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• We report the first use of tracer

The analysis of tracer efflux kinetics is fundamental to membrane transport studies, but requires the rigorous identification of subcellular tracer sources. We present a solution to this problem through the analysis of sharp increases in 13NH4+ efflux from roots of radiolabelled barley (Hordeum vulgare L.) seedlings, in response to a 100-fold increase in external [NH4+]. By comparing these trans-stimulation data with a mathematical model incorporating changes in subcellular NH4+ fluxes and pool sizes, we show that the cytosol of root cells is the origin of the tracer efflux. Our analysis provides new insight into the rapidly occurring events underlying compensatory flux regulation during transitions from one nutritional steady state to another, and confirms the validity of compartmental analysis by tracer efflux (CATE) in this important model system.

A systematic analysis of NaCl-dependent, plasma-membrane depolarization (∆∆Ψ) in rice roots calls into question the current leading model of rapid membrane cycling of Na+ under salt stress. To investigate the character and mechanisms of Na+ influx into roots, Na+-dependent changes in plasma-membrane electrical potentials (∆∆Ψ) were measured in root cells of intact rice (Oryza sativa L., cv. Pokkali) seedlings. As external sodium concentrations ([Na+]ext) were increased in a step gradient from 0 to 100 mM, membrane potentials depolarized in a saturable manner, fitting a Michaelis–Menten model and contradicting the linear (non-saturating) models developed from radiotracer studies. Clear differences in saturation patterns were found between plants grown under low- and high-nutrient (LN and HN) conditions, with LN plants showing greater depolarization and higher affinity for Na+ (i.e., higher Vmax and lower Km) than HN plants. In addition, counterion effects on ∆∆Ψ were pronounced in LN plants (with ∆∆Ψ decreasing in the order: Cl− > SO42− > HPO 4 2- ), but not seen in HN plants. When effects of osmotic strength, Cl− influx, K+ efflux, and H+-ATPase activity on ∆∆Ψ were accounted for, resultant Km and Vmax values suggested that a single, dominant Na+-transport mechanism was operating under each nutritional condition, with Km values of 1.2 and 16 mM for LN and HN plants, respectively. Comparing saturating patterns of depolarization to linear patterns of 24Na+ radiotracer influx leads to the conclusion that electrophysiological and tracer methods do not report the same phenomena and that the current model of rapid transmembrane sodium cycling may require revision.

Provision of silicon (Si) to roots of rice (Oryza sativa L.) can alleviate salt stress by blocking apoplastic, transpirational bypass flow of Na+ from root to shoot. However, little is known about how Si affects Na+ fluxes across cell membranes. Here, we measured radiotracer fluxes of 24Na+, plasma membrane depolarization, tissue ion accumulation, and transpirational bypass flow, to examine the influence of Si on Na+ transport patterns in hydroponically grown, salt-sensitive (cv. IR29) and salt-tolerant (cv. Pokkali) rice. Si increased growth and lowered [Na+] in shoots of both cultivars, with minor effects in roots; neither root nor shoot [K+] were affected. In IR29, Si lowered shoot [Na+] via a large reduction in bypass flow, while in Pokkali, where bypass flow was small and not affected by Si, this was achieved mainly via a growth dilution of shoot Na+. Si had no effect on unidirectional 24Na+ fluxes (influx and efflux), or on Na+-stimulated plasma-membrane depolarization, in either IR29 or Pokkali. We conclude that, while Si can reduce Na+ translocation via bypass flow in some (but not all) rice cultivars, it does not affect unidirectional Na+ transport or Na+ cycling in roots, either across root cell membranes or within the bulk root apoplast.

Although the global nitrogen (N) cycle is largely driven by soil microbes, plant root exudates can profoundly modify soil microbial communities and influence their N transformations. A detailed understanding is now beginning to emerge regarding the control that root exudates exert over two major soil N processes - nitrification and N2 fixation. We discuss recent breakthroughs in this area, including the identification of root exudates as nitrification inhibitors and as signaling compounds facilitating N-acquisition symbioses. We indicate gaps in current knowledge, including questions of how root exudates affect newly discovered microbial players and N-cycle components. A better understanding of these processes is urgent given the widespread inefficiencies in agricultural N use and their links to N pollution and climate change.

Nitrogen (N) and potassium (K) are the two most abundantly acquired mineral elements by plants, and their acquisition pathways interact in complex ways. Here, we review pivotal interactions with respect to root acquisition, storage, translocation and metabolism, between the K+ ion and the two major N sources, ammonium (NH4+ ) and nitrate (NO3- ). The intersections between N and K physiology are explored at a number of organizational levels, from molecular-genetic processes, to compartmentation, to whole plant physiology, and discussed in the context of both N-K cooperation and antagonism. Nutritional regulation and optimization of plant growth, yield, metabolism and water-use efficiency are also discussed.

A systematic analysis of NaCl-dependent, plasma-membrane depolarization (∆∆Ψ) in rice roots calls into question the current leading model of rapid membrane cycling of Na

The nitrogen (N)-use efficiency of agricultural plants is notoriously poor. Globally, about 50% of the N fertilizer applied to cropping systems is not absorbed by plants, but lost to the environment as ammonia (NH3), nitrate (NO3-), and nitrous oxide (N2O, a greenhouse gas with 300 times the heat-trapping capacity of carbon dioxide), raising agricultural production costs and contributing to pollution and climate change. These losses are driven by volatilization of NH3 and by a matrix of nitrification and denitrification reactions catalysed by soil microorganisms (chiefly bacteria and archaea). Here, we discuss mitigation of the harmful and wasteful process of agricultural N loss via biological nitrification inhibitors (BNIs) exuded by plant roots. We examine key recent discoveries in the emerging field of BNI research, focusing on BNI compounds and their specificity and transport, and discuss prospects for their role in improving agriculture while reducing its environmental impact.

