Polygalacturonase isozyme 2 binding and catalysis in cell walls from tomato fruit: pH and β-subunit effects

The catalytic activity of endopolygalacturonase (PG, EC 3.2.1.15) against pectic polymers in vitro is typically not expressed in vivo. In the present study, the binding and catalytic properties of PG isozyme 2 and the influence of the β-subunit protein were investigated in cell walls prepared from tomato fruit expressing an antisense gene to the β-subunit protein. Cell walls prepared from mature-green fruit were employed for binding and assay of PG2. Walls were provided with rate-limiting quantities of purified PG2 and incubated at 100 mM KCl, pH 4.5, or 25 mM KCl, pH 6.0. Cell walls of both β-subunit antisense and wild-type fruit retained comparable quantities of added PG2. The release of pectin from PG2-loaded walls was proportional to the quantity of added enzyme, consistent with a finite catalytic capacity of individual PG proteins. β-Subunit-antisense cell walls released 2- to 3-fold higher levels of pectin in response to PG2 than did wild-type walls. Cell walls incubated at pH 6.0 released lower quantities and showed less extensive depolymerization of pectins than did walls incubated at pH 4.5. Pectins recovered from ripe fruit were similar in size distribution to polymers released by PG2 at pH 6.0, indicating that pH can influence both quantitative and qualitative aspects of pectin metabolism and may be responsible for the restricted hydrolysis of pectins in vivo. Molecular mass differences were not evident in the polymers rendered freely soluble in response to PG2-mediated hydrolysis of β-subunit-antisense compared with wild-type cell walls. The solubilization of pectin from cell walls was not the sole indicator of the extent of PG-mediated cell wall hydrolysis. Hydrolytic modifications were also evident in a pectic fraction extracted from postcatalytic cell walls with 50 mM CDTA (trans-1,2-cyclohexanediamine-N,N,N′,N′-tetraacetic acid), and were more extensive for the β-subunit-antisense cell walls compared with the wild-type walls. Pectic polymers derived from ethanol insoluble-powders showed molecular mass downshifts during ripening but differences between the β-subunit-antisense and wild-type fruits were not observed.     

Osmolytes Contribute to pH Homeostasis of Escherichia coli

Background

Cytoplasmic pH homeostasis in Escherichia coli includes numerous mechanisms involving pH-dependent catabolism and ion fluxes. An important contributor is transmembrane K⁺ flux, but the actual basis of K⁺ compensation for pH stress remains unclear. Osmoprotection could mediate the pH protection afforded by K⁺ and other osmolytes.

Methods and Principal Findings

The cytoplasmic pH of E. coli K-12 strains was measured by GFPmut3 fluorimetry. The wild-type strain Frag1 was exposed to rapid external acidification by HCl addition. Recovery of cytoplasmic pH was enhanced equally by supplementation with NaCl, KCl, proline, or sucrose. A triple mutant strain TK2420 defective for the Kdp, Trk and Kup K⁺ uptake systems requires exogenous K⁺ for steady-state pH homeostasis and for recovery from sudden acid shift. The K⁺ requirement however was partly compensated by supplementation with NaCl, choline chloride, proline, or sucrose. Thus, the K⁺ requirement was mediated in part by osmolarity, possibly by relieving osmotic stress which interacts with pH stress. The rapid addition of KCl to strain TK2420 suspended at external pH 5.6 caused a transient decrease in cytoplasmic pH, followed by slow recovery to an elevated steady-state pH. In the presence of 150 mM KCl, however, rapid addition of another 150 mM KCl caused a transient increase in cytoplasmic pH. These transient effects may arise from secondary K⁺ fluxes occurring through other transport processes in the TK2420 strain.

Conclusions

Diverse osmolytes including NaCl, KCl, proline, or sucrose contribute to cytoplasmic pH homeostasis in E. coli, and increase the recovery from rapid acid shift. Osmolytes other than K⁺ restore partial pH homeostasis in a strain deleted for K⁺ transport.     

Insulin-induced Stimulation of Na+,K+-ATPase Activity in Kidney Proximal Tubule Cells Depends on Phosphorylation of the α-Subunit at Tyr-10

Phosphorylation of the α-subunit of Na⁺,K⁺-ATPase plays an important role in the regulation of this pump. Recent studies suggest that insulin, known to increase solute and fluid reabsorption in mammalian proximal convoluted tubule (PCT), is stimulating Na⁺,K⁺-ATPase activity through the tyrosine phosphorylation process. This study was therefore undertaken to evaluate the role of tyrosine phosphorylation of the Na⁺,K⁺-ATPase α-subunit in the action of insulin. In rat PCT, insulin and orthovanadate (a tyrosine phosphatase inhibitor) increased tyrosine phosphorylation level of the α-subunit more than twofold. Their effects were not additive, suggesting a common mechanism of action. Insulin-induced tyrosine phosphorylation was prevented by genistein, a tyrosine kinase inhibitor. The site of tyrosine phosphorylation was identified on Tyr-10 by controlled trypsinolysis in rat PCTs and by site-directed mutagenesis in opossum kidney cells transfected with rat α-subunit. The functional relevance of Tyr-10 phosphorylation was assessed by 1) the abolition of insulin-induced stimulation of the ouabain-sensitive ⁸⁶Rb uptake in opossum kidney cells expressing mutant rat α1-subunits wherein tyrosine was replaced by alanine or glutamine; and 2) the similarity of the time course and dose dependency of the insulin-induced increase in ouabain-sensitive ⁸⁶Rb uptake and tyrosine phosphorylation. These findings indicate that phosphorylation of the Na⁺,K⁺-ATPase α-subunit at Tyr-10 likely participates in the physiological control of sodium reabsorption in PCT.     

