Compartmentation and transport of zinc in barley primary leaves as basic mechanisms involved in zinc tolerance

The heavy metal zinc was administered to barley seedlings by increasing its concentration in the hydroponic medium. The most dramatic effect was a severe inhibition of root elongation with little effect on root biomass production. The growth of primary leaves was little affected although the zinc content of the primary leaves increased several-fold. A detailed compartment analysis was performed for 10-d-old barley primary leaves. Under low zinc nutrition (2mmol m ⁻³), highest zinc contents were observed in the cytoplasm of mesophyll protoplasts. At inhibitory zinc concentrations in the hydroponic medium (400 μmol m ⁻³), zinc levels dramatically and preferentially increased in the apoplastic space. Elevated zinc levels were also observed in the epidermal cells, and to a lesser extent, in mesophyll vacuoles. The cytoplasmic content of mesophyll protoplasts was unchanged, indicating perfect zinc homeostasis within the leaf. In order to understand the transport mechanisms underlying the steady-state distribution profile, we used ⁶⁵Zn to conduct uptake experiments with leaves whose lower epidermis had been stripped. The leaves were placed on zinc solutions of varying concentrations containing ⁶⁵Zn for 5 min to 6 h. After the incubation, the leaves were fractionated into mesophyll and epidermis protoplasts and residue, the latter mainly representing cell wall. Adsorption of Zn to the extracellular matrix was 100 times faster than Zn uptake into the cells. By far the largest portion taken up into the mesophyll protoplasts rapidly appeared in the vacuolar compartment. These results demonstrate the importance of compartmentation and transport as homeostatic mechanisms within the leaves to handle high, possibly toxic, zinc levels in the shoot.     

Implication of ABA and proline on cell membrane injury of water deficit stressed barley seedlings

The aim of this work was to examine the ability of ABA and proline to counteract the deleterious effect of water deficit stress on cell membrane injuries. Six-day-old seedlings of two barley genotypes (cv. Aramir, line R567) were treated with ABA (2·10⁻⁴ M) or proline (0.1 M) for 24 h, and then subjected to osmotic stress for 24h, by immersing their roots in polyethylene glycol (PEG 6000) solution of osmotic potential of −1.0 MPa and −1.5 MPa or by submerging the leaf pieces in PEG solution of osmotic potential of −1.6 MPa. Pretreatment of plants with ABA and proline caused an increase of free proline level in the leaves. Plants treated with ABA exhibited a lower membrane injury index under water stress conditions than those untreated even when no effect of this hormone on RWC in the leaves of stressed plants was observed. Pretreatment of plants with proline prevented to some extent membrane damage in leaves of the stressed seedlings, but only in the case when stress was imposed to roots. Improvement in water status of leaves was also observed in seedlings pretreatment with proline. The protective effect of both ABA and proline was more pronounced in line R567 that exhibited higher membrane injury under water deficit stress conditions.     

Isolation and characterization of variant wheat cultivars for ABA sensitivity

Genetic variants for abscisic acid (ABA) sensitivity are important for investigating the role of ABA sensitivity in conditioning plant response to environmental stress, and especially to those soil conditions that may elicit a root-mediated hormonal signal. This study was performed in order to isolate variation in ABA sensitivity among wheat (Triticum aestivum and T. durum) cultivars, as characterized by two plant responses: (i) shoot growth reduction in response to 5×10^?2mol m^?3 ABA (racemic) in the root medium of hydroponically grown plants, and (ii) changes in transpiration and gas exchange in a bioassay of detached leaves (leaflaminac) infused with 10^?4mol m^?3 ABA. Very significant (P≤0.01) and repeatable differences were found among 36 wheat cultivars and 19 landraces in the growth rate in ABA-containing nutrient solutions, expressed as a percentage of the growth rate in control nutrient solutions (ABA/control ratio). In duplicate experiments, the ABA/control ratio ranged between 60 and 83% for the least sensitive cultivars (V2151-3, Bethlehem, K1056 and Sunstar) and between 9 and 19% for the most sensitive cultivars (Sundor, Comet, Barkaec and V5). In the transpiration bioassay, performed with seven selected cultivars, it was found that the reductions in transpiration of ABA-infused leaves corresponded very well with the reductions in growth in response to ABA in the root media. Measurement of gas exchange in the detached leaves of two cultivars differing in ABA sensitivity (Bethlehem and Sundor) showed that variable ABA sensitivity was expressed very well in the stomatal conductance, carbon exchange rate (CER) and photosynthetic capacity (CER/C_i ratio) of the leaf. These results therefore allowed us to isolate wheat variants for ABA sensitivity and to conclude that, while ABA sensitivity is expressed in the growth of plants challenged by ABA in the root medium, the control of sensitivity resides, at least partly, in the leaf.     

