Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Studies on aluminum toxicity in an aluminum-sensitive cultivar of barley: impact on ion fluxes and calcium… Nichol, Brian E. 1993

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata


831-ubc_1993_spring_phd_nichol_brian.pdf [ 4.56MB ]
JSON: 831-1.0086385.json
JSON-LD: 831-1.0086385-ld.json
RDF/XML (Pretty): 831-1.0086385-rdf.xml
RDF/JSON: 831-1.0086385-rdf.json
Turtle: 831-1.0086385-turtle.txt
N-Triples: 831-1.0086385-rdf-ntriples.txt
Original Record: 831-1.0086385-source.json
Full Text

Full Text

STUDIES ON ALUMINUM TOXICITY IN AN ALUMINUM-SENSITIVE CULTIVAR OFBARLEY. IMPACT ON ION FLUXES AND CALCIUM DISTRIBUTIONbyBRIAN EDWIN NICHOLB.Sc., UNIVERSITY OF VICTORIA, VICTORIA, B.C., 1981M.Sc., QUEEN'S UNIVERSITY, KINGSTON, ONTARIO, 1986A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFDOCTOR OF PHILOSOPHYinTHE DEPARTMENT OF BOTANYWe accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIASeptember 1992© Brian E. Nichol, 1992In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature) Department of^Co-rThe University of British ColumbiaVancouver, CanadaDate /172-DE-6 (2/88)iiAbstractThe effects of aluminum (Al) on root growth and developmentin the aluminum-sensitive cultivar of barley, Hordeum vulgare Lemend. Lam. var. Kearney was studied using low (1 AM) and highconcentrations (50 AM) of Al. To analyze the impact of thealuminum treatments, changes in the anatomy, respiration, weightand growth rate of roots were analyzed. Aluminum-induced changesin cell division and cell elongation were also measured in orderto determine the importance of each one of these factors in rootgrowth inhibition. These studies were correlated withmeasurements of the root surface area to determine the impact ofaluminum treatment on apoplasmic ion absorption (i.e. potassium(K)) and distribution visualized by dispersive X-raymicroanalysis. Both levels of Al completely inhibited theinitiation of secondary roots and inhibited primary root growthbut had no effect on germination in Hordeum vulgare var Kearney.The growth rate was progressively reduced as the aluminumconcentration increased. Growth was essentially arrested after 4days treatment with 0.1 gM Al, while growth ceased after 2 daystreatment with 50 AM Al. However, respiration was found to beunaffected by treatment with 50 AM Al for 2 days. Aluminum at 50gM and 1 AM dramatically reduced cell elongation. However, thesame Al treatments appeared to stimulate cell division. Thereduction in root length was thus due to an Al-induced inhibitionof root cell elongation and not cell division. Root width wasincreased by Al treatment as was the average width of root cells.iiiAluminum dramatically reduced K in the apical region of the root(90% inhibition) as shown by energy dispersive X-raymicroanalysis and reduced the [P] concentration in the root tipby 20-30%.The effects of short (10 minutes) and long term (5 day)treatments with Al on the influx and efflux of K ( 86Rb) inHordeum vulgare var. Kearney were studied. The speed at whichinhibition occurred and the persistence of any inhibition in theabsence of aluminum was also investigated. The influx of K wasinhibited by 55% when treated for 5 days with 5 AM Al at pH 4.2.Plants treated with 50 gM Al for 30 seconds showed a significantreduction in K influx. The Al-induced inhibition of K influx wasfound to be reversible since plants treated for 10 minutes with50 AM Al returned to control influx levels when transferred toAl-free medium. The K inhibition from long term (5 days)treatment with 5 gM Al was also reversible since K influxreturned immediately to control levels when Al was not present.The half times for K efflux from plants treated with 5 AM Al for5 days was found to be similar to controls. When K influx wasmeasured with increasing potassium in solution, it was found thatK appear to competitively reduce the impact of Al.The mechanism by which aluminum interferes with ion influxis not known. In this study, the effects of aluminum on theinflux of the cations, calcium, potassium and ammonium and theanions, nitrate and phosphate was measured in an aluminumsensitive cultivar of barley. Aluminum (100 AM) was found toinhibit the influx of the cations, calcium (69%), ammonium (40%)ivand potassium (13%) while enhancing the influx of the anionsnitrate (44%) and phosphate (17%). Aluminum interfered with thebinding of the cations in the cell wall by the same order ofmagnitude as influxes were reduced, while phosphate binding wasstrongly enhanced. Aluminum was found to have a direct effect oncation transport since the treatment time of less than 20 minuteswould not probably be long enough to cause indirect effects dueto energy-related inhibition. The results could not be caused byAl-induced inhibition in proton pumping and proton motive force(pmf), since nitrate transport, which is believed to be directlycoupled to the H -1--ATPase was not inhibited by Al. These resultsare consistent with a mechanism where Al binds to plasma membranephospholipids, forming a positive charge layer influencing ionmovement to the binding sites of the transport proteins. Apositive charge layer could retard the movement of cations to theplasma membrane proportionally to the charge carried by the ionand increase the movement of anions again in proportion to thecharge they carried.Chlorotetracycline (CTC) and Fluo-3 were used to study thedistribution and concentration of membrane-bound and cytosolicfree Ca in the Al-sensitive var. Kearney of Hordeum vulgare.Both Ca-indicator dyes revealed substantial increases influorescence in both root apices and root hair cells after Al-treatment. Mn was used to quench the Fluo-3 fluorescence andensure that the signal was located in the cytosol and not thecell wall. Confocal scanning laser microscopic observations wereused to ascertain that the Fluo-3 signal was restricted to thecytoplasm and it was not the result of the compartmentalizationof the indicator within organelles, such as the vacuole.Confocal line plots were utilized to determine the relativeconcentration of Ca throughout the different regions of the rootand root hairs. The effects of Al on the viability of the cellswas monitored by differential contrast microscopy and neutral redaccumulation within the vacuoles. The results indicate that Alinterferes with both Ca distribution and concentration, with someregions of the root being particularly sensitive to its effects.Aluminum increased the CTC and Fluo-3 fluorescence in root apexcells and in the cortical cytoplasm of root hairs. This Al-induced disturbance in cellular calcium could interfere with Ca-dependent processes involved in cell elongation and division andplay a major role in the establishment of well known Al-inducedtoxicity symptoms.viTABLE OF CONTENTSPageAbstract^Table of contents^  viList of tables  viiiList of figures^  ixAcknowledgements  xiiGENERAL INTRODUCTION^  1CHAPTER 1: Effects of aluminum on