Use of intercellular washing fluid to investigate the secreted proteome of the rice–<Emphasis Type="Italic">Magnaporthe</Emphasis> interaction

Early interactions between invading penetration hyphae of the pathogenic fungus Magnaporthe oryzae and rice cells occur at the apoplast, the free diffusional space outside the plasma membrane of leaves. After initial colonization, intercellular hyphae are again in intimate contact with the rice apoplast. While several studies have looked at proteomics in rice–Magnaporthe interactions, none have focused on apoplast localized proteins. We adjusted a protocol for intercellular washing fluids (IWF) to rice leaves infected with Magnaporthe oryzae for proteomic analysis. In our IWF extract, we identified several proteins associated with compatible or incompatible pathogen interactions. Three DUF26 domain proteins were identified as changing in abundance 12 h after inoculation, confirming DUF26 domain-containing proteins are among early, pathogen stress-responsive proteins induced by infection with Magnaporthe oryzae. A Magnaporthe cyclophilin, previously identified as a virulence factor was also identified in the intercellular washing fluid.     

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.

     

Abscisic acid in apoplastic sap can account for the restriction in leaf conductance of white lupins during moderate soil drying and after rewatering

We studied the effects of drought on leaf conductance (g) and on the concentration of abscisic acid (ABA) in the apoplastic sap of Lupinus albus L. leaves. Withholding watering for 5d resulted in complete stomatal closure and in severe leaf water deficit. Leaf water potential fully recovered immediately after rewatering, but the aftereffect of drought on stomata persisted for 2d. ABA and sucrose were quantified in pressurized leaf xylem extrudates. We assumed that the xylem sucrose concentration is negligible and hence that the presence of sucrose in leaf extrudates indicated that they were contaminated by phloem. To eliminate this interference, the concentration of ABA in leaf apoplast was estimated by extrapolation to zero sucrose concentration, using the regression between ABA and sucrose concentrations. The estimated apoplastic ABA concentration increased by 100-fold with soil drying and did not return to pre-stress values immediately following rewatering. g was closely related to the concentration of ABA in leaf apoplast. Furthermore, the feeding of exogenous ABA to leaves detached from well-watered plants brought about the same degree of depression in g as resulted from the drought-induced increase in ABA concentration. We therefore conclude that the observed changes in the concentration of ABA in leaf apoplast were quantitatively adequate to explain drought-induced stomatal closure and the delay in stomatal reopening following rewatering.     

Ascorbate transport from the apoplast to the symplast in intact leaves

Infiltration of reduced ascorbate (ASC) into the leaves of Betula pendula Roth and subsequent measurement of its loss therein after incubation allowed us to follow ascorbate transport from apoplast to symplast in intact leaves. All of the ascorbate extracted from the native apoplast was in fully oxidized form, dehydroascorbate (DHA). When 5 m M of ASC was infiltrated into the leaves, its intense decay occurred, but only 55% of ASC lost was recovered in apoplast as DHA. When ASC was added to the freshly extracted intercellular washing fluid (IWF), ASC oxidation occurred as well. However, all oxidized ASC was recovered as DHA, indicating that further decomposition of DHA did not occur. Similarly, all of the ASC infiltrated into the leaves was found therein either as ASC or DHA after incubation of leaves for up to 60 min. On this base the ascorbate infiltrated into the leaves and not recovered in the IWF was interpreted as ascorbate taken up into the symplast. The calculated uptake rates of ascorbate at different ASC concentrations followed saturation kinetics with the maximum uptake rate of 300 nmol m⁻² plasma membrane (PM) area min⁻¹ and Michaelis constant of 12.8 m M . The uptake of ascorbate was significantly inhibited by the addition of dithiothreitol or by PM H⁺ ATPase inhibitor erythrosin B. Thus, our results support the previous observations that DHA is preferably transported from the apoplastic to the cytoplasmic side of the membrane and show that this process is dependent upon PM proton gradient.     

