Locating active proton extrusion pumps in leaves

Abstract Stabilized microscopic preparations of an apoplastic fluorescent tracer, sulphorhodamine G (SR), have previously shown it confined to leaf cell walls. SR has a pK of 3.2, is dissociated at normal wall pH, and therefore does not enter cells. In transpiring soybean leaves, the SR showed a major internal water pathway in the walls of the paraveinal mesophyll (PVM), which has been implicated in the temporary storage of protein. Also the SR penetrated the PVM and bundle sheath cells, staining organelles and vacuoles, but not other leaf cells. This implies that sufficient SR is undissociated in these walls to allow penetration, and that the pH of the PVM walls is lower than that of most other cells. It is proposed that proton extrusion pumps are revealed by the low wall pH, and that these pumps are probably involved in collecting ammo acids from the transpiration stream.     

Proton pump activity in bundle sheath tissues of broad-leaved trees in relation to leaf age

The fluorescent probe sulphorhodamine G (SR) has been previously used as an indicator of low extra-cellular pH and, by inference, of proton extrusion activity in living leaves. In legumes the SR uptake and proton extrusion was characteristic of the extended bundle sheath system (EBS) or paraveinal mesophyll, composed of bundle sheath cells and the related network of bridging cells between veins. This system has been identified as a site of temporary storage of amino carbon in soybean. A tree species. Populus deltoides Bartr. ex Marsh, was known both to have the EBS system in its leaves and to carry organic nitrogen in its xylem sap. It is now shown that P. deltoides also accumulates the SR probe in the EBS system. This association has been explored in 8 other broad-leaved tree species. Seven of the 8 species have EBS systems and accumulate SR in them in early summer. The 8th species, Tilia americana L. has no EBS system and shows weak SR accumulation. The capacity to accumulate SR (and by inference to scavenge solutes from the transpiration stream) disappeared in all species at various stages in late summer. In two species, in addition, SR accumulation is interrupted for several weeks during fruit growth. It is proposed that EBS systems will be found in many dicotyledonous leaves, and will be found to scavenge solutes, especially organic nitrogen, from the xylem sap.     

Leaf teeth, transpiration and the retrieval of apoplastic solutes in balsam poplar

The rapid flow of the transpiration stream through major veins to leaf teeth was followed in leaves of Populus balsamifera L., using the tracer sulphorhodamine G (SR), which probes for cells with H⁺-extrusion pumps. The tracer accumulated quickly in the hydathodes of the teeth. It was shown by freeze-substitution and anhydrous processing that SR was taken up by phloem parenchyma and epithem cells of the hydathode. When ¹⁴C-labelled aspartate was fed to the leaves in the transpiration stream, it also was taken up most strongly by the same phloem parenchyma and epithem cells. It is proposed that one function of the hydathodes in leaf teeth is the retrieval of solutes from the transpiration stream.     

Water pathways in wheat leaves. III. The passage of the mestome sheath and the function of the suberised lamellae

The fluorochrome sulphorhodamine G, when present in the transpiration stream in wheat leaves, passes rapidly out of the veins and produces fluorescence in the mesophyll and epidermal cell walls. The path of movement of the dye out of the tracherary elements and across the mestome sheath to the parenchyma sheath cells was followed by rapid freezing, freeze-subsitution, dry embedding in resin, sectioning and epifluorescence microscopy. The sulphorhodamine solution was visible in tracheary elements, and, where it had passed out of the tracheary elements, strongly fluorescent in some of the cell walls. The patterns of wall fluorescence are used to chart the movements of water from the xylem through some of the radial walls of mestome sheath cells near the xylem to the free space of the mesophyll. The suberised lamellae of the mestome sheath cells must form an incomplete barrier near the xylem to permit passage of the dye. A hypothesis is formulated that the function of the suberised lamellae is to keep separate the oppositely directed fluxes of water and assimilates through the sheath. It is further proposed that the function of pits in living cells is a similar insulation of the symplastic traffic from the wayward waters of the apoplast.     