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.     

The relationship between leaf growth and ABA accumulation in the grass leaf elongation zone

Detached barley (Hordeum vulgare L.) shoots, maintained at different air temperatures and VPDs, were fed ABA via the sub-crown internode in a leaf elongation assay. Analysis of variance of leaf elongation rate (LER) showed significant effects of temperature (T), fed [ABA] and the interaction T × [ABA]. However, the interaction became non-significant when LER was modelled against the [ABA] of the elongation zone, [EZ-ABA] When detached barley shoots were fed sap from droughted maize (Zea mays L.) plants, sap [ABA] could not explain the growth inhibitory activity. Measurement of [EZ-ABA] accounted for this ‘unexplained’ growth inhibition. The detached shoot experiments indicated that [EZ-ABA], and not xylem sap [ABA], was an appropriate explanatory variable to measure in droughted plants. However, ABA accumulation in the elongation zone could not explain a 35% growth reduction in intact droughted plants; thus we considered an interaction of water status and ABA. Using a coleoptile growth assay, we applied mild osmotic stresses (ψ=0 to ?0.06 MPa) and 10^?4 mol m^?3 ABA. Individually, these treatments did not inhibit growth. However, osmotic stress and ABA applied together significantly reduced growth. This interaction may be an important mechanism in explaining leaf growth inhibition of droughted plants.     

Cell biological mechanism for triggering of ABA accumulation under water stress inVicia faba leaves

Water stress-induced ABA accumulation is a cellular signaling process from water stress perception to activation of genes encoding key enzymes of ABA biosynthesis, of which the water stress-signal perception by cells or triggering mechanism of the ABA accumulation is the center in the whole process of ABA related-stress signaling in plants. The cell biological mechanism for triggering of ABA accumulation under water stress was studied in leaves ofVicia faba. Mannitol at 890 mmol ·kg^-1 osmotic concentration induced an increase of more than 5 times in ABA concentration in detached leaf tissues, but the same concentration of mannitol only induced an increase of less than 40 % in ABA concentration in protoplasts. Like in detached leaf tissues, ABA concentration in isolated cells increased more than 10 times under the treatment of mannitol at 890 mmol · kg^-1 concentration, suggesting that the interaction between plasmalemma and cell wall was essential to triggering of the water stress-induced ABA accumulation. Neither Ca²⁺-chelating agent EGTA nor Ca²⁺channel activator A23187 nor the two cytoskeleton inhibitors, colchicine and cytochalasin B, had any effect on water stress-induced ABA accumulation. Interestingly water stress-induced ABA accumulation was effectively inhibited by a non-plasmalemma-permeable sulfhydryl-modifier PCMBS (p-chloromercuriphenyl-sulfonic acid), suggesting that plasmalemma protein(s) may be involved in the triggering of water stress-induced ABA accumulation, and the protein may contain sulfhydryl group at its function domain.     

Abscisic acid introduced into the transpiration stream accumulates in the guard-cell apoplast and causes stomatal closure

Petioles of water‐sufficient intact Vicia faba L. plants were infused with 1 µm abscisic acid (ABA) to simulate the import of root‐source ABA. This protocol permitted quantitative ABA delivery, up to 300 pmol ABA over 60 min, to the leaf without ambiguities associated with perturbations in plant–water status. The ABA concentrations in whole‐leaf samples and in apoplastic sap increased with the amount infused; ABA degradation was not detected. The ABA concentration in apoplastic sap was consistent with uptake of imported ABA into the leaf symplast, but this interpretation is qualified. Our focus was quantitative cellular compartmentation of imported ABA in guard cells. Unlike when leaves are stressed, the guard‐cell symplast ABA content did not increase because of ABA infusion (P = 0·48; 3·0 ± 0·5 versus 4·0 ± 1·2 fg guard‐cell‐pair^?1). However, the guard‐cell apoplast ABA content increased linearly (R² = 0·98) from ?0·2 ± 0·5 to 3·1 ± 1·3 fg guard‐cell‐pair^?1 (≈ 3·1 µm ) and was inversely related to leaf conductance (R² = 0·82). Apparently, xylem ABA accumulates in the guard‐cell wall as a result of evaporation of the apoplast solution. This mechanism provides for integrating transpiration rate and ABA concentration in the xylem solution.     

