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La Trobe University Faculty of Science, Technology and Engineering Murray Darling Freshwater Research CentreMDFRC item.The ability to measure directly individual protoplast ion fluxes is a valuable addition to patch clamp and other techniques when using protoplasts to study membrane transporters. Before interpreting observations on protoplasts in terms of behaviour of intact cells and tissues, some methodological questions should be addressed. These include effects of space and time variations of transporter activities over the membrane, the osmotic dependence of specific ion transporters and the effect of the regenerating cell wall. In this study net H+ and Ca2+ fluxes were measured from individual corn (Zea mays L.) coleoptile protoplasts using a noninvasive microelectrode technique for ion flux measurements. For Ca2+, the flux distribution was almost symmetrical, ranging +/- 30 nmol.m(-2).s(-1) around zero. For H+ it was skewed towards efflux ranging from -100 to + 10 nmol.m(-2).s(-1). The distribution of H+ fluxes through the protoplast surface was a complex mosaic which changed with time, sometimes showing oscillations. These flux variations with time and position around the surface, apparently driven by endogenous mechanisms, may be relevant to protoplast pH homeostasis. When the new cell wall was partially regenerated on the next day, the correlation between H+ and Ca2+ fluxes increased. which is consistent with the weak-acid Donnan-Manning model of cell wall ion exchange.

This chapter considers Macklin’s relationship to song by considering the role that song plays in several of Macklin’s most successful theatrical productions. Beginning with the songs that Thomas Arne wrote for Macklin’s Merchant of Venice, the chapter argues that song was a powerful conduit for affect that forged complicated alliances between author, actor, character and audience. The chapter also considers the association between drinking and singing in theatrical representation. It examines the legacies of the songs that Macklin included in Love à la Mode, showing how the songs moved from the theatre to wider areas of cultural production, and suggesting that song forged in the London theatre, played an important role in the development of national song traditions.

This chapter gives a survey of Macklin's life and career, tracing his trajectory from unknown actor to one of the best-known figures in Georgian theatre. Looking at Macklin's many activities over decades of London life, the chapter argues that considering Macklin’s life as a performance of Enlightenment allows us an unusually appropriate metaphor for considering the many facets to this theatrical practitioner’s career while retaining an appropriate degree of critical scepticism as to the magnitude of his intellectual achievement. The chapter pays attention to Macklin's conflicts with others, his place within the Irish diaspora, and his associational activities. But while previous accounts have focused on Macklin's obdurate cantankerousness, this chapter places emphasis on his sociability and argues that the the Enlightenment is a commitment to enjoyment as much as to improvement. By paying attention to all of Macklin's activities, this chapter—and the volume as a whole—proposes Macklin as an exemplary if unorthodox figure of Enlightenment.

Welcome to the fifth collection by Wyvern Poets, in collaboration with the University of Dundee. 2021 marks the 250th anniversary of the birth of Sir Walter Scott (1771-1832), father of the historical novel and, effectively, of a new kind of mass ‘time travel’. Scott’s prolific output exported an image of his homeland with global appeal, if not always scrupulous authenticity. Stuart Kelly’s 2011 biography, Scott-land, is subtitled The Man Who Invented a Nation, perhaps without too much exaggeration. Scott’s antiquarian vision transformed a turbulent past into a pre-industrial landscape for the Romantic imagination, virtually overwhelming its place of origin or at least melding with it, as he rapidly became one the best-selling authors on earth. John Davidson’s ‘The Salvation of Nature’ (1891), fantasised a future Scotland bought out by an entertainment conglomerate. The World’s Pleasance Company, Ltd. demolishes anything built after 1700, ‘rewilding’ Scotland into a kind of neo-medieval theme park re-staging the past for tourists. Davidson’s story was both satirical exaggeration and backhanded tribute to Scott’s work for bringing history to life in a certain form. Hence this collection considers the many ways in which Scott’s evocative, but also problematic reimagining of his homeland remains relevant to our time and beyond.

The Keats Letters Project was conceived as an ongoing bicentennial celebration of John Keats’s correspondence. On the 200th anniversary of the original composition of each of the letters we recircu...