Adaptation of Active Proton Pumping and Plasmalemma ATPase Activity of Corn Roots to Low Root Medium pH

Corn (Zea mays L.) root adaptation to pH 3.5 in comparison with pH 6.0 (control) was investigated in long-term nutrient solution experiments. When pH was gradually reduced, comparable root growth was observed irrespective of whether the pH was 3.5 or 6.0. After low-pH adaptation, H⁺ release of corn roots in vivo at pH 5.6 was about 3 times higher than that of control. Plasmalemma of corn roots was isolated for investigation in vitro. At optimum assay pH, in comparison with control, the following increases of the various parameters were caused by low-pH treatment: (a) hydrolytic ATPase activity, (b) maximum initial velocity and Michaelis constant (c) activation energy of H⁺-ATPase, (d) H⁺-pumping activity, (e) H⁺ permeability of plasmalemma, and (f) pH gradient across the membranes of plasmalemma vesicles. In addition, vanadate sensitivity remained unchanged. It is concluded that plasmalemma H⁺-ATPase contributes significantly to the adaptation of corn roots to low pH. A restricted net H⁺ release at low pH in vivo may be attributed to the steeper pH gradient and enhanced H⁺ permeability of plasmalemma but not to deactivation of H⁺-ATPase. Possible mechanisms responsible for adaptation of plasmalemma H⁺-ATPase to low solution pH during plant cultivation are discussed.     

The Arabidopsis HKT1 Gene Homolog Mediates Inward Na+ Currents in Xenopus laevis Oocytes and Na+ Uptake in Saccharomyces cerevisiae

The Na⁺-K⁺ co-transporter HKT1, first isolated from wheat, mediates high-affinity K⁺ uptake. The function of HKT1 in plants, however, remains to be elucidated, and the isolation of HKT1 homologs from Arabidopsis would further studies of the roles of HKT1 genes in plants. We report here the isolation of a cDNA homologous to HKT1 from Arabidopsis (AtHKT1) and the characterization of its mode of ion transport in heterologous systems. The deduced amino acid sequence of AtHKT1 is 41% identical to that of HKT1, and the hydropathy profiles are very similar. AtHKT1 is expressed in roots and, to a lesser extent, in other tissues. Interestingly, we found that the ion transport properties of AtHKT1 are significantly different from the wheat counterpart. As detected by electrophysiological measurements, AtHKT1 functioned as a selective Na⁺ uptake transporter in Xenopus laevis oocytes, and the presence of external K⁺ did not affect the AtHKT1-mediated ion conductance (unlike that of HKT1). When expressed in Saccharomyces cerevisiae, AtHKT1 inhibited growth of the yeast in a medium containing high levels of Na⁺, which correlates to the large inward Na⁺ currents found in the oocytes. Furthermore, in contrast to HKT1, AtHKT1 did not complement the growth of yeast cells deficient in K⁺ uptake when cultured in K⁺-limiting medium. However, expression of AtHKT1 did rescue Escherichia coli mutants carrying deletions in K⁺ transporters. The rescue was associated with a less than 2-fold stimulation of K⁺ uptake into K⁺-depleted cells. These data demonstrate that AtHKT1 differs in its transport properties from the wheat HKT1, and that AtHKT1 can mediate Na⁺ and, to a small degree, K⁺ transport in heterologous expression systems.     

The inactivation of pectin depolymerase associated with isolated tomato fruit cell wall: implications for the analysis of pectin solubility and molecular weight

An approach commonly employed to assess the potential role of the enzyme polygalacturonase (PG, EC 3.2.1.15) in tomato fruit cell-wall pectin metabolism includes correlating levels of extractable PG with changes in specific characteristics of cell wall pectins, most notably solubility and molecular weight. Since information on these features of pectins is generally derived from analyses of subfractions of isolated cell wall, assurance of inactivation of the various isoforms of wall-associated PG is imperative. In the present study, cell wall prepared from ripe tomato (Lycopersicon esculentum Mill. cv. Rutgers) fruit was examined for the presence of active PG and for the ability of phenolic solvents to inactivate the enzyme. Using pectin solubility and M_r (relative molecular mass) changes as criteria for the presence of wall-associated PG activity, pectins from phenol-treated and nonphenol-treated (enzymically active) cell wall from ripe fruit incubated in 50 mM Na-acetate, 50 mM cyclohexanetrans-1,2-diamine tetraacetic acid (CDTA), pH 6.5 (outside the catalytic range of PG), were of similar M_r and exhibited no change in size with incubation time. Wall prepared without exposure to the phenolic protein-denaturants exhibited extensive pectin solubilization and depolymerization when incubated in 50 mM Na-acetate, 50 mM CDTA at pH 4.5, indicating the presence of active PG. Based on the changes in the M_r of pectins solubilized in 50 mM Na-acetate, 50 mM CDTA, pH 4.5, active PG was also detected in wall exposed during isolation to phenolacetic acid-water (PAW, 2:1:1, w/v/v), a solvent commonly employed as an enzyme denaturant. Although the depolymerization of pectins in PAW-treated wall was extensive, oligouronides constituted minor reaction products. Interestingly, PAW-treated wall did not exhibit PG-mediated pectin release when incubated under conditions (30 mM Na-acetate, 150 mM NaCl, pH 4.5) in which nonphenol-treated cell wall exhibited high autolytic activity. In an alternative protocol designed to inactivate PG, cell wall was exposed to Tris-buffered phenol (BP). In contrast to pectins released from PAW-treated wall, pectins solubilized from BP-treated wall at pH 4.5 were indistinguishable in M_r from those recovered from BP-treated wall at pH 6.5 Even when incubated at pH 4.5 at 34°C, conditions under which pectins from PAW-treated wall underwent more rapid and extensive depolymerization, pectins from BP-treated wall exhibited no change in M_r, providing evidence that active PG was not present in these wall preparations. The implications of this study in interpreting the solubility and M_r of pectin in cell wall from ripening fruit are discussed.     