Palisade mesophyll cell expansion during leaf development in Zinnia elegans (Asteraceae)

Temporal and spatial patterns of palisade mesophyll cell expansion in Zinnia elegans were characterized as a basis for developing a suspension culture model for mesophyll cell expansion. Our objectives were to 1) identify the leaf regions from which cells in various stages of expansion could be selectively isolated for culture, and 2) develop a basis for comparison of rate and extent of mesophyll cell expansion in culture with that in the leaf. Palisade mesophyll cells were isolated from expanding leaves by gentle physical maceration without the use of enzymes. Isolated cells from leaves in different stages of expansion were then measured by computer image analysis. Analysis of size frequency distributions showed that unexpanded cells can be isolated from the entire blade of small leaves or the basal regions of partially expanded leaves. Fully expanded cells can be obtained from the apical and middle regions of partially expanded leaves. Within the leaf, Zinnia mesophyll cells expanded from about 400 μm² to about 2.300 μm² at an estimated rate of 160 μm² d^-1. The percent increase in cell length exceeded the percent increase in cell width. Expansion of mesophyll cells continued for 6–8 d after epidermal expansion ceased. This difference in the timing of cell expansion in epidermal and mesophyll cells indicates that different regulatory factors may be operating in these adjacent tissues and underscores the importance of investigating the regulation of mesophyll cell expansion at the cellular level.     

The permeability of the epidermal cell plasma membrane of barley leaves to abscisic acid

Uptake of ³H-labelled (±)-abscisic acid (ABA) into isolated barley (Hordeum vulgare L.) epidermal cell protoplasts (ECP) was followed over a range of pH values and ABA concentrations. The present results show that ABA uptake is not always linearly correlated with the external concentration of undissociated ABA (ABAH). At pH 7.25, ABA uptake exhibited saturation kinetics with an apparent K _m value of 75 mmol·m^–3 to tal ABA. This saturable transport component was inhibited by pretreating the protoplasts with 1 mol·m^–3 p-chloromercuribenzenesulfonic acid at pH 8.0, conditions that minimized the uptake of this acid sulfhydryl reagent. Moreover, the rate of (±)-[^3]HABA uptake was reduced by addition of 0.1 mol·m^–3 (±)-ABA to 41%, whereas the same concentration of (±)-ABA was approximately half as effective (46% of the inhibitory effect). Thus, it was concluded that only (±)-ABA competes for an ABA carrier that is located in the epidermal cell plasma membrane. The permeability of the epidermal cell plasma membrane was studied by performing a Collander analysis. At pH 6 the overall plasma-membrane permeability of epidermal cells was similar to that of guard cells but was about two times higher than that of mesophyll cells.Abbreviations ABA abscisic acid - ABA^– anion of ABA - ABAH undissociated ABA - 2,4-D 2,4-dichlorophenoxyacetic acid - DMO 5,5-dimethyloxazolidine-2,4-dione - ECP deepidermal cell protoplast - K_r partition coefficient - M_r relative molecular mass - NEM N-ethylmaleimide - PCMBS p-chloromercuriben zenesulfonic acid - P_s permeability coefficient We are grateful to Barbara Dierich for expert technical assistance, to Prof. H. Gimmler (Lehrstuhl für Botanik I, Universität Würzburg, FRG) for helpful discussions and to the Deutsche Forschungsgemeinschaft (SFB 251, TP 3) for financial support.     

Characterization of the epidermis from barley primary leaves

A method is described for isolating epidermal protoplasts from the primary leaves of barley (Hordeum vulgare L.). Epidermal protoplasts are lighter than mesophyll protoplasts because of their smaller ratio of cytoplasm to vacuole, and can be separated from the latter by density-gradient centrifugation after complete digestion of the leaves. We have started a basic characterization of the epidermal protoplast fraction in comparison with mesophyll protoplasts. Epidermal protoplasts had a mean diameter of 63.5 m, whereas that of mesophyll protoplasts was 35.7 m. Their respiratory oxygen consumption was not influenced by light. They contained acid hydrolases and cytoplasmic enzymes in relative activities different from those of mesophyll protoplasts. Their polypeptide pattern as judged from two-dimensional separations was, in principle, similar to that of mesophyll cells after elimination of the plastids from the latter by the preparation of vacuoplasts. However, in addition, a considerable number of epidermis-specific polypeptides were observed. Isolated epidermal protoplasts were viable and efficiently incorporated [³⁵S]methionine into newly synthesized proteins. The results show that epidermal protoplasts are suitable for the investigation of the physiological and molecular properties of epidermal cells in leaves.Abbreviation SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis We are grateful to Professor U. Heber (Lehrstuhl Botanik 1, Würzburg) for his continuous support. This work was supported by the DFG and the University of Würzburg within the Sonderforschungsbereich 176.     