IN A GENERALLY FAVORABLE REVIEW OF THE 1800 EDITION OF THE LYRICAL Ballads published in The British Critic, John Stoddart took exception to the title of Wordsworth's collection for two reasons. Firstly, there were many compositions in blank verse that were not at all lyrical, and secondly, the title was a tautology: "For what Ballads are not Lyrical?" he asked. (1) Precisely what Stoddart understood by the terms "lyrical" and "ballad" is not entirely clear, but his suggestion that ballads had always been lyrical challenges the frequent supposition that in combining these terms Wordsworth and Coleridge had developed a revolutionary new form of poetic expression. Marilyn Butler, for example, wrote that "by adding the implicitly genteel 'Lyrical' to the plebeian 'Ballad,' "Wordsworth and Coleridge signaled a "change of direction" for English radical poetry. (2) As Stoddart's review implies, however, a decade after the French Revolution it was not at all self-evident that these poetic forms mapped as cleanly onto social hierarchies as Butler implies. (3) Whether the term "lyrical ballad" was a contradiction, a tautology, or an unexceptional generic description is significant given the claims frequently made for the revolutionary nature of Wordsworth's and Coleridge's collection, and its monumental position in the canon of British poetry. The nature of the lyric has been a touchstone for Romanticist scholarship since well before M. H. Abrams first described the "Greater Romantic Lyric" in 1965. (4) The ballad, however, has received a similar kind of attention only more recently but, as I hope to demonstrate, it is worth our trouble to be precise about what was understood by the term "ballad" in the period leading up to the publication of Wordsworth's and Coleridge's collection. By mapping the landscape of popular balladry in the 1790s, taking into account both radical and conservative valuations, I will show how the ballad form had become suffused with anxieties about class, literacy, piety and the interaction of orality and print. Seen in the context of ongoing controversies about the ballad, it becomes clear how careful Wordsworth is in the Preface to present his collection neither as revolutionary, nor as a withdrawal from the political domain, but as cautiously, complexly moderate--a thorny middle ground between radical zeal and sanctimonious piety. My main interest here is in Wordsworth's theory of balladry, and how the "Preface" participates in contemporary debates about the aesthetics and politics of the ballad and the stakes involved in engaging those debates. As critics from Wordsworth's contemporaries on have frequently pointed out, however, the Preface and the poems it precedes are not necessarily coextensive, and the theory is often unevenly deployed in practice. But seen in the light of the ballad debates of the 1790s, certain poems in the collection that have received scant critical attention, such as "The Two Thieves," appear newly significant for the way they engage with ballad debates. A detailed excavation of these controversies helps us understand that Stoddart's objection to the tautology of the collection's title was a calculated stance, itself an intervention into fiercely contested disputes over the meaning of the ballad form. As I will show, no one who had lived through the 1790s--not Stoddart, and certainly not Wordsworth--could have been insensible to the powerfully contested social, moral, and political valances that balladry carried during the revolutionary decade. Before exploring this terrain in more detail, however, it will be helpful to understand in broad terms what is meant by the term ballad. 1. Defining the Ballad Commonly defined as "songs sung about the streets," Anglo-American ballad scholarship recognizes, broadly speaking, two different kinds of ballad that are distinct, but connected. (5) The first are the "traditional" ballads collected by antiquaries such as Thomas Percy, Joseph Ritson, Charlotte Brooke, and Sir Walter Scott, the appeal of which was that they were ancient, a kind of poetry that tied the present to the past in ways that writers then, as now, found fascinating. …

The association of the public house with the plebeian radical movement in the 1790s has diverted attention from the function of the eighteenth-century tavern, which (unlike the alehouse) was typically a highly refined space for elegant dining. The London Tavern in Bishopsgate Street, where Richard Price and the Revolution Society gathered after hearing Price's sermon, was one of the grandest, most elegant taverns in all of Europe, providing dinners to London's wealthy commercial classes, and to East India Company employees in particular. In his writings on the French Revolution, Burke understands the tavern as the headquarters of a commercial class whose bureaucratic regime has begun to compromise English freedoms. Burke's concern about metropolitan tavern meetings is not with the threat of an incipient democratic movement prompted by the “lower orders,” but with the consequences of a public sphere dominated by wealthy merchants who patronize London's exclusive tavern societies. Burke's concerns about the...

Daniel Defoe’s The Life, Adventures, and Pyracies, of the Famous Captain Singleton (1720) features a protagonist who has no family and no home and whose existence consists of a series of journeys. In contrast to Robinson Crusoe (1719), Defoe’s later novel explores the subjectivity of an adventurer who has not issued from the “middle way” and has no centering place that provides meaning to his actions. Singleton’s lack of self-conscious reflection and his indifference to the acquisition of property provide a suggestive model of identity that operates outside familiar paradigms of possessive individualism.

