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 of intact leaves of Vicia faba as influenced by light

The fluorochrome FITC-dextran was used to measure the effectof light on the apoplastic pH of intact Vicia faba leaves withthe ratio imaging technique. In darkadapted leaves the apoplasticpH varied depending on the leaf between 5.2 and 5.9. Red light(660 nm, 4–12 W m^–2) leads to multiphasic responses:in the first seconds an alkalinization ({small tilde}0.3 pHunits), and thereafter an acidification of the leaf apoplast({small tilde}0.4 pH units) were observed. Both effects couldbe inhibited by DCMU. While variation of CO₂ concentration revealedno effect on light-induced apoplastic pH changes, a decreasein O₂ concentration decreased the effect. On the basis of ourdata it is suggested that the influence of photosynthesis onplasmalemma H⁺ ATPase is responsible for the observed effects,rather than altered CO₂ uptake. Key words: Leaf apoplast, apoplastic pH, light, ratio imaging, pH-sensitive fluorescent dye, Vicia fab     

Salinity Stiffens the Epidermal Cell Walls of Salt-Stressed Maize Leaves: Is the Epidermis Growth-Restricting?

As a result of salt (NaCl)-stress, sensitive varieties of maize (Zea mays L.) respond with a strong inhibition of organ growth. The reduction of leaf elongation investigated here has several causes, including a modification of the mechanical properties of the cell wall. Among the various tissues that form the leaf, the epidermis plays a special role in controlling organ growth, because it is thought to form a rigid outer leaf coat that can restrict elongation by interacting with the inner cell layers. This study was designed to determine whether growth-related changes in the leaf epidermis and its cell wall correspond to the overall reduction in cell expansion of maize leaves during an osmotic stress-phase induced by salt treatment. Two different maize varieties contrasting in their degree of salt resistance (i.e., the hybrids Lector vs. SR03) were compared in order to identify physiological features contributing to resistance towards salinity. Wall loosening-related parameters, such as the capacity of the epidermal cell wall to expand, β-expansin abundance and apoplastic pH values, were analysed. Our data demonstrate that, in the salt-tolerant maize hybrid which maintained leaf growth under salinity, the epidermal cell wall was more extensible under salt stress. This was associated with a shift of the epidermal apoplastic pH into a range more favourable for acid growth. The more sensitive hybrid that displayed a pronounced leaf growth-reduction was shown to have stiffer epidermal cell walls under stress. This may be attributable to the reduced abundance of cell wall-loosening β-expansin proteins following a high salinity-treatment in the nutrient solution (100 mM NaCl, 8 days). This study clearly documents that salt stress impairs epidermal wall-loosening in growth-reduced maize leaves.     

Determination of apoplastic K+ in intact leaves by ratio imaging of PBFI fluorescence

The tetraammonium salt of the K⁺ binding fluorescent dye benzofuranisophthalate (PBFI) was used to investigate the influence ofpotassium nutrition (0.1–2.1 mol m^–3) on apoplasticK⁺ inVicia faba leaves by means of ratio imaging. As a referencethe infiltration-centrifugation method was used. Both methodsreflected the influence of K⁺ supply on apoplastic K⁺ concentration.The abaxial leaf side revealed significantly higher K⁺ concentrations(20-25 mol m^–3) than the adaxial side (5–8 mol m^–3).Application of CCCP led to an immediate increase in apoplasticK⁺ demonstrating the reliability of the PBFI method. Key words: Vicia faba, leaf, apoplast, K⁺, PBFI, ratio imaging, ratiometric fluorescence microscopy     

