Evidence for the existence of a sulfonylurea-receptor-like protein in plants: Modulation of stomatal movements and guard cell potassium channels by sulfonylureas and potassium channel openers

Limitation of water loss and control of gas exchange is accomplished in plant leaves via stomatal guard cells. Stomata open in response to light when an increase in guard cell turgor is triggered by ions and water influx across the plasma membrane. Recent evidence demonstrating the existence of ATP-binding cassette proteins in plants led us to analyze the effect of compounds known for their ability to modulate ATP-sensitive potassium channels (K-ATP) in animal cells. By using epidermal strip bioassays and whole-cell patch-clamp experiments with Vicia faba guard cell protoplasts, we describe a pharmacological profile that is specific for the outward K+ channel and very similar to the one described for ATP-sensitive potassium channels in mammalian cells. Tolbutamide and glibenclamide induced stomatal opening in bioassays and in patch-clamp experiments, a specific inhibition of the outward K+ channel by these compounds was observed. Conversely, application of potassium channel openers such as cromakalim or RP49356 triggered stomatal closure. An apparent competition between sulfonylureas and potassium channel openers occurred in bioassays, and outward potassium currents, previously inhibited by glibenclamide, were partially recovered after application of cromakalim. By using an expressed sequence tag clone from an Arabidopsis thaliana homologue of the sulfonylurea receptor, a 7-kb transcript was detected by Northern blot analysis in guard cells and other tissues. Beside the molecular evidence recently obtained for the expression of ATP-binding cassette protein transcripts in plants, these results give pharmacological support to the presence of a sulfonylurea-receptor-like protein in the guard-cell plasma membrane tightly involved in the outward potassium channel regulation during stomatal movements.

Correlation of breath ammonia with blood urea nitrogen and creatinine during hemodialysis

We have spectroscopically determined breath ammonia levels in seven patients with end-stage renal disease while they were undergoing hemodialysis at the University of California, Los Angeles, dialysis center. We correlated these measurements against simultaneously taken blood samples that were analyzed for blood urea nitrogen (BUN) and creatinine, which are the accepted standards indicating the level of nitrogenous waste loading in a patient's bloodstream. Initial levels of breath ammonia, i.e., at the beginning of dialysis, are between 1,500 ppb and 2,000 ppb (parts per billion). These levels drop very sharply in the first 15–30 min as the dialysis proceeds. We found the reduction in breath ammonia concentration to be relatively slow from this point on to the end of dialysis treatment, at which point the levels tapered off at 150 to 200 ppb. For each breath ammonia measurement, taken at 15–30 min intervals during the dialysis, we also sampled the patient's blood for BUN and creatinine. The breath ammonia data were available in real time, whereas the BUN and creatinine data were available generally 24 h later from the laboratory. We found a good correlation between breath ammonia concentration and BUN and creatinine. For one of the patients, the correlation gave an R2 of 0.95 for breath ammonia and BUN correlation and an R2 of 0.83 for breath ammonia and creatinine correlation. These preliminary data indicate the possibility of using the real-time breath ammonia measurements for determining efficacy and endpoint of hemodialysis.

Equilibration of the terrestrial water, nitrogen, and carbon cycles

Recent advances in biologically based ecosystem models of the coupled terrestrial, hydrological, carbon, and nutrient cycles have provided new perspectives on the terrestrial biosphere’s behavior globally, over a range of time scales. We used the terrestrial ecosystem model Century to examine relationships between carbon, nitrogen, and water dynamics. The model, run to a quasi-steady-state, shows strong correlations between carbon, water, and nitrogen fluxes that lead to equilibration of water/energy and nitrogen limitation of net primary productivity. This occurs because as the water flux increases, the potentials for carbon uptake (photosynthesis), and inputs and losses of nitrogen, all increase. As the flux of carbon increases, the amount of nitrogen that can be captured into organic matter and then recycled also increases. Because most plant-available nitrogen is derived from internal recycling, this latter process is critical to sustaining high productivity in environments where water and energy are plentiful. At steady-state, water/energy and nitrogen limitation “equilibrate,” but because the water, carbon, and nitrogen cycles have different response times, inclusion of nitrogen cycling into ecosystem models adds behavior at longer time scales than in purely biophysical models. The tight correlations among nitrogen fluxes with evapotranspiration implies that either climate change or changes to nitrogen inputs (from fertilization or air pollution) will have large and long-lived effects on both productivity and nitrogen losses through hydrological and trace gas pathways. Comprehensive analyses of the role of ecosystems in the carbon cycle must consider mechanisms that arise from the interaction of the hydrological, carbon, and nutrient cycles in ecosystems.