This paper reviews the debate surrounding the value of specialised treatment facilities for people with seriously challenging behaviours. Distinctions are made between advantages and disadvantages resulting from specialisation, and those resulting from specalised services being unit based. Distinctions are also made between the inherent disadvantages of services and observed but remediable disadvantages arising from inadequate implementation. Results from a survey of specialised units in England and Wales are presented.

Salinity is a major abiotic stress affecting agricultural production. To understand the genetic behavior of salinity tolerance traits, a half-diallel cross was made among six barley cultivars (Hordeum vulgare L.), with contrasting levels of known tolerance, to study the combining ability of salinity tolerance on the basis of K+ loss from plant roots under saline conditions. The glasshouse pot experiments showed that the six parents were significantly different in salinity tolerance and those tolerances were highly correlated with the K+ flux measurements. The combining ability analysis showed that the variances of both general combining ability (GCA) and specific combining ability (SCA) were highly significant. Two tolerant cultivars, CM72 and Numar, showed significantly higher GCA for salinity tolerance (less K+ loss under salinity stress). Cultivars with medium GCA were YU6472 and Yan90260. Salinity tolerance was mainly controlled by additive effects with the tolerance allele showing partial dominance. High positive SCA was also found between two tolerant cultivars and between tolerant and medium-tolerant cultivars, indicating possible different tolerant genes or some minor genes in these cultivars. The combination of these genes from different sources of tolerant cultivars should produce cultivars with even greater tolerance. © Crop Science Society of America. All rights reserved.

Plants may suffer from ammonium (NH4+) toxicity when NH4+ is the sole nitrogen source. Nitrate (NO3-) is known to alleviate NH4+ toxicity, but the mechanisms are unknown. This study has evaluated possible mechanisms of NO3- alleviation of NH4+ toxicity in canola (Brassica napus L.). Dynamics of net fluxes of NH4+, H+, K+ and Ca2+ were assessed, using a non-invasive microelectrode (MIFE) technique, in plants having different NO3- supplies, after single or several subsequent increases in external NH4Cl concentration. After an increase in external NH4Cl without NO3-, NH4+ net fluxes demonstrated three distinct stages: release (tau1), return to uptake (tau2), and a decrease in uptake rate (tau3). The presence of NO3- in the bathing medium prevented the tau1 release and also resulted in slower activation of the tau3 stage. Net fluxes of Ca2+ were in the opposite direction to NH4+ net fluxes, regardless of NO3- supply. In contrast, H+ and K+ net fluxes and change in external pH were not correlated with NH4+ net fluxes. It is concluded that (i) NO3- primarily affects the NH4+ low-affinity influx system; and (ii) NH4+ transport is inversely linked to Ca2+ net flux.

Calcium can ameliorate Na+ toxicity in plants by decreasing Na+ influx through nonselective cation channels. Here, we show that elevated external [Ca2+] also inhibits Na+-induced K+ efflux through outwardly directed, K+-permeable channels. Noninvasive ion flux measuring and patch-clamp techniques were used to characterize K+ fluxes from Arabidopsis (Arabidopsis thaliana) root mature epidermis and leaf mesophyll under various Ca2+ to Na+ ratios. NaCl-induced K+ efflux was not related to the osmotic component of the salt stress, was inhibited by the K+ channel blocker TEA+, was not mediated by inwardly directed K+ channels (tested in the akt1 mutant), and resulted in a significant decrease in cytosolic K+ content. NaCl-induced K+ efflux was partially inhibited by 1 mm Ca2+ and fully prevented by 10 mm Ca2+. This ameliorative effect was at least partially attributed to a less dramatic NaCl-induced membrane depolarization under high Ca2+ conditions. Patch-clamp experiments (whole-cell mode) have demonstrated that two populations of Ca2+-sensitive K+ efflux channels exist in protoplasts isolated from the mature epidermis of Arabidopsis root and leaf mesophyll cells. The instantaneously activating K+ efflux channels showed weak voltage dependence and insensitivity to external and internal Na+. Another population of K+ efflux channels was slowly activating, steeply rectifying, and highly sensitive to Na+. K+ efflux channels in roots and leaves showed different Ca2+ and Na+ sensitivities, suggesting that these organs may employ different strategies to withstand salinity. Our results suggest an additional mechanism of Ca2+ action on salt toxicity in plants: the amelioration of K+ loss from the cell by regulating (both directly and indirectly) K+ efflux channels.

