K+ channels of stomatal guard cells: Abscisic-acid-evoked control of the outward rectifier mediated by cytoplasmic pH

The activation by abscisic acid (ABA) of current through outward-rectifying K⁺ channels and its dependence on cytoplasmic pH (pH_i) was examined in stomatal guard cells of Vicia faba L. Intact guard cells were impaled with multibarrelled and H⁺-selective microelectrodes to record membrane potentials and pH_i during exposures to ABA and the weak acid butyrate. Potassium channel currents were monitored under voltage clamp and, in some experiments, guard cells were loaded with pH buffers by iontophoresis to suppress changes in pH_i. Following impalements, stable pH_i values ranged between 7.53 and 7.81 (7.67±0.04, n = 17). On adding 20 M ABA, pH_i rose over periods of 5–8 min to values 0.27±0.03 pH units above the pH_i before ABA addition, and declined slowly thereafter. Concurrent voltage-clamp measurements showed a parallel rise in the outward-rectifying K⁺ channel current (I_{K, out}) and, once evoked, both pH_i and I_{K, out} responses were unaffected by ABA washout. Acid loads, imposed with external butyrate, abolished the ABA-evoked rise in I_{K, out}. Butyrate concentrations of 10 and 30 mM (pH₀ 6.1) caused pH_i to fall to values near 7.0 and below, both before and after adding ABA, consistent with a cytoplasmic buffer capacity of 128±12 mM per pH unit (n = 10) near neutrality. Butyrate washout was characterised by an appreciable alkaline overshoot in pH_i and concomitant swell in the steady-state conductance of I_{K, out}. The rise in pH_i and i_{K, out} in ABA were also virtually eliminated when guard cells were first loaded with pH buffers to raise the cytoplasmic buffer capacity four- to sixfold; however, buffer loading was without appreciable effect on the ABA-evoked inactivation of a second, inward-rectifying class of K⁺ channels (I_{K, in}). The pH_i dependence of I_{K, out} was consistent with a cooperative binding of at least 2H⁺ (apparent pK_a = 8.3) to achieve a voltage-independent block of the channel. These results establish a causal link previously implicated between cytoplasmic alkalinisation and the activation of I_{K, out} in ABA and, thus, affirm a role for H⁺ in signalling and transport control in plants distinct from its function as a substrate in H⁺-coupled transport. Additional evidence implicates a coordinate control of I_{K, in} by cytoplasmic-free [Ca²⁺] and pH_i.Abbreviations ABA abscisic acid - [Ca²⁺]_i cytoplasmic free [Ca²⁺]_i - E_K K⁺ equilibrium potential - I_{K, out}, I_{K, in} outward-, inward-rectifying K⁺ channel (current) - I-V current-voltage (relation) - Mes 2-(N-morpholino)ethanesulfonic acid - pH_i cytoplasmic pH - Tes 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]-amino}ethanesulfonic acid - V_m membrane potential We are grateful to G. Thiel (Pflanzenphysiologisches Institut, Universität Göttingen, Germany) for helpful discussions. This work was possible with equipment grants-in-aid from the Gatsby Charitable Foundation, the Royal Society and the University of London Central Research Fund. F.A. holds a Sainsbury Studentship.     

Cytological and molecular characterization of a chromosome interchange and addition lines in Cadet involving chromosome 5B of wheat and 6Ag of Lophopyrum ponticum

Efforts to transfer wheat curl mite (Eriophyes tulipae Keifer) resistance from Lophopyrum ponticum 10X (Podb.) Love to bread wheat (Triticum aestivum L.) have resulted in the production of a number of cytogenetic stocks, including an addition line of 6Ag, a ditelo addition line, and a wheat-Lophopyrum translocation line. Characterization of these lines with C-banding, in situ hybridization with a Lophopyrum species-specific repetitive DNA probe (pLeUCD2), and Southern blotting with pLeUCD2 and a 5S ribosomal DNA probe (pScT7) confirmed that the distal portion of the short arm of 6Ag was translocated onto the distal portion of 5BS (5BL. 5BS-6AgS). It was also determined that the ditelo addition was an acrocentric chromosome of 6AgS.     

Exotoxin production by Aeromonas spp. in foods

A psychrotrophic toxin-producing strain of Aeromonas hydrophila grew well in a range of food slurries (scallop, prawn, fish, chicken liver paté, liverwurst, chicken luncheon slice and commercial baby food preparations) held at refrigeration temperatures. In most foods, excluding the baby food preparations, exotoxins were produced at levels comparable with production in bacteriological broth without apparent food spoilage (all but prawn and fish). Addition of ultra-heat treated (UHT) milk to toxin-containing broth culture supernatants markedly decreased or removed haemolytic and cytotoxic activities, explaining low levels of toxins found in milk in a previous study. Baby food preparations did not inactivate exotoxins under similar conditions suggesting production of toxins rather than their inactivation was inhibited in these foods.     