Britto, D. T., Balkos, K. D., Becker, A., Coskun, D., Huynh, W. Q. and Kronzucker, H. J. 2014. Potassium and nitrogen poising: Physiological changes and biomass gains in rice and barley. Can. J. Plant Sci. 94: 1085–1089. Soil nitrogen, potassium, and water are three of the most important factors influencing, often interdependently, the growth of plants. Maximizing plant growth is not simply a matter of maximizing the availability of these and other nutrients; indeed, excess supply can be deleterious to plant performance. Rather, optimal performance may come about by adjusting the supply of each of the disparate factors required for plant growth, not only individually, but in relation to one another. In our work investigating the nutritional maximization of plant growth, we have found that altering the ratios of N and K provided to seedlings of cereal grasses can result in very substantial increases in vegetative biomass accrual, e.g., >220% of low-K+controls, in short-term studies with rice, the world's most important cereal grain, and even greater gains in grain yield, in the longer term. Hence, the findings in our laboratory are of direct relevance to the aim of NSERC's Green Crop Network, which was to contribute to the amelioration of climate change by improvement of carbon capture and sequestration in crop plants. In addition, these findings may help to increase the world's food supply, the security of which is sometimes at odds with proposed means to thwart climate change. Our work in this area has also led to a potential breakthrough of a more fundamental sort in plant nutritional biology, which may in itself have important practical implications: evidence that aquaporin-type transport proteins conduct rapid NH3fluxes into roots at toxic levels of external ammonia/ammonium.

Vascular plants are major intermediaries in the global water cycle, and are highly adapted to both facilitate and resist water fluxes, such as during root uptake, translocation in the xylem, and transpiration by leaves. Here, we summarize the contributions to a Special Issue on water in the Journal of Plant Physiology, which cluster around the theme of control and facilitation of water movement in plants. We conclude with an editorial view of the need for plant physiologists to consider larger cultural issues surrounding water use, especially in terms of the increasing agricultural demand for water to produce animal feed, with its associated trophic nutritive losses and environmental damage.

Futile transmembrane NH3/NH4 + cycling in plant root cells, characterized by extremely rapid fluxes and high efflux to influx ratios, has been successfully linked to NH3/NH4 + toxicity. Surprisingly, the fundamental question of which species of the conjugate pair (NH3 or NH4 +) participates in such fluxes is unresolved. Using flux analyses with the short-lived radioisotope 13N and electrophysiological, respiratory, and histochemical measurements, we show that futile cycling in roots of barley (Hordeum vulgare) seedlings is predominately of the gaseous NH3 species, rather than the NH4 + ion. Influx of 13NH3/13NH4 +, which exceeded 200 µmol g–1 h–1, was not commensurate with membrane depolarization or increases in root respiration, suggesting electroneutral NH3 transport. Influx followed Michaelis-Menten kinetics for NH3 (but not NH4 +), as a function of external concentration (K m = 152 µm, V max = 205 µmol g–1 h–1). Efflux of 13NH3/13NH4 + responded with a nearly identical K m. Pharmacological characterization of influx and efflux suggests mediation by aquaporins. Our study fundamentally revises the futile-cycling model by demonstrating that NH3 is the major permeating species across both plasmalemma and tonoplast of root cells under toxicity conditions.

Sodium (Na+) is one of the most intensely researched ions in plant biology and has attained a reputation for its toxic qualities. Following the principle of Theophrastus Bombastus von Hohenheim (Paracelsus), Na+ is, however, beneficial to many species at lower levels of supply, and in some, such as certain C4 species, indeed essential. Here, we review the ion’s divergent roles as a nutrient and toxicant, focusing on growth responses, membrane transport, stomatal function, and paradigms of ion accumulation and sequestration. We examine connections between the nutritional and toxic roles throughout, and place special emphasis on the relationship of Na+ to plant potassium (K+) relations and homeostasis. Our review investigates intriguing connections and disconnections between Na+ nutrition and toxicity, and concludes that several leading paradigms in the field, such as on the roles of Na+ influx and tissue accumulation or the cytosolic K+/Na+ ratio in the development of toxicity, are currently insufficiently substantiated and require a new, critical approach.

The role of potassium (K+) transporters in high- and low-affinity K+ uptake was examined in roots of intact barley (Hordeum vulgare) and Arabidopsis (Arabidopsis thaliana) plants by use of 42K radiotracing, electrophysiology, pharmacology, and mutant analysis. Comparisons were made between results from barley and five genotypes of Arabidopsis, including single and double knockout mutants for the high-affinity transporter, AtHAK5, and the Shaker-type channel, AtAKT1. In Arabidopsis, steady-state K+ influx at low external K+ concentration ([K+]ext = 22.5 µm) was predominantly mediated by AtAKT1 when high-affinity transport was inhibited by ammonium, whereas in barley, by contrast, K+ channels could not operate below 100 µm. Withdrawal of ammonium resulted in an immediate and dramatic stimulation of K+ influx in barley, indicating a shift from active to passive K+ uptake at low [K+]ext and yielding fluxes as high as 36 µmol g (root fresh weight)−1 h−1 at 5 mm [K+]ext, among the highest transporter-mediated K+ fluxes hitherto reported. This ammonium-withdrawal effect was also established in all Arabidopsis lines (the wild types, atakt1, athak5, and athak5 atakt1) at low [K+]ext, revealing the concerted involvement of several transport systems. The ammonium-withdrawal effect coincided with a suppression of K+ efflux and a significant hyperpolarization of the plasma membrane in all genotypes except athak5 atakt1, could be sustained over 24 h, and resulted in increased tissue K+ accumulation. We discuss key differences and similarities in K+ acquisition between two important model systems and reveal novel aspects of K+ transport in planta.

Carbon dioxide (CO2) concentrations in the earth's atmosphere are projected to rise from current levels near 400ppm to over 700ppm by the end of the 21st century. Projections over this time frame must take into account the increases in total net primary production (NPP) expected from terrestrial plants, which result from elevated CO2 (eCO2) and have the potential to mitigate the impact of anthropogenic CO2 emissions. However, a growing body of evidence indicates that limitations in soil nutrients, particularly nitrogen (N), the soil nutrient most limiting to plant growth, may greatly constrain future carbon fixation. Here, we review recent studies about the relationships between soil N supply, plant N nutrition, and carbon fixation in higher plants under eCO2, highlighting key discoveries made in the field, particularly from free-air CO2 enrichment (FACE) technology, and relate these findings to physiological and ecological mechanisms.

