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.     

Modification of leaf apoplastic pH in relation to stomatal sensitivity to root-sourced abscisic acid signals

The confocal microscope was used to determine the pH of the leaf apoplast and the pH of microvolumes of xylem sap. We quantified variation in leaf apoplast and sap pH in relation to changes in edaphic and atmospheric conditions that impacted on stomatal sensitivity to a root-sourced abscisic acid signal. Several plant species showed significant changes in the pH of both xylem sap and the apoplast of the shoot in response to environmental perturbation. Xylem sap leaving the root was generally more acidic than sap in the midrib and the apoplast of the leaf. Increasing the transpiration rate of both intact plants and detached plant parts resulted in more acidic leaf apoplast pHs. Experiments with inhibitors suggested that protons are removed from xylem sap as it moves up the plant, thereby alkalinizing the sap. The more rapid the transpiration rate and the shorter the time that the sap resided in the xylem/apoplastic pathway, the smaller the impact of proton removal on sap pH. Sap pH of sunflower (Helianthus annuus) and Commelina communis did not change significantly as soil dried, while pH of tomato (Lycopersicon esculentum) sap increased as water availability in the soil declined. Increasing the availability of nitrate to roots also significantly alkalinized the xylem sap of tomato plants. This nitrogen treatment had the effect of enhancing the sensitivity of the stomatal response to soil drying. These responses were interpreted as an effect of nitrate addition on sap pH and closure of stomata via an abscisic acid-based mechanism.     

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.     

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 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.     

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.     

Long-distance ABA Signaling and Its Relation to Other Signaling Pathways in the Detection of Soil Drying and the Mediation of the Plant’s Response to Drought

In this article we review evidence for a variety of long-distance signaling pathways involving hormones and nutrient ions moving in the xylem sap. We argue that ABA has a central role to play, at least in root-to-shoot drought stress signaling and the regulation of functioning, growth, and development of plants in drying soil. We also stress the importance of changes in the pH of the leaf cell apoplast as influenced both by edaphic and climatic variation, as a regulator of shoot growth and functioning, and we show how changes in xylem and apoplastic pH can affect the way in which ABA regulates stomatal behavior and growth. The sensitivity to drought of the pH/ABA sensing and signaling mechanism is emphasized. This allows regulation of plant growth, development and functioning, and particularly shoot water status, as distinct from stress lesions in growth and other processes as a reaction to perturbations such as soil drying.     

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.     

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.     

An effector-targeted protease contributes to defense against Phytophthora infestans and is under diversifying selection in natural hosts

Since the leaf apoplast is a primary habitat for many plant pathogens, apoplastic proteins are potent, ancient targets for apoplastic effectors secreted by plant pathogens. So far, however, only a few apoplastic effector targets have been identified and characterized. Here, we discovered that the papain-like cysteine protease C14 is a new common target of EPIC1 and EPIC2B, two apoplastic, cystatin-like proteins secreted by the potato (Solanum tuberosum) late blight pathogen Phytophthora infestans. C14 is a secreted protease of tomato (Solanum lycopersicum) and potato typified by a carboxyl-terminal granulin domain. The EPIC-C14 interaction occurs at a wide pH range and is stronger than the previously described interactions of EPICs with tomato defense proteases PIP1 and RCR3. The selectivity of the EPICs is also different when compared with the AVR2 effector of the fungal tomato pathogen Cladosporium fulvum, which targets PIP1 and RCR3, and only at apoplastic pH. Importantly, silencing of C14 increased susceptibility to P. infestans, demonstrating that this protease plays a role in pathogen defense. Although C14 is under conservative selection in tomato, it is under diversifying selection in wild potato species (Solanum demissum, Solanum verrucosum, and Solanum stoliniferum) that are the natural hosts of P. infestans. These data reveal a novel effector target in the apoplast that contributes to immunity and is under diversifying selection, but only in the natural host of the pathogen.     