root growth and development inan aluminum-sensitive cultivar of barley.INTRODUCTION^  12MATERIALS AND METHODS^  13RESULTS^  17DISCUSSION  19CHAPTER 2 The effects of short and long term aluminum treatmenton potassium influx in Hordeum vulgare  variety Kearney.INTRODUCTION^  38MATERIALS AND METHODS^  39RESULTS^  41DISCUSSION  42CHAPTER 3 The effects of aluminum on the influx of calcium,potassium, ammonium, nitrate and phosphate in an aluminum-sensitive cultivar of barley.INTRODUCTION^  55MATERIALS AND METHODS^  57RESULTS^  59DISCUSSION  60viiCHAPTER 4 The effects of aluminum on cellular calciumdistribution as monitored by Fluo-3 and Chlorotetracycline inroot hair cells and different regions of the root of an Al—sensitive variety of barley.INTRODUCTION^  67MATERIALS AND METHODS^  69RESULTS^  72DISCUSSION  76CONCLUSIONS^  96LITERATURE CITED  100viiiLIST OF TABLESPageTable 1.1. Estimates of cell numbers in barley roots treatedwith 1 and 50 AM Al for 2 days^  24Table 1.2. Effect of aluminum on the axial length and widthof root cells^  25Table 1.3. Effect of 50 gM aluminum on the root surface areaof Hordeum vulgare var. Kearney^  26Table 1.4. Measurement of respiration rates in barleyroots treated with 0 (control) and 50 gM aluminum for2 and 10 days^  27Table 2.1. Half times for the slow (vacuole), intermediate(cytoplasm) and fast (cell wall) components of K effluxfrom roots treated with or without 5 AM Al for 5 days^ 47Table 2.2. The effect of Al on the influx of K ( 86Rb) inplants grown in Al for 5 days^  48Table 2.3. The effect of brief exposure to 50 gM Al onthe influx of K ( 86Rb) in plants grown without Al^ 49Table 2.4. Parameters Km and Vmax calculated from theK isotherm in Figure 2.2^  50Table 3.1. Rates of Ca, NH4, K, NO3, and PO4 ion influx with50 and 100 gM Al^  65Table 3.2. Aluminum-induced changes in Ca, NH4, K,NO3, and PO 4 ion binding to the cell wall^ 66Table 4.1. Confocal laser scanning fluorescence intensitymeasurements of Fluo-3 fluorescence in controls andAl-treated roots^  83ixLIST OF FIGURESPageFig.1.1. Roots of barley plants treated with 0.1, 0.5, 1.0and 50 gM Al for 5 days^  29Fig.1.2. Root growth in control and Al-treated roots(0.1, 0.5, 1.0 and 50 AM) over 7 days^  31Fig.1.3. SEM image and energy dispersive X-ray maps ofAl, K, P, and background counts in control root tip^ 33Fig.1.4. SEM image and energy dispersive X-ray maps ofAl, K, P, and background counts in 1 AM Al treatedroot tip^  35Fig.1.5. SEM image and energy dispersive X-ray maps ofAl, K, P, and background counts in 50 AM Altreated root tip^  37Fig.2.1. Linear regression on semi-log plot of K-ion (86Rb)tracer content vs time in roots of barley, var. Kearney(+Al), after elution to unlabeled solution^ 52Fig.2.2. K-ion ( 86Rb) influx from labelled solutionscontaining K-ion concentrations from 10 to 500 AMwith and without 50 gM Al. Also Lineweaver-Burke plotof the data^  54Fig.4.1. Neutral red staining of vacuoles in root hairs(control root) ^  85Fig.4.2. Neutral red staining of vacuoles in root hairs(Al treated material) ^  85xFig.4.3. Differential contrast observations of the generalstructure of root hairs (control root)^ 85Fig.4.4. Differential contrast observations of the generalstructure of Al treated root hairs^  85Fig.4.5. CTC staining of the root apex in control root^ 87Fig.4.6. CTC staining of the root apex of an Al treated root 87Fig.4.7. OTC staining of the root apex in control root  87Fig.4.8. OTC staining of the root apex of an Al treated root 87Fig.4.9. Fluo-3 staining of root apex (control root)  87Fig.4.10. Fluo-3 staining of the root apex of an Altreated root^  87Fig.4.11. Longitudinal confocal line plots of Fluo-3fluorescence (control root) ^  89Fig.4.12. Longitudinal confocal line plot of Fluo-3fluorescence (Al treated root) ^  89Fig.4.13. Transverse confocal line plots of Fluo-3fluorescence through the elongation zone(control root) ^  89Fig.4.14 Transverse confocal line plot of Fluo-3fluorescence through the elongation zone (Al-treatedroot) ^  89Fig.4.15. Confocal observations of Fluo-3 fluorescence inroot cap cells (control root) ^  91Fig.4.16. Confocal observations of Fluo-3 fluorescence inAl treated root cap cells^  91Fig.4.17. Confocal observations of Fluo-3 fluorescence inmeristematic cells (control root) ^  91xiFig.4.18. Confocal observations of the Fluo-3 fluorescencein Al treated meristematic cells^  91Fig.4.19. Epifluorescence observations of control roothair cells stained with CTC^  93Fig.4.20. Epifluorescence observations of Al-treated roothair cell stained with CTC^  93Fig.4.21. Epifluorescence observations of control roothair cell stained with OTC^  93Fig.4.22. Epifluorescence observations of Al-treated roothair cell stained with OTC^  93Fig.4.23. Epifluorescence observations of control roothair cell stained with Fluo-3^  93Fig.4.24. Epifluorescence observations of Al-treatedroot hair cell stained with Fluo-3^  93Fig.4.25. Epifluorescence observations of control roothair cell stained with Fluo-3 to study the effect offluorescence quenching by Mn2+^  93Fig.4.26. Epifluorescence observations of root haircell treated with 0.1 mM Mn2+ and stained with Fluo-3. 93Fig.4.27. Confocal observations of fluorescence in controlroot hair stained with Fluo-3^  95Fig.4.28. Confocal observations of fluorescence inAl treated root hair stained with Fluo-3^  95Fig.4.29. Confocal line plot of control root hairstained with Fluo-3^  95Fig.4.30. Confocal line plot through Al treated cellstained with Fluo-3^  95AcknowledgementsI would like to thank my supervisor, Dr Luis Oliveira forall of the help he has given me, both academic and financial,throughout this thesis. I would also like to thank Dr. Glass,Dr. Siddiqi, Micheal Weiss, Dr. Lacy Samuels, Dr. Harold Wegerand Dr. Jolliffe for the technical assistance they gave me duringthe research. Special thanks go to my running buddies, Dr.Robert DeWreede, Lacy Samuels and Libby John who helped to keepme calmer than expected during these stressful times. Thanksalso to the fellow graduate students in my lab. Laurie Tornbom,Lucy Peat and James Bailey for the support and discussions overthe last few years.xi i1General IntroductionAluminum (Al) is the most abundant metal in the earth'scrust (Haug 1985). Aluminum has been implicated as the probablecause of growth inhibition in plants as early as 1918. Hartwelland Pember (1918) suggested that Al is responsible for the root-growth retardation seen in barley and rye plants grown in acidsoils. Now, aluminum toxicity is recognized as a severe problemlimiting global crop productivity because significant areas ofthe world, especially in the subtropic and topic zones, inwestern Europe and in the eastern United States, suffer from soilacidity which results in the mobilization of soil aluminum (Haug1985). It is estimated that 40% of the world's that is undercultivation and as much as 70% of the potential arable land areacidic (Haug 1984). The subsoil is often acidic which limitsroot growth and