Extracellular beta-glucosidase activity in barley involved in the hydrolysis of ABA glucose conjugate in leaves

Abscisic acid conjugate concentrations increased in barley xylem sap under salinity, whereas it remained at a low level in the intercellular washing fluid (IWF) of barley primary leaves (Hordeum vulgare cv. Gerbel). Here it is shown that IWF contains beta-glucosidase activity which releases abscisic acid (ABA) from the physiologically inactive ABA-glucose conjugate pool in the leaf apoplast. The following data support this conclusion and give the first biochemical and physiological characterization of the extracellular glucosidase activity in barley. Free ABA was released by the incubation of ABA glucose ester with IWF. The product exhibited the retention time of authentic ABA upon separation by thin layer chromatography and was identified by ABA-ELISA. p-Nitrophenol-beta-D-glucopyranoside (pNPG) was used as the substrate for beta-glucosidases. The K(M)(pNPG) was 1.8 mmol l(-1). The activity was affected by ABA glucopyranoside in a competitive type of inhibition with a K(I) of 400 micromol l(-1). Various hormone conjugates were compared with respect to their inhibitory effect on beta-glucosidase activity. Inhibition was highest for the ABA glucopyranoside and the zeatin riboside, but insignificant for ABA methyl ester and zeatin-9-beta-D-glucoside. The specific activity of the beta-glucosidase was 16-fold greater in IWF as compared to crude leaf extracts confirming its extracellular compartmentation. The activity of beta-glucosidase was strongly increased after growth in hydroponic medium supplemented with NaCl. The data support the hypothesis that the glucose conjugate is a long-distance transport form of ABA.     

A fast method to detect the occurrence of nonhomogeneous distribution of stomatal aperture in heterobaric plant leaves

Summary Pressure infiltration of water into a leaf via the stomatal pores can be used to quickly determine whether all stomata are open, or as recently described for several mesophytic and xerophytic species, whether there is a non-homogeneous distribution of stomatal opening (stomatal patchiness) on the leaf surface. Information about this phenomenon is important since the commonly used algorithms for calculation of leaf conductance from water vapor exchange measurements imply homogeneously open stomata, which in the occurrence of stomatal patchiness will lead to erroneous results. Infiltration experiments in a growth chamber with leaves of the Mediterranean evergreen shrub Arbutus unedo, carried out under simulated Mediterranean summer day conditions, where the species typically exhibits a strong midday stomatal closure, revealed a temporary occurrence of stomatal patchiness during the phase of stomatal closure in the late morning and during the stomatal reopening in the afternoon. Leaves were, however, found to be fully (i.e. homogeneously) infiltratable in the morning and in the evening. At midday during maximum stomatal closure, leaves were almost non-infiltratable. During the day, the infiltrated amount of water was found to be linearly correlated with porometer measurements of leaf conductance of the same leaves, carried out with the attached leaves immediately before infiltration.     

FITC-dextran for measuring apoplast pH and apoplastic pH gradients between various cell types in sunflower leaves

The liquid in the free space of leaf cell walls, the apoplast, is in direct contact with the plasma membrane and its nutrient uptake systems. Therefore, the pH of the apoplast is of utmost interest. We have elaborated a non-destructive method by which excised sunflower leaves ( Helianthus annuus cv. Erika) were perfused with fluorescein isothiocyanate-dextran (FITC-dextran) (4 000 Da) via the transpiration stream. We showed that leaf apoplast pH can be measured by using the fluorescence ratio technique together in conjunction with this dye. Evidence is provided that FITC-dextran does not penetrate the plasma membrane over a period of ca 17 h from the beginning of dye perfusion. Dye enrichment in the leaf apoplast did not cause an 'inner filter effect' and thus the fluorescence ratio was only dependent on pH. In vivo calibration yielded a pKa of 5.92, which was virtually identical to the pKa of 5.93 calculated for dye solutions. Hence, FITC-dextran can be detected in complex environments and covers a pH range prevailing in the leaf apoplast.
Based on this method we developed a microscope image technique visualizing pH gradients between various cell types. The pH in the lumen of the xylem vessel was ca 0.3–0.5 units lower than that of the apoplast of surrounding cells. Nitrate present in the leaf apoplast caused an increase in pH, especially in the dark. Under these conditions, in the intercostal area, the apoplast pH around the stomata was ca 0.5–1.0 units higher than that of the surrounding epidermal cells.     