Soil sodium, while toxic to most plants at high concentrations, can be beneficial at low concentrations, particularly when potassium is limiting. However, little is known about Na(+) uptake in this 'high-affinity' range. New information is provided here with an insight into the transport characteristics, mechanism, and ecological significance of this phenomenon. High-affinity Na(+) and K(+) fluxes were investigated using the short-lived radiotracers (24)Na and (42)K, under an extensive range of measuring conditions (variations in external sodium, and in nutritional and pharmacological agents). This work was supported by electrophysiological, compartmental, and growth analyses. Na(+) uptake was extremely sensitive to all treatments, displaying properties of high-affinity K(+) transporters, K(+) channels, animal Na(+) channels, and non-selective cation channels. K(+), NH(4)(+), and Ca(2+) suppressed Na(+) transport biphasically, yielding IC(50) values of 30, 10, and

The heavy metals silver, gold, and mercury can strongly inhibit aquaporin-mediated water flow across plant cell membranes, but critical examinations of their side effects are rare. Here, the short-lived radiotracer (42)K is used to demonstrate that these metals, especially silver, profoundly change potassium homeostasis in roots of intact barley (Hordeum vulgare L.) plants, by altering unidirectional K(+) fluxes. Doses as low as 5 μM AgNO(3) rapidly reduced K(+) influx to 5% that of controls, and brought about pronounced and immediate increases in K(+) efflux, while higher doses of Au(3+) and Hg(2+) were required to produce similar responses. Reduced influx and enhanced efflux of K(+) resulted in a net loss of40% of root tissue K(+) during a 15 min application of 500 μM AgNO(3), comprising the entire cytosolic potassium pool and about a third of the vacuolar pool. Silver also brought about major losses of UV-absorbing compounds, total electrolytes, and NH(4)(+). Co-application, with silver, of the channel blockers Cs(+), TEA(+), or Ca(2+), did not affect the enhanced efflux, ruling out the involvement of outwardly rectifying ion channels. Taken together with an examination of propidium iodide staining under confocal microscopy, the results indicate that silver ions affect K(+) homeostasis by directly inhibiting K(+) influx at lower concentrations, and indirectly inhibiting K(+) influx and enhancing K(+) efflux, via membrane destruction, at higher concentrations. Ni(2+), Cd(2+), and Pb(2+), three heavy metals not generally known to affect aquaporins, did not enhance K(+) efflux or cause propidium iodide incorporation. The study reveals strong and previously unknown effects of major aquaporin inhibitors and recommends caution in their application.

Potassium (K(+) ) flux into plant cells is a well-characterized ion transport phenomenon. By contrast, little is known about the mechanisms and regulation of K(+) flux from the cell. Here, we present a radioisotopic analysis of K(+) fluxes from roots of intact barley (Hordeum vulgare), in the context of recent discoveries in the molecular biology and electrophysiology of this process. Plants were labelled with (42)K(+), and kinetics of its release from roots were monitored at low (0.1 mM) or high (1.0 mM) external K concentration, [K(+)](ext), and with the application of channel modulators and nutrient shifts. At 0.1 (but not 1.0) mM [K(+)], where K(+) efflux is thought to be mediated by K(+)-outward-rectifying channels, (42)K(+) efflux was inhibited by the channel blockers barium (Ba(2+)), caesium (Cs(+)), tetraethylammonium (TEA(+)), and lanthanum (La(3+)). Ammonium and nitrate (10 mM) stimulated and inhibited (42)K(+) efflux, respectively, while 10 mM [K(+)](ext) or [Rb(+) ](ext) decreased it. No evidence for the involvement of ATP-binding cassettes, nonselective cation channels, or active K(+)-efflux pumps was found. Our study provides new evidence for the thermodynamic transition between high- and low-affinity transport, from the efflux perspective, identifying the operation of channels at low [K(+)], and the cessation of transmembrane efflux at high [K(+)].

Plant transport physiologists have developed a range of models describing the movement of ions across cell membranes. However, while substantial progress has been made towards providing precise descriptions of the mechanisms underlying these fluxes, important instances remain in which the prevailing models cannot account for repeated observations, particularly in terms of energy transformations. As we shall show, disagreements with experimental findings may entail a revision of the proposed models, similarly to what has been required in animal transport studies (Ussing, 1994; see below). We present, as a key example, the futile cycling of sodium under toxic conditions (see Britto & Kronzucker, 2006, and discussed later), and show that unidirectional flux magnitudes measured by several groups, including our own, cannot be explained energetically by current models. We attempt to explain these observations by proposing alternative mechanisms of Na + transport across the root-cell plasma membrane. The influx/efflux cycle of Na + in plants has been attributed to the sophisticated activity of distinct transport proteins in

International audience; Globally, over one-third of irrigated land is affected by salinity, including much of the land under lowland rice cultivation in the tropics, seriously compromising yields of this most important of crop species. However , there remains an insufficient understanding of the cellular basis of salt tolerance in rice. Here, three methods of 24 Na + tracer analysis were used to investigate primary Na + transport at the root plasma membrane in a salt-tolerant rice cultivar (Pokkali) and a salt-sensitive cultivar (IR29). Futile cycling of Na + at the plasma membrane of intact roots occurred at both low and elevated levels of steady-state Na + supply ([Na + ] ext ¼1 mM and 25 mM) in both cultivars. At 25 mM [Na + ] ext , a toxic condition for IR29, unidirectional influx and efflux of Na + in this cultivar, but not in Pokkali, became very high [>100 mmol g (root FW) 21 h 21 ], demonstrating an inability to restrict sodium fluxes. Current models of sodium transport energetics across the plasma membrane in root cells predict that, if the sodium efflux were mediated by Na + /H + antiport, this toxic scenario would impose a substantial respiratory cost in IR29. This cost is calculated here, and compared with root respiration, which, however, comprised only ;50% of what would be required to sustain efflux by the antiporter. This suggests that either the conventional 'leak-pump' model of Na + transport or the energetic model of proton-linked Na + transport may require some revision. In addition, the lack of suppression of Na + influx by both K + and Ca 2+ , and by the application of the channel inhibitors Cs + , TEA + , and Ba 2+ , questions the participation of potassium channels and non-selective cation channels in the observed Na + fluxes.