Cadmium uptake and sequestration kinetics in individual leaf cell protoplasts of the Cd/Zn hyperaccumulator Thlaspi caerulescens

Hyperaccumulators store accumulated metals in the vacuoles of large leaf epidermal cells (storage cells). For investigating cadmium uptake, we incubated protoplasts obtained from leaves of Thlaspi caerulescens (Ganges ecotype) with a Cd-specific fluorescent dye. A fluorescence kinetic microscope was used for selectively measuring Cd-uptake and photosynthesis in different cell types, so that physical separation of cell types was not necessary. Few minutes after its addition, cadmium accumulated in the cytoplasm before its transport into the vacuole. This demonstrated that vacuolar sequestration is the rate-limiting step in cadmium uptake into protoplasts of all leaf cell types. During accumulation in the cytoplasm, Cd-rich vesicle-like structures were observed. Cd uptake rates into epidermal storage cells were higher than into standard-sized epidermal cells and mesophyll cells. This shows that the preferential heavy metal accumulation in epidermal storage cells, previously observed for several metals in intact leaves of various hyperaccumulator species, is due to differences in active metal transport and not differences in passive mechanisms like transpiration stream transport or cell wall adhesion. Combining this with previous studies, it seems likely that the transport steps over the plasma and tonoplast membranes of leaf epidermal storage cells are driving forces behind the hyperaccumulation phenotype.     

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.     

Establishment of low extracellular pH is essential for uptake of the fluorescent anionic dye hydroxypyrenetrisulfonate by suspension-cultured carrot cells

Uptake of the fluorescent dye hydroxypyrenetrisulfonate by carrot (Daucus carota L.) suspension cells only occurs when the external (medium) pH falls to below 4.0. Uptake of the dye was shown to be inhibited by a range of compounds, including the ‘Good’ buffers MES, HEPES, HEPPSO and HEPPS, the growth medium component coconut water (CW) and probenecid, an organic anion translocator inhibitor. Tris(hydroxymethyl)aminomethane stimulated transport. Buffer effects were not correlated with pK_a values. Inhibitors of dye uptake (MES, probenecid, CW) also inhibited medium acidification by the cells and Tris stimulated acidification and uptake. Uptake of HPTS correlated strongly with external pH and was restored when external pH was experimentally reduced to below 4.0 even in the presence of inhibitors. This suggests that inhibitors of HPTS uptake at the plasma membrane act primarily by preventing the establishment of a low external pH required for transport. The implications of this on the mechanism of HPTS transport are discussed.     

Solute sorting in grass leaves: the transpiration stream

Solutes distribute differentially between leaf tissues and cells. The present study tested the hypothesis that certain solutes are supplied preferentially to the epidermis in the transpiration stream, by-passing mesophyll cells along bundle sheath extensions. Using energy dispersive X-ray analysis of extracted cell sap, the distribution of solutes was studied in the emerged zone (transpiring) and the elongation zone (non-transpiring) of the developing leaf three of barley (Hordeum vulgare L.). The basic distribution of Cl, K, P and Ca between epidermis and bulk tissue, and between cells within the epidermis, was similar in the two leaf regions. However, in the emerged zone differences in solute concentrations between tissues and cells were greater. A local reduction in transpiration rate along the emerged portion of the blade specifically prevented Ca from accumulating to high levels in epidermal cells close to stomata. It is concluded that differences in solute concentrations between epidermal cells and other leaf tissues can be established in the absence of transpiration, but that they require transpiration for their full expression. Peristomatal transpiration appears to be responsible for high Ca in interstomatal cells.Abbreviations EDX-analysis Energy-dispersive X-ray analysis - IS-cell Interstomatal cell - R-cell Ridge cell - TR-cell Trough cell     

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.     