therefore will render the plants susceptible todrought as they will have reduced access to water and subsoilnutrients (Foy et al. 1978).It is not clear, due to the complexities of Al chemistry andsoil composition, at what pH Al becomes soluble in soils (Haugand Caldwell, 1985). However, plants do not usually display Altoxicity symptoms at pH values above 5, which indicates thatsoluble Al is probably present at toxic levels only below thisvalue. The speciation of Al changes dramatically between pH 3and 7 (Macdonald and Martin 1988, Parker et al. 1989). Aluminumis predominantly in the insoluble Al(OH)4 - form at pH 7 while atpH 4 Al is predominantly in the soluble A1 3+ form (Macdonald and2Martin 1988). The mole fraction of free A1 3+ relative to thetotal Al ion species increases from less than 10 -6 at pH 7 to 1at pH 4. Within this range, small changes in pH can dramaticallychange the A1 31- activity. There has been considerablespeculation in the literature as to the identity of Al toxicspecies (Kinraide 1988). Mononuclear hydroxyl-Al species such asA1(OH) 2+ , Al(OH)2 + and A1(OH)4 - which are in equilibrium withA13+ , most likely are not toxic (Parker et al. 1988). Oneunstable polynuclear hydroxy-Al species is more toxic than A1 3+ ,but it is not known whether it is present in soils. It is likelythat A13+ then is the only important rhizotoxic species and Altoxicity might simply be the result of a general phenomenon ofrhizotoxicity caused by polyvalent cations (Clarkson 1965, Parkeret al. 1988).Root systems of trees grown in soils subjected toacidification showed dramatic changes in morphology, whichincreased with soil depth (Hutterman 1985). Development of fineroots occurred in the top soil with its higher organic content,while at soil depths below 20 cm, there was no fine rootdevelopment. No loss of fine root development was seen atequivalent soil depths at a higher pH. When acid-grown fineroots were examined, necrosis was found in the endodermis leadingin extreme cases to complete detachment of the central cylinderfrom the root cortex. These symptoms could only be reproduced inthe laboratory when spruce seedlings were grown under hydroponicconditions in the presence of Al and the pH of the nutrientsolution was below 4.2 (Hutterman 1985). In general, Al-injuredroot tips and lateral roots were short, thickened and brownish in3colour (Foy et al. 1978).Aluminum impairs numerous root processes. For example, ithas been found to inhibit the net uptake of potassium (Clarksonand Sanderson 1971, Miyasaka et al. 1989, Pettersson and Strid1989, Nichol et al. 1991) and calcium ions (Johnson and Jackson1964, Lance and Pearson 1969, Clarkson and Sanderson 1971,Godbold 1991, Huang et al. 1992a,b). It has also been found toinhibit cell division (Horst et al. 1983, Clarkson 1965,Morimura et al. 1978) and cell elongation (Matsumoto et al.1977). As yet, it is unclear which, if any of these, areresponsible for the observed Al toxicity symptoms.The development of aluminum tolerant crops could savebillions of dollars in purchases of lime now used to raise thesoil pH (Haug 1985). Also crop yields could be dramaticallyincreased by the development of such plants. Understanding themolecular mechanisms of metal tolerance and the processes bywhich aluminum results in the disruption of cellular functionswould greatly assist the development of aluminum-tolerantcultivars of major crops (Haug 1985). Aluminum tolerance hasbeen an extensively studied topic in recent years (Taylor 1988).Many plants have natural Al tolerance and often closely relatedcultivars of the same species can differ considerably in their Altolerance. For example, cultivars of wheat show a nine-folddifference in growth under conditions of Al stress (Taylor andFoy 1985a,b). Differential tolerance to Al was also found to alesser extent in barley (Hordeum vulgare) (Foy et al. 1987).Many mechanisms have been proposed to account for this observedaluminum tolerance. These proposed mechanisms include: 1) Al4exclusion from cells, 2) chelation of Al within the symplast and3) that transport proteins of tolerant plants might by moreresistant to Al inhibition.In wheat and barley, Foy et al., (1965) found that Alconcentrations were higher in sensitive varieties than intolerant varieties, while Niedziela and Aniol (1983) foundsimilar Al concentrations in sensitive and tolerant varieties ofwheat. No explanation has been given for the contradiction.Foy et al., (1965) have proposed that tolerant varietiesincreased the pH around the root zone which reduces thesolubility and availability of aluminum around the root. Taylorand Foy (1985a) suggested that this pH change was due todifferential preference for NH4+ and NO3+ as nitrogen source.Al-tolerant cultivars would have a preference for NO3 + over NH4 +resulting in a higher solution pH than Al-sensitive cultivars.Subsequent experiments supported these trends (Taylor and Foy1985b). However, later experiments did not support thisinterpretation since the apparent aluminum tolerance of asensitive and tolerant cultivar of wheat was not influenced bythe NO3 -/N ratio or the rhizophere pH (Taylor 1987). Thepossibility was raised that the pH change associated with Alsensitivity and tolerance might have been simply a consequence ofdifferential necrosis in the roots (Foy et al. 1978).Other mechanisms of Al exclusion from the cell have beenproposed. For instance, the Al-absorptive ability of the cellwalls might differ in sensitive and resistant varieties. Theextent of methylation was suggested as a possible variable in thecell wall's ability to bind Al although no interaction between Al5and uronic acids was found (Matsumoto et al. 1977).The plasmamembrane was thought to be less permeable to Alions in tolerant than sensitive cultivars of wheat (Foy et al.1978). I was found that a 200 times higher Al concentration wasrequired for Al to cross the plasmamembrane of meristematic rootcells of the Al sensitive cultivar "Breyer" than the equivalentcells of the tolerant cultivar "Atlas 66". It was concluded thatmolecular differences in the plasmamembrane were responsible forthe differential Al tolerance which reduced the permeability ofthe plasmamembrane of the tolerant cultivar to Al ions. Moore(1974) suggested that changes in the membrane proteins, undergenetic control of a dominant allele in tolerant wheat cultivars,might reduce the membrane permeability to Al.The chelation of Al by organic acids is also thought to playan important role in tolerance by preventing Al ions frominteracting with and disrupting enzymes and/or internal membranesand other cellular structures. Some plants, such as tea,accumulate extremely high concentrations of Al, up to 16000 ppm(Haug and Caldwell 1985). Since these plants contain highconcentrations of aluminum-chelating compounds such as organicacids, these chelates might have a role in protecting the cellsfrom Al injury. Al-tolerant cultivars of pea and barley alsocontain elevated