The interaction of sodium and potassium ions in the context of the primary entry of Na(+) into plant cells, and the subsequent development of sodium toxicity, has been the subject of much recent attention. In the present study, the technique of compartmental analysis with the radiotracers (42)K(+) and (24)Na(+) was applied in intact seedlings of barley (Hordeum vulgare L.) to test the hypothesis that elevated levels of K(+) in the growth medium will reduce both rapid, futile Na(+) cycling at the plasma membrane, and Na(+) build-up in the cytosol of root cells, under saline conditions (100 mM NaCl). We reject this hypothesis, showing that, over a wide (400-fold) range of K(+) supply, K(+) neither reduces the primary fluxes of Na(+) at the root plasma membrane nor suppresses Na(+) accumulation in the cytosol. By contrast, 100 mM NaCl suppressed the cytosolic K(+) pool by 47-73%, and also substantially decreased low-affinity K(+) transport across the plasma membrane. We confirm that the cytosolic [K(+)]:[Na(+)] ratio is a poor predictor of growth performance under saline conditions, while a good correlation is seen between growth and the tissue ratios of the two ions. The data provide insight into the mechanisms that mediate the toxic influx of sodium across the root plasma membrane under salinity stress, demonstrating that, in the glycophyte barley, K(+) and Na(+) are unlikely to share a common low-affinity pathway for entry into the plant cell.

Futile plasma membrane cycling of ammonium (NH4+) is characteristic of low-affinity NH4+ transport, and has been proposed to be a critical factor in NH4+ toxicity. Using unidirectional flux analysis with the positron-emitting tracer 13N in intact seedlings of barley (Hordeum vulgare L.), it is shown that rapid, futile NH4+ cycling is alleviated by elevated K+ supply, and that low-affinity NH4+ transport is mediated by a K+-sensitive component, and by a second component that is independent of K+. At low external [K+] (0.1 mM), NH4+ influx (at an external [NH4+] of 10 mM) of 92 micromol g(-1) h(-1) was observed, with an efflux:influx ratio of 0.75, indicative of rapid, futile NH4+ cycling. Elevating K+ supply into the low-affinity K+ transport range (1.5-40 mM) reduced both influx and efflux of NH4+ by as much as 75%, and substantially reduced the efflux:influx ratio. The reduction of NH4+ fluxes was achieved rapidly upon exposure to elevated K+, within 1 min for influx and within 5 min for efflux. The channel inhibitor La3+ decreased high-capacity NH4+ influx only at low K+ concentrations, suggesting that the K+-sensitive component of NH4+ influx may be mediated by non-selective cation channels. Using respiratory measurements and current models of ion flux energetics, the energy cost of concomitant NH4+ and K+ transport at the root plasma membrane, and its consequences for plant growth are discussed. The study presents the first demonstration of the parallel operation of K+-sensitive and -insensitive NH4+ flux mechanisms in plants.

Potassium (K(+)) acquisition in roots is generally described by a two-mechanism model, consisting of a saturable, high-affinity transport system (HATS) operating via H(+)/K(+) symport at low (1mM) external [K(+)] ([K(+)]ext), and a linear, low-affinity system (LATS) operating via ion channels at high (1mM) [K(+)]ext. Radiotracer measurements in the LATS range indicate that the linear rise in influx continues well beyond nutritionally relevant concentrations (10mM), suggesting K(+) transport may be pushed to extraordinary, and seemingly limitless, capacity. Here, we assess this rise, asking whether LATS measurements faithfully report transmembrane fluxes. Using (42)K(+)-isotope and electrophysiological methods in barley, we show that this flux is part of a K(+)-transport cycle through the apoplast, and masks a genuine plasma-membrane influx that displays Michaelis-Menten kinetics. Rapid apoplastic cycling of K(+) is corroborated by an absence of transmembrane (42)K(+) efflux above 1mM, and by the efflux kinetics of PTS, an apoplastic tracer. A linear apoplastic influx, masking a saturating transmembrane influx, was also found in Arabidopsis mutants lacking the K(+) transporters AtHAK5 and AtAKT1. Our work significantly revises the model of K(+) transport by demonstrating a surprisingly modest upper limit for plasma-membrane influx, and offers insight into sodium transport under salt stress.

Nitrogen (N) and potassium (K) are the two most abundantly acquired mineral elements by plants, and their acquisition pathways interact in complex ways. Here, we review pivotal interactions with respect to root acquisition, storage, translocation and metabolism, between the K

The efflux of sodium (Na(+)) ions across the plasma membrane of plant root cells into the external medium is surprisingly poorly understood. Nevertheless, Na(+) efflux is widely regarded as a major mechanism by which plants restrain the rise of Na(+) concentrations in the cytosolic compartments of root cells and, thus, achieve a degree of tolerance to saline environments. In this review, several key ideas and bodies of evidence concerning root Na(+) efflux are summarized with a critical eye. Findings from decades past are brought to bear on current thinking, and pivotal studies are discussed, both "purely physiological", and also with regard to the SOS1 protein, the only major Na(+) efflux transporter that has, to date, been genetically characterized. We find that the current model of rapid transmembrane sodium cycling (RTSC), across the plasma membrane of root cells, is not adequately supported by evidence from the majority of efflux studies. An alternative hypothesis cannot be ruled out, that most Na(+) tracer efflux from the root in the salinity range does not proceed across the plasma membrane, but through the apoplast. Support for this idea comes from studies showing that Na(+) efflux, when measured with tracers, is rarely affected by the presence of inhibitors or the ionic composition in saline rooting media. We conclude that the actual efflux of Na(+) across the plasma membrane of root cells may be much more modest than what is often reported in studies using tracers, and may predominantly occur in the root tips, where SOS1 expression has been localized.

