separation and Immunological Characterization of Membrane Fractions from Barley Roots

Tonoplast and plasma membranes (PM) were isolated from barley roots (Hordeum vulgare L. cv California Mariout 72) using sucrose step gradients. The isolation procedure yielded sufficient quantities of PM and tonoplast vesicles that were sealed and of the right orientation to measure ATP-dependent proton transport in vitro. The proteins of the endoplasmic reticulum, tonoplast-plus-Golgi membrane (TG) and PM fractions were separated on sodium dodecyl sulfate gels, and immunoblots were used to test for cross-contamination between the fractions. Proteins that cross-reacted with antibodies to the PM ATPase from corn roots and Neurospora were greatly enriched in the PM fraction, as were proteins that cross-reacted with monoclonal antibodies to an arabinogalactan protein from the PM of tobacco cells. Proteins that cross-reacted with antibodies to the 58- and 72-kilodalton subunits of the tonoplast ATPase of red beet storage tissue were greatly enriched in the TG fraction. The results with immunoblots and enzyme assays indicated that there was little cross-contamination between the tonoplast and PM vesicles. The molecular weights and isoelectric points of the PM ATPase and the tonoplast ATPase subunits were also determined using immunoblots of two-dimensional gels of the PM and TG proteins.     

Phosphate Transport and Apoplastic Phosphate Homeostasis in Barley Leaves

Levels of apoplastic inorganic phosphate (P_i) in leaves andP_i-transport activities of mesophyll cells were measured insitu in control and P_i-deficient plants. When detached leaveswere fed a solution that contained 10 mM P_i, the apoplasticP_i levels, as measured by an infiltration method, remained almostconstant. When the leaves were immersed in pure water, the apoplasticP_i level gradually decreased. With 50 mM P_i in the feeding solution,the level increased dramatically. The apoplastic P_i levels inP_i-deficient leaves were somewhat, but not very much lower thanthose in controls. When the immersion medium was changed topure water 60 mm after feeding with 10 mM P_i, the apoplasticP_i levels started to decrease and then returned to the initiallevel. It is suggested that intracellular P_i may be transportedback to the apoplast to maintain the apoplastic P_i levels ata constant value. Changes in cytoplasmic pH were measured during feeding of P_ito the leaves by use of the pH-sensitive fluorescent dye, pyranineafter Yin et al. (l990a, b). On feeding of P_i the cytoplasmicpH decreased in P_i-deficient plants as a result of co-transportof P_i and protons in situ. After removal of P_i from the immersionmedium, the cytoplasmic pH returned to the original value. ³ Present address: Institute für Biochemische Pflanzenpathologie,GSF-München, D-8042 Neuherberg, Germany.     

In vivo Treatments That Modulate PPi-Dependent H+ Transport Activity of Tonoplast-Enriched Membrane Vesicles from Barley Roots

The PP_i-dependent H⁺ transport activity of tonoplast-enrichedmembrane vesicles prepared from barley roots was greatly reducedwhen the plants were grown for 4 or 5 days with an additional3 raM KC1 in growth medium that contained only 0.1 mM CaCl₂in water. To characterize the mechanism of this reduction inactivity, we attempted to treat barley roots with K⁺ ions, Cl^-ions(or acetate), and A23187 [GenBank] (with or without Ca²⁺ ions), whichmight be expected to cause alkalization, acidification and mobilizationof Ca²⁺ ions in the cytoplasm, respectively. One-day treatmentof barley roots with K⁺ ions significantly decreased PP_i--dependentH⁺ transport activity of prepared tonoplast-enriched membranevesicles, while treatment with Cl^- ions or acetate significantlyincreased the activity. A similar increase in the activity alsooccurred by treatment with Ca²⁺ ions alone or in combinationwith A23187 [GenBank] . Determination of the PP_i-hydrolyzing activity ofmembrane vesicles showed that changes in this activity by thevarious treatments were similar to those in the PP_i-dependentH⁺ transport activity. The changes in ATP-dependent H⁺ transportactivity of membrane vesicles caused by these treatments weresmall. These results indicate that the in vivo treatments hadsignificant effects on the H⁺ transport activity of H⁺-PP_i-ase,one of the two active vacuolar H⁺-pumps (H⁺-PP_iase and H⁺-ATPase).In addition, these results suggest the possibility that changesin levels of cytoplasmic H⁺ or Ca²⁺ ions may be involved inmodulation of the H⁺ transport activity of the vacuolar H⁺-PP_iaseduring plant growth. (Received September 14, 1992; Accepted March 1, 1993)     

Inhibition of Nitrate Transport by Anti-Nitrate Reductase IgG Fragments and the Identification of Plasma Membrane Associated Nitrate Reductase in Roots of Barley Seedlings