Comparative genomic analysis of soybean flowering genes

Flowering is an important agronomic trait that determines crop yield. Soybean is a major oilseed legume crop used for human and animal feed. Legumes have unique vegetative and floral complexities. Our understanding of the molecular basis of flower initiation and development in legumes is limited. Here, we address this by using a computational approach to examine flowering regulatory genes in the soybean genome in comparison to the most studied model plant, Arabidopsis. For this comparison, a genome-wide analysis of orthologue groups was performed, followed by an in silico gene expression analysis of the identified soybean flowering genes. Phylogenetic analyses of the gene families highlighted the evolutionary relationships among these candidates. Our study identified key flowering genes in soybean and indicates that the vernalisation and the ambient-temperature pathways seem to be the most variant in soybean. A comparison of the orthologue groups containing flowering genes indicated that, on average, each Arabidopsis flowering gene has 2-3 orthologous copies in soybean. Our analysis highlighted that the CDF3, VRN1, SVP, AP3 and PIF3 genes are paralogue-rich genes in soybean. Furthermore, the genome mapping of the soybean flowering genes showed that these genes are scattered randomly across the genome. A paralogue comparison indicated that the soybean genes comprising the largest orthologue group are clustered in a 1.4 Mb region on chromosome 16 of soybean. Furthermore, a comparison with the undomesticated soybean (Glycine soja) revealed that there are hundreds of SNPs that are associated with putative soybean flowering genes and that there are structural variants that may affect the genes of the light-signalling and ambient-temperature pathways in soybean. Our study provides a framework for the soybean flowering pathway and insights into the relationship and evolution of flowering genes between a short-day soybean and the long-day plant, Arabidopsis.     

A Comparison of the Uptake and Translocation of Some Organic Herbicides and a Systemic Fungicide by Barley: I. ABSORPTION IN EELATION TO PHYSICO-CHEMICAL PROPERTIES

A comparative study has been made of the uptake by and translocationfrom roots of intact barley plants of six herbicides and a systemicfungicide (four triazines, diuron, 2,4-dichloro-phenoxyaceticacid (2,4-D) and ethirimol). Relationships between uptake andtranspiration rate are discussed in the light of the physico-chemicalproperties of these compounds, notably their partition coefficientsin oil/water systems and their dissociation constants. Apartfrom 2,4-D, sorption of these compounds appears to be a passiveprocess. At pH4 the uptake of 2,4-D seems to be influenced bymetabolism; not only may the concentration of this compoundin the transpiration stream be considerably greater than thatin the medium surrounding the roots but absorption by rootsis markedly reduced at low temperatures and by sodium azide. The initial rate of uptake of these compounds correlates reasonablywell with their partition coefficients in olive oil/water orn-dodecane/water systems; likewise the concentration in thetranspiration stream is greater for lipophilic than for lipophobicsubstances. Whereas the hydrogen ion and calcium concentrations of the ambientmedium appear to have no effect on the uptake of compounds withlow pK's, the uptake of those substances which protonate betweenpH4 and pH6 is affected by them. These findings are discussedfrom the viewpoint that the pathways of transport of lipophilicand lipophobic compounds across the roots may differ. Although there is some evidence that retention by roots canlimit transport to shoots, there is no simple inverse correlationbetween the total concentration of the different substancesin the roots and that in the transpiration stream. This questionis discussed in a subsequent paper.     

9-Aminoacridine, a fluorescent probe of the thiamine carrier in yeast cells

The uptake of 9-aminoacridine is studied in the yeast Saccharomyces cerevisiae by fluorescence and absorbance measurements of the dye. Uptake of the dye proceeds via two pathways. One pathway consists of a diffusion of the non-protonated form. At high pH (7.5) this pathway is the predominant one, and the dye distributes between the cell inner and the medium according to the ratio of the proton concentrations in the two compartments. In other words, at high pH 9-aminoacridine behaves as a probe of the H⁺ gradient across the yeast cell membrane. At low external pH (4.5) a second pathway is involved. Much greater accumulation ratios for the dye are observed than can be accounted for by the H⁺ gradient across the membrane. The transport system predominantly responsible for the great accumulation of the dye appears to be inducible, to require metabolic energy and to be saturable. This transport system is competitively inhibited by thiamine, and also by dibenzyldimethylammonium and thiaminedisulfide, two specific inhibitors of the thiamine carrier in the yeast. On the other hand, the thiamine uptake by the yeast cells is competitively inhibited by 9-aminoacridine. In addition, uptake of 9-aminoacridine is greatly reduced in the thiamine transport-negative mutant of S. cerevisiae, PT-R2. It is concluded that at low pH 9-aminoacridine is taken up by yeast via the thiamine carrier of the cell and that, consequently, the dye may be applied as a probe of this transport system.     