levels of citric acid compared to Al-sensitivevarieties of the same species (Haug and Caldwell 1985). Foy etal. (1987), found reduced concentrations of citric and succinicacids in the roots of the Al-sensitive barley cultivar "Kearney",while normal to higher levels of these organic acids weremaintained in the tolerant cultivar "Dayton" when stressed by Al.6Malate and to a lesser extent trans-aconitic acid were also foundto have quite strong affinities for Al. An Al-tolerant varietyof corn maintained higher levels of malic acid and trans-aconiticacid when stressed with 150 gM Al at pH 5 than did a sensitivevariety and it was proposed that the maintenance of higherorganic acid concentrations reduced the toxicity of Al in thetolerant variety (Suhayda and Haug 1986).Al tolerance in certain pea cultivars was found to beassociated with higher phosphate concentrations in the roots (Foyet al. 1978), while in snapbean this has been linked to theability to resist an Al-induced reduction in Ca transport ortotal cellular Ca (Foy et al. 1972). Ca levels were found to behigher in Al tolerant cultivars than in sensitive cultivars.Thus Al reduction of P and Ca concentrations in the cytoplasm ofsensitive varieties may be one of the causes of their lowertolerance.Aluminum tolerance might be related to ability to maintainhigher net uptake of important ions in the presence of aluminum.Miyasaka et al. (1989) have found that a tolerant cultivar ofwheat was able to maintain normal net K uptake and proton effluxwhile a sensitive cultivar was not.Taylor and his research group have investigated therelationship between aluminum tolerance and the uptake of Al intocells. As discussed above (Foy et al. 1965), tolerant plants areoften found to take up less Al than sensitive plants. Thepossibility that Al-tolerance involves the restriction of Aluptake has been studied in some detail. It was found that thedifferences in growth of sensitive and tolerant cultivars of7wheat under Al stress conditions were not reflected bydifferences in Al uptake or distribution at the whole plant ortissue level (Zhang and Taylor 1988). However, Zhang and Taylor(1990) have shown that the kinetics of Al uptake into Al tolerantexcised roots consists of a rapidly-exchanging saturable phase ofuptake (30 min.) superimposed over a continuous slowly-exchanginglinear phase. In tolerant cultivars, the linear phase uptakeincreased when roots were treated with the metabolic inhibitorDNP. This DNP-induced stimulation of Al uptake was absent in anAl sensitive cultivar. These results suggest the possibleoperation of a metabolism-dependent exclusion mechanism in Al-tolerant cultures. It was not clear whether this metabolism-dependent exclusion resulted from reduced net uptake into thesymplast and resided at the plasma membrane or whether thisactive immobilization of Al resides in the cell wall.Aluminum has also known to be toxic in animal cells. It hasbeen implicated in diseases such as Alzheimer's disease, Guamianamyotrophic lateral sclerosis, Parkinson's dementia (Perl et al.1982, Spencer et al. 1987) and dialysis encephalopathy (Arieff1985). Aluminum has been found to be present in differentregions of the brain in Alzheimer's disease victims at a muchhigher concentration than in normal individuals (Ross 1987). Ifthe brains of certain animals are injected with aluminum salts,progressive dementia results. In addition, aluminum has beenimplicated in the dementia that occurs in patients receiving longterm serum hemodialysis. Patients with kidney failure have up toseven times as much aluminum associated with their brain tissue.Residents of the island of Guam, the Kii Peninsula of Japan and8the Irian area of New Guinea have high levels of aluminum in thesoil and a high incidence of Guam Parkinsonism dementia caused byneuron degeneration in either the cerebral cortex and brain stemor Guam Amyotrophic lateral sclerosis resulting from degenerationof motor neurons. The two conditions are similar to Alzheimer'sdisease in that neurofibrillary tangles, which are abnormalneurofibres concentrated in the cytoplasm of nerve cells andhaving the appearance of a tangle, are found in all threediseases (Ross 1987). There has also been a suggestion that lowcalcium in the diet might contribute to these diseases. In theabsence of calcium, aluminum might bind to the calcium receptorsin the calcium transport system and subsequently travel to thebrain. Rats fed with a diet deficient in calcium and magnesiumshowed a considerable increase in brain aluminum (Ross 1987).In addition, aluminum was found to affect neurocognativeability (Bolla et al. 1992). Higher Al levels resulted indiminished visual memory. Patients with lower vocabulary scoresshowed a decline in attention/concentration, frontal lobefunctions, and on several neurocognitive measures. In contrast,those with higher vocabulary scores showed no Al-related decline.The authors suggested that individuals with lower verbalintelligence may possess less well-developed compensatorystrategies to overcome the Al-induced neurocognitive effects.Aluminum is often found in elevated concentrations in freshwater aquatic systems due to the increases in mobilization oftrace metals including aluminum due to acidic precipitation(LaZerte 1986). This was often found to result in aluminumtoxicity in a variety of fresh water aquatic taxa (Campbell and9Stokes 1985). Aluminum (at concentrations found in lake water)was found to reduce the mean length, total cell area and volumeup to 50% in the freshwater acidophilic diatom Asterionella ralfsii at pH 6 (Gensemer 1990). Ecologically relevantconcentrations of aluminum reduced the growth rate of the greenalgae Monoraphidium dybowskii (aluminum-tolerant) andStichococcus sp. (aluminum-sensitive)(Claesson and Tornqvist1988). Changes in ultrastructure induced by Al treatment werealso studied in these algae (Tornqvist 1989). The two alga wereaffected by Al in different ways. The plasmalemma was detachedfrom the cell wall in Monoraphidium cells but not in Stichococcuscells. Also in Monoraphidium cells, the cell wall was brokendown or not synthesized and were highly vacuolized. Aluminumalso reduced the growth of the acid-tolerant green alga Chlorellasaccarphila (Folsom et al. 1986) Protection from aluminumtoxicity in this alga was found to be provided by calcium,magnesium, sodium and potassium. In addition, aluminum was foundto have a significant negative effect on fish (Driscoll et al.1980) and amphibians (Clark and LaZerte 1985).The plant material used in this study was an Al-sensitivevariety of barley, Hordeum vulgare L. emend. Lam. var. Kearney.It was chosen because barley has been extensively used in iontransport research (Siddiqi pers. comm.) and root growth is verysensitive to low concentrations of Al. Many of the previousstudies on Al-toxicity utilized Al concentrations in themillimolar range (Clarkson 1965, Sampson et al. 1965, Morimura etal. 1978). This is ten times