Identification of an Na+-Dependent Malonate Transporter of Malonomonas rubra and Its Dependence on Two Separate Genes

Two membrane proteins encoded by the malonate fermentation gene cluster of Malonomonas rubra, MadL and MadM, have been synthesized in Escherichia coli. MadL and MadM were shown to function together as a malonate transport system, whereas each protein alone was unable to catalyze malonate transport. Malonate transport by MadLM is Na⁺ dependent, and imposition of a ΔpNa⁺ markedly enhanced the rate of malonate uptake. The kinetics of malonate uptake into E. coli BL21(DE3) cells synthesizing MadLM at different pH values indicated that Hmalonate⁻ is the transported malonate species. The stimulation of malonate uptake by Na⁺ ions showed Michaelis-Menten kinetics, and a K_m for Na⁺ of 1.2 mM was determined. These results suggest that MadLM is an electroneutral Na⁺/Hmalonate⁻ symporter and that it is dependent on two separate genes.     

Isolation and characterization of a tomato non-specific lipid transfer protein involved in polygalacturonase-mediated pectin degradation

An important aspect of the ripening process of tomato fruit is softening. Softening is accompanied by hydrolysis of the pectin in the cell wall by pectinases, causing loss of cell adhesion in the middle lamella. One of the most significant pectin-degrading enzymes is polygalacturonase (PG). Previous reports have shown that PG in tomato may exist in different forms (PG1, PG2a, PG2b, and PGx) commonly referred to as PG isoenzymes. The gene product PG2 is differentially glycosylated and is thought to associate with other proteins to form PG1 and PGx. This association is thought to modulate its pectin-degrading activity in planta. An 8 kDa protein that is part of the tomato PG1 multiprotein complex has been isolated, purified, and functionally characterized. This protein, designated 'activator' (ACT), belongs to the class of non-specific lipid transfer proteins (nsLTPs). ACT is capable of 'converting' the gene product PG2 into a more active and heat-stable form, which increases PG-mediated pectin degradation in vitro and stimulates PG-mediated tissue breakdown in planta. This finding suggests a new, not previously identified, function for nsLTPs in the modification of hydrolytic enzyme activity. It is proposed that ACT plays a role in the modulation of PG activity during tomato fruit softening.     

Differential Sodium and Potassium Transport Selectivities of the Rice OsHKT2;1 and OsHKT2;2 Transporters in Plant Cells