Water stress effects on leaf elongation,leaf water potential,transpiration, and nutrient uptake of rice,maize, and soybean

A pot experiment was conducted in the greenhouse to determine and compare the responses of rice (Oryza sativa L. var, IR 36), maize (Zea mays L. var. DMR-2), and soybean (Glycine max [L.] Merr. var. Clark 63) to soil water stress. Leaf elongation, dawn leaf water potential, transpiration rate, and nutrient uptake in stressed rice declined earlier than in maize and soybean. Maize and soybean, compared with rice, maintained high dawn leaf water potential for a longer period of water stress before leaf water potential. Nutrient uptake under water stress conditions was influenced more by the capacity of the roots to absorb nutrients than by transpiration. Transport of nutrients to the shoots may occur even at reduced transpiration rate It is concluded that the ability of maize and soybean to grow better than rice under water stress conditions may be due to their ability to maintain turgor as a result of the slow decline in leaf water potential brought about by low, transpiration rate and continued uptake of nutrient, especially K, which must have allowed osmotic adjustment to occur.     

Quantitative Determination of Dye Uptake by Bacterial Cells

A colorimetric method is presented for the quantitative determination of dye uptake by bacterial cells. Experiments showed that the dye to cell ratio was of major importance in controlling the amount of dye taken up per weight of bacterial cells. Approximate dye saturation of cells could be obtained (at pH 6.1 to 6.3) although the dye uptake curves did not absolutely level off.     

Amino Acid transport in protoplasts isolated from soybean leaves

We isolated large quantities of mesophyll protoplasts from source and sink leaves of soybean plants and examined them for amino acid uptake. Accumulation of amino acids in isolated protoplasts was linear for at least 40 minutes. Uptake kinetics revealed the presence of both saturable and linear components. Increasing external pH decreases the uptake. The uncoupler, carbonyl cyanide p-trifluoromethoxyphenylhydrazone at 15 micromolar inhibited and fusicoccin at 10 micromolar stimulated amino acid uptake. Our data are consistent with a proton-cotransport mechanism for the uptake of l-glutamine and α-amino isobutyric acid into soybean mesophyll cells.     

Regulation of Soybean Nitrogen Fixation in Response to Rhizosphere Oxygen: II. Quantification of Nodule Gas Permeability

Nodule nitrogen fixation rates are regulated by a mechanism which is responsive to the rhizosphere oxygen concentration. In some legumes, this oxygen-sensitive mechanism appears to involve changes in the gas permeability of a diffusion barrier in the nodule cortex. In soybean evidence for such a mechanism has not been found. The purpose of this research was to make quantitative measurements of soybean nodule gas permeability to test the hypothesis that soybean nodule gas permeability is under physiological control and responsive to the rhizosphere oxygen concentration. Intact hydroponically grown soybean plants were exposed to altered rhizosphere oxygen concentrations, and the nodule gas permeability, acetylene reduction and nodule respiration rates were repeatedly assayed. After a change in the external oxygen concentration, nitrogenase activity and nodule respiration rates displayed a short-term transient response after which the values returned to rates similar to those observed under ambient oxygen conditions. In contrast to steady-state nitrogenase activity and nodule respiration, nodule gas permeability was dramatically affected by the change in oxygen concentration. Decreasing the external oxygen concentration to 0.1 cubic millimeter per cubic millimeter resulted in a mean increase in nodule gas permeability of 63%. Increasing the rhizosphere oxygen concentration resulted in decreased nodule gas permeability. These data are consistent with the hypothesis that soybean nodules are capable of regulating nitrogen fixation and nodule respiration rates in response to changes in the rhizosphere oxygen concentration and indicate that the regulatory mechanism involves physiological control of the nodule gas permeability.     