Hypoxia-induced changes in net H +, K + and O 2 fluxes across the plasma membrane (PM) of epidermal root cells were measured using the non-invasive microelectrode ion flux measurement (MIFE) system in elongation, meristem and mature root zones of two barley (Hordeum vulgare L.) varieties contrasting in their waterlogging (WL) tolerance. The ultimate goal of this study was to shed light on the mechanisms underlying effects of WL on plant nutrient acquisition and mechanisms of WL tolerance in barley. Our measurements revealed that functionally different barley root zones have rather different O 2 requirements, with the highest O 2 influx being in the elongation zone of the root at about 1 mm from the tip. Oxygen deprivation has qualitatively different effects on the activity of PM ion transporters in mature and elongation zones. In the mature zone, hypoxic treatment caused a very sharp decline in K + uptake in the WL sensitive variety Naso Nijo, but did not reduce K + influx in the WL tolerant TX9425 variety. In the elongation zone, onset of hypoxia enhanced K + uptake from roots of both cultivars. Pharmacological experiments suggested that hypoxia-induced K + flux responses are likely to be mediated by both K + -inward- (KIR) and non-selective cation channels (NSCC) in the elongation zone, while in the mature zone K + -outward- (KOR) channels are the key contributors. Overall, our results suggest that oxygen deprivation has an immediate and substantial effect on root ion flux patterns, and that this effect is different in WL-sensitive and WL-tolerant cultivars. To what extent this difference in ion flux response to hypoxia is a factor conferring WL tolerance in barley remains to be answered in future studies. © 2006 Blackwell Publishing Ltd.

Although oscillations in membrane-transport activity are ubiquitous in plants, the ionic mechanisms of ultradian oscillations in plant cells remain largely unknown, despite much phenomenological data. The physiological role of such oscillations is also the subject of much speculation. Over the last decade, much experimental evidence showing oscillations in net ion fluxes across the plasma membrane of plant cells has been accumulated using the non-invasive MIFE technique. In this study, a recently proposed feedback-controlled oscillatory model was used. The model adequately describes the observed ion flux oscillations within the minute range of periods and predicts: (i) strong dependence of the period of oscillations on the rate constants for the H 1 pump; (ii) a substantial phase shift between oscillations in net H 1 and K 1 fluxes; (iii) cessation of oscillations when H 1 pump activity is suppressed; (iv) the existence of some ‘window’ of external temperatures and ionic concentrations, where nondamped oscillations are observed: outside this range, even small changes in external parameters lead to progressive damping and aperiodic behaviour; (v) frequency encoding of environmental information by oscillatory patterns; and (vi) strong dependence of oscillatory characteristics on cell size. All these predictions were successfully confirmed by direct experimental observations, when net ion fluxes were measured from root and leaf tissues of various plant species, or from single cells. Because oscillatory behaviour is inherent in feedback control systems having phase shifts, it is argued from this model that suitable conditions will allow oscillations in any cell or tissue. The possible physiological role of such oscillations is discussed in the context of plant adaptive responses to salinity, temperature, osmotic, hypoxia, and pH stresses.

Development of salt-tolerant genotypes is central both to remediation of salinity-affected land and to meet increasing global food demand, which has been driving expansion of cropping into marginal areas. The bottleneck of any breeding programme is the lack of a reliable screening technique. This study tested the hypothesis that the ability of plants to retain K+ under saline conditions is central to their salt tolerance. Using seven barley cultivars contrasting in salt tolerance (CM72, Numar, ZUG293, ZUG95, Franklin, Gairdner, ZUG403), a comprehensive study was undertaken of whole-plant (growth rate, biomass, net CO2 assimilation, chlorophyll fluorescence, root and leaf elemental and water content) and cellular (net fluxes of H+, K+, Na+ and Ca2+) responses to various concentrations of NaCl (20–320 m m). Na+ selective microelectrodes were found to be unsuitable for screening purposes because of non-ideal selectivity of the commercially available Na+ LIX. At the same time, our results show very strong negative correlation between the magnitude of K+ efflux from the root and salt tolerance of a particular cultivar. K+ efflux from the mature root zone of intact 3-day-old seedlings following 40 min pretreatment with 80 m m NaCl was found to be a reliable screening indicator for salinity tolerance in barley. As a faster and more cost-effective alternative to microelectrode measurements, a procedure was developed enabling rapid screening of large numbers of seedlings, based on amount of K+ leaked from plant roots after exposure to NaCl.