Na⁺ and K⁺ homeostasis are crucial for plant growth and development. Two HKT transporter/channel classes have been characterized that mediate either Na⁺ transport or Na⁺ and K⁺ transport when expressed in Xenopus laevis oocytes and yeast. However, the Na⁺/K⁺ selectivities of the K⁺-permeable HKT transporters have not yet been studied in plant cells. One study expressing 5′ untranslated region-modified HKT constructs in yeast has questioned the relevance of cation selectivities found in heterologous systems for selectivity predictions in plant cells. Therefore, here we analyze two highly homologous rice (Oryza sativa) HKT transporters in plant cells, OsHKT2;1 and OsHKT2;2, that show differential K⁺ permeabilities in heterologous systems. Upon stable expression in cultured tobacco (Nicotiana tabacum) Bright-Yellow 2 cells, OsHKT2;1 mediated Na⁺ uptake, but little Rb⁺ uptake, consistent with earlier studies and new findings presented here in oocytes. In contrast, OsHKT2;2 mediated Na⁺-K⁺ cotransport in plant cells such that extracellular K⁺ stimulated OsHKT2;2-mediated Na⁺ influx and vice versa. Furthermore, at millimolar Na⁺ concentrations, OsHKT2;2 mediated Na⁺ influx into plant cells without adding extracellular K⁺. This study shows that the Na⁺/K⁺ selectivities of these HKT transporters in plant cells coincide closely with the selectivities in oocytes and yeast. In addition, the presence of external K⁺ and Ca²⁺ down-regulated OsHKT2;1-mediated Na⁺ influx in two plant systems, Bright-Yellow 2 cells and intact rice roots, and also in Xenopus oocytes. Moreover, OsHKT transporter selectivities in plant cells are shown to depend on the imposed cationic conditions, supporting the model that HKT transporters are multi-ion pores.Intracellular Na⁺ and K⁺ homeostasis play vital roles in growth and development of higher plants (Clarkson and Hanson, 1980). Low cytosolic Na⁺ and high K⁺/Na⁺ ratios aid in maintaining an osmotic and biochemical equilibrium in plant cells. Na⁺ and K⁺ influx and efflux across membranes require the function of transmembrane Na⁺ and K⁺ transporters/channels. Several Na⁺-permeable transporters have been characterized in plants (Zhu, 2001; Horie and Schroeder, 2004; Apse and Blumwald, 2007). Na⁺/H⁺ antiporters mediate sequestration of Na⁺ into vacuoles under salt stress conditions in plants (Blumwald and Poole, 1985, 1987; Sze et al., 1999). Na⁺ (cation)/H⁺ antiporters are encoded by six AtNHX genes in Arabidopsis (Arabidopsis thaliana; Apse et al., 1999; Gaxiola et al., 1999; Yokoi et al., 2002; Aharon et al., 2003). A distinct Na⁺/H⁺ antiporter, Salt Overly Sensitive1, mediates Na⁺/H⁺ exchange at the plasma membrane and mediates cellular Na⁺ extrusion (Shi et al., 2000, 2002; Zhu, 2001; Ward et al., 2003). Electrophysiological analyses reveal that voltage-independent channels, also named nonselective cation channels, mediate Na⁺ influx into roots under high external Na⁺ concentrations (Amtmann et al., 1997; Tyerman et al., 1997; Buschmann et al., 2000; Davenport and Tester, 2000); however, the underlying genes remain unknown.Potassium is the most abundant cation in plants and an essential nutrient for plant growth. The Arabidopsis genome includes 13 genes encoding KUP/HAK/KT transporters (Quintero and Blatt, 1997; Santa-María et al., 1997; Fu and Luan, 1998; Kim et al., 1998), and 17 genes have been identified encoding this family of transporters in rice (Oryza sativa ‘Nipponbare’; Bañuelos et al., 2002). Several KUP/HAK/KT transporters have been characterized as mediating K⁺ uptake across the plasma membrane of plant cells (Rigas et al., 2001; Bañuelos et al., 2002; Gierth et al., 2005).Ionic balance, especially the Na⁺/K⁺ ratio, is a key factor of salt tolerance in plants (Niu et al., 1995; Maathuis and Amtmann, 1999; Shabala, 2000; Mäser et al., 2002a; Tester and Davenport, 2003; Horie et al., 2006; Apse and Blumwald, 2007; Chen et al., 2007; Gierth and Mäser, 2007). Salinity stress is a major problem for agricultural productivity of crops worldwide (Greenway and Munns, 1980; Zhu, 2001). The Arabidopsis AtHKT1;1 transporter plays a key role in salt tolerance of plants by mediating Na⁺ exclusion from leaves (Mäser et al., 2002a; Berthomieu et al., 2003; Gong et al., 2004; Sunarpi et al., 2005; Rus et al., 2006; Davenport et al., 2007; Horie et al., 2009). athkt1;1 mutations cause leaf chlorosis and elevated Na⁺ accumulation in leaves under salt stress conditions in Arabidopsis (Mäser et al., 2002a; Berthomieu et al., 2003; Gong et al., 2004; Sunarpi et al., 2005). AtHKT1;1 and its homolog in rice, OsHKT1;5 (SKC1), mediate leaf Na⁺ exclusion by removing Na⁺ from the xylem sap to protect plants from salinity stress (Ren et al., 2005; Sunarpi et al., 2005; Horie et al., 2006, 2009; Davenport et al., 2007).The land plant HKT gene family is divided into two classes based on their nucleic acid sequences and protein structures (Mäser et al., 2002b; Platten et al., 2006). Class 1 HKT transporters have a Ser residue at a selectivity filter position in the first pore loop, which is replaced by a Gly in all but one known class 2 HKT transporter (Horie et al., 2001; Mäser et al., 2002b; Garciadeblás et al., 2003). While the Arabidopsis genome includes only one HKT gene, AtHKT1;1 (Uozumi et al., 2000), seven full-length OsHKT genes were found in the japonica rice cv Nipponbare genome (Garciadeblás et al., 2003). Members of class 1 HKT transporters, AtHKT1;1 and SKC1/OsHKT1;5, have a relatively higher Na⁺-to-K⁺ selectivity in Xenopus laevis oocytes and yeast