Systemic signalling in barley through action potentials

Using apoplastic voltage- and ion selective microprobes, in barley leaves action potentials (APs) have been measured, which propagate acropetally as well as basipetally from leaf to leaf or from root to leaf following the application of mild salt stress (e.g. 30–50 mM KCl or NH₄Cl) or amino acids (e.g. 1 mM glutamic acid or 5 mM GABA). Voltage changes were biphasic, followed an ‘all-or-none’ characteristic, and propagated at 20–30 cm min⁻¹ irrespective of the direction. With the salt-induced APs, a strong initial depolarization is the main AP-releasing factor that first causes Ca²⁺ influx and then anion efflux. Ca²⁺ influx coincides with an initial slower depolarization, the rapid anion efflux causes the typical voltage ‘break-through’. Subsequently, K⁺-efflux starts after the depolarizing voltage has passed the K⁺ equilibrium potential (inversion of the K⁺ driving force). Glutamic acid and GABA induce APs not through membrane depolarization, but presumably by binding to a putative receptor or to ligand-gated Ca²⁺-conducting channels, respectively, followed by Ca²⁺ induced activation of anion efflux. APs are accompanied by transient apoplastic pH increase (about 1 unit), and by cytoplasmic pH decrease (about 0.5 units). The apoplastic pH change is interpreted as an indicator of stress, the cytoplasmic pH change as a prerequisite for defence related gene activation. Since APs are released by agents added in a moderate concentration range, it is suggested that they may serve as first and fast systemic signals following attack from pathogens.     

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.     

Abscisic Acid Movement into the Apoplastic solution of Water-Stressed Cotton Leaves: Role of Apoplastic pH

Leaves of cotton (Gossypium hirsutum L.) were subjected to overpressures in a pressure chamber, and the exuded sap was collected and analyzed. The exudate contained low concentrations of solutes that were abundant in total leaf extracts, and photosynthetic rates and stomatal conductance were completely unaffected by a cycle of pressurization and rehydration. These criteria and others indicate that the experimental techniques inflicted no damage upon the leaf cells. The pH and abscisic acid (ABA) content of the apoplastic fluid both increased greatly with pressure-induced dehydration. Although ABA concentrations did not reach a steady state, the peak levels were above 1 micromolar, an order of magnitude greater than bulk ABA concentrations of the leaf blades. Treatment of leaves with fusicoccin decreased the K⁺ concentration, greatly reduced the pH rise, and completely eliminated the increase in ABA in the apoplast upon dehydration. When water-stressed leaves were pressurized, the pH of the exuded sap was increased by 0.2 units per 1 megapascal decrease in initial leaf water potential. Buffer capacity of the sap was least in the pH range of interest (6.5-7.5), allowing extremely small changes in H⁺ fluxes to create large changes in apoplastic pH. The data indicate that dehydration causes large changes in apoplastic pH, perhaps by effects on ATPases; the altered pH then enhances the release of ABA from mesophyll cells into the apoplastic fluid.     

Regulation of Apoplastic pH in Source Leaves of Vicia faba by Gibberellic Acid

Leaf discs of broad bean (Vicia faba L.), peeled on the spongy mesophyll side, rapidly altered the pH of the surrounding medium (apoplast). Using pH indicator paper appressed against the leaf, immediately after peeling, initial apoplastic pH was estimated to be 4.5. Changes in the apoplastic pH were measured with a microelectrode placed into a 100-microliter drop of an unbuffered solution (2 millimolar KCl, 0.5 millimolar CaCl₂, and 200 millimolar mannitol) on the peeled surface. Discs acidified the medium until the pH stabilized at about 5.0 (about 10 minutes). Acidification was inhibited by 50 micromolar sodium vanadate, an inhibitor of the plasmalemma H⁺-ATPase and attenuated by omitting the osmoticum or potassium ions from the medium. Fusicoccin (10 micromolar) greatly enhanced the rate of acidification. The presence of 0.1 to 1 micromolar gibberellic acid resulted in a slower rate of medium acidification. Gibberellic acid appeared to modulate the activity of the H⁺-translocating ATPase located at the plasma membrane of the mesophyll cells.     