Potassium transport was investigated in the root elongation zone of Arabidopsis seedlings during the first minutes of Al3+ exposure, using the non-invasive MIFE microelectrode technique. To prevent pH changes during Al3+ application, and to separate aluminium from acidic stress, plants were pre-treated with 5 mM homoPIPES before addition of AlCl3 (pH 4.2). The 30-min treatment with 50 or 500 μM AlCl3 led to a significant increase in K+ efflux in solutions containing 100 μM CaCl2. This efflux was suppressed by high concentrations of Ca2+ (10 mM) in the bathing solution. Our results suggest that elevated external Ca2+ activities can sustain K+ influx in the root elongation zone during Al3+ exposure either by maintaining [Ca2+]cyt or by affecting Al3+ uptake across the plasma membrane.

Photosynthetic characteristics, leaf ionic content, and net fluxes of Na(+), K(+), and Cl(-) were studied in barley (Hordeum vulgare L) plants grown hydroponically at various Na/Ca ratios. Five weeks of moderate (50 mM) or high (100 mM) NaCl stress caused a significant decline in chlorophyll content, chlorophyll fluorescence characteristics, and stomatal conductance (g(s)) in plant leaves grown at low calcium level. Supplemental Ca(2+) enabled normal photochemical efficiency of PSII (F(v)/F(m) around 0.83), restored chlorophyll content to 80-90% of control, but had a much smaller (50% of control) effect on g(s). In experiments on excised leaves, not only Ca(2+), but also other divalent cations (in particular, Ba(2+) and Mg(2+)), significantly ameliorated the otherwise toxic effect of NaCl on leaf photochemistry, thus attributing potential targets for such amelioration to leaf tissues. To study the underlying ionic mechanisms of this process, the MIFE technique was used to measure the kinetics of net Na(+), K(+), and Cl(-) fluxes from salinized barley leaf mesophyll in response to physiological concentrations of Ca(2+), Ba(2+), Mg(2+), and Zn(2+). Addition of 20 mM Na(+) as NaCl or Na(2)SO(4) to the bath caused significant uptake of Na(+) and efflux of K(+). These effects were reversed by adding 1 mM divalent cations to the bath solution, with the relative efficiency Ba(2+)Zn(2+)=Ca(2+)Mg(2+). Effect of divalent cations on Na(+) efflux was transient, while their application caused a prolonged shift towards K(+) uptake. This suggests that, in addition to their known ability to block non-selective cation channels (NSCC) responsible for Na(+) entry, divalent cations also control the activity or gating properties of K(+) transporters at the mesophyll cell plasma membrane, thereby assisting in maintaining the high K/Na ratio required for optimal leaf photosynthesis.

Both parts of the actin-myosin complex involved in cytoplasmic streaming could be regulated by mineral ions. The main goal of this study was to find a relationship between cyclosis and ion transport across the cell wall and plasma membrane. The transport of K(+) and Ca(2+) along pH bands in Chara branchlet internodal cells was characterized by using the MIFE system for non-invasive microelectrode measurement of ion fluxes. Branchlets formed acidic and alkaline bands with the pH ranging from 5 to 8. Different pH patterns were observed for different sides of the branchlets. Sides with cyclosis streaming acropetally generally showed greater variation in the profiles of pH and H(+) fluxes. Although a high correlation was not found between pH bands and Ca(2+) or K(+) fluxes, there was a positive correlation between Ca(2+) and K(+) fluxes themselves for both sides of the branchlets. Application of cytochalasin D, an inhibitor of cyclosis, had no immediate effect on pH and ion fluxes, however, the time of cyclosis cessation corresponded with a dramatic change in Ca(2+) and K(+) fluxes; pH profiles and H(+) fluxes were affected within 2 h. The evidence suggests that, in Chara branchlets, pH band formation and Gd(3+)-insensitive Ca(2+) transport systems are linked to the cyclosis machinery: (i) the pH band amplitude for the acropetally streaming side was larger than that for the basipetally streaming side; (ii) cessation of cytoplasmic streaming after cytochalasin D application resulted in changed pH banding profiles and H(+), Ca(2+) and K(+) fluxes; and (iii) the application of GdCl(3) or incubation in GdCl(3) solutions did not lead to the cessation of cytoplasmic streaming, although external Ca(2+) fluxes changed.