It has long been believed that maintenance of low Na+ : K+ ratios in the cytosol of plant cells is critical to the plant's ability to tolerate salinity stress. Direct measurements of such ratios, however, have been few. Here we apply the non-invasive technique of compartmental analysis, using the short-lived radiotracers 42K+ and 22Na+, in intact seedlings of barley (Hordeum vulgare L.), to evaluate unidirectional plasma membrane fluxes and cytosolic concentrations of K+ and Na+ in root tissues, under eight nutritional conditions varying in levels of salinity and K+ supply. We show that Na+ : K+ ratios in the cytosol of root cells adjust significantly across the conditions tested, and that these ratios are poor predictors of the plant's growth response to salinity. Our study further demonstrates that Na+ is subject to rapid and futile cycling at the plasma membrane at all levels of Na+ supply, independently of external K+, while K+ influx is reduced by Na+, from a similar baseline, and to a similar extent, at both low and high K+ supply. We compare our results to those of other groups, and conclude that the maintenance of the cytosolic Na+ : K+ ratio is not central to plant survival under NaCl stress. We offer alternative explanations for sodium sensitivity in relation to the primary acquisition mechanisms of Na+ and K+.

The existence of distinct high- and low-affinity transport systems (HATS and LATS) is well established for major nutrient ions. However, influx mediated by these systems is usually estimated using uniformly simple tracer protocols. Two (42)K radiotracer methods to measure potassium influxes in the HATS and LATS ranges in intact barley (Hordeum vulgare L.) roots are compared here: a direct influx (DI) method, and an integrated flux analysis (IFA), which is designed to account for tracer efflux from labelled roots and differential tracer accumulation along the plant axis. Methods showed only minor discrepancies for influx values in the HATS range, but large discrepancies in the LATS range, revealing striking distinctions in the cellular exchange properties dominated by the operation of the two transport systems. It is shown that accepted DI protocols are associated with very large errors in the high-conductance LATS range, underestimating influx at least 6-fold due to four characteristics of this transport mode: (i) accelerated cellular (42)K exchange; (ii) a greatly increased ratio of efflux to influx; (iii) increased (42)K loss during the removal of water from roots in preweighing centrifugation or blotting protocols; and (iv) increased (42)K retention at the root-shoot interface, a region of the plant frequently disregarded in DI determinations. The findings warrant a re-evaluation of a large body of literature reporting influx in the LATS range, and are of fundamental importance to ion flux experimentation in plant physiology.

Summary In this chapter, we shall review plant responses to changes in supply of the two inorganic N sources nitrate (N03 -) and ammonium (NH4 +), with an emphasis on primary mechanisms of transport, and their regulation. The topics discussed here include: soil N resources and their ecological implications for plant life; parallel responses in plant growth and N transport activity as functions of N supply; transport kinetics; inducibility and downregulation of N03 - and NH4 + acquisition; plant sensing of N03 -; and low-affinity N transport. Throughout, we attempt to identify areas that are controversial, and those in need of further examination.

An isotope procedure for the tracing of ion fluxes and rate constants in intact plants is presented and applied to 42K-labelled potassium fluxes in cells of intact barley (Hordeum vulgare L.) roots. This procedure differs from conventional tracer efflux protocols in that tracer accrual in the external solution bathing the labelled roots is continually monitored by solution subsampling, whereas conventional protocols involve monitoring the specific-activity decline in a sequence of eluates that wash out tracer released by roots. The new technique minimizes physical disturbance to the plant system, while permitting excellent time resolution of efflux kinetics. In the high-affinity transport (HATS) range, the flux and exchange parameters determined using this method showed close agreement with those found using a conventional protocol. However, in the low-affinity transport (LATS) range, substantially higher influx and efflux were seen than are normally observed with conventional tracer techniques. It is shown that this difference is attributable to the greater disturbance-sensitivity of LATS transport, and conclude that the measurement of fluxes is much more difficult in this transport range than in the disturbance-resistant HATS range.

The classic biochemical pH-stat model of cytosolic pH regulation in plant cells presupposes a pH-dependent biosynthesis and degradation of organic acids, specifically malic acid, in the cytosol. This model has been used to explain the higher tissue accumulation of organic acids in nitrate (NO 3 ‐ )-grown, relative to ammonium (NH 4 + )-grown, plants, the result of proposed cytosolic alkalinization by NO 3 ‐ metabolism, and acidification by NH 4 + metabolism. Here, a critical examination of the model shows that its key assumptions are fundamentally problematic, particularly in the context of the effects on cellular pH of nitrogen source differences. Specifically, the model fails to account for proton transport accompanying inorganic nitrogen transport, which, if considered, renders the H + production of com‐ + provided N sources. We show that the model’s evidentiary basis in total-tissue mineral ion and organic acid analysis is not directly relevant to subcellular (cytosolic) pH homeostasis, while the analysis of the ionic components of the cytosol is relevant to this process. A literature analysis further shows that the assumed greater activity of the enzyme phosphoenolpyruvate (PEP) carboxylase under nitrate nutrition, which is a key characteristic of the biochemical pH-stat model as it applies to nitrogen source, is not borne out in numerous instances. We conclude that this model is not tenable in its current state, and propose an alternative model that reaffirms the anaplerotic role of PEP carboxylase within the context of N nutrition, in the production of carbon skeletons for amino acid synthesis.

Ratios of ammonium (NH 4 +) to nitrate (NO 3 -) in soils are known to increase during forest succession. Using evidence from several previous studies, we hypothesize that a malfunction in NH 4 + transport at the membrane level might limit the persistence of early successional tree species in later seral stages. In those studies, 13 N radiotracing was used to determine unidirectional fluxes and pool sizes of NH 4 + and NO 3 -in seedlings of the late-successional species white spruce (Picea glauca) and in the early successional species Douglas-fir (Pseudotsuga menziesii var. glauca) and trembling aspen (Populus tremuloides). At high external NH 4 +, the two early successional species accumulated excessive NH 4 + in the root cytosol, and exhibited high-velocity, low-efficiency (15% to 22%), membrane fluxes of NH 4 +. In sharp contrast, white spruce had low cytosolic NH 4 + accumulation, and lower-velocity but much higher-efficiency (65%), NH 4 + fluxes. Because these divergent responses parallel known differences in tolerance and toxicity to NH 4 + amongst these species, we propose that they constitute a significant driving force in forest succession, complementing the discrimination against NO 3 - documented in white spruce (Kronzucker et al. 1997).