Evaluation of an aphid‐rearing method using excised leaves and agar medium

The present study evaluated the effectiveness of an aphid‐rearing method devised by Milner in 1981 using Acyrthosiphon pisum and its host plant Vicia faba. In the “agar‐leaf method,” excised leaves of V. faba were attached to the surface of 1% agar gel containing nutrient solution, and test aphids were transferred onto the leaves. Excised leaves grew in size and weight on the agar medium. Fecundity, longevity, body size and developmental time to adulthood were compared between aphids reared using the agar‐leaf method vs. those reared on V. faba seedlings under the same conditions. No significant difference was detected between the two treatments for any of the four parameters, suggesting that the aphids grew and reproduced on excised leaves as successfully as on V. faba seedlings. This method was also useful for inducing males and oviparous females at lower temperature and in short days. Therefore, the present study confirms the effectiveness of using excised leaves on agar and suggests that this method could be applied to the rearing of other aphids, phytophagous mites, leaf miners and leaf‐gall formers.     

Response of Vicia faba L. to metal toxicity on mine tailing substrate: Geochemical and morphological changes in leaf and root

Vicia faba L. seeds were grown in a pot experiment on soil, mine tailings, and a mixture of both to mimic field situations in cultivated contaminated areas near mining sites. Metals in the substrates and their translocation in root, stem and leaf tissues were investigated, including morphological responses of plants growing on mine tailings. Metal concentration – and generally bioaccumulation – was in the order: roots > leaves > stems, except Pb and Cd. Translocation was most significant for Zn and Cd, but limited for Pb. Metal concentration in root and leaf was not proportional to that in the substrates, unexpectedly the minimum being observed in the mixed substrate whilst plant growth was retarded by 20% (38% on tailings). Calcium, pH, organic matter and phosphorus were the main influencing factors for metal translocation. The ultrastructure of V. faba L. cells changed a lot in the mine tailings group: root cell walls were thickened with electron dense Pb, Zn and C particles; in chloroplasts, the number of plastoglobuli increased, whereas the thylakoids were swollen and their number decreased in grana. Finally, needle-shaped crystalline concretions made of Ca and P, with Zn content, were formed in the apoplast of the plants. The stratagems of V. faba L. undergoing high concentrations of toxic metals in carbonate substrate, suggest root cell wall thickening to decrease uptake of toxic metals, a possible control of metal transport from roots to leaves by synthesizing phytochelators–toxic metal complexes, and finally a control of exceeded Ca and metal concentration in leaves by crystal P formation as ultimate response to stress defense. The geochemical factors influencing metal availability, guaranty a reduction of metal content in plant growing on mixed tailing/soil substrate as far as carbonate are not completely dissolved.     

Movement of Abscisic Acid into the Apoplast in Response to Water Stress in Xanthium strumarium L

The effect of water stress on the redistribution of abcisic acid (ABA) in mature leaves of Xanthium strumarium L. was investigated using a pressure dehydration technique. In both turgid and stressed leaves, the ABA in the xylem exudate, the `apoplastic' ABA, increased before `bulk leaf' stress-induced ABA accumulation began. In the initially turgid leaves, the ABA level remained constant in both the apoplast and the leaf as a whole until wilting symptoms appeared. Following turgor loss, sufficient quantities of ABA moved into the apoplast to stimulate stomatal closure. Thus, the initial increase of apoplastic ABA may be relevant to the rapid stomatal closure seen in stressed leaves before their bulk leaf ABA levels rise.

Following recovery from water stress, elevated levels of ABA remained in the apoplast after the bulk leaf contents had returned to their prestress values. This apoplastic ABA may retard stomatal reopening during the initial recovery period.

     

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.     

Slow-growth phenotype of transgenic tomato expressing apoplastic invertase

The growth of transgenic tomato (Lycopersicon esculentum) plants that express in their apoplast yeast invertase under the control of the cauliflower mosaic virus 35S promoter is severely inhibited. The higher the level of invertase, the greater the inhibition of growth. A second phenotypic characteristic of these transgenic plants is the development of yellow and necrotic spots on the leaves, and leaf curling. Again the severity of the symptoms is correlated with the level of invertase. These symptoms do not develop in shaded leaves indicating the need for photosynthesis. Keeping the plants in the dark for a prolonged period (24 hours) results in the disappearance of leaf starch from the control plants, but not from the plants with apoplastic invertase. These results are consistent with the interpretation that apoplastic invertase prevents photosynthate export from source leaves and that phloem loading includes an apoplastic step.     