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.     

Abscisic acid added to the nutrient solution induces a water deficit-like response in Trifolium subterraneum

The response of subterranean clover (Trifolium subterraneum) to the addition of increasing concentrations of abscisic acid (ABA) in the nutrient solution (10^?3, 10^?2 and 10^?1 mol m^?3) was studied under growth room conditions. Six cultivars of contrasting yield capacity were compared. Plants were grown in Hoagland's solution until they had produced at least four fully developed leaves. ABA was then added and its effect on the fresh weight, leaf number and longest root length was determined, 1 day, 4 days, 7 days and 11 days after addition. The addition of ABA caused significant reductions in all the measured growth parameters, as well as a significant decrease in leaf water potential, which was dependent on the ABA concentration. The average growth reduction after 11 days under 10^?1 mol m^?3 ABA were within previously reported ranges for this crop under drought, in field conditions. The average leaf number, area of a fully developed leaf and the dry weight per plant of the six cultivars decreased by approximately 50%, whereas the root/shoot ratio increased by 80%. The variation and ranking for this treatment resembled closely those obtained for the same cultivars in field experiments. The cvs Clare, Nuba and Seaton Park, showed the best results under both control and ABA treated conditions. The correlation between the response to ABA in nutrient solution and previous water deficit studies raises the possibility of using this approach as an alternative way to quantify the drought sensitivity of subterranean clover cultivars.     

Movement of Abscisic Acid Across the Plasmalemma and the Tonoplast of Guard Cells of Valerianella locusta

Uptake experiments and efflux compartmental analyses of abscisic acid (ABA) with acid treated epidermal peels of Valerianella locusta were performed to elucidate the mechanisms of transport of ABA across the plasmalemma and tonoplast of guard cells. ABA uptake across the plasmalemma is linearly correlated with external ABA concentration in the incubation medium. Under alkaline conditions ABA-uptake was not significantly above background, indicating that ABA uptake occurs mainly by diffusion of undissociated ABAH as the most permeable species, which is trapped afterwards in the alkaline cytosol as impermeable ABA^?. Efflux analysis of ABA revealed a saturable component of ABA transfer across the tonoplast. A Woolf-Augustinsson-Hofstee analysis suggested the existence of two transport systems for ABA at the tonoplast. The high affinity transport system had a K_M of 0.21 mol m^?3 and a V_max 85.8 amol ABA cell^?1 h^?1. Using the data of the uptake and efflux experiments we calculated the permeability coefficients of ABA for the plasmalemma and the tonoplast of guard cells, which are 2.46 10^?7 m s–¹ and 1.26 10^?8m s^?1, respectively. The distribution of the pH-probe (¹⁴C)-DMO between medium, cytosol and vacuole was investigated and used to calculate cytosolic and vacuolar pH. The vacuolar pH is too low to explain the high vacuolar ABA concentration by trapping of ABA^?, whereas the cytosol is sufficiently alkaline to act as an efficient anion trap. Therefore we conclude that ABA transport across the guard cell tonoplast is catalyzed by a saturable uptake component.     

Xylem Sap pH Increase: A Drought Signal Received at the Apoplastic Face of the Guard Cell That Involves the Suppression of Saturable Abscisic Acid Uptake by the Epidermal Symplast