Linear sucrose transport in protoplasts from developing soybean cotyledons

Previous studies with isolated soybean cotyledon protoplasts revealed the presence of a saturable, simple diffusion, and nonsaturating carrier-mediated uptake of sucrose into soybean cotyledon cells. A proton/sucrose cotransport may be involved in the saturable sucrose uptake (Lin et al. 1984 Plant Physiol 75: 936-940 and Schmitt et al. 1984 Plant Physiol 75: 941-946). In this study, we investigated the linear sucrose uptake mechanism by treating isolated protoplasts with 15 micromolar p-trifluoromethoxy-carbonylcyanide phenylhydrazone (FCCP) or 100 micromolar p-chloromecuribenzenesulfonic acid to eliminate the saturable uptake. We found: (a) increasing external pH decreases the linear sucrose uptake; (b) fusicoccin at 20 micromolar stimulates and FCCP at 15 micromolar inhibits this linear sucrose uptake; and (c) the ratio of the initial influx of proton to sucrose is close to one in both saturable and nondiffusive linear (difference between the total linear and diffusive components) uptakes. The results suggest that a proton/sucrose cotransport is also involved in the nondiffusive linear sucrose uptake into soybean cotyledon cells.     

HELIOTROPIC LEAF MOVEMENT RESPONSE TO H+/ATPase activation,H+/ATPase inhibition,AND K+ CHANNEL INHIBITION IN VIVO

The pulvinus, located at the base of soybean leaflets, is both the light perception and motor organ for heliotropic leaf movements. Our objective was to investigate the role of plasma membrane H⁺/ATPase and TEA-sensitive K⁺ channels in mediating pulvinar response to light. The plasma membrane H+/ATPase activator, fusicoccin, plasma membrane H⁺/ATPase inhibitors, vanadate and erythrosin-B, and the K⁺ channel blocker TEA were introduced to the intact pulvinus through the transpiration stream. The pulvinus was illuminated by a vertical light beam of 1,400 μmol m^-2 s^-1 to stimulate leaf movement. Leaf orientation was measured every 5 min for 60 min of illumination. All compounds tested inhibited pulvinar bending, but concentration and uptake time required for inhibition varied: 12.5 μM fusicoccin reduced leaf movement after 3 hr uptake, 2 mM vanadate reduced leaf movement after 6 hr uptake, 100 μM erythrosin-B reduced leaf movement after 3 hr uptake, and 15 mM TEA reduced leaf movement after 6 hr uptake. In all cases final leaf angle was reduced by higher concentrations and/or increased time for uptake of the chemical into the pulvinus. Results support the hypothesis that the proximal mechanism of heliotropic movement is similar to that of nyctinastic movements.     

Uptake of the fluorescent indicator atebrin into acidic vacuoles in the halotolerant alga Dunaliella satina

The fluorescent indicator atebrin (3-chloro-9-(4-diethylamino-1-methylbutyl)-7-methyoxy-acridine) is taken up by Dunaliella salina cells at alkaline external pH and accumulates in acidic vacuoles. The uptake is unaffected by light, by photosynthetic inhibitors, by protonophores or by ionophores; however, the dye can be released by amines, indicating that it is specifically accumulating in acidic vacuoles. Amines induce a biphasic enhancement of atebrin fluorescence — a fast phase, accompanied by redistribution within the cell, consistent with release of the dye from the vacuoles to the cytoplasm, and a slow phase, correlated with release of atebrin from the cells. These results are interpreted to indicate a slow equilibration of atebrin across the plasma membrane and a fast equilibration across the vacuolar membrane. Part of the dye cannot be released by the amines, and appears to be internally bound. Atebrin uptake is inhibited by cholesteryl hemisuccinate and is stimulated by lysophosphatidylcholine, indicating that modification of the lipid composition of the plasma membrane affects the permeability to atebrin. Analysis of the pH dependence of atebrin uptake indicates that the dye enters the cells by fluid-phase permeation. Different stresses enhance the rate of atebrin uptake and release, indicating that they modify plasma-membrane structure or composition. Atebrin may serve as a specific marker for acidic vacuoles, as an indicator for amine uptake, and as a probe for subtle changes in the permeability of the plasma membrane.Abbreviations Atebrin 3-chloro-9-(4-diethylamino-1-methylbutyl)-7-methoxy-acridine - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl-urea - SF-6847 3,5-ditertbutyl-4-hydroxybenzylidenemalonitrile