Chitinases and beta-1,3-Glucanases in the Apoplastic Compartment of Oat Leaves (Avena sativa L.)

To isolate chitinases and β-1,3-glucanases from the intercellular space of oats (Avena sativa L.), primary leaves were infiltrated with buffer and subjected to gentle centrifugation to obtain intercellular washing fluid (IWF). Approximately 5% of the chitinase and 10% of the β-1,3-glucanase activity of the whole leaf were released. Only small amounts (0.01-0.03%) of the intracellular marker malate-dehydrogenase were released into the IWF during infiltration. Activities of chitinase and β-1,3-glucanase in the IWF and in the leaf extract were compared by different chromatographic methods. On Sephadex G-75, chitinase appeared as a single peak (M_r 29.8 kD) both in IWF and homogenate. β-1,3-Glucanase, however, showed two peaks in the IWF (M_r 52 and 31.3 kD), whereas the elution pattern of the homogenate showed only one major peak at 22 kD. Chromatofocusing indicated that the IWF contained four chitinases and five β-1,3-glucanases. The elution pattern of the homogenate and IWF were similar with regard to the elution pH, but the peak intensities were distinctly different. Our results demonstrate that extracellular β-1,3-glucanases are different from those located intracellularly. Extracellular and intracellular chitinases do not differ in molecular properties, except for one isozyme which seems to be confined to the extracellular space. We suggest that both enzymes might play a special role in pathogenesis during fungal infection.     

Light-flecks cause non-uniform stomatal opening – studies with special emphasis on Fagus sylvatica L.

The appearance of stomatal patchiness in response to rapid (seconds) changes in light has been studied in European beech, Fagus sylvatica L., and, by comparison, in a further 17 different woody species from the understorey of a European beech forest, using a simple water infiltration method. Water infiltrated areoles indicate open stomata. Since infiltration changes optical characteristics of a leaf section it can be analysed by photography, computer-aided image analysis and by weighing. For F. sylvatica clear differences were found between infiltration of cotyledons (no patchy pattern) and any other leaf type. Despite identical cultivation, leaves of the same type and age from different individual plants responded differently to application of 30 s of light after darkness. In contrast, the patchiness patterns were very similar for leaves of the same type originating from the same plant. Infiltration patterns after a light-fleck, observed on different leaves as a series of momentary clusters, probably indicate waves of opening stomata moving across the leaf blade. During and after a 30 s light-fleck infiltration increased and it continued to increase in the dark up to 10 min, indicating increasing stomatal opening over that period. In general, shade leaves became more infiltrated (by weight) than half-shade or sun leaves, due to larger intercellular air spaces. All species, without exception, showed patchy infiltration and, thus, non-uniform stomatal opening. Measuring leaf gas exchange (as ”quasi-steady states” using a fast responding system) during photosynthetic induction resulted in very similar CO₂ responses of net photosynthesis (A/c _i) as in the true steady state, proving that, in shade and half-shade leaves, the presence of stomatal patchiness does not necessarily affect the calculation of intercellular CO₂ concentrations. Causes and consequences of stomatal patchiness are discussed. Received: 18 November 1998 / Accepted: 1 July 1999     

Plasmalemma protection by the apoplast as assessed from above-zero ozone concentrations in leaf intercellular air spaces

To evaluate reactive absorption of ozone (O3) in the leaf apoplast, amphistomatous leaves of Phaseolus vulgaris L. were allowed to take up O3 through the stomata on the lower leaf surface at high rates for 3-5 min. Up to 5% of the O3 taken up diffused through the leaf and emerged from the stomata on the upper surface, suggesting above-zero O3 concentrations in the leaf intercellular air space, [O3]i. Moreover, measurements revealed that [O3]i increased during exposure to the pollutant. Time patterns of O3 fluxes through the gas phase and into the aqueous apoplast indicated that the increase in [O3]i was the result of a decrease in the diffusion-reaction conductance of the aqueous apoplast, gaq. Under an intense O3 pulse, gaq approached the value of the pure diffusional conductance within 2.5 min of exposure, suggesting the exhaustion of protective resources in the leaf apoplast. Toward the end of the exposure gaq tended to increase, suggesting either a recovery in the protective resources in the leaf apoplast and/or the induction of new defences. The possibility of estimating the degree of protection afforded by apoplast constituents and the rate of recovery of these protective systems in intact leaves using brief O3 pulses is discussed.     