D-amino acid levels in human physiological fluids

Plasma, urine, cerebrospinal fluid (CSF), and amniotic fluid were examined to determine whether free D-amino acids were present and if so at what levels. It was found that D-amino acids exist in all physiological fluids tested, but that their level varied, considerably. The lowest levels of D-amino acids were usually found in amniotic fluid or CSF (almost always <1% of the corresponding L-amino acid). The highest levels were found in urine (usually tenth percent to low percent levels). Pipecolic acid seemed to be different from the other amino acids tested in that it was excreted primarily as the D-enantiomer (often >90%). Correspondingly high levels of D-pipecolic acid were not found in plasma. Some of the trends found in this work seemed to be analogous to those found in a recent rodent study. © 1993 Wiley-Liss, Inc.     

MINIMAL MALE/FEMALE TRADEOFFS IN ZIZANIA PALUSTRIS,A MONOECIOUS ANNUAL GRASS

Because a male/female resource tradeoff is a basic assumption of many models of sex allocation in cosexual plants, statistical and manipulative methods were used to look for evidence of intersexual resource conflicts in Zizania palustris. In this monoecious grass, male and female investments overlap in time within each panicle and on successive panicles, and sex allocation quickly responds to environmental changes. Nevertheless, no negative correlations were found between the numbers of florets of each sex within panicles, on consecutive panicles, or on whole plants. Removing immature fruits or florets of either sex did not significantly increase subsequent investment in the other sex. The one significant tradeoff was slightly lower total fruit production on plants with exceptionally large male investment. Wild rice, therefore, fits the tradeoff assumption of sex allocation models at the population level but rarely at the individual level.     

Efficient enzymatic synthesis of 13 C,15N-labeled DNA for NMR studies

The power of heteronuclear NMR spectroscopy to study macromoleculesand their complexes has been amply demonstrated over the last decade. Theobstacle to routinely applying these techniques to the study of DNA has beenthe synthesis of ¹³C,¹⁵N-labeled DNA. Here wepresent a simple and efficient method to generate isotope-labeled DNA forNMR studies that is as easy as that for isotope labeling of RNA. The methodwas used to synthesize a uniformly¹³ C,¹⁵N-labeled 32-nucleotide DNA that binds tohuman basic fibroblast growth factor with high affinity and specificity.Isotope-edited experiments were applied to the¹³ C,¹⁵N-labeled DNA bound to unlabeled protein,and the ¹³ C,¹⁵N-labeled DNA was also examined incomplex with ¹⁵N-labeled protein. The NMR experiments showthat the DNA adopts a well-defined stable structure when bound to theprotein, and illustrate the potential of¹³ C,¹⁵N-labeled DNA for structural studies ofDNA–protein complexes.     

Intravenous vs. oral rehydration: effects on subsequent exercise-heat stress

Castellani, John W., Carl M. Maresh, Lawrence E. Armstrong,Robert W. Kenefick, Deborah Riebe, Marcos Echegaray, Douglas Casa, andV. Daniel Castracane. Intravenous vs. oral rehydration: effects onsubsequent exercise-heat stress. J. Appl.Physiol. 82(3): 799-806, 1997.This studycompared the influence of intravenous vs. oral rehydration afterexercise-induced dehydration during a subsequent 90-min exercisebout. It was hypothesized that cardiovascular, thermoregulatory, and hormonal variables would be the same between intravenous and oral rehydration because of similar restoration ofplasma volume (PV) and osmolality (Osmo). Eight non-heat-acclimated menreceived three experimental treatments (counterbalanced design) immediately after exercise-induced dehydration (33°C) to 4%body weight loss. Treatments were intravenous 0.45% NaCl (iv; 25 ml/kg), no fluid (NF), and oral saline (Oral; 25 ml/kg).After rehydration and rest (2 h total), subjects walked at 50% maximalO₂ consumption for up to 90 min at36°C. The following observations were made: 1) heart rate was higher(P < 0.05) in Oral vs. ivat minutes 45, 60, and75 of exercise;2) rectal temperature, sweat rate, percent change in PV, and change in plasma Osmo were similar between ivand Oral; 3) change in plasmanorepinephrine decreased less (P < 0.05) in Oral compared with iv at minute45; 4) changes in plasma adrenocorticotropic hormone and cortisol were similar between ivand Oral after exercise was initiated; and5) exercise time was similar betweeniv (77.4 ± 5.4 min) and Oral (84.2 ± 2.3 min). These datasuggest that after exercise-induced dehydration, iv and Oral wereequally effective as rehydration treatments. Thermoregulation, changein adrenocorticotropic hormone, and change in cortisol were notdifferent between iv and Oral after exercise began; this is likely dueto similar percent change in PV and change in Osmo.

     

Identification of Possible Artifacts in the Association of Official Analytical Chemists Sporicidal Test

Two laboratories tested four different brands of alkaline 2% glutaraldehyde sterilants by the Association of Official Analytical Chemists sporicidal test. Each laboratory found survival of Clostridium sporogenes spores on spore-labeled unglazed porcelain penicylinders (cylinders) to vary from test to test, and survival did not always correlate with increasing sterilant exposure time. These results were consistent with a theory that there may be random conditions within the test that prevent the sterilant from contacting all spores. Further studies indicated that the prior history of the unglazed porcelain cylinders and whether the C. sporogenes culture grown in egg-meat media had been processed (homogenized) to eliminate visible pieces of egg-meat media were important factors affecting the results and repeatability of this test.