Changes induced by blue light (BL) in ion fluxes and pH in etiolated seedlings of dicotyledons (Cucumis sativus L., Phaseolus lunatus L., Pisum sativum L.) were studied using a non-invasive ion-selective microelectrode technique (MIFE). BL induced significant Ca2+ uptake within the first 5 min after onset in upper parts of the whole cucumber seedling. To locate the sites of the BL perception, studies on excised segments were performed. The data obtained demonstrated that the observed Ca2+ uptake is associated with the very top of the hypocotyl hook of cucumber and butter bean, and with the whole epicotyl of pea, although the response was stronger for the upper parts of the epicotyl. Addition of Gd3+ and La3+ into the bathing solution completely prevented Ca2+ influx, implicating the involvement of Ca2+ channels in the regulation of Ca2+ transport during BL exposure. BL-induced changes in H+ and Ca2+ fluxes were not likely to be linked to the one mechanism, as they were separated spatially and pharmacologically.

Introduction This paper addresses a subject which, at first sight, may not seem directly relevant to IT education. The first part of the paper, therefore, explains what we mean by empowerment and why we think it is highly relevant, worthwhile and fun! The remainder of the paper shows how students can be empowered with examples drawn from our combined teaching experiences over many years (Dawson 2000; Dawson and Newsham, 1997a & b; Dawson, Newsham and Fernley, 1997; Jackson and Dawson, 1999; Newman, Dawson and Parks 2000; Newman, 2001a & b; Newsham and Dawson, 1996, Parish and Newman, 1999). Most of our experience relates to university students undertaking programmes with a significant IT content, so this provides the main focus for the paper. However, we also have experience of teaching, and using IT to empower, University students for whom IT is not a major element of their degree and also people in industry and children in schools. This paper presents three related messages: * 'Empowerment' is as important an issue in education as acquiring 'knowledge', although less time is devoted to it in the curriculum; * IT provides an ideal environment in which to promote empowerment; * Empowering students provides a means of 'protecting' them against the rapid changes that take place in IT. Background: What is Empowerment and Why is it Important? This section explains what we mean by 'empowerment' and why we think it should be a focus for IT related teaching. We argue that empowerment is at least as important as knowledge acquisition and that IT is an ideal vehicle to empower people studying a variety of subjects at different educational levels. A dictionary definition taken from a Pocket Oxford Dictionary originally published in 1947 is: "Empower: to authorize or enable; empowerment: the act of enabling". Definitions in other dictionaries are similar and have not varied significantly over time. In our case we are enabling students to cope with the unexpected in an IT context, a skill that seems to be desirable (Dyba, 2000; Sandburg and Vinberg, 2000). The relative stability of the dictionary definition, contrasts sharply with rapidly changing experiences of IT. This creates a problem for IT teachers and is the main reason that we are championing the empowerment approach. Basically, because of the rapid changes that are taking place in all IT related subjects, we believe that the most useful attribute we can give students is the confidence to find their own solutions to any given IT problem. The pragmatic value of promoting this idea was first recognized when it was observed that students undertaking an intern year in industry in the middle of their computing degree programmes were frequently being asked to carry out tasks for which they had not specifically been educated. For example, interns were expected to be proficient in a range of programming languages other than the languages they had been taught. However, it was simply not possible to add extra teaching to cover all the programming languages and other computing topics that they might encounter in industry. Yet, despite this, we found that, according to the employers, our interns were, in the main, rising to the challenge. The employers also indicated that our students were more capable, in this respect, than those from some of the other 'computing' degree programmes. Questioning the employers and the students to try to discover the reasons for this suggested that at least part of the reason was a second year project based course (module). In this module the students were set tasks which involved them writing a program in a language they had not been taught and working as part of a group to accomplish an open ended task. In both cases they were expected to find out the information they required for themselves and were, therefore, neither surprised nor worried when they were expected to do this again in their intern year. …

This study introduced the use of a non-invasive ion-selective microelectrode (MIFE) technique to study membrane-transport processes in bacteria. Net ion fluxes and changes in the extracellular concentrations of H+, Ca2+, K+ and NH4+ in adherent bacteria, isolated from cultures at different growth stages (exponential, late exponential, and stationary phases), were monitored. With the exception of Ca2+, a significant (P=0.05) difference was found in the magnitude of net fluxes of the ions measured from bacterial cells at different stages of the population growth curve. The magnitude of the H+ response was glucose-dependent with maximum changes occurring at the highest concentration. There was a progressive increase in H+ extrusion followed by a gradual return to zero at late stationary phase. Measurements of net ion fluxes crossing the bacterial cytoplasmic membrane, demonstrated here for the first time, may offer insight into underlying mechanisms of ion transport kinetics. Applications of the non-invasive ion-selective microelectrode technique in microbiology are discussed.