Production of hydrogen peroxide and nitric oxide following introduction of nitrate and nitrite into wheat leaf apoplast

Infiltration of wheat (Triticum aestivum L.) seedling leaves with excess of nitrate, nitrite, or the NO donor sodium nitroprusside leads to increase both in content of hydroperoxide and activity of peroxidase and decrease in superoxide dismutase (SOD) activity in the leaf apoplast. Polymorphism of extracellular peroxidases and the presence of Cu/Zn-SOD have been shown in apoplast. Using an ESR assay, a considerable increase in the level of NO following infiltration of leaf tissues with nitrite has been demonstrated. These data suggest development of both oxidative and nitrosative stresses in leaves exposed to high levels of nitrate or nitrite. A possible interplay of NO and reactive oxygen species in plant cells is discussed.     

水稻叶片气孔导度与冠层反射光谱的定量关系分析

研究了不同土壤水氮条件下水稻(Oryza sativa)叶片气孔导度与冠层光谱反射特征的量化关系。结果表明,不同水分处理下,水稻不同叶位气孔导度变化趋势为:GsL1>GsL2>GsL3>GsL4。高于W3水分条件下,高氮处理的叶片气孔导度高于低氮处理,而低于W3水分条件下,高低氮处理条件下叶片气孔导度差异不显著。发现比值指数R(1 650,760)与不同叶位叶片及不同层次叶片气孔导度的相关性大小为:GsL1>GsL12>GsL123>GsL1234>GsL2>GsL3>GsL4(水稻顶部自上而下第一、二、三、四叶以及自上而下顶部2张、3张、4张叶片的气孔导度值分别表示为:GsL1、GsL2、GsL3、GsL4、GsL12、GsL123和GsL1234),而顶1叶气孔导度与叶面积指数的乘积(冠层叶片气孔导度)同比值指数R(1 650,760)相关程度更高。R(1 650,760)与顶1叶和冠层叶片气孔导度之间皆呈极显著的幂函数负相关。利用不同年份的不同水稻试验对两者的监测模型进行了检验,模型的检验误差RMSE分别为0.05和0.24,表明比值指数R(1 650,760)可以较好地监测不同水氮条件下水稻叶片的气孔开闭特征。     

Proteins in Intercellular Washing Fluids from Leaves of Barley (Hordeum vulgare L.)

Primary leaves of barley were detached, infiltrated with variousbuffers, and centrifuged to yield ‘intercellular washingfluid’ (IWF). Effective pH control of the IWF was obtainedonly with Tris, among all buffers tried. In these liquids, upto 30 proteins were detected by gradient gel electrophoresis.Intracellular protein from injured cells at the cut ends ofleaves was present in IWF but did not contribute significantlyto the total protein recovered in this liquid. The yield ofprotein in the IWF depended on the buffer used for infiltrationand on the concentration of the buffer. Higher concentrationsof buffer yielded more protein. In other experiments leaves were infiltrated with Tris, centrifuged,and then infiltrated a second time with this buffer containingvarious concentrations of the zwitterionic detergent CHAPS,a sulphobetaine derivative of cholate. Gel electrophoresis ofthe IWF obtained after the second centrifugation revealed protein‘bands’ not detected when the detergent had beenomitted from the infiltration buffer. The electrophoretic patternsof protein ‘bands’ in the gels differed dependingon the CHAPS concentration used for infiltration. The effect of CHAPS on plasmalemma integrity was studied byobserving infiltrated tissue with the electron microscope andby treating isolated protoplasts with the detergent. After infiltrationwith CHAPS at 0.6 mM or 2.0 mM no plasmalemma breaks were detectedin leaves, and isolated protoplasts survived exposure to CHAPSat these concentrations for 2 h without bursting. Evidently,CHAPS at these low concentrations did not destroy the integrityof the plasmalemma; the additional protein recovered in theIWF under these conditions probably originated in the cell wall.Infiltration of leaves with 6.0 mM CHAPS resulted in breaksof the plasmalemma, in tissue collapse and leaf tip necrosis.Isolated protoplasts burst within minutes after being exposedto CHAPS at this concentration. Key words: Cell wall permeability, Intercellular space, Detergent, CHAPS, Protoplasts     