than class 2 HKT transporters (Uozumi et al., 2000; Horie et al., 2001; Mäser et al., 2002b; Ren et al., 2005). The first identified plant HKT transporter, TaHKT2;1 from wheat (Triticum aestivum), is a class 2 HKT transporter (Schachtman and Schroeder, 1994). TaHKT2;1 was found to mediate Na⁺-K⁺ cotransport and Na⁺ influx at high Na⁺ concentrations in heterologous expression systems (Rubio et al., 1995, 1999; Gassmann et al., 1996; Mäser et al., 2002b). Thus, class 1 HKT transporters have been characterized as Na⁺-preferring transporters with a smaller K⁺ permeability (Fairbairn et al., 2000; Uozumi et al., 2000; Su et al., 2003; Jabnoune et al., 2009), whereas class 2 HKT transporters function as Na⁺-K⁺ cotransporters or channels (Gassmann et al., 1996; Corratgé et al., 2007). In addition, at millimolar Na⁺ concentrations, class 2 HKT transporters were found to mediate Na⁺ influx, without adding external K⁺ in Xenopus oocytes and yeast (Rubio et al., 1995, 1999; Gassmann et al., 1996; Horie et al., 2001). However, the differential cation transport selectivities of the two types of HKT transporters have not yet been analyzed and compared in plant cells.A study of the barley (Hordeum vulgare) and wheat class 2 transporters has suggested that the transport properties of HvHKT2;1 and TaHKT2;1 expressed in yeast are variable, depending on the constructs from which the transporter is expressed, and have led to questioning of the K⁺ transport activity of HKT transporters characterized in Xenopus oocytes and yeast (Haro et al., 2005). It was further proposed that the 5′ translation initiation of HKT proteins in yeast at nonconventional (non-ATG) sites affects the transporter selectivities of HKT transporters (Haro et al., 2005), although direct evidence for this has not yet been presented. However, recent research has shown a K⁺ permeability of OsHKT2;1 but not of OsHKT1;1 and OsHKT1;3 in Xenopus oocytes. These three OsHKT transporters show overlapping and also distinctive expression patterns in rice (Jabnoune et al., 2009).The report of Haro et al. (2005) has opened a central question addressed in this study: are the Na⁺/K⁺ transport selectivities of plant HKT transporters characterized in heterologous systems of physiological relevance in plant cells, or do they exhibit strong differences in the cation transport selectivities in these nonplant versus plant systems? To address this question, we analyzed the Na⁺/K⁺ transport selectivities of the OsHKT2;1 and OsHKT2;2 transporters expressed in cultured tobacco (Nicotiana tabacum ‘Bright-Yellow 2’ [BY2]) cells. OsHKT2;1 and OsHKT2;2 are two highly homologous HKT transporters from indica rice cv Pokkali, sharing 91% amino acid and 93% cDNA sequence identity (Horie et al., 2001). OsHKT2;1 mediates mainly Na⁺ uptake, which correlates with the presence of a Ser residue in the first pore loop of OsHKT2;1 (Horie et al., 2001, 2007; Mäser et al., 2002b; Garciadeblás et al., 2003). In contrast, OsHKT2;2 mediates Na⁺-K⁺ cotransport in Xenopus oocytes and yeast (Horie et al., 2001). Furthermore, at millimolar Na⁺ concentrations, OsHKT2;2 mediates Na⁺ influx in the absence of added K⁺ (Horie et al., 2001). Recent research on oshkt2;1 loss-of-function mutant alleles has revealed that OsHKT2;1 from japonica rice mediates a large Na⁺ influx component into K⁺-starved roots, thus compensating for lack of K⁺ availability (Horie et al., 2007). But the detailed Na⁺/K⁺ selectivities of Gly-containing, predicted K⁺-transporting class 2 HKT transporters have not yet been analyzed in plant cells.Here, we have generated stable OsHKT2;1- and OsHKT2;2-expressing tobacco BY2 cell lines and characterized the cell lines by ion content measurements and tracer influx studies to directly analyze unidirectional fluxes (Epstein et al., 1963). These analyses showed that OsHKT2;1 exhibits Na⁺ uptake activity in plant BY2 cells in the absence of added K⁺, but little K⁺ (Rb⁺), influx activity. In contrast, OsHKT2;2 was found to function as a Na⁺-K⁺ cotransporter/channel in plant BY2 cells, showing K⁺-stimulated Na⁺ influx and Na⁺-stimulated K⁺ (Rb⁺) influx. The differential K⁺ selectivities of the two OsHKT2 transporters were consistently reproduced by voltage clamp experiments using Xenopus oocytes here, as reported previously (Horie et al., 2001). OsHKT2;2 was also found to mediate K⁺-independent Na⁺ influx at millimolar external Na⁺ concentrations. These findings demonstrate that the cation selectivities of OsHKT2;1 and OsHKT2;2 in plant cells are consistent with past findings obtained from heterologous expression analyses under similar ionic conditions (Horie et al., 2001; Garciadeblás et al., 2003; Tholema et al., 2005). Furthermore, the shift in OsHKT2;2 Na⁺-K⁺ selectivity depending on ionic editions is consistent with the model that HKT transporters/channels are multi-ion pores (Gassmann et al., 1996; Corratgé et al., 2007). Classical studies of ion channels have shown that ion channels, in which multiple ions can occupy the pore at the same time, can change their relative selectivities depending on the ionic conditions (Hille, 2001). Moreover, the presence of external K⁺ and Ca²⁺ was found here to down-regulate OsHKT2;1-mediated Na⁺ influx both in tobacco BY2 cells and in rice roots. The inhibitory effect of external K⁺ on OsHKT2;1-mediated Na⁺ influx into intact rice roots, however, showed a distinct difference in comparison with that of BY2 cells, which indicates a possible posttranslational regulation of OsHKT2;1 in K⁺-starved rice roots.     