Membrane associated nitrate reductase (NR) was detected in plasma membrane (PM) fractions isolated by aqueous two-phase partitioning from barley (Hordeum vulgare L. var CM 72) roots. The PM associated NR was not removed by washing vesicles with 500 millimolar NaCl and 1 millimolar EDTA and represented up to 4% of the total root NR activity. PM associated NR was stimulated up to 20-fold by Triton X-100 whereas soluble NR was only increased 1.7-fold. The latency was a function of the solubilization of NR from the membrane. NR, solubilized from the PM fraction by Triton X-100 was inactivated by antiserum to Chlorella sorokiniana NR. Anti-NR immunoglobulin G fragments purified from the anti-NR serum inhibited NO₃⁻ uptake by more than 90% but had no effect on NO₂⁻ uptake. The inhibitory effect was only partially reversible; uptake recovered to 50% of the control after thorough rinsing of roots. Preimmune serum immunoglobulin G fragments inhibited NO₃⁻ uptake 36% but the effect was completely reversible by rinsing. Intact NR antiserum had no effect on NO₃⁻ uptake. The results present the possibility that NO₃⁻ uptake and NO₃⁻ reduction in the PM of barley roots may be related.     

大麦根质膜微囊的制备及其H~ ,Ca~(2 )转运活性

用蔗糖密度梯度离心法制备出密闭程度较高的大麦根细胞质膜微囊。喹吖咽荧光猝灭和~(45)Ca~(2 )同位素示踪测定表明所制备的微囊具H~ ,Ca~(2 )转运活性。对制备出的质膜制剂纯度和膜朝向进行了分析,并探讨了质膜纯化中影响膜微囊密闭性的因素。匀浆液和悬浮液巾的单价离子盐有利于密闭膜微囊的形成。蔗糖密度梯度和葡聚糖密度睇度离心法均可得到密闭性较高的膜微囊,但后者的纯化效果较差。     

Inhibition of Ion Transport in Excised Barley Roots by Abscisic Acid; Relation to Water Permeability of the Roots

Previous papers have shown that abscisic acid can inhibit transportof ions across the root to the xylem vessels, resulting in reducedexudation from excised roots or inhibiting guttation from intactplants. However, it has not been established whether the inhibitionwas due to a reduction in salt transport (J_s) or in permeabilityof the roots to water (L_p). This paper investigates the effectof ABA on L_p and J_s separately. It is shown that L_p increasedin ABA and then fell, but was about the same as in control rootswhen transport was inhibited. The effect of ABA on exudationtherefore appeared to be mainly due to reduction in J_s. Inhibitionof J_s was also present in intact, transpiring plants and sowas not due to reduced water flow. The inhibition of ion releaseto the xylem affected Na⁺, Mg²⁺, Ca²⁺, and phosphate as wellas the major ion in the exudate, K⁺. It is concluded that ABAinhibits salt transport to the shoot by acting on ion transportinto the xylem, and not by reducing water flow coupled withsalt transport.     

The Accumulation and Transport of Calcium in Barley Roots

The accumulation and transport of Ca by various zones of 6-day old barley roots (Hordeum vulgare L.) were examined with special reference to their relationship to the salt status of the root. The initial salt content had a profound effect on Ca transport and a lesser effect on Ca accumulation. High-salt roots transported Ca in much larger amounts than did low salt roots. In low salt roots the apical zone was more active in transporting Ca to the conducting tissue than were the mid or basal zones However, in high salt roots all zones were about equally active in transporting Ca. The metabolic inhibitor, DNP, had little effect on accumulation but inhibited transport very effectively. The effect of DNP was more pronounced on transport from the apical zone than from the other root zones. Calcium applied anywhere along the root length moved only basally and its polarized longitudinal movement was maintained irrespective of the salt status of the root. The movement of Ca was characterized by a rapid release of preabsorbed Ca and a ready exchange of apoplastic Ca. The hypothesis is presented that cellular Ca is in a relatively mobile state. Its entry into the symplasm is the rate limiting step in longitudinal transport and its overall movement is metabolically controlled.     

Salt-induced Pinocytosis in Barley and Bean

The ultrastructure of the halophyte, Salicornia europea L. wascompared with barley and bean plants grown under saline conditions.S. europea exhibited frequent pinocytotic vesiculation in thehypocotyl and stem but less vesiculation in the root and cotyledon.No vesiculation was observed in barley and bean plants grownin the absence of sodium chloride though plants grown in thepresence of sodium chloride demonstrated vesiculation, morein barley than in bean. Both the halophyte and the glycophytesdemonstrated more pinocytosis in the plant tops than in theroot system. The multivesiculate structures observed were connectedto the wall free space at the early stages of their formationand have not been reported in plants under normal physiologicalconditions. The observations on pinocytosis are discussed in relation totoxic ion compartmentation and salt resistance in plants.     

Cadmium-Induced Inhibition of Apoplastic Ascorbate Oxidase in Barley Roots

The effect of excess cadmium (Cd: 0.0, 0.25, 0.5, 1.0 and 2.0 mM) on growth and ascorbate oxidase (AO) activity was investigated in barley (Hordeum vulgare L. cv. Jubilant) roots. The study employed a filter-paper technique to germinate and grow the germinating seeds following imbibition with respective Cd treatments for 4 h at 25 °C in darkness. Cd was required at 1.0 mM to affect 50% root growth inhibition 72 h after the treatment. This Cd-induced root growth inhibition was accompanied by a corresponding loss of plasma membrane integrity in root cells as evaluated by Evans blue uptake. Excess Cd (1.0 and 2.0 mM) significantly inhibited the AO activity in all the analysed fractions of barley roots such as extracellular, soluble, cell wall (CW)- and membrane-bound fractions. AO was localized in the apoplast, and its highest specific activity was detected in the CW II fraction obtained by extraction with 1.0 M NaCl from purified cell walls. The analysis of AO isozyme profile showed that besides the reduced activity of two anionic and two cationic isozymes, one cationic AO isozyme was activated during excess Cd treatment, which could be detected in cell wall fractions CW II, III and IV.     