Pathway of phloem loading in the C3 tropical orchid hybrid Oncidium Goldiana

The apoplast of mature leaves of the tropical orchid OncidiumGoldiana was perfused with 0.5 mM p-chloromercuribenzenesulphonicacid (PCMBS) via the transpiration stream in order to test themode of phloem loading. The efficacy of introducing PCMBS byperfusion was shown by saffranin O dye movement in the veinsand leaf apoplast in control experiments. Photoassimilate exportas the result of phloem loading was measured by collection of¹⁴CO₂-derived photoassimilates from the basal cut-ends of intactleaves. Phloem loading and translocation of photoassimilates was inhibitedby 89% in leaves perfused with PCMBS for 1 h. The effect ofPCMBS on leaf photosynthesis was minimal. The amount of radiocarbonfixed by PCMBS-treated leaves averaged 89% of control leavesperfused with distilled water. A negative correlation betweenthe total amount of photoassimilate exuded and the calculatedconcentration of PCMBS in the leaf apoplast was also observed.The results indicate that phloem loading in Oncidium Goldianaoccurs via the apoplastic pathway. Key words: Phloem loading, apoplast, PCMBS, tropical orchid     

The guard-cell apoplast as a site of abscisic acid accumulation in Vicia faba L.

Abscisic acid (ABA) integrates the water status of a plant and causes stomatal closure. Physiological mechanisms remain poorly understood, however, because guard cells flanking stomata are small and contain only attomol quantities of ABA. Here, pooled extracts of dissected guard cells of Vicia faba L. were immunoassayed for ABA at sub‐fmol sensitivity. A pulse of water stress was imposed by submerging the roots in a solution of PEG. The water potentials of root and leaf declined during 20 min of water stress but recovered after stress relief. During stress, the ABA concentration in the root apoplast increased, but that in the leaf apoplast remained low. The ABA concentration in the guard‐cell apoplast increased during stress, providing evidence for intra‐leaf ABA redistribution and leaf apoplastic heterogeneity. Subsequently, the ABA concentration of the leaf apoplast increased, consistent with ABA import via the xylem. Throughout, the ABA contents of the guard‐cell apoplast, but not the guard‐cell symplast, were convincingly correlated with stomatal aperture size, identifying an external locus for ABA perception under these conditions. Apparently, ABA accumulates in the guard‐cell apoplast by evaporation from the guard‐cell wall, so the ABA signal in the xylem is amplified maximally at high transpiration rates. Thus, stomata will display apparently higher sensitivity to leaf apoplastic ABA if stomata are widely open in a relatively dry atmosphere.     

Protoplast volume:water potential relationship and bound water fraction in spinach leaves

Methods used to estimate the (nonosmotic) bound water fraction (BWF) (i.e. apoplast water) of spinach (Spinacia oleracea L.) leaves were evaluated. Studies using three different methods of pressure/volume (P/V) curve construction all resulted in a similar calculation of BWF; approximately 40%. The theoretically derived BWF, and the water potential (Ψ_w)/relative water content relationship established from P/V curves were used to establish the relationship between protoplast (i.e. symplast) volume and Ψ_w. Another method of establishing the protoplast volume/Ψ_w relationship in spinach leaves was compared with the results from P/V curve experiments. This second technique involved the vacuum infiltration of solutions at a range of osmotic potentials into discs cut from spinach leaves. These solutions contained radioactively labeled H₂O and sorbitol. This dual label infiltration technique allowed for simultaneous measurement of the total and apoplast volumes in leaf tissue; the difference yielded the protoplast volume. The dual label infiltration experiments and the P/V curve constructions both showed that below −1 megapascals, protoplast volume decreases sharply with decreasing water potential; with 50% reduction in protoplast volume occurring at −1.8 megapascals leaf water potential.     

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.     