Drought increased the pH of Commelina communis xylem sap from 6.1 to 6.7. Conductances of transpiring leaves were 50% lower in pH 7.0 than in pH 6.0 buffers, but bulk leaf abscisic acid (ABA) concentration and shoot water status were unaffected by pH. Stomatal apertures of isolated abaxial epidermis incubated on simple buffers increased with external pH, so in vivo this must be overridden by alternative pH effects. Reductions in leaf transpiration rate at pH 7.0 were dependent on the presence of 10-8 mol dm-3 ABA in the xylem stream. We inferred that at pH 7.0 leaf apoplastic ABA concentrations increased: pH did not affect distributions of ABA among leaf tissues, but isolated epidermis and mesophyll tissue took up more 3H-ABA from pH 6.0 than from pH 7.0 buffers. The apoplastic ABA increase at pH 7.0 may result from reduced symplastic sequestration. A portion of 3H-ABA uptake by the epidermis was saturable at pH 6.0 but not at pH 7.0. An ABA uptake carrier may contribute to ABA sequestration by the leaf symplast of well-watered plants, and its inactivity at pH 7.0 may favor apoplastic ABA accumulation in draughted plants. Effects of external pH on stomatal apertures in the isolated epidermis indicate that published data supporting a role for internal guard cell ABA receptors should be reassessed.     

Cell biological mechanism for triggering of ABA accumulation under water stress inVicia faba leaves

Water stress-induced ABA accumulation is a cellular signaling process from water stress perception to activation of genes encoding key enzymes of ABA biosynthesis, of which the water stress-signal perception by cells or triggering mechanism of the ABA accumulation is the center in the whole process of ABA related-stress signaling in plants. The cell biological mechanism for triggering of ABA accumulation under water stress was studied in leaves ofVicia faba. Mannitol at 890 mmol ·kg^-1 osmotic concentration induced an increase of more than 5 times in ABA concentration in detached leaf tissues, but the same concentration of mannitol only induced an increase of less than 40 % in ABA concentration in protoplasts. Like in detached leaf tissues, ABA concentration in isolated cells increased more than 10 times under the treatment of mannitol at 890 mmol · kg^-1 concentration, suggesting that the interaction between plasmalemma and cell wall was essential to triggering of the water stress-induced ABA accumulation. Neither Ca²⁺-chelating agent EGTA nor Ca²⁺channel activator A23187 nor the two cytoskeleton inhibitors, colchicine and cytochalasin B, had any effect on water stress-induced ABA accumulation. Interestingly water stress-induced ABA accumulation was effectively inhibited by a non-plasmalemma-permeable sulfhydryl-modifier PCMBS (p-chloromercuriphenyl-sulfonic acid), suggesting that plasmalemma protein(s) may be involved in the triggering of water stress-induced ABA accumulation, and the protein may contain sulfhydryl group at its function domain.     

Changes in cell ultrastructure and endogenous abscisic acid and indole-3-actic acid concentrations in Fatsia japonica leaves under polyethylene glycol-induced water stress

Ultrastructural alterations in epidermal and mesophyll cells as well as variations in bulk leaf endogenous ABA and IAA concentrations were studied in PEG-treated plants of Fatsia japonica Decne & Plank. Under stress induced by PEG vesicles containing fibrous material and electron-dense bodies associated with plasma membranes were observed. Cytochemical examination indicated that electron-dense bodies corresponded to lipids and the fibrous material of the vesicles were polysaccharides. Chloroplasts, mitochondria, nuclei and Golgi apparatus also showed modifications. A strong relationship was found between increasing PEG-induced water stress, increasing endogenous ABA and ultrastructural changes. In relation with leaf ontogeny and ABA concentration a higher ABA level was observed in younger than in older leaves. The differences in the endogenous concentrations of indole-3-acetic acid are unclear, except after 7 days of PEG-treatment. The increase in the endogenous abscisic acid concentration could be related with the ultrastructural changes.Abbreviations ABA = abscisic acid - IAA = indole-3-acetic acid - PEG = polyethylene glycol - = leaf water potential - TEM = transmission electron microscope     

Changes in Chloride Fluxes and Cytosolic pH Induced by Abscisic Acid in Elodea densa Leaves

The effects of ABA on intracellular pH, net H⁺ extrusion, Cl^? fluxes and E_m values were studied in Elodea densa leaves, and the possible relationships between the ABA-induced changes of cytosolic pH and of Cl^? and H⁺ fluxes were investigated. Cytosolic and vacuolar pH were calculated by the weak acid and weak base distribution method. The data show that, also in this material (a water plant without stomata), ABA induces a decrease in both net H⁺ extrusion and intracellular pH, and strongly inhibits Cl^? efflux. No significant effect of ABA is detectable on E_m values, either at short or long intervals in the presence or absence of K⁺. Cl^? efflux is apparently independent of the activity of the plasmalemma H⁺ pump and of the E_m values. Conversely, it strongly depends on the value of cytosolic pH, a larger efflux occurring for the lower pH values both in the presence and in the absence of ABA. These results indicate that the ABA-induced cytosolic acidification cannot be the cause but, possibly, a consequence of the decrease in Cl^? efflux, and are consistent with the hypothesis of a primary role of ABA in regulating Cl^? efflux, presumably by directly affecting a class of Cl^?-permeable channels.     