higher than the concentration usedin the present studies and well above those found in acid soils10(Haug and Caldwell 1985). In the present studies, the maximumconcentration of Al employed was 100 AM and in most of theresearch, growth solutions containing no more than 50 AM Al wereused. This was done to approximate as close as possible thegrowth conditions observed in acid soils.The major objectives of these studies were to assess theeffects of Al on the growth of both entire root system and rootcells, and the uptake and distribution of some of the ionsconsidered to be physiologically important. In chapter 1, theeffects of Al on root growth are determined. Previous studies inthe literature mostly described root inhibition only in generaldescriptive terms. This study examines this phenomenon ingreater detail by measuring cell size (to estimate rootelongation) and cell numbers (to estimate cell division). Theeffects of Al on the elemental content of the root is alsoinvestigated. This data was then be compared with the ion fluxdata in chapters 2 and 3 to ascertain whether changes in ionfluxes of potassium correlate with changes in [K] in the root.In chapters 2 and 3, the effects of Al on ion influx wereexamined. Influx was chosen as the major parameter for thesestudies because the literature suggested Al had a greater impacton influx than efflux [preliminary experiments with potassium andcalcium fluxes confirmed this (data not shown)]. Chapter 2 alsosets out to document the effects of Al on potassium influx andwhether its interaction with Al is competitive, noncompetitive oruncompetitive in nature. Such information could suggest apossible mechanism by which aluminum interacts with cationinflux. The literature also suggested that Al affected cations11more than anions (Clarkson 1966, Clarkson and Sanderson 1971, Lee1971, Clark 1977, Pettersson and Strid 1989, Nichol et al. 1991).To clarify whether this interpretation is correct, Chapter 3examined the interactions between Al and both cations and anions,to determine if a possible mechanism by where by Al interactswith ion transport processes could be elucidated. Particularattention was given to calcium, with the distribution, cytosolicconcentration (chapter 4) and transport of this ion (chapter 3)considered in detail. The reason(s) for the calcium studiesarise from the growing evidence indicative of its potential inameliorating Al-induced toxicity symptoms (Taylor 1988).Furthermore, the recent discovery of Al-interference with thecalcium regulatory protein calmodulin (Haug and Caldwell 1985)raises the possibility of Al-toxicity resulting from interferencewith cellular processes regulated by this calcium-dependentprotein. Also recent work has clearly indicated that in the rootapex, which appears to be the critical site for Al toxicity, Alspecifically blocks Ca 2+ influx rapidly and reversibly (Huang etal. 1992a,b, Ryan et al. 1992a,b). This was found to becorrelated to Al tolerance. Transport of other important ions(K+ , H+ , C1 -) were not affected rapidly.The results obtained from these different studies arecompared to see if they support any of the proposed mechanismsdescribed in the literature or if they suggest a new mechanism(s)of Al toxicity.12CHAPTER 1Effects of aluminum on root growth and development in analuminum-sensitive cultivar of barleyIntroductionThe most notable effect of aluminum (Al, predominantly inthe form of Al 3+) toxicity on roots is the inhibition of rootelongation. This was considered to be mainly due to theinhibition of cell division in cowpea (Horst et al. 1983) andonion plants (Clarkson 1965, Morimura et al. 1978). However,the effects of Al upon the cells of the zone of elongation ofplant root tips was never adequately studied. Matsumoto et al.(1977) reported inhibition of cell elongation by Al, but not inany detail.In a recent study using Fluo-3, it was demonstrated that Alsignificantly increased the cytosolic calcium (Ca, in the form ofCa2+) fluorescence of the cells in the zone of elongation of anAl-sensitive variety of barley. These observations suggested thepossibility that Al disturbed Ca homeostasis in these cells andas a result a number of cellular processes responsible for cellexpansion might have been perturbed (Nichol and Oliveira,unpublished). For example, the stability of microtubules whichare thought to participate in cell wall synthesis by acting astemplates for cellulose microfibril deposition (Hepler 1985) andexocytosis of cell wall materials might have been disturbed(Steer 1988). Other studies have also addressed the importance13of Ca on the regulation of the osmotic balance of plant cells(Okazaki and Tazawa 1990, Okazaki and Iwasaki 1991), a primaryfactor in the regulation of cell expansion. In this study we aimat determining to what extent cell division and inhibition ofcell elongation are contributing factors responsible foraluminum-induced root growth inhibition in the Al-sensitive var.Kearney of Hordeum vulqare (barley). Previous studies on Al-induced inhibition of cell division (Clarkson 1965, Sampson etal. 1965, Morimura et al. 1978), utilized high concentrations ofAl (millimolar range). In this study, the levels of Al employedare within the range found in agricultural soils (Haug andCaldwell 1985). Of interest also is the potential impact ofgrowth inhibition on the reduction of ion uptake by calculatingits effect on the root surface area with aluminum treatment andwhether aluminum-induced changes in ion distribution (K, P),studied by X-ray microanalysis, are correlated with theinhibition of root growth.Materials and MethodsPlant Materials Seeds of Hordeum vulgare L. emend. Lam. var. Kearney, analuminum-sensitive cultivar, were surface-sterilized for 10 minin 1% NaOC1 and rinsed 5 times in distilled water. Inexperiments to determine aluminum-induced inhibition of celldivision, cell elongation and root growth rates, seeds weregerminated by being soaked in aerated distilled water for 24 h.The seeds were subsequently placed onto plastic mesh supported by14plexiglas rings so that the emerging roots grew downward, incontact with the nutrient medium in 3.7 liter hydroponic pots.The aerated hydroponic solution contained (in MM), 127 Ca, 27 Mg,82 K, 32 NH4, 370 NO3, 13 PO4, 13 SO4, 1.8 FeEDTA, 3.6 Na, 1 Cl,1.3 B03, 0.5 Mn, 0.1 Zn, 0.03 Cu and 0.02 Mo. Seeds weregerminated in the absence of Al. To assess the effects of Al ongermination, seeds were germinated in aerated distilled watercontaining 1 or 50 AM Al adjusted to pH 4 with HC1. The numberof seedlings that germinated from 100 seeds was counted in thetwo treatments to assess the germination rate. Only sampleswhere more than 70% of the seeds germinated under controlconditions were utilized for these and other studies.In experiments designed to determine aluminum-inducedreduction of root surface area as well as its effect on rootrespiration, the seeds were soaked in aerated distilled water for4 h then spread onto