Seasonal variation in ammonia compensation point and nitrogen pools in beech leaves (Fagus sylvatica)

Ammonia (NH₃) fluxes between beech leaves (Fagus sylvatica) and the atmosphere were investigated in a 90-year-old forest canopy and related to leaf nitrogen (N) pools and glutamine synthetase (GS) activities. The stomatal ammonia compensation point, ?? _NH3, was measured by both a twig cuvette and bioassay techniques involving measurements of pH and ammonium (NH ₄ ⁺ ) concentration in the leaf apoplastic solution. The ?? _NH3 determined on the basis of the gas exchange measurements followed a seasonal variation with early-season peaks during leaf expansion (9.6 nmol NH₃ mol^?1 air) and late-season peaks during leaf senescence (7.3 nmol NH₃ mol^?1 air). In the mid-season, the ?? _NH3 of mature green leaves was much lower (around 3 nmol NH₃ mol^?1 air) and dropped below the NH₃ concentration in the ambient atmosphere. For comparison, ?? _NH3 obtained by the apoplastic bioassay were 7.0, 3.7 and 6.4 nmol NH₃ mol^?1 air in early-, mid-, and late -season, thus agreeing reasonably well with ?? _NH3 values derived from the gas exchange measurements. Potential NH₃ emission fluxes during early and late season were 1.31 and 0.51 nmol m^?2 leaf surface area s^?1, respectively, while leaves were a sink for NH₃ during mid-season. During leaf establishment and senescence, both apoplastic and bulk tissue NH ₄ ⁺ concentrations were relatively high coinciding with low activities of glutamine synthetase, which is a key enzyme in leaf N metabolism. In conclusion, the exchange of NH₃ between beech leaves and the atmosphere followed a seasonal variation with NH₃ emission peaks being related to N mobilization during early leaf establishment and remobilization during late leaf senescence.     

Ion Relations of Symplastic and Apoplastic Space in Leaves from Spinacia oleracea L. and Pisum sativum L. under Salinity

Salt tolerant spinach (Spinacia oleracea) and salt sensitive pea (Pisum sativum) plants were exposed to mild salinity under identical growth conditions. In order to compare the ability of the two species for extra- and intracellular solute compartmentation in leaves, various solutes were determined in intercellular washing fluids and in aqueously isolated intact chloroplasts. In pea plants exposed to 100 millimolar NaCl for 14 days, apoplastic salt concentrations in leaflets increased continuously with time up to 204 (Cl⁻) and 87 millimolar (Na⁺), whereas the two ions reached a steady concentration of only 13 and 7 millimolar, respectively, in spinach leaves. In isolated intact chloroplasts from both species, sodium concentrations were not much different, but chloride concentrations were significantly higher in pea than in spinach. Together with data from whole leaf extracts, these measurements permitted an estimation of apoplastic, cytoplasmic, and vacuolar solute concentrations. Sodium and chloride concentration gradients across the tonoplast were rather similar in both species, but spinach was able to maintain much steeper sodium gradients across the plasmamembrane compared with peas. Between day 12 and day 17, concentrations of other inorganic ions in the pea leaf apoplast increased abruptly, indicating the onset of cell disintegration. It is concluded that the differential salt sensitivity of pea and spinach cannot be traced back to a single plant performance. Major differences appear to be the inability of pea to control salt accumulation in the shoot, to maintain steep ion gradients across the leaf cell plasmalemma, and to synthesize compatible solutes. Perhaps less important is a lower selectivity of pea for K⁺/Na⁺ and NO₃⁻/Cl⁻ uptake by roots.     