Knowledge of the preferred source of N for Eucalyptus nitens will lead to improved fertiliser management practices in plantations. Ion selective microelectrodes were used non-invasively to measure simultaneously net fluxes of NH4+, NO3− and H+ along the tap root of solution-cultured E. nitens. Measurements were conducted in solutions containing 100 μm NH4NO3. The pattern of fluxes was such that there was a large influx of NH4+, a smaller influx of NO3− and large H+ efflux. The ratio of these fluxes was constant, according to the ratio 3:1:−6 (NH4+:NO3−:H+). Within the region 20–60 mm from the root apex of E. nitens seedlings there was spatial and temporal variation in fluxes but flux patterns remained constant. Root hair density did not affect fluxes nor did proximity to lateral roots. Variation was less than that found in previous studies of localised root fluxes using similar high-resolution measurement techniques. It was concluded that small-scale spatial variation in fluxes may have confounded previous studies. There were associations between fluxes of all three ions, the strongest associations being between NH4+ and H+, and NH4+ and NO3−. Overall, these results are consistent with NH4+ being the preferred source N for E. nitens.

and productivity by imposing osmotic stress on plants. To cope with the problem, plant cells must readjust their Transient kinetics of net H+ ,K + ,C a2+, and Cl− fluxes osmotic potential to prevent water losses. That can be were measured non-invasively, using an ion-selective achieved by either uptake of inorganic ions from the microelectrode technique, for bean (Vicia faba L.) leaf external solution, or by de novo synthesis of compatible mesophyll in response to 150 mM mannitol treatment. solutes (amino acids, sugars, polyoles, quaternary amines) In a parallel set of experiments, changes in the plasma acting as osmolytes ( Wyn Jones and Pritchard, 1989; membrane potential and the total proline content in Delauney and Verma, 1993; Bohnert et al., 1995; Bohnert leaves were monitored. Regardless of the ionic com- and Shen, 1999; Serrano et al., 1999). These two processes position of the bath solution, hyperosmotic stress differ significantly in their time scales. Immediate changes caused a significant increase in the K+ and Cl− uptake in ion fluxes are believed to provide quick (within a few into mesophyll cells. At the same time, no significant minutes) osmotic adjustment while a fine ‘tuning’ by proline changes were observed for at least 16 h after means of biochemical synthesis of compatible solutes has the onset of stress. Experiments with inhibitors sug- a scale of hours and days ( Wyn Jones and Pritchard, gested that potassium inward rectifier (KIR) channels, 1989; Lew, 1996). exhibiting mechanosensitive properties and acting as Recent progress in molecular genetics has made bioprimary receptors of osmotic stress, are likely to be chemical mechanisms of osmolyte biosynthesis the priminvolved. Due to the coupling by membrane potential, ary target for genetic engineering of salt and drought changes in K+ and Cl− transport may modify activity tolerance in crops (see Yeo, 1998, for a review). However, of the plasma membrane H+-pump. Such coupling may the progress is disappointingly slow, and an increase in also be responsible for the mannitol-induced oscilla- the osmotic tolerance in a field situation is only marginal tions (period of about 4 min) in net ion fluxes observed (Bohnert and Shen, 1999). It appears that membrane in 90% of plants. Calculations show that influx of K+ transport processes play a more crucial role in plant and Cl− observed in response to hyperosmotic treat- osmoregulation than was believed so far. ment may provide an adequate osmotic adjustment in There is much evidence supporting this statement. bean mesophyll, which suggests that the activity of Cerda et al. concluded that accumulation of inorganic the plasma membrane transporters for these ions ions was sufficient for osmotic adjustment in salt-stressed should be targeted to improve osmotolerance, at least maize cultivars, and that no single organic solute appeared in this crop. to be important in this process (Cerda et al., 1995). Similar conclusions were made by Huang and Redmann

Although salinity is one of the major problems limiting agricultural production around the world, the underlying mechanisms of high NaCl perception and tolerance are still poorly understood. The effects of different bathing solutions and fusicoccin (FC), a known activator of plasma membrane ATPase, on plasma membrane potential (Em) and net fluxes of Na+, K+and H+were studied in wheat suspension cells (Triticum aestivum) in response to different NaCl treatments. Emof cells in Murashige and Skoog (MS) medium was less negative than in cells exposed to a medium containing 10 mM KCl + 0.1 m M CaCl2(KSM) and to a basic salt medium (BSM), containing 1 m M KCl and 0.1 m M CaCl2. Multiphasic Na+accumulation in cells was observed, peaking at 13 min after addition of 120 m M NaCl to MS medium. This time scale was in good agreement with net Na+flux changes measured non-invasively by moving ion-selective microelectrodes (the MIFE system). When 120 m M NaCl was added to all media studied, a quick rise of Na+influx was reversed within the first 20 min. In both 120 and 20 m M NaCl treatments in MS medium, net Na+efflux was observed, indicating that active Na+transporters function in the plant cell response to salt stress. Lower external K+concentrations (KSM and BSM) and FC pre-treatment caused shifts in Na+fluxes towards net influx at 120 m M NaCl stress.