Summary • We report the first use of tracer 13NH4+ (13N-ammonium) efflux and retention data to analyse subcellular fluxes and compartmentation of NH4+ in the leaves of a higher plant (wheat, Triticum aestivum). • Leaf segments, 1–2 mm, were obtained from 8-d-old seedlings. The viability of the segments, and stability of NH 4+ acquisition over time, were confirmed using oxygen-exchange and NH 4+ -depletion measurements. Fluxes of NH 4+ and compartment sizes were estimated using tracer efflux kinetics and retention data. • Influx and efflux across the plasma membrane, half-lives of exchange and cytosolic pool sizes were broadly similar to those in root systems. As the external concentration of NH 4+ ([NH 4+ ] o ) increased from 10 µ m to 10 m m , both influx and efflux greatly increased, with a sixfold increase in the ratio of efflux to influx. Half-lives were similar among treatments, except at [NH 4+ ] o = 10 m m , where they declined. Concentrations of NH 4+ in the cytosol ([NH 4+ ] c ) increased from 2.6 to 400 m m . • Although [NH 4+ ] c became large as [NH 4+ ] o increased, the ratio of [NH 4+ ] c to [NH 4+ ] o decreased more than sixfold. The apparently futile cycling of NH 4+ at high [NH 4+ ] o suggested by the large fluxes of NH 4+ in both directions across the membrane indicate that leaf cells respond to potentially toxic NH 4+ concentrations in a manner similar to root cells.

Summary Ammonium (NH 4 + ) toxicity is an issue of global ecological and economic importance. In this review, we discuss the major themes of NH 4 + toxicity, including the occurrence of NH 4 + in the biosphere, response differences to NH 4 + nutrition among wild and domesticated species, symptoms and proposed mechanisms underlying toxicity, and means by which it can be alleviated. Where possible, nitrate (NO 3 − ) nutrition is used as point of comparison. Particular emphasis is placed on issues of cellular pH, ionic balance, relationships with carbon biochemistry, and bioenergetics of primary NH 4 + transport. Throughout, we attempt to identify areas that are controversial, and areas that are in need of further examination.

Glutamate receptors and related proteins are important players in plant ion transport, cellular signalling, and ion toxicity, as substantial recent work has shown (see reviews by Dietrich et al. 2010; Kronzucker and Britto 2011; Maathuis 2007; Zhang et al. 2010). A new article on the subject, published in the journal Science, however, comes with the unsettling title “Glutamate Receptor–Like Genes Form Ca Channels in Pollen Tubes and Are Regulated by Pistil D-Serine” (Michard et al. 2011). This paper discusses the role of “ionotropic GLRs [glutamate-receptor-like genes]...identified in the genome of Arabidopsis”, and declares in its final paragraph that “Genes for putative cyclic nucleotidegated channels were the first reported as plausible Ca channels....” Thus, at several junctures, the manuscript rather boldly conflates genetic and functional attributes. This requires some editorial comment. Surely, the authors of this paper, its referees, and the journal’s editors must be aware that genes do not form channels, mediate ion fluxes (i.e. behave “ionotropically”), or perform any known catalytic or transport function in living organisms. Yet, the very term “glutamate receptor-like gene” is itself a misnomer: surely, a gene cannot be likened to a glutamate receptor. Moreover, the genes in question are not themselves regulated by the amino acid Dserine, contrary to what the paper’s title unequivocally states. To be fair, this sort of category error is not without precedent in the literature; for example, it can be seen in other articles on GLR channels by Chiu et al. (2002), Li et al. (2005), Meyerhoff et al. (2005), and Roy et al. (2008). While the conflation of gene and protein seems unusually rife in this area, it is by no means restricted to it, nor is it restricted to plant biology. Examples abound of statements that genes “transport” substances (Dean et al. 2003), “synthesize” or “produce” other substances (Malik et al. 2009; Pulkkinen et al. 2000; Weiner et al. 1993), and “catalyze” chemical reactions (Chen et al. 2005; Drakas et al. 2005; Lewinsohn et al. 2001; Metherall et al. 1996; Ono et al. 1999; Powell et al. 2008; Ullrich and van Putten 1995). Common usage of an error does not make it any less erroneous. However, the example of Michard et al. is particularly egregious in that it has introduced this manner of speech into the pages of one of the world’s most highly esteemed scientific journals. With this stamp of approval, it propagates a rather stunning Plant Soil (2011) 346:15–17 DOI 10.1007/s11104-011-0872-1

Unidirectional influx and efflux of nutrients and toxicants, and their resultant net fluxes, are central to the nutrition and toxicology of plants. Radioisotope tracing is a major technique used to measure such fluxes, both within plants, and between plants and their environments. Flux data obtained with radiotracer protocols can help elucidate the capacity, mechanism, regulation, and energetics of transport systems for specific mineral nutrients or toxicants, and can provide insight into compartmentation and turnover rates of subcellular mineral and metabolite pools. Here, we describe two major radioisotope protocols used in plant biology: direct influx (DI) and compartmental analysis by tracer efflux (CATE). We focus on flux measurement of potassium (K(+)) as a nutrient, and ammonia/ammonium (NH3/NH4(+)) as a toxicant, in intact seedlings of the model species barley (Hordeum vulgare L.). These protocols can be readily adapted to other experimental systems (e.g., different species, excised plant material, and other nutrients/toxicants). Advantages and limitations of these protocols are discussed.

Summary • A comprehensive and pragmatic approach to the design of unidirectional ion transport experiments in plants is presented here, revising and simplifying classical models. • The kinetic constant for cytosolic ion exchange ( kc ) is critical to the understanding of the interrelated flux processes occurring simultaneously at the cellular level. This constant is most effectively estimated using the compartmental analysis by efflux method, which, by providing values for additional kinetic parameters (i.e. unidirectional influx and efflux) can be used to determine the extent of distortion inherent in assessments of influx at, or close to, the steady state. • Focusing on the kinetics of nitrogen exchange, with tracer efflux experiments in barley ( Hordeum vulgare ) using 13NH4+, conducted under perturbation conditions, it was demonstrated that a transitional state was rapidly established following a concentration shift, characterized by a restoration of the preperturbational (steady-state) kc value. It is concluded that a reasonably accurate estimate of unidirectional influx can be made when influx measurements are conducted subsequent to the establishment of this transitional state. • A mathematical treatment of unidirectional flux processes allows the exact determination of errors caused by ionic counterfluxes under steady-state conditions.