Inhibition of Loading of C Assimilates by p-Chloromercuribenzenesulfonic Acid : Localization of the Apoplastic Pathway in Vicia faba

The apoplast of mature leaves excised from broadbean (Vicia faba L.) plants was infiltrated with 2 millimolar p-chloromercuribenzenesulfonic acid (PCMBS) via the transpiration stream, and the ability of the tissues to take up sugars was tested. An infiltration time of 75 minutes was sufficient to obtain a maximal (75%) inhibition of exogenous [¹⁴C]sucrose (1 millimolar) uptake. This infiltration affected neither CO₂ assimilation nor the transmembrane potential difference of leaf cells but strongly inhibited phloem loading of endogenous [¹⁴C] assimilates. The study of the symplastic relations between the different cell types of the mature leaf showed that the density of the plasmodesmata is generally very low in comparison with other species investigated so far, particularly when considering the mesophyll/bundle sheath and the bundle sheath/phloem cells connections, as well as the connections of the transfer cell-sieve tube complex with the surrounding cells. These three successive barriers therefore strongly limit the possibilities of symplastic transit of the assimilates to the conducting cells. The comparison of the densities of plasmodesmata in an importing and an exporting leaf suggests that the maturation of the leaf is characterized by a marked symplastic isolation of the phloem, and, within the phloem itself, by the isolation of the conducting complex. As a consequence, these physiological and cytological data demonstrate the apoplastic nature of loading in the mature leaf of Vicia faba, this species undoubtedly presenting a typical model for apoplastic loading.     

Stress-dependent redistribution of abscisic acid (ABA) in Hordeum vulgare L leaves: the role of epidermal ABA metabolism, tonoplastic transport and the cuticle

When ¹⁴C-labelled abscisic acid ([¹⁴C]ABA) was supplied to isolated protoplasts of the barley leaf at pH 6, initial rates of metabolism were about five times higher in epidermal cell protoplasts than in mesophyll cell protoplasts if equal cytosolic volumes were considered. In spite of the fact that epidermal cells make up only about 35% of the total water space in barley leaves, and despite the small cytosolic volume of these cells, in intact leaves all epidermal cells would thus metabolize half as much ABA per unit time as the mesophyll cells (0–27 and 0–51 mmol h^?1 m^?3 leaf water). Therefore, under these conditions epidermal cells seem to be a stronger sink than mesophyll cells for ABA that arrives via the transpiration stream. However, at an apoplastic pH of 7–25, which occurs in stressed leaves, the proportion of total metabolized ABA would be much smaller in epidermal than in mesophyll cells (0–029 and 0–204 mmolh^?l m^?3 leaf water). Our results indicate that under conditions of slightly alkaline apoplastic pH the epidermis may serve as the main source for fast stress-dependent ABA redistribution into the guard cell apoplast. This is partly the result of ABA transport across the epidermal tonoplast, which is dependent on the apoplastic pH and possibly on the cytosolic calcium concentration. The cuticle seems to be of no particular importance in stress-induced apoplastic ABA shifts and cannot be regarded as a significant sink for high ABA concentrations under stress.     

Water Stress and Leaf Growth of Field Beans (Vicia faba L.) in the Field: Water Potentials and Laminar Expansion

Field beans were grown in three different irrigation treatments.The growth of each leaf was followed and estimates made of plantwater potential at dawn and of plant water potential, solutepotential, and pressure potential in the afternoon. The growthin area of the leaves against time was fitted with a logisticcurve from which the parameters of leaf growth were defined.The parameters were the area at which a leaf unfolds, the meangrowth rate, and the duration of growth, which combine to givethe final leaf size. Water stress reduced the final leaf sizeby reducing both the area at unfolding and the mean growth rate.The duration of growth was not consistently altered. Final leafsize was closely correlated with the plant water potential inthe afternoon and apparently more with pressure than solutepotential. Vicia faba, field bean, water stress, leaf area, leaf growth, water potential     

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