Drought-Induced Effects on Nitrate Reductase Activity and mRNA and on the Coordination of Nitrogen and Carbon Metabolism in Maize Leaves1

Maize (Zea mays L.) plants were grown to the nine-leaf stage. Despite a saturating N supply, the youngest mature leaves (seventh position on the stem) contained little NO3− reserve. Droughted plants (deprived of nutrient solution) showed changes in foliar enzyme activities, mRNA accumulation, photosynthesis, and carbohydrate and amino acid contents. Total leaf water potential and CO2 assimilation rates, measured 3 h into the photoperiod, decreased 3 d after the onset of drought. Starch, glucose, fructose, and amino acids, but not sucrose (Suc), accumulated in the leaves of droughted plants. Maximal extractable phosphoenolpyruvate carboxylase activities increased slightly during water deficit, whereas the sensitivity of this enzyme to the inhibitor malate decreased. Maximal extractable Suc phosphate synthase activities decreased as a result of water stress, and there was an increase in the sensitivity to the inhibitor orthophosphate. A correlation between maximal extractable foliar nitrate reductase (NR) activity and the rate of CO2 assimilation was observed. The NR activation state and maximal extractable NR activity declined rapidly in response to drought. Photosynthesis and NR activity recovered rapidly when nutrient solution was restored at this point. The decrease in maximal extractable NR activity was accompanied by a decrease in NR transcripts, whereas Suc phosphate synthase and phosphoenolpyruvate carboxylase mRNAs were much less affected. The coordination of N and C metabolism is retained during drought conditions via modulation of the activities of Suc phosphate synthase and NR commensurate with the prevailing rate of photosynthesis.

Role of sodium and potassium ions in regulation of glucose metabolism in cultured astroglia.

Effects of increasing extracellular K+ or intracellular Na+ concentrations on glucose metabolism in cultures of rat astroglia and neurons were examined. Cells were incubated in bicarbonate buffer, pH 7.2, containing 2 mM glucose, tracer amounts of [14C]deoxyglucose ([14C]dGlc), and 5.4, 28, or 56 mM KCl for 10, 15, or 30 min, and then for 5 min in [14C]dGlc-free buffer to allow efflux of unmetabolized [14C]dGlc. Cells were then digested and assayed for labeled products, which were shown to consist of 96-98% [14C]deoxyglucose 6-phosphate. Increased K+ concentrations significantly raised [14C]deoxyglucose 6-phosphate accumulation in both neuronal and mixed neuronal-astroglial cultures at 15 and 30 min but did not raise it in astroglial cultures. Veratridine (75 microM), which opens voltage-dependent Na+ channels, significantly raised rates of [14C]dGlc phosphorylation in astroglial cultures (+20%), and these elevations were blocked by either 1 mM ouabain, a specific inhibitor of Na+,K(+)-ATPase (EC 3.6.1.37), or 10 microM tetrodotoxin, which blocks Na+ channels. The carboxylic sodium ionophore, monensin (10 microM), more than doubled [14C]dGlc phosphorylation; this effect was only partially blocked by ouabain and unaffected by tetrodotoxin. L-Glutamate (500 microM) also stimulated [14C]dGlc phosphorylation in astroglia--not through N-methyl-D-aspartate or non-N-methyl-D-aspartate receptor mechanisms but via a Na(+)-dependent glutamate-uptake system. These results indicate that increased uptake of Na+ can stimulate energy metabolism in astroglial cells.

Nitrogen management and the future of food: Lessons from the management of energy and carbon

The food system dominates anthropogenic disruption of the nitrogen cycle by generating excess fixed nitrogen. Excess fixed nitrogen, in various guises, augments the greenhouse effect, diminishes stratospheric ozone, promotes smog, contaminates drinking water, acidifies rain, eutrophies bays and estuaries, and stresses ecosystems. Yet, to date, regulatory efforts to limit these disruptions largely ignore the food system. There are many parallels between food and energy. Food is to nitrogen as energy is to carbon. Nitrogen fertilizer is analogous to fossil fuel. Organic agriculture and agricultural biotechnology play roles analogous to renewable energy and nuclear power in political discourse. Nutrition research resembles energy end-use analysis. Meat is the electricity of food. As the agriculture and food system evolves to contain its impacts on the nitrogen cycle, several lessons can be extracted from energy and carbon: (i) set the goal of ecosystem stabilization; (ii) search the entire production and consumption system (grain, livestock, food distribution, and diet) for opportunities to improve efficiency; (iii) implement cap-and-trade systems for fixed nitrogen; (iv) expand research at the intersection of agriculture and ecology, and (v) focus on the food choices of the prosperous. There are important nitrogen-carbon links. The global increase in fixed nitrogen may be fertilizing the Earth, transferring significant amounts of carbon from the atmosphere to the biosphere, and mitigating global warming. A modern biofuels industry someday may produce biofuels from crop residues or dedicated energy crops, reducing the rate of fossil fuel use, while losses of nitrogen and other nutrients are minimized.