In tomato (Lycopersicon esculentum L. cv Trust Fl), effects of various nutrient treatments on growth, fruit yield and quality, nutrient uptake and accumulation were studied in a hydroponic system. Reductions of macronutrient concentrations to 50% (0.5 × C) or 25% (0.25 × C) of the control (C) levels as well as cessation of replenishment of the feed solution for the last 16 days after 7 months growth at control levels, had no adverse effect on growth, fruit yield and fruit quality. However, reduction of macronutrient concentration to 10% of control (0.1 × C) reduced fruit yield by ‐30%. Steady‐state influx and net flux of NO3 ‐ into the roots of 4–6 week‐old seedlings had not acclimated and showed concentration dependence from 1.1 mM (0.1 × C) to 11 mM (C). Whereas, Pi and K+ fluxes were similar at 0.5 × C and C levels, at 0.1 × C they were significantly lower than the fluxes at higher concentrations, showing lack of acclimation at this concentration. This lack of flux acclimation may account for ...

K(+) channels are among the best-characterized classes of membrane protein in plants. Nevertheless, in-planta demonstrations of traits emerging from molecular characterizations have often been insufficient or lacking altogether. Such linkages are, however, critical to our basic understanding of plant nutrition and to addressing 'real-world' issues that are faced in environmental and agricultural settings. Here, we cover some of the recent advances in K(+) acquisition with particular focus on voltage-gated K(+) channel functioning and regulation in roots, and highlight where linkages to in-planta behavior have been successfully made and, conversely, where such linkages are yet to be made.

Standard procedures for the tracing of ion fluxes into roots of plants are described here, with emphasis on cations, especially potassium (K(+)). We focus in particular on the measurement of unidirectional influx by use of radiotracers and provide a brief introduction to compartmental analysis by tracer efflux (CATE).

Radioisotopes (particularly (22)Na, (24)Na, (42)K, and (86)Rb) have been used for many decades to trace the fluxes and accumulation of sodium and potassium ions in plant tissues. In this article, standard procedures for the tracing of ion fluxes are described, with emphasis on special problems encountered when examining K(+) and Na(+) transport under salinity conditions. We focus in particular on unidirectional influx measurements, while also providing a brief introduction to compartmental analysis by tracer efflux.

Radioisotopes (particularly (22)Na, (24)Na, (42)K, and (86)Rb) have been used for many decades to trace the fluxes and accumulation of sodium and potassium ions in plant tissues. In this article, standard procedures for the tracing of ion fluxes are described, with emphasis on special problems encountered when examining K(+) and Na(+) transport under salinity conditions. We focus in particular on unidirectional influx measurements, while also providing a brief introduction to compartmental analysis by tracer efflux.

Recently, it has been proposed that ammonium is toxic to barley because of the energetic cost of pumping ammonium that has leaked into root cells back into the soil. This does not occur in rice because high levels of ammonium reduce the potential difference across the plasma membrane of rice – whereas the potential difference in barley appears to be ammonium insensitive. These results highlight the potentially high costs of membrane transport, and thus the central importance of transport processes in plants.

Sodium (Na) toxicity is one of the most formidable challenges for crop production world-wide. Nevertheless, despite decades of intensive research, the pathways of Na(+) entry into the roots of plants under high salinity are still not definitively known. Here, we review critically the current paradigms in this field. In particular, we explore the evidence supporting the role of nonselective cation channels, potassium transporters, and transporters from the HKT family in primary sodium influx into plant roots, and their possible roles elsewhere. We furthermore discuss the evidence for the roles of transporters from the NHX and SOS families in intracellular Na(+) partitioning and removal from the cytosol of root cells. We also review the literature on the physiology of Na(+) fluxes and cytosolic Na(+) concentrations in roots and invite critical interpretation of seminal published data in these areas. The main focus of the review is Na(+) transport in glycophytes, but reference is made to literature on halophytes where it is essential to the analysis.

*Stimulation of potassium (K(+)) efflux by sodium (Na(+)) has been the subject of much recent attention, and its mechanism has been attributed to the activities of specific classes of ion channels. *The short-lived radiotracer (42)K(+) was used to test this attribution, via unidirectional K(+)-flux analysis at the root plasma membrane of intact barley (Hordeum vulgare), in response to NaCl, KCl, NH(4)Cl and mannitol, and to channel inhibitors. *Unidirectional K(+) efflux was strongly stimulated by NaCl, and K(+) influx strongly suppressed. Both effects were ameliorated by elevated calcium (Ca(2+)). As well, K(+) efflux was strongly stimulated by KCl, NH(4)Cl and mannitol , and NaCl also stimulated (13)NH(4)(+) efflux. The Na(+)-stimulated K(+) efflux was insensitive to cesium (Cs(+)) and pH 4.2, weakly sensitive to the K(+)-channel blocker tetraethylammonium (TEA(+)) and quinine, and moderately sensitive to zinc (Zn(2+)) and lanthanum (La(3+)). *We conclude that the stimulated efflux is: specific neither to Na(+) as effector nor K(+) as target; composed of fluxes from both cytosol and vacuole; mediated neither by outwardly-rectifying K(+) channels nor nonselective cation channels; attributable, alternatively, to membrane disintegration brought about by ionic and osmotic components; of limited long-term significance, unlike the suppression of K(+) influx by Na(+), which is a greater threat to K(+) homeostasis under salt stress.