Apoplastic pH and growth in expanding leaves of Vicia faba under salinity

Salinity affects water availability in the soil and subsequently the plant uptake capacity. Upon exposure to salt stress, leaf growth in monocot plants has been shown to be reduced instantaneously, followed by a gradual acclimation. The growth reactions are caused by an initial water deficit and an accompanied osmotic effect, followed by an IAA-induced sequestration of protons into the apoplast that increases leaf growth again as explained by the acid growth theory. In this study, we investigated the dynamics of growth reactions and apoplastic pH in leaves of the dicot Vicia faba in the presence of NaCl during the initiation of salt stress. Concurrent changes in apoplastic pH were detected by ratiometric fluorescence microscopy using the fluorescent dye fluorescein tetramethylrhodamine dextran. To elucidate the possible relation between the dynamics of leaf growth and apoplastic pH, results of the ratio imaging technique were combined with an in vivo growth analysis imaging approach. Leaf growth rate of V. faba was highest in the dusk and the early night phase; at this time a concomitant decrease of the apoplastic pH was observed. Under salinity, the apoplastic pH in leaves of V. faba increased with a simultaneous decrease of leaf growth towards increasing developmental stages, but with complex aberrations in the 24-h-leaf-growth pattern compared to control leaves. In conclusion, these results show that salt stress leads to an increase in apoplastic pH and to a declined leaf growth activity with complex 24-h-interactions of growth and pH in V. faba.     

High apoplastic solute concentrations in leaves alter water relations of the halophytic shrub, Sarcobatus vermiculatus

Predawn plant water potential (Psi(w)) is used to estimate soil moisture available to plants because plants are expected to equilibrate with the root-zone Psi(w). Although this equilibrium assumption provides the basis for interpreting many physiological and ecological parameters, much work suggests predawn plant Psi(w) is often more negative than root-zone soil Psi(w). For many halophytes even when soils are well-watered and night-time shoot and root water loss eliminated, predawn disequilibrium (PDD) between leaf and soil Psi(w) can exceed 0.5 MPa. A model halophyte, Sarcobatus vermiculatus, was used to test the predictions that low predawn solute potential (Psi(s)) in the leaf apoplast is a major mechanism driving PDD and that low Psi(s) is due to high Na+ and K+ concentrations in the leaf apoplast. Measurements of leaf cell turgor (Psi(p)) and solute potential (Psi(s)) of plants grown under a range of soil salinities demonstrated that predawn symplast Psi(w) was 1.7 to 2.1 MPa more negative than predawn xylem Psi(w), indicating a significant negative apoplastic Psi(s). Measurements on isolated apoplastic fluid indicated that Na+ concentrations in the leaf apoplast ranged from 80 to 230 mM, depending on salinity, while apoplastic K+ remained around 50 mM. The water relations measurements suggest that without a low apoplastic Psi(s), predawn Psi(p) may reach pressures that could cause cell damage. It is proposed that low predawn apoplastic Psi(s) may be an efficient way to regulate Psi(p) in plants that accumulate high concentrations of osmotica or when plants are subject to fluctuating patterns of soil water availability.     

Do pH changes in the leaf apoplast contribute to rapid inhibition of leaf elongation rate by water stress? Comparison of stress responses induced by polyethylene glycol and down‐regulation of root hydraulic conductivity

We have dissected the influences of apoplastic pH and cell turgor on short-term responses of leaf growth to plant water status, by using a combination of a double-barrelled pH-selective microelectrodes and a cell pressure probe. These techniques were used, together with continuous measurements of leaf elongation rate (LER), in the (hidden) elongating zone of the leaves of intact maize plants while exposing roots to various treatments. Polyethylene glycol (PEG) reduced water availability to roots, while acid load and anoxia decreased root hydraulic conductivity. During the first 30 min, acid load and anoxia induced moderate reductions in leaf growth and turgor, with no effect on leaf apoplastic pH. PEG stopped leaf growth, while turgor was only partially reduced. Rapid alkalinization of the apoplast, from pH 4.9 ± 0.3 to pH 5.8 ± 0.2 within 30 min, may have participated to this rapid growth reduction. After 60 min, leaf growth inhibition correlated well with turgor reduction across all treatments, supporting a growth limitation by hydraulics. We conclude that apoplastic alkalinization may transiently impair the control of leaf growth by cell turgor upon abrupt water stress, whereas direct hydraulic control of growth predominates under moderate conditions and after a 30-60 min delay following imposition of water stress.     