Reduction of Hydraulic Conductivity during Inhibition of Exudation from Excised Maize and Barley Roots

The uncoupler, carbonyl cyanide m-chlorophenyl hydrazone (CCCP) is shown to reduce the hydraulic conductivity of barley, maize, mung bean, and onion roots. In barley and maize, the reduction in exudation from excised roots is partly due to the reduction in the permeability of the root to water (I_p), but it can be inferred that the rate of salt release to the xylem, is also inhibited. The action of CCCP on L_p is suggested to be mainly in blocking the symplasmic pathway at the plasmodesmata.     

The Fructose Transport Mechanism in Microsomal Membrane Vesicles from Rye Roots

In a fructose-containing medium in which rye root-microsomal membrane vesicles had reached the equilibrium of uptake of fructose, the presence of both Mg²⁺ and ATP caused the efflux of fructose from the vesicles. Among nucleotides examined, ATP caused the largest efflux of fructose. The efflux of fructose dependent on Mg²⁺ and ATP was quite insensitive to a protonophore, carbonylcyanide m-chlorophenylhydrazone (CCCP), which actually abolished MgATP-dependent proton accumulation in the vesicles, while it was largely inhibited by vanadate, which inhibits ATP-binding cassette transporters (ABCTs). The Michaelis-Menten constant (Km) of the efflux of fructose was 0.4 mM. It was observed that fructose stimulated the ATPase activity of the vesicles and that vanadate markedly decreased the fructose-stimulated ATPase activity. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effect of Exogenous Glycinebetaine on Na+ Transport in Barley Roots

Ahmad, N., Wyn Jones, R. G. and Jeschke, W. D. 1987. Effectof exogenous glycinebetaine on Na⁺ transport in barley roots.—J.exp. Bot. 38: 913–921. A comparison has been made of the kinetics of 22Na⁺ uptake intoexcised barley roots and roots pre-loaded with glycinebetaine.The elevated intracellular glycinebetaine or a metabolic consequencethereof increased the Na⁺ influx, and the effect was relatedto the level of internal glycinebetaine and or Na⁺ [Cl^–].The quasi-steady-state Na⁺ influx at the tonoplast rather thanthe plasmalemma influx was apparently influenced by glycinebetaineloading. The tonoplast fluxes and vacuolar Na⁺ content wereconsistently higher in glycinebetaine-loaded roots than unloadedroots. A membrane-modifying role of glycinebetaine in relationto ion compartmentation is discussed. Key words: Excised roots, glycinebetaine, Na⁺, ion fluxes, barley     

Characterization of an Anion Transporter in the Plasma Membrane of Barley Roots

To examine the relationship between H⁺-ATPase and the transportof anions, we investigated the effects of various inhibitorson the activity of the H⁺-ATPase, the transport of protons,and the transport of Cl^- ions using plasma membrane vesiclesprepared from barley roots. Some inhibitors, namely, 4,4-diisothiocyano-2,2-stilbenedisulfonate (DIDS) and Zn²⁺ ions markedly inhibited H⁺- ATPaseactivity. Other compounds, such as phenylglyoxal (PGO) and niflumicacid (NIF), inhibited H⁺-ATPase activity by 20-30%, while anthracene-9-carboxylate(A-9-C) and tetraethylammonium chloride (TEA-Cl) had littleeffect on this activity. The ATP-dependent acidification ofthe interior of vesicles was strongly dependent on the presenceof permeant anions, such as chloride (Cl^-) and nitrate (NO₃^-),and it was completely inhibited by 0.2 mM NIF. Other compounds,namely, A-9-C of 0.1 mM and TEA-Cl of 10 mM, did not affectH⁺-transport activity. The inhibition of H⁺-transport activityby NIF was observed even when the activity was assayed in thepresence of KCl, KNO₃, or bis-tris-propane (BTP)-Cl. Using ³⁶cl,we quantified Cl^--transport activity by measuring the uptakeof Cl^- ions into the plasma membrane vesicles. The uptake dependedon the potential difference across the membrane that was generatedby H⁺-ATPase; it was enhanced by an inside-positive potentialgradient. At 0.1 mM, NIF completely blocked the voltage-dependentCl^--transport activity. From these properties of the Cl^- transporterand the inhibition of H⁺-transport activity by NIF, we suggestthat H⁺-transport activity across the plasma membrane mightbe modulated by the transport of anions via a NIF-sensitiveanion-permeable transporter that acts to collapse the inside-positivepotential generated by H⁺-ATPase. (Received September 7, 1995; Accepted July 23, 1996)