We present the first characterization of K(+) optimization of N uptake and metabolism in an NH(4)(+)-tolerant species, tropical lowland rice (cv. IR-72). (13)N radiotracing showed that increased K(+) supply reduces futile NH(4)(+) cycling at the plasma membrane, diminishing the excessive rates of both unidirectional influx and efflux. Pharmacological testing showed that low-affinity NH(4)(+) influx may be mediated by both K(+) and non-selective cation channels. Suppression of NH(4)(+) influx by K(+) occurred within minutes of increasing K(+) supply. Increased K(+) reduced free [NH(4)(+)] in roots and shoots by 50-75%. Plant biomass was maximized on 10 mm NH(4)(+) and 5 mm K(+), with growth 160% higher than 10 mm NO(3)(-)-grown plants, and 220% higher than plants grown at 10 mm NH(4)(+) and 0.1 mm K(+). Unlike in NH(4)(+)-sensitive barley, growth optimization was not attributed to a reduced energy cost of futile NH(4)(+) cycling at the plasma membrane. Activities of the key enzymes glutamine synthetase and phosphoenolpyruvate carboxylase (PEPC) were strongly stimulated by elevated K(+), mirroring plant growth and protein content. Improved plant performance through optimization of K(+) and NH(4)(+) is likely to be of substantial agronomic significance in the world's foremost crop species.

Potassium (K(+)) is an essential nutrient and the most abundant cation in plant cells. Plants have a wide variety of transport systems for K(+) acquisition, catalyzing K(+) uptake across a wide spectrum of external concentrations, and mediating K(+) movement within the plant as well as its efflux into the environment. K(+) transport responds to variations in external K(+) supply, to the presence of other ions in the root environment, and to a range of plant stresses, via Ca(2+) signaling cascades and regulatory proteins. This review will summarize the molecular identities of known K(+) transporters, and examine how this information supports physiological investigations of K(+) transport and studies of plant stress responses in a changing environment.

The disruption of K(+) transport and accumulation is symptomatic of NH(4)(+) toxicity in plants. In this study, the influence of K(+) supply (0.02-40 mM) and nitrogen source (10 mM NH(4)(+) or NO(3)(-)) on root plasma membrane K(+) fluxes and cytosolic K(+) pools, plant growth, and whole-plant K(+) distribution in the NH(4)(+)-tolerant plant species rice (Oryza sativa L.) was examined. Using the radiotracer (42)K(+), tissue mineral analysis, and growth data, it is shown that rice is affected by NH(4)(+) toxicity under high-affinity K(+) transport conditions. Substantial recovery of growth was seen as [K(+)](ext) was increased from 0.02 mM to 0.1 mM, and, at 1.5 mM, growth was superior on NH(4)(+). Growth recovery at these concentrations was accompanied by greater influx of K(+) into root cells, translocation of K(+) to the shoot, and tissue K(+). Elevating the K(+) supply also resulted in a significant reduction of NH(4)(+) influx, as measured by (13)N radiotracing. In the low-affinity K(+) transport range, NH(4)(+) stimulated K(+) influx relative to NO(3)(-) controls. It is concluded that rice, despite its well-known tolerance to NH(4)(+), nevertheless displays considerable growth suppression and disruption of K(+) homeostasis under this N regime at low [K(+)](ext), but displays efficient recovery from NH(4)(+) inhibition, and indeed a stimulation of K(+) acquisition, when [K(+)](ext) is increased in the presence of NH(4)(+).

Potassium (K(+)) is the most abundant ion in the plant cell and is required for a wide array of functions, ranging from the maintenance of electrical potential gradients across cell membranes, to the generation of turgor, to the activation of numerous enzymes. The majority of these functions depend more or less directly upon the activities and regulation of membrane-bound K(+) transport proteins, operating over a wide range of K(+) concentrations. Here, we review the physiological aspects of potassium transport systems in the plasma membrane, re-examining fundamental problems in the field such as the distinctions between high- and low-affinity transport systems, the interactions between K(+) and other ions such as NH(4)(+) and Na(+), the regulation of cellular K(+) pools, the generation of electrical potentials and the problems involved in measurement of unidirectional K(+) fluxes. We place these discussions in the context of recent discoveries in the molecular biology of K(+) acquisition and produce an overview of gene families encoding K(+) transporters.

Using the short-lived radiotracer 42K+, we present a comprehensive subcellular flux analysis of low-affinity K+ transport in plants. We overturn the paradigm of cytosolic K+ pool-size homeostasis and demonstrate that low-affinity K+ transport is characterized by futile cycling of K+ at the plasma membrane. Using two methods of compartmental analysis in intact seedlings of barley (Hordeum vulgare L. cv Klondike), we present data for steady-state unidirectional influx, efflux, net flux, cytosolic pool size, and exchange kinetics, and show that, with increasing external [K+] ([K+]ext), both influx and efflux increase dramatically, and that the ratio of efflux to influx exceeds 70% at [K+]ext ≥ 20 mm. Increasing [K+]ext, furthermore, leads to a shortening of the half-time for cytosolic K+ exchange, to values 2 to 3 times lower than are characteristic of high-affinity transport. Cytosolic K+ concentrations are shown to vary between 40 and 200 mm, depending on [K+]ext, on nitrogen treatment (NO3− or NH4+), and on the dominant mode of transport (high- or low-affinity transport), illustrating the dynamic nature of the cytosolic K+ pool, rather than its homeostatic maintenance. Based on measurements of trans-plasma membrane electrical potential, estimates of cytosolic K+ pool size, and the magnitude of unidirectional K+ fluxes, we describe efflux as the most energetically demanding of the cellular K+ fluxes that constitute low-affinity transport.

Rice is the most important crop species on earth, providing staple food for 70% of the world's human population. Over the past four decades, successes in classical breeding, fertilization, pest control, irrigation and expansion of arable land have massively increased global rice production, enabling crop scientists and farmers to stave off anticipated famines. If current projections for human population growth are correct, however, present rice yields will be insufficient within a few years. Rice yields will have to increase by an estimated 60% in the next 30 years, or global food security will be in danger. The classical methods of previous green revolutions alone will probably not be able to meet this challenge, without being coupled to recombinant DNA technology. Here, we focus on the promise of these modern technologies in the area of nitrogen acquisition in rice, recognizing that nitrogen deficiency compromises the realization of rice yield potential in the field more than any other single factor. We summarize rice-specific advances in four key areas of research: (1). nitrogen fixation, (2). primary nitrogen acquisition, (3). manipulations of internal nitrogen metabolism, and (4). interactions between nitrogen and photosynthesis. We develop a model for future plant breeding possibilities, pointing out the importance of coming to terms with the complex interactions among the physiological components under manipulation, in the context of ensuring proper targeting of intellectual and financial resources in this crucial area of research.