plastic mesh supported by plexiglas ringswhich were firmly bedded into moist sand, covered with aluminumfoil and allowed to germinate in the dark for 3 days at 20 °C.The seedlings were then washed to remove adhering sand particlesand placed in 3.7 liter hydroponic pots containing aeratednutrient solution for an additional period of 5 days.Measurements of cell division, cell elongation and root growthRoot growth rates were estimated from daily measurements ofthe length of the roots during 6 days from the time ofgermination. Each plant was briefly removed from the plasticmesh support and placed on a ruler for this purpose.To assess the effects of Al on cell division and cellelongation, the roots of control and Al-treated seedlings,15exposed to 1 and 50 gM Al were fixed in 3% glutaraldehyde/0.3 Mpipes buffer (pH 7.2) then dehydrated with an ethanol series (70%to 90%) for 30 minutes during each step. The roots weresubsequently embedded in JB-4 plastic (JB EM services inc.) usingstandard methods (Botti and Vasil 1984). Longitudinal sections(2 gm in thickness) were cut through the central part of the rootand subsequently photographed after staining with 1% toluidineblue. To examine the effects of Al on cell division, the totalcell number in control and Al-treated roots was calculated byestimating the average cell volume, then dividing this figureinto the total root volume. Root volume was estimated, byassuming the root to be a cylinder, from measurements of the rootradius and length. The total cell number was also calculated bycounting the number of cells in medium longitudinal rootsections, estimating the volume of that cell layer, thenextrapolation to the total root volume. The effects of Al onroot cell elongation was assessed by measuring the length andwidth of root cells in the meristematic and elongation zones.Absolute measurements were obtained by photographing a micrometerscale then using this as a ruler by superimposing it onmicrographs of the root sections.Root (primary plus secondary roots) surface areameasurements were calculated from photographs of JB-4 sections.Root surface area, excluding root hairs, was calculated frommeasurements of root length and width assuming the root to be acylinder. To this value, the surface area of root hairs wasadded. To calculate the last value, the surface area of theaverage root hair (mm 2 ) was estimated from the average root hair16length and width (again assuming a root hair to approximate acylinder). The root hair surface area per root system was thenobtained by estimating the number of root hairs by sampling 5zones of 1 mm 2 , and multiplying this number by the average roothair surface area. The total root surface area per root systemwas then the sum of the root surface plus root hair surface area.The effects of Al on root respiration were evaluated usingplants treated with 50 AM Al for 2 and 10 days. Root tips ofapproximately 5 mm length were placed in a 800 Al reaction vesselfilled with oxygen saturated growth medium at 20 °C and fittedwith an oxygen electrode (Hansatech, King's Lynn, U.K.). Oxygenconsumption was recorded until a steady value was obtained(approx. 5 minutes) and this rate was considered to be therespiration rate.Energy dispersive X-ray microanalysis was conducted onfreeze-dryed material. Roots mounted on aluminum stubs wereplunged into liquid nitrogen and faced off on a cryomicrotome(Reichert-Jung Ultracut E) so that the root was cut in halflongitudinally exposing an internal root surface. The roots werethen freeze-dryed at -80 0C for 8 hours, mounted on carbon stubsand a layer of carbon evaporated (Balzers 080T vacuum evaporator)over the specimens prior to analysis with a scanning electronmicroscope (Cambridge Instruments, SEM 250), operating at 20 kV,and equipped with X-ray (Link AN 10000) and image analysis(Kontron Image Processing System) instrumentation.Semiquantitative values were estimates of the relativeintensities (density of dots) from X-ray maps.17ResultsGeneral effect of aluminum on root growth and anatomyAluminum (A1)(50 AM) had no effect on the rate of seedgermination, while both 1 and 50 pM Al completely inhibitedsecondary root growth (not shown). Inhibition of root growthresulted in very short, thickened roots (Fig. 1.1). Theinhibition was concentration-dependent, with larger effectsobserved at higher concentrations (Fig. 1.2). Root growth ceasedafter 4 to 5 days of treatment with 0.1 pM Al, and after 2 dayswith 50 pM Al. Root weight was reduced more (72%) than shootweight (23%) by Al treatment compared to controls (data notshown).Aluminum-induced changes in the number of cells and in theirlength and width Table 1.1 summarizes estimates of the total number of cellsobtained from sections of control and Al treated roots. Thenumber of cells in the whole root was not reduced with Altreatment. In fact with 1 and 50 pM Al treatments, these numbersincreased. This increase in cell numbers was apparent whethercell volume or cell count estimates were employed. Changes incell length and width in the root meristem and elongation zoneswith 1 pM Al are illustrated in Table 1.2. Al reduced celllength by approximately 40% in both the elongation andmeristematic zones. Cell width remained unchanged in themeristematic zone and increased in the elongation zone.18The effect of Al on root surface area. Table 1.3 shows that 50 AM Al reduced the total root surfacearea by approximately 59%. Root hairs accounted forapproximately 10% of the total root surface area in controls.Aluminum reduced the root surface area by approximately 55% whenthe root hairs were excluded from the calculations.Aluminum-induced effect on root respiration Root respiration was unaffected by treatment with 50 AM Alfor 2 days, and even after 10 days of treatment, it was inhibitedby only 25% (Table 1.4).Effects of Al on ion distribution as determined by dispersive X-ray microanalysis Figures 1.3, 1.4 and 1.5 show SEM images and energydispersive X-ray maps of Al, potassium (K) and phosphorus (P) incontrol, 1 gM and 50 AM Al treated roots, respectively.Semiquantitative estimates show that aluminum (1 and 50 AM)reduced the K concentration in the meristematic and elongationzones by approximately 90 % (Figs. 1.4c and 1.5c) compared to thecontrol (Fig. 1.3c). The level of K in the root cap of thecontrol was less than 50% of that in the meristematic andelongation zones (Fig. 1.3c), while in Al (both 1 and 50 AM)treated roots K was absent from the root cap (Figs. 1.4c and1.5c). The distribution of K in the control root was homogeneousthroughout the meristem and elongation zones (Fig 1.3c), andremained unchanged with both Al treatments (Figs. 1.4c and 1.5c).The phosphorus concentration (semiquantitative estimate) declinedby 30 to 40% with both levels of Al treatment (Figs. 1.4d and1.5d) compared to the control (Fig. 1.3d). Aluminum associated19with the root tissue increased as the concentration of Al in thetreatment solution increased (Fig 