Structural and Thermodynamic Properties of Selective Ion Binding in a K+ Channel

Thermodynamic measurements of ion binding to the Streptomyces lividans K⁺ channel were carried out using isothermal titration calorimetry, whereas atomic structures of ion-bound and ion-free conformations of the channel were characterized by x-ray crystallography. Here we use these assays to show that the ion radius dependence of selectivity stems from the channel's recognition of ion size (i.e., volume) rather than charge density. Ion size recognition is a function of the channel's ability to adopt a very specific conductive structure with larger ions (K⁺, Rb⁺, Cs⁺, and Ba²⁺) bound and not with smaller ions (Na⁺, Mg²⁺, and Ca²⁺). The formation of the conductive structure involves selectivity filter atoms that are in direct contact with bound ions as well as protein atoms surrounding the selectivity filter up to a distance of 15 Å from the ions. We conclude that ion selectivity in a K⁺ channel is a property of size-matched ion binding sites created by the protein structure.     

Direct Measurement of Calcium Transport across Chloroplast Inner-Envelope Vesicles

The initial rate of Ca²⁺ movement across the inner-envelope membrane of pea (Pisum sativum L.) chloroplasts was directly measured by stopped-flow spectrofluorometry using membrane vesicles loaded with the Ca²⁺-sensitive fluorophore fura-2. Calibration of fura-2 fluorescence was achieved by combining a ratiometric method with Ca²⁺-selective minielectrodes to determine pCa values. The initial rate of Ca²⁺ influx in predominantly right-side-out inner-envelope membrane vesicles was greater than that in largely inside-out vesicles. Ca²⁺ movement was stimulated by an inwardly directed electrochemical proton gradient across the membrane vesicles, an effect that was diminished by the addition of valinomycin in the presence of K⁺. In addition, Ca²⁺ was shown to move across the membrane vesicles in the presence of a K⁺ diffusion potential gradient. The potential-stimulated rate of Ca²⁺ transport was slightly inhibited by diltiazem and greatly inhibited by ruthenium red. Other pharmacological agents such as LaCl₃, verapamil, and nifedipine had little or no effect. These results indicate that Ca²⁺ transport across the chloroplast inner envelope can occur by a potential-stimulated uniport mechanism.     

D-glucose Stimulates the Na+/K+ Pump in Mouse Pancreatic Islet Cells

To determine the effect of D-glucose on the β-cell Na⁺/K⁺ pump, ⁸⁶Rb⁺ influx was studied in isolated, -cell-rich islets of Umeå-ob/ob mice in the absence or presence of lmM ouabain. D-glucose (20 mM) stimulated the ouabain-sensitive portion of ⁸⁶Rb⁺ influx by 65%, whereas the ouabain-resistant portion was inhibited by 48%. The Na⁺/K⁺ ATPase activity in homogenates of islets of Umeå-ob/ob mice or normal mice was determined to search for direct effects of D-glucose. Thus, ouabain-sensitive ATP hydrolysis in islet homogenates was measured in the presence of different D-glucose concentrations. No effect of D-glucose (3–20 mM) was observed in either ob/ob or normal islets at the optimal Na⁺/K⁺ ratio for the enzyme (135 mM Na⁺ and 20 mM K⁺). Neither D-glucose (3–20 mM) nor L-glucose or 3-O-methyl-D-glucose (20 mM) affected the enzyme activity at a high Na⁺/K⁺ ratio (175 mM Na⁺ and 0.7mM K⁺). Diphenylhydantoin (150 μM) decreased the enzyme activity at optimal Na⁺/K⁺ ratio, whereas 50 μM of the drug had no effect. The results suggest that D-glucose induces a net stimulation the Na⁺/K⁺ pump of β-cells in intact islets and that D-glucose does not exert any direct effect on the Na⁺/K⁺ ATPase activity.     

Development, Characterization, and Application of a Cadmium-Selective Microelectrode for the Measurement of Cadmium Fluxes in Roots of Thlaspi Species and Wheat

A Cd²⁺-selective vibrating microelectrode was constructed using a neutral carrier-based Cd ionophore to investigate ion-transport processes along the roots of wheat (Triticum aestivum L.) and two species of Thlaspi, one a Zn/Cd hyperaccumulator and the other a related nonaccumulator. In simple Cd(NO₃)₂ solutions, the electrode exhibited a Nernstian response in solutions with Cd²⁺ activities as low as 50 nm. Addition of Ca²⁺ to the calibration solutions did not influence the slope of the calibration curve but reduced the detection limit to a solution activity of 1 μm Cd²⁺. Addition of high concentrations of K⁺ and Mg²⁺ to the calibration solution to mimic the ionic composition of the cytoplasm affected neither the slope nor the sensitivity of the electrode, demonstrating the pH-insensitive electrode's potential for intracellular investigations. The electrode was assayed for selectivity and was shown to be at least 1000 times more selective for Cd²⁺ than for any of those potentially interfering ions tested. Flux measurements along the roots of the two Thlaspi species showed no differences in the pattern or the magnitude of Cd²⁺ uptake within the time frame considered. The Cd²⁺-selective microelectrode will permit detailed investigations of heavy-metal ion transport in plant roots, especially in the area of phytoremediation.     

Diversity in Expression Patterns and Functional Properties in the Rice HKT Transporter Family