The nature of heterogeneity in the stomatal behaviour of Phaseolus vulgaris L. primary leaves

The aim of this research was to investigate the nature of heterogeneity in stomatal conductance and, in particular, to determine whether the characteristic 'patchy' pattern of water infiltration is reflected in measurements on individual stomata. Silicone rubber replicas were made of primary leaves of glasshouse-grown Phaseolus vulgaris L. plants, and the leaves were then infiltrated with water at controlled sub-atmospheric gas pressures according to their estimated or measured stomatal conductance. Seven leaves examined in detail all showed patchy infiltration, and the mean sire of infiltrated areas was negatively correlated with the prevailing stomatal conductance. In four of the leaves, a one millimetre wide transect across the leaf was selected for further detailed study. Measurements of mean peristomatal groove distance (PGD) and stomatal frequency were made along the transect and related to the state of infiltration. Analysis of variance indicated that, in all four cases, variation in PGD among patches was highly significant, but there was no significant difference between patches of different infiltration categories. Thus, local (patch-level) variation in stomatal aperture appeared to bear no relation to the infiltration status of the patches. The dominant source of stomatal variability was between individual pores in the same locality, which accounted for 82% or more of the total variability. Taking into account variation in stomatal frequency, correlations between predicted stomatal conductance and the extent of infiltration were significant in only one out of the seven leaves studied. Possible reasons for these results are discussed. It is suggested that the infiltration method misrepresents the underlying state of the stomata as being either open or closed, when there is little evidence for this from measurements of stomatal dimensions. For these unstressed plants under relatively stable conditions, it is concluded that the 'unit of variability' in stomatal heterogeneity may rest at the individual pore ('micro') scale, rather than at the areolar patch ('macro') scale, or above.     

Leaf age-related differences in apoplastic NH(4)(+) concentration, pH and the NH(3) compensation point for a wild perennial.

Extracts of the foliar apoplast of leaves of different ages of Luzula sylvatica (Huds.) Gaud. were prepared by vacuum infiltration and centrifugation. Measurements of pH and concentration were performed on extracts. From these bioassay measurements the relative magnitude of NH(3) compensation points for leaves of different ages were inferred. Young leaves were found to have much higher apoplast pH than old leaves, leading to the calculation of 4-10-fold higher NH(3) compensation points. Such age-related differences in the NH(3) compensation point are considerably larger than those previously reported. Apoplast pH and concentration were found to increase during leaf expansion before declining prior to senescence. Bulk foliar tissue pH, and total N concentrations were also found to be generally higher in young leaves than in old leaves. Where a significant correlation was found, total foliar N, bulk tissue foliar and the calculated NH(3) compensation point were all found to increase with N supplied to roots, whilst apoplast and bulk tissue H(+) concentrations were found to decline. The potential of bulk foliar tissue measurements to act as simple predictors of the NH(3) compensation point is discussed.     

Avoiding high relative air humidity during critical stages of leaf ontogeny is decisive for stomatal functioning

Plants of several species, if grown at high relative air humidity (RH ≥85%), develop stomata that fail to close fully in case of low leaf water potential. We studied the effect of a reciprocal change in RH, at different stages of leaf expansion of Rosa hybrida grown at moderate (60%) or high (95%) RH, on the stomatal closing ability. This was assessed by measuring the leaf transpiration rate in response to desiccation once the leaves had fully expanded. For leaves that started expanding at high RH but completed their expansion after transfer to moderate RH, the earlier this switch took place the better the stomatal functioning. Leaves initially expanding at moderate RH and transferred to high RH exhibited poor stomatal functioning, even when this transfer occurred very late during leaf expansion. Applying a daily abscisic acid (ABA) solution to the leaves of plants grown at continuous high RH was effective in inducing stomatal closure at low water potential, if done before full leaf expansion (FLE). After FLE, stomatal functioning was no longer affected either by the RH or ABA level. The results indicate that the degree of stomatal adaptation depends on both the timing and duration of exposure to high RH. It is concluded that stomatal functionality is strongly dependent on the humidity at which the leaf completed its expansion. The data also show that the effect of ambient RH and the alleviating role of ABA are restricted to the period of leaf expansion.