Net fluxes of H a and Ca 2a were measured in the mesophyll tissue of broad bean (Vicia faba L.) leaves and in protoplasts derived from these cells. NaCl at 90 mM enhanced H a extrusion in both protoplasts and tissue, but in diAerent ways. Proton extrusion was inhibited by vanadate, suggesting the involvement of the plasma membrane H a ATPase in cell responses to salinity. There was virtually no eAect of NaCl on the net Ca 2a flux in protoplasts, while in the tissue a large transient Ca 2a eCux followed the salt treatment. Salt-induced Ca 2a eCux was essentially independent of external Ca2a concentrations in the range 0. 1t o 10 mM. Also, Ca2a flux responses were ‘saturated’ above 50 mM NaCl. It is suggested that almost all the measured Ca2a flux originates from Naa/Ca2a and Ha/Ca2a ion exchange in the cell wall. This conclusion was supported by the results of modelling cation exchange in the cell wall. # 2000 Annals of Botany Company

Noninvasive, ion-selective vibrating microelectrodes were used to measure the kinetics of H+, Ca2+, K+, and Cl− fluxes and the changes in their concentrations caused by illumination near the mesophyll and attached epidermis of bean (Vicia fabaL.). These flux measurements were related to light-induced changes in the plasma membrane potential. The influx of Ca2+ was the main depolarizing agent in electrical responses to light in the mesophyll. Changes in the net fluxes of H+, K+, and Cl− occurred only after a significant delay of about 2 min, whereas light-stimulated influx of Ca2+ began within the time resolution of our measurements (5 s). In the absence of H+ flux, light caused an initial quick rise of external pH near the mesophyll and epidermal tissues. In the mesophyll this fast alkalinization was followed by slower, oscillatory pH changes (5–15 min); in the epidermis the external pH increased steadily and reached a plateau 3 min later. We explain the initial alkalinization of the medium as a result of CO2 uptake by photosynthesizing tissue, whereas activation of the plasma membrane H+ pump occurred 1.5 to 2 min later. The epidermal layer seems to be a substantial barrier for ion fluxes but not for CO2diffusion into the leaf.

The effects of auxin on net ion fluxes near parenchyma of oat coleoptiles were studied using the non-invasive MIFE system to measure specific ion fluxes using ion selective microelectrodes. Application of 10 μm1-naphthaleneacetic acid (NAA) for 3 h caused doubling of coleoptile segment growth, without changing the pH of the unbuffered bathing solution from pH 5.4 during that time. Short term experiments revealed that auxin led to an immediate three-fold increase of chloride influx to 1200 nmol m−2s−1, maintained for at least 1 h. In the first minutes after auxin application, proton fluxes were small (-25 nmol m−2s−1, an efflux) and tended to decrease, whereas potassium and calcium fluxes changed little, fluctuating from -100 to 0 nmol m−2s−1and from -15 to 0 nmol m−2s−1, respectively. It is suggested that one target of auxin action in plant cells is the plasma membrane chloride transport system mediating increased chloride uptake.

H + flux kinetics were measured in solution around the roots of chilling-tolerant pea (Pisum sativum) and bean (Vicia faba), chilling-sensitive cucumber (Cucumis sativus) and pumpkin (Cucurbita pepo), and intermediate corn (Zea mays) species using a microelectrode technique to measure net flux. As a root warmed to room temperature after 90 min at 4 °C, at which temperature the H + flux was near zero, the flux rose (influx) and then fell. These changes occurred at two apparent critical temperatures, which were higher for the more chilling-sensitive species. The first, lower, apparent critical temperature may represent the start of passive inward H + transport. The higher critical temperature may represent the start of active H + extrusion. From these apparent critical temperatures we have calculated the real critical temperature and the time delay of the chilling signal transduction process. Passive and active H + transporters appear to have the same real critical temperature of chilling sensitivity, about 9 °C, but have, respectively, 4 min and 11 min time delays. Measurement of these apparent critical temperatures may provide quick and reliable screening for chilling sensitivity in plant breeding programmes. Future ion flux studies may show the cellular location of chilling stress perception and the signal transduction pathways.