Signal Integration by ABA in the Blue Light-Induced Acidification of Leaf Pavement Cells in Pea (Pisum sativum L. var. Argenteum)

Leaf pavement cell expansion in light depends on apoplastic acidification by a plasma membrane proton-pumping ATPase, modifying cell wall extensibility and providing the driving force for uptake of osmotically active solutes generating turgor. This paper shows that the plant hormone ABA inhibits light-induced leaf disk growth as well as the blue light-induced pavement cell growth in pea (Pisum sativum L.). In the phytochrome chromophore-deficient mutant pcd2, the effect of ABA on the blue light-induced apoplastic acidification response, which exhibits a high fluence phase via phytochrome and a low fluence phase via an unknown blue light receptor, is still present, indicating an interaction of ABA with the blue light receptor pathway. Furthermore, it is shown that ABA inhibits the blue light-induced apoplastic acidification reversibly. These results indicate that the effect of ABA on apoplastic acidification can provide a mechanism for short term, reversible adjustment of leaf growth rate to environmental change.Key Words: ABA, apoplastic acidification, blue light, epidermal pavement cell growth, leaf growth, pea (Pisum sativum L.), signal integration     

Iron deficiency decreases the Fe(III)-chelate reducing activity of leaf protoplasts

The ferric-chelate reductase (FC-R) activity of mesophyll protoplasts isolated from Fe-sufficient (control) and Fe-deficient sugar beet (Beta vulgaris L.) leaves has been characterized. Measurements were made in an ionic environment similar to that in the apoplastic space of the sugar beet mesophyll cells. The FC-R activity of Fe-sufficient and Fe-deficient protoplasts was dependent on light. Fe deficiency decreased markedly the FC-R activity per protoplast surface unit. The optimal pH for the activity of the FC-R in mesophyll protoplasts was in the range 5.5 to 6.0, typical of the apoplastic space. Beyond pH 6.0, the activity of the FC-R in mesophyll protoplasts decreased markedly in both Fe-sufficient and Fe-deficient protoplasts. These data suggest that both the intrinsic decrease in FC-R activity per protoplast surface and a possible shift in the pH of the apoplastic space could lead to the accumulation of physiologically inactive Fe pools in chlorotic leaves.     

Comparison of exportation and metabolism of xylem-delivered ABA in maize leaves at different water status and xylem sap pH

³H-ABA was introduced into the xylem stream of maize ( Zea mays}) leaves on intact plants by incubation of a semi-attached flap of the sheath in solutions. The relative contribution of exportation and metabolism to the fate of xylem-delivered ABA was assessed in leaves which were either kept at different water potentials through soil drying treatments or subjected to different xylem pHs (pH 7.4 vs. pH 5.5) through a phosphate buffer in the feeding solutions. Xylem-delivered ABA was rapidly metabolised in well-watered leaves with a half-life of 2.19 h in the relatively mature leaves used in this study. Re-exportation of xylem-delivered ABA from leaves was much slower than metabolism. It took 24 h for half of the fed radioactivity to disappear from the well-watered leaves, and very possibly this radioactivity was in the form of metabolites of fed ³H-ABA. Although soil drying usually increases the output of ABA through phloem as reported in previous studies, it greatly reduced the re-exportation of xylem-fed ABA and/or its metabolites. Metabolism was also significantly reduced by the treatment of soil drying (half-life extended from 2.19 to 3.63 h), although the magnitude of change was much less than that of exportation. Manipulation of the pH in the feeding solution also had its effect on the re-exportation. A shift of pH from 5.5 to 7.4 reduced the rate of disappearance of the total radioactivity fed into the attached leaves, but showed no significant effect on the rate of ABA metabolism. It was concluded that it was the ABA metabolism, rather than a re-exportation from leaves, which was mainly responsible for the disposal of the ABA signal from the xylem and therefore preventing an accumulation in leaves. Water stress and pH increase of xylem sap would increase the time of such ABA's presence in the leaves. Since xylem-imported ABA is unlikely to be re-exported from leaves in its intact form, we believe a recycling of ABA from xylem to phloem through leaves plays only a minor role.