A critical review on methods to measure apoplastic pH in plants

Various attempts have been made to estimate or measure apoplastic pH over the last few decades. These approaches include pH indicators in agar or beads, measurement of the pH of apoplastic fluid, weak acid influx, ion-selective electrodes and optical probes. Each of these has its own applications and limitations, and has contributed to the understanding of the processes taking place in the apoplast in relation to pH changes. However, convincing methods allowing us to probe this cell compartment are still lacking and invite exploration. The distinction between apoplast components is also discussed to clarify the indiscriminate use of the term `apoplastic pH'.     

Long-distance signalling of abscisic acid (ABA): the factors regulating the intensity of the ABA signal

Abscisic acid (ABA) is a stress signal, which moves in the xylem from the roots to the aerial parts of the plant, where it regulates stomatal movement and the activity of shoot meristems. Root growth-promoting microorganisms in the rhizosphere, lateral ABA flows in the root cortex across apoplastic barriers, ABA redistribution in the stem, leaf apoplastic pH values, and the action of beta-glucosidases, both in the apoplast and the cytosol of the mesophyll, play an important role in the regulation of signal intensity. The significance of abscisic acid glucose ester as a long-distance stress signal is discussed.     

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.     

Apoplastic Sugar Concentration and pH in Barley Leaves Infected with Brown Rust

Soluble sugars were extracted by low speed centrifugation fromthe apoplast of leaves of barley (Hordeum distichum L.) infiltratedwith water. Infection of the leaf with the brown rust fungus(Puccinia hordeii) resulted in a reduction in the concentrationof sucrose, glucose and fructose in the apoplast. Sugars werepresent in an apoplastic space occupying 12 and 17 cm³ m^–2of leaf area in healthy and infected tissue, respectively. Uptakeof hexoses by intercellular hyphae is suggested as a cause ofthis reduction. The pH of apoplastic sap extracted from rust-infectedleaves was increased to pH 7·3 from pH 6·6 incontrols. The effect of a reduced apoplastic sugar pool andincreased pH on export from infected leaves is discussed. Key words: Apoplast, barley (Hordeum distichum L.), brown rust (Puccinia hordeii Otth.), pH, sucrose, hexose     

pH biosensing in the plant apoplast—a focus on root cell elongation

The pH parameter of soil plays a key role for plant nutrition as it is affecting the availability of minerals and consequently determines plant growth. Although the mechanisms by which root perceive the external pH is still unknown, the impact of external pH on tissue growth has been widely studied especially in hypocotyl and root. Thanks to technological development of cell imaging and fluorescent sensors, we can now monitor pH in real time with at subcellular definition. In this focus, fluorescent dye-based, as well as genetically-encoded pH indicators are discussed especially with respect to their ability to monitor acidic pH in the context of primary root. The notion of apoplastic subdomains is discussed and suggestions are made to develop fluorescent indicators for pH values below 5.0.

The pH parameter of soil plays a key role for plant nutrition as it is affecting the availability of minerals and consequently determines plant growth. Although the mechanisms by which root perceive the external pH is still unknown, the impact of external pH on tissue growth has been widely studied especially in hypocotyl and root. Thanks to technological development of cell imaging and fluorescent sensors, we can now monitor pH in real time with at subcellular definition. In this focus, fluorescent dye-based, as well as genetically-encoded pH indicators are discussed especially with respect to their ability to monitor acidic pH in the context of primary root. The notion of apoplastic subdomains is discussed and suggestions are made to develop fluorescent indicators for pH values below 5.0.     

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.