1.3b, Fig 1.4b and Fig 1.5b).The aluminum associated with roots treated with 50 pM Al wassignificantly greater than that with 1 pM treatment (Fig. 1.5band 1.4b), with the highest concentrations being present in theepidermal cells (Fig. 1.5b).DiscussionPrevious studies of the effects of aluminum (Al) on rootgrowth utilized concentrations in the millimolar range (Clarkson1965, Sampson et al. 1965, Morimura et al. 1978). In this studywe used concentrations of Al that are found in agricultural soils(Haug and Caldwell, 1985). Aluminum (Al) treatment (50 gM) for 2days had no effect on root respiration (Table 1.4). Thissuggested that the fundamental energy metabolism of the root wasunaffected after two days of exposure to toxic levels ofaluminum. Root growth was dramatically affected by this level ofaluminum after 2 days (Fig 1.2), indicating that growthinhibition was not due to a decline in the energy supply. Thisis supported by the fact that even after 10 days of treatmentwith 50 pM Al, respiration was reduced by only 25%.One of the most striking effects of aluminum toxicity is theshortening and thickening of roots (Foy et al. 1978). This hasbeen explained as the result of an Al-induced inhibition of bothcell division and elongation (Taylor 1988). Aluminum (1 gM)reduced cell elongation but not cell division, since the numberof cells in Al treated roots was actually higher than in20controls. The reduction in cell length is particularly apparentin the meristematic and elongation zones (Table 1.2) and it canalmost entirely account for the shortening of the roots seen withAl treatment in the present study. Typically, Al-treated rootsin these experiments are 45% of the control length, while thelength of root cells is 57-63% of that of control cells (Table1.2).Aluminum treated roots were always approximately 30% widerthan controls. Coincidentally, cells in the elongation zone ofaluminum treated roots were 18% wider than control cells (Table1.2). This increase in cell width, in conjunction with a largernumber of cells, seems to account for most of the increase inroot width.It is clear from these observations that the parameter mostaffected by Al treatment is cell growth. Instead of the cellsgrowing predominantly in the direction of the root axis, theyfail to elongate correctly and grow inappropriately in width.Calcium (Ca) is known to be an important factor in the regulationof many cellular processes involved in cell growth andelongation. These include cell wall synthesis (Hepler 1985),exocytosis of cell wall materials (Steer 1988) and regulation ofthe osmotic balance of plant cells (Okazaki and Tazawa 1990,Okazaki and Iwasaki 1991). Aluminum is known to interfere withCa homeostasis (Chapter 4) and could, through the disruption ofsome or all of these factors, be responsible for the changesobserved in the cell growth pattern.Cell division was not inhibited by the concentrations ofaluminum used in this study (50 gM or less). In fact cell21division appeared to increase with both 1 and 50 AM Al treatments(Table 1.2). This result is in contrast with those reported byother authors showing cell division inhibition to be one of theconsistent effects of Al treatment (Sampson et al. 1965, Clarkson1965, Morimura et al. 1978). The discrepancy between their andour observations could result from the fact that they utilize intheir study much higher concentrations of Al. This indicatesthat Al most likely only inhibits cell division in barley at highconcentrations, above those typically found in most soils (Haugand Caldwell 1985). In contrast, cell elongation was stronglyinhibited at lower Al concentrations. Probably then under fieldconditions, the Al-induced root shortening is more likely due toan inhibition of cell elongation than division in most species.However, more studies are required in this area before moreprecise conclusions can be reached.Al reduces root surface area by at least 50% (Table 1.3) andtherefore has the potential to reduce the uptake of ions such aspotassium (K). X-ray analysis showed that 1 and 50 AM Al,reduced K in the root meristematic and elongation zones (Figs.1.3 to 1.5). The location of this K could be apoplastic orintracellular, as this analysis did not discriminate between thetwo localizations. Since cells accumulate substantial amounts ofK, loss of cellular potassium may affect cell turgor which is thedriving force behind cell and root elongations (Glass 1989). Thereduction of K in cells undergoing elongation could then be amajor contributing factor of growth inhibition seen in Al-treatedplants. The reduction of K in Al treated roots suggests that Alinhibited the uptake of K. This would be another factor, in22addition to the reduction in root surface area, that would limitthe potential of Al-treated roots to take up K. Ion influxexperiments (Chapter 3) demonstrated that K influx was reduced byAl, which is consistent with the X-ray analysis results.Aluminum was found in high concentrations in the epidermal cells.It is likely then that Al-impairment of cellular functions ismore severe in cells of the epidermis than those of the cortexand vascular cylinder. This might be particularly deleterious tothe root since the epidermis is important in the uptake ofnutrients from the soil (Glass 1989), and it is also consistentwith an observation of the occurrence of severe Al-effects on theuptake and distribution of K. Miyasaka et al. (1991) found thatthere was a net efflux of K from the root apex of wheat rootswhile there was a net influx in the rest of the root. If this isalso the case in barley, if would be difficult to explain thereduction of root tip K as an inhibition of K uptake.Aluminum showed a lesser effect on the concentration ofphosphorus (P)(semiquantitative estimate). Phosphorus reductionwas less than K, and no effect on its distribution was apparent.Most of this P was probably intracellular with little associatedwith the apoplast since the concentration of phosphate in thegrowth medium was low (13 gM) and the cell wall has a poor anionbinding capacity since its components carry mostly negativelycharged carboxyl groups (Noggle and Fritz, 1983). This isinconsistent with observations (Chapter 3) showing that Al doesnot reduce phosphate influx over a short treatment period (10minutes). Therefore, the reduction in P seen in the root apex isnot likely due to a reduction in uptake, but to other unknown23factor(s). One possibility might be that Al causes a change inmembrane permeability resulting in an increased phosphate efflux.Aluminum induced potassium efflux from intact roots of Trifoliumrepens when grown without ammonium (Lee et al., 1984) whilepotassium efflux in Agrostis capillaris was increased when grownin low phosphate (McCain and Davies, 1984). However, potassium(Chapter 2) and calcium (not shown) efflux in this study wasunaffected by Al.24Table 1.1. Estimates of cell numbers in barley roots treated with1 and 50 gM Al for 2 days. Values represent duplicateexperiments + SEM (except where stated). Values in bracketsare % increase over the control. Note: experiments A and Bwere conducted over different time scales; hence cellnumbers in experiment B are greater than in experiment A.Experiment^ Al(1M) 0^ 1^503930+48 1^5144+11341 (31)4291 2 7230 2 (68)B^7165+423 1^13390+3031(87)*1 Calculated from average cell size extrapolated to the wholeroot volume.2 Calculated by counting the number of cells in the whole rootsection then extrapolating to the whole root. One valuecalculated only.