)(-) in the apoplastic solution. Finally, there are no indications in the present study that NO(3)(-) supply to roots results in the postulated inactivation of Fe in the leaf apoplast. It is concluded that NO(3)(-) nutrition results in Fe deficiency chlorosis exclusively by inhibited Fe acquisition by roots due to high pH at the root surface.     

Proteins accumulate in the apoplast of winter rye leaves during cold acclimation

During cold acclimation, winter rye ( Secale cereale L.) plants develop the ability to tolerate freezing temperatures by forming ice in intercellular spaces and xylem vessels. In this study, proteins were extracted from the apoplast of rye leaves to determine their role in controlling extracellular ice formation. Several polypeptides in the 15 to 32 kDa range accumulated in the leaf apoplast during cold acclimation at 5°C and decreased during deacclimation at 20°C. A second group of polypeptides (63, 65 and 68 kDa) appeared only when the leaves were maximally frost tolerant. Ice nucleation activity, as well as the previously reported antifreeze activity, was higher in apoplastic extracts from cold-acclimated than from nonacclimated rye leaves. These results indicate that apoplastic proteins exert a direct influence on the growth of ice. In addition, freezing injury was greater in extracted cold-acclimated leaves than in unextracted cold-acclimated leaves, which suggests that the proteins present in the apoplast are an important component of the mechanism by which winter rye leaves tolerate ice formation     

Apoplastic pH in corn root gravitropism: a laser scanning confocal microscopy measurement

The ability to measure the pH of the apoplast in situ is of special interest as a test of the cell wall acidification theory. Optical sectioning of living seedlings of corn roots using the laser scanning confocal microscope (LSCM) permits us to make pH measurements in living tissue. The pH of the apoplast of corn roots was measured by this method after infiltration with CI-NERF, a pH-sensitive dye, along with Texas Red Dextran 3000, a pH-insensitive dye, as an internal standard. In the elongation zone of corn roots, the mean apoplastic pH was 4.9. Upon gravitropic stimulation, the pH on the convex side of actively bending roots was 4.5. The lowering of the apoplastic pH by 0.4 units appears to be sufficient to account for the increased growth on that side. This technique provides site-specific evidence for the acid growth theory of cell elongation. The LSCM permits measurements of the pH of living tissues, and has a sensitivity of approximately 0.2 pH units.     

The apoplastic pH of the Zea mays root cortex as measured with pH-sensitive microelectrodes: aspects of regulation

In the root cortex of Zea mays the apoplastic pH and aspects of its regulation were investigated using pH-sensitive microelectrodes. To measure the pH directly in different cell layers of the apoplast sharp double-barrelled electrodes were applied, whereas blunt pH-electrodes were used simultaneously to measure the pH at the root surface. Recordings carried out 8-10 mm behind the root tip show that the apoplastic pH is maintained between 5.1 and 5.6, depending on the given experimental conditions, i.e. varying external [K⁺], [Ca²⁺], pH, weak buffering, as well as perfusion of the test medium. When the medium pH (bulk) differs considerably from the apoplastic pH, a small pH gradient is built up between the root surface (unstirred layer) and the outer cortex layers. In a standing medium these gradients equilibrate. The apoplastic pH responds to increases in external [K⁺] and [CA²⁺] with an acidification, which is attributed to ion-exchange properties of the cell wall constituents. Stimulation of proton pump activity with fusicoccin acidifies the apoplast from pH 5.6 to pH 4.8, while deactivation of the pump with cyanide/salicylhydroxamic acid increases the pH of the apoplast from 5.6 to 6.2, and further to pH 6.6 with CCCP. The Ca²⁺ channel antagonists nifedipine and La³⁺ also increase the apoplastic pH. It is suggested that not only the proton pump, but also the cation channels may contribute to the regulation of the apoplastic pH.Keywords: Apoplast, ion-selective microelectrodes, pH, unstirred layer, Zea mays, root.      

Role of the Apoplast in the Control of Assimilate Transport,Photosynthesis, and Plant Productivity

A concept is suggested, which supposes that assimilates are transferred within the plant downward through phloem sieve tubes and, after entering the stem apoplast, are carried up with the ascending flow of transpiration water. After entering the apoplast of fully expanded leaves, these solutes are reexported through the phloem. Thus, a common pool of assimilates with uniform concentration is formed in the plant apoplast. According to this concept, the mechanism of assimilate demand represents a response of photosynthetic apparatus to changes in the apoplastic level of metabolites consumed by sink organs. The ratios of labeled photoassimilates differ between the apoplast and mesophyll cells. Most of the apoplastic labeled carbon is contained in sucrose, less in amino acids, and even less in hexoses. The ¹⁴C-labeling of amino acids increases and the sucrose/hexose labeling ratio decreased under conditions of enhanced nitrate supply. The well-known effect of relative inhibition of assimilate export from leaves under conditions of enhanced nitrogen supply is explained by an enhanced hydrolysis of apoplast-derived sucrose due to the increase in invertase activity, rather than by diversion of primary photosynthetic products from sucrose synthesis to other pathways required for activated growth processes in leaves. This notion is based on observations that the sucrose/hexose ratio is reduced to a greater extent in the apoplast than in the symplast. The last assumption was supported by data obtained after artificial changes in the apoplastic pH. In these experiments intact plants were placed in the atmosphere of NH₃ or HCl vapors, which induced opposite changes in relative content of labeled assimilates in the apoplast and in the photosynthetic rate.     

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     

Compartmentation of Assimilate Fluxes in Leaves

Abstract: Sugar levels in the apoplast of assimilate exporting leaves were studied in two groups of plant species with contrasting structures of companion cells in minor veins. These species are termed either "symplastic" (with intermediary cells) or "apoplastic" (with transfer or ordinary cells). Sugars were measured in intercellular washing fluid after extracting the apoplast by an infiltration-centrifugation technique. During the course of a day, sugar contents in the apoplast were, in general, lower in species with intermediary cells than in species with transfer or ordinary cells. In "symplastic" species, apoplastic sucrose concentrations were between 0.3 and 1 mM. In "apoplastic" species with transfer cells, they ranged between 2 and 6 mM. Apoplastic hexose contents were between 0.3 and 1 mM irrespective of presumed transport mode. "Symplastic" and "apoplastic" plants differed markedly in their response to a'translocation block. In "symplastic" plants, inhibition of assimilate export left apoplastic concentrations of sucrose and hexoses unchanged, whereas in "apoplastic" plants sugar levels increased, the maximal increase being observed with sucrose. In these plants, concentrations of sucrose were two to six times higher in the apoplast under export inhibition than in control leaves. The data suggest a different role of the leaf apoplast in the compartmentation and export of assimilates in the two plant groups under study.