Plant growth under low K⁺ availability or salt stress requires tight control of K⁺ and Na⁺ uptake, long-distance transport, and accumulation. The family of membrane transporters named HKT (for High-Affinity K⁺ Transporters), permeable either to K⁺ and Na⁺ or to Na⁺ only, is thought to play major roles in these functions. Whereas Arabidopsis (Arabidopsis thaliana) possesses a single HKT transporter, involved in Na⁺ transport in vascular tissues, a larger number of HKT transporters are present in rice (Oryza sativa) as well as in other monocots. Here, we report on the expression patterns and functional properties of three rice HKT transporters, OsHKT1;1, OsHKT1;3, and OsHKT2;1. In situ hybridization experiments revealed overlapping but distinctive and complex expression patterns, wider than expected for such a transporter type, including vascular tissues and root periphery but also new locations, such as osmocontractile leaf bulliform cells (involved in leaf folding). Functional analyses in Xenopus laevis oocytes revealed striking diversity. OsHKT1;1 and OsHKT1;3, shown to be permeable to Na⁺ only, are strongly different in terms of affinity for this cation and direction of transport (inward only or reversible). OsHKT2;1 displays diverse permeation modes, Na⁺-K⁺ symport, Na⁺ uniport, or inhibited states, depending on external Na⁺ and K⁺ concentrations within the physiological concentration range. The whole set of data indicates that HKT transporters fulfill distinctive roles at the whole plant level in rice, each system playing diverse roles in different cell types. Such a large diversity within the HKT transporter family might be central to the regulation of K⁺ and Na⁺ accumulation in monocots.Although it is not clear what levels of Na⁺ are toxic in the plant cell cytosol and actually unacceptable in vivo, the hypothesis that this cation must be excluded from the cytoplasm is widely accepted. The most abundant inorganic cation in the cytosol is K⁺, in plant as in animal cells. This cation has probably been selected during evolution because it is less chaotropic than Na⁺ (i.e. more compatible with protein structure even at high concentrations; Clarkson and Hanson, 1980). Its selection might also be due to the fact that in primitive cells, which originated in environmental conditions (seawater) where Na⁺ was more abundant than K⁺, a straightforward process to energize the cell membrane was to accumulate the less abundant cation and to exclude the most abundant one.In the cell, K⁺ plays a role in basic functions, such as regulation of cell membrane polarization, electrical neutralization of anionic groups, and osmoregulation. Concerning the latter function, K⁺ uptake or release is the usual way through which plant cells control their water potential and turgor. Although toxic at high concentrations, Na⁺ can be used as osmoticum and substituted for K⁺, mainly in the vacuole, when the plant is facing low K⁺ conditions and Na⁺ is available in the soil solution. This use of Na⁺, however, requires a tight regulation of K⁺ and Na⁺ transport and compartmentalization that becomes crucial in conditions of high Na⁺ concentrations in the soil solution. Control of Na⁺ and K⁺ uptake, long-distance transport in the xylem and phloem vasculatures, accumulation in aerial parts, and compartmentalization at the cellular and tissue levels have actually been shown to be essential in plant adaptation to salt stress (Greenway and Munns, 1980; Flowers, 1985; Hasegawa et al., 2000; Mühling and Läuchli, 2002). Thus, accumulation of Na⁺ as osmoticum during K⁺ shortage or plant adaptation to salt stress requires integration at the whole plant level of Na⁺ and K⁺ membrane transport system activities (Apse et al., 1999; Shi et al., 2002; Qi and Spalding, 2004; Ren et al., 2005; Maathuis, 2006; Pardo et al., 2006; Horie et al., 2007).This report concerns transport systems named HKT upon first identification (for High-Affinity K⁺ Transporters) that are active at the plasma membrane and permeable to either K⁺ and Na⁺ or to Na⁺ only (Schachtman and Schroeder, 1994; Rodríguez-Navarro and Rubio, 2006). Several members of the HKT family have already been shown, by genetic approaches, to play important roles in plant salt tolerance (Berthomieu et al., 2003; Ren et al., 2005; Huang et al., 2006; Byrt et al., 2007) or growth in conditions of K⁺ shortage (Horie et al., 2007). In Arabidopsis (Arabidopsis thaliana), the HKT family comprises a single member, AtHKT1;1, which is permeable to Na⁺ only (Uozumi et al., 2000) and contributes to Na⁺ removal from the ascending xylem sap and recirculation from the leaves to the roots via the phloem vasculature (Berthomieu et al., 2003; Sunarpi et al., 2005). Interestingly, the HKT family comprises a much larger number of members in rice (Oryza sativa), with seven to nine genes depending on the cultivar (Garciadeblás et al., 2003). In line with previous reports using rice as a model species to decipher the roles that HKT transporters can play in the plant, we have analyzed the expression patterns of three rice HKT genes, OsHKT2;1, OsHKT1;1, and OsHKT1;3, and investigated the functional properties of these transporters after heterologous expression, revealing new patterns of expression for HKT transporters and striking functional diversity.     

Platinum cross-linking of adenines and guanines on the quadruplex structures of the AG3(T2AG3)3 and (T2AG3)4 human telomere sequences in Na+ and K+ solutions

The quadruplex structures of the human telomere sequences AG₃(T₂AG₃)₃ I and (T₂AG₃)₄ II were investigated in the presence of Na⁺ and K⁺ ions, through the cross-linking of adenines and guanines by the cis- and trans-[Pt(NH₃)₂(H₂O)₂](NO₃)₂ complexes 1 and 2. The bases involved in chelation of the cis- and trans-Pt(NH₃)₂ moieties were identified by chemical and 3′-exonuclease digestions of the products isolated after denaturing gel electrophoresis. These are the four adenines of each sequence and four out of the 12 guanines. Two largely different structures have been reported for I: A from NMR data in Na⁺ solution and B from X-ray data of a K⁺-containing crystal. Structure A alone agrees with our conclusions about the formation of the A1–G10, A13–G22, A1–A13 platinum chelates at the top of the quadruplex and A7–A19, G4–A19 and A7–G20 at the bottom, whether the Na⁺ or K⁺ ion is present. At variance with a recent proposal that structures A and B could be the major species in Na⁺ and K⁺ solutions, respectively, our results suggest that structure A exists predominantly in the presence of both ions. They also suggest that covalent platinum cross-linking of a human telomere sequence could be used to inhibit telomerase.     

Improved Salinity Tolerance of Rice Through Cell Type-Specific Expression of AtHKT1;1

Previously, cell type-specific expression of AtHKT1;1, a sodium transporter, improved sodium (Na⁺) exclusion and salinity tolerance in Arabidopsis. In the current work, AtHKT1;1, was expressed specifically in the root cortical and epidermal cells of an Arabidopsis GAL4-GFP enhancer trap line. These transgenic plants were found to have significantly improved Na⁺ exclusion under conditions of salinity stress. The feasibility of a similar biotechnological approach in crop plants was explored using a GAL4-GFP enhancer trap rice line to drive expression of AtHKT1;1 specifically in the root cortex. Compared with the background GAL4-GFP line, the rice plants expressing AtHKT1;1 had a higher fresh weight under salinity stress, which was related to a lower concentration of Na⁺ in the shoots. The root-to-shoot transport of ²²Na⁺ was also decreased and was correlated with an upregulation of OsHKT1;5, the native transporter responsible for Na⁺ retrieval from the transpiration stream. Interestingly, in the transgenic Arabidopsis plants overexpressing AtHKT1;1 in the cortex and epidermis, the native AtHKT1;1 gene responsible for Na⁺ retrieval from the transpiration stream, was also upregulated. Extra Na⁺ retrieved from the xylem was stored in the outer root cells and was correlated with a significant increase in expression of the vacuolar pyrophosphatases (in Arabidopsis and rice) the activity of which would be necessary to move the additional stored Na⁺ into the vacuoles of these cells. This work presents an important step in the development of abiotic stress tolerance in crop plants via targeted changes in mineral transport.     

A Saccharomyces cerevisiae Mutant Lacking a K+/H+ Exchanger

The KHA1 gene corresponding to the open reading frame YJL094c (2.62 kb) encoding a putative K⁺/H⁺ antiporter (873 amino acids) in Saccharomyces cerevisiae was disrupted by homologous recombination. The core protein is similar to the putative Na⁺/H⁺ antiporters from Enterococcus hirae (NAPA gene) and Lactococcus lactis (LLUPP gene) and the putative K⁺/H⁺ exchanger from Escherichia coli (KEFC gene). Disruption of the KHA1 gene resulted in an increased K⁺ accumulation and net influx without a significant difference in efflux, as well as an increased growth rate, smaller cells, and twice the cell yield per glucose used. Flow cytometry analysis showed an increase of the DNA duplication rate in the mutant. Kinetic studies of ⁸⁶Rb⁺ uptake showed the same saturable system for wild-type and disruptant strains. Mutant cells also produced a greater acidification of the medium coincident with an internal pH alkalinization and showed a higher oxygen consumption velocity. We speculate that higher K⁺ accumulation and increased osmotic pressure accelerate the cell cycle and metabolic activity.     

Characterization of SKT1, an Inwardly Rectifying Potassium Channel from Potato, by Heterologous Expression in Insect Cells

A cDNA encoding a novel, inwardly rectifying K⁺ (K⁺_in) channel protein, SKT1, was cloned from potato (Solanum tuberosum L.). SKT1 is related to members of the AKT family of K⁺_in channels previously identified in Arabidopsis thaliana and potato. Skt1 mRNA is most strongly expressed in leaf epidermal fragments and in roots. In electrophysiological, whole-cell, patch-clamp measurements performed on baculovirus-infected insect (Spodoptera frugiperda) cells, SKT1 was identified as a K⁺_in channel that activates with slow kinetics by hyperpolarizing voltage pulses to more negative potentials than −60 mV. The pharmacological inhibitor Cs⁺, when applied externally, inhibited SKT1-mediated K⁺_in currents half-maximally with an inhibitor concentration (IC₅₀) of 105 μm. An almost identical high Cs⁺ sensitivity (IC₅₀ = 90 μm) was found for the potato guard-cell K⁺_in channel KST1 after expression in insect cells. SKT1 currents were reversibly activated by a shift in external pH from 6.6 to 5.5, which indicates a physiological role for pH-dependent regulation of AKT-type K⁺_in channels. Comparative studies revealed generally higher current amplitudes for KST1-expressing cells than for SKT1-expressing insect cells, which correlated with a higher targeting efficiency of the KST1 protein to the insect cell's plasma membrane, as demonstrated by fusions to green fluorescence protein.     

The Two Major Types of Plant Plasma Membrane H+-ATPases Show Different Enzymatic Properties and Confer Differential pH Sensitivity of Yeast Growth

The proton-pumping ATPase (H⁺-ATPase) of the plant plasma membrane is encoded by two major gene subfamilies. To characterize individual H⁺-ATPases, PMA2, an H⁺-ATPase isoform of tobacco (Nicotiana plumbaginifolia), was expressed in Saccharomyces cerevisiae and found to functionally replace the yeast H⁺-ATPase if the external pH was kept above 5.0 (A. de Kerchove d'Exaerde, P. Supply, J.P. Dufour, P. Bogaerts, D. Thinès, A. Goffeau, M. Boutry [1995] J Biol Chem 270: 23828–23837). In the present study we replaced the yeast H⁺-ATPase with PMA4, an H⁺-ATPase isoform from the second subfamily. Yeast expressing PMA4 grew at a pH as low as 4.0. This was correlated with a higher acidification of the external medium and an approximately 50% increase of ATPase activity compared with PMA2. Although both PMA2 and PMA4 had a similar pH optimum (6.6–6.8), the profile was different on the alkaline side. At pH 7.2 PMA2 kept more than 80% of the maximal activity, whereas that of PMA4 decreased to less than 40%. Both enzymes were stimulated up to 3-fold by 100 μg/mL lysophosphatidylcholine, but this stimulation vanished at a higher concentration in PMA4. These data demonstrate functional differences between two plant H⁺-ATPases expressed in the same heterologous host. Characterization of two PMA4 mutants selected to allow yeast growth at pH 3.0 revealed that mutations within the carboxy-terminal region of PMA4 could still improve the enzyme, resulting in better growth of yeast cells.