*significantly  different at the 95% confidence level.25Table 1.2. Effect of aluminum on the axial length and width ofroot cells. Values are expressed in gm and representaverages of 25 cells + SEM.Region of the rootTreatment^first lmm at apex^elongation zonelength^width^length^widthControl 84.7+5.7 32.6+1.8 154.1+8.1 28.8+1.51 ihM Al 48.1+4.8 * 32.2+2.6 97.8+5.8 * 33.9+2.2 **significantly  different at the 95% confidence level.26Table 1.3. Effect of 50 gM aluminum on the root surface area ofHordeum vulgare var. Kearney. Values are expressed in mm 2and represent the average of 5 calculations + SEM. Valuesin brackets are % reduction compared to the control.surface areaTreatment Excluding^ Includingroot hairs root hairsControl^992 ± 90^ 1190 ± 10550 pM Al^448 ± 17 (55%) *^486 ± 18 (59%) ** represents significantly different at the 95% confidence level.27Table 1.4. Measurement of respiration rates (gmols 02. g(drywt.) -1 . hr-1 ) in barley roots treated with 0 (Control) and50 gM aluminum for 2 and 10 days. Values represent averagesof three measurements ± SEM.Treatment Days2 10Control 252 ± 9 154 ± 1050 gM Al 260 ± 13 115 + 5 **significantly different at the 95% confidence level.28Figure 1.1. Roots of barley plants treated with 0.1, 0.5, 1.0 and50 pM Al for 5 days.29Control 0.1 AtM Al 0 . 5 AM Al 1 AM Al 50 4M Al30Figure 1.2. Root growth of control and Al-treated roots (0.1,0.5, 1.0 and 50 gM) over 7 days. SE is the StandardError of the Mean.1.^3.^4.^7DAYSLegendII SO AM Al1 AM Al0.5 AM Al0.1 AM AlControl3132Figure 1.3. SEM image (a) and X-ray maps of Al (b), K (c), P (d)and background (e) in control root3 334Figure 1.4. SEM image and X-ray maps of Al, K, P and backgroundin a root treated with 1 AM Al.3 536Figure 1.5. SEM image and X-ray maps of Al, K, P and backgroundin a root treated with 50 AM Al.3 738CHAPTER 2The effects of short and long term aluminum treatmenton potassium fluxes in Hordeum vulgare variety Kearneycultivar of barleyIntroductionAluminum (Al) inhibits the net uptake of calcium, magnesiumand nitrogen (Huang et al. 1992a,b, Godbold 1991, Gomes et al.1985). Phosphate uptake, in contrast, was found to be unaffectedby Al treatment (Clarkson 1966). There are conflicting reportsas to the effects of prolonged exposure to Al on the rates of netuptake and accumulation of potassium (K).^Mugwira et al. (1980)found increased K concentrations in roots and shoots of sixcultivars of wheat, while Foy and Fleming (1982) found that Kdeclined in the shoots of an Al-sensitive cultivar of wheat.Clarkson and Sanderson (1971) demonstrated that in barley, netuptake of K ( 86Rb) over 24 hours was severely inhibited (by 83%at pH 4.2) in plants grown in the presence of 100 gM Al. Inaddition to studies looking at net K uptake and status, K fluxeshave also been studied in wheat. Pettersson and Strid (1989)reported an inhibition of influx as a result of pretreatment with50 gM Al. In this study, however, the inhibition of K influx wasevident only after 2 days exposure to Al. Maximum inhibition (by40%) required at least 9 days pretreatment with Al. It isnoteworthy that in this study of Pettersson and Strid (1989), Aldid not affect K efflux. In contrast, Miyasaka et al. (1989)found that K influx in the Al-sensitive wheat cultivar Scout was39rapidly and dramatically inhibited by Al.The major objective of this study was to investigate theeffect of Al on K fluxes in the Al-sensitive cultivar of barley,Hordeum vulgare L. var. Kearney. Miyasaka et al.(1989), havedemonstrated that Al-tolerance in wheat is associated with theability to maintain normal K net uptake and proton efflux.Aluminum toxicity and tolerance might then be partially relatedto the inhibition of ion fluxes by Al. Therefore, this studyexamines the effects of low concentrations of Al on K influx andefflux after prolonged treatment (5 days), and K influx overshort periods of exposure (minutes). It also studies thepersistence of these effects in the absence of Al. Finally, itconsiders whether Al-induced inhibition of K fluxes iscompetitive or non-competitive in nature.Materials and MethodsSeeds of Hordeum vulgare L. emend. Lam. var. Kearney weresurface-sterilized for 10 minutes in 1% NaOC1, rinsed 5 times indistilled water and then soaked in aerated distilled water for 3-5 hours. The seeds were then spread onto plastic mesh supportedby plastic rings which were firmly bedded into moist sand,covered with aluminum foil and allowed to germinate in the darkfor 3 days at 20 °C. The seedlings were then washed to removesurplus sand and placed in 3.7 liter hydroponic pots. Theaerated hydroponic solution contained (in MM), 127 Ca, 27 Mg, 82K, 32 NH4, 370 NO3, 13 PO4, 13 SO4, 1.8 FeEDTA, 3.6 Na, 1 Cl, 1.3B03, 0.5 Mn, 0.1 Zn, 0.03 Cu and 0.02 Mo. The plants were grown40for 5 days prior to use in experiments.In K influx and efflux experiments, the uptake solutionswere labelled with 86Rb (180 MBq 1-1 ). In influx experiments,plants were preincubated in fresh unlabeled growth medium asdescribed above for 15 minutes. The pH of the solutions used inthis and subsequent experiments was 4.2 (adjusted by adding HC1).The plants were then placed in 86Rb labelled growth medium withor without Al2(SO4)3 at concentrations shown in the text, for 10minutes. This was followed by a 5 minute wash in unlabeledsolution. The roots were excised, weighed and ashed at 400 0Covernight. Distilled water (10 ml) was then added before theradioactivity was measured by Cerenkov counting in a SearleIsocap 300 liquid scintillation counter. The influx rate wasestimated using the equation: counts / (specific activity ( 86Rb).root weight).In experiments where the potassium influx was measured withvariable potassium ion concentrations, the 86Rb/K ratio was keptconstant so that the label present varied between 30 and 1500 MBq1-1 .Efflux was measured using 5 day old plants which were loadedin a solution containing 86Rb (180 MBq 1 -1 ) for 3 days. Rootswere then allowed to exchange K ( 86Rb) into unlabeled washsolutions (growth medium) and these solutions were collectedafter 1,2,3,4,5,7.5,10,15,20,30,45,60,90 and 120 minutes, thenafter every hour up to a total of 7 hours. The counts in thewash solutions were measured as above. The kinetics of the K +efflux were analyzed with a program written in BASIC for a HP87microcomputer (Rygiewicz et al. 1984). All experiments were41repeated at least once.ResultsLong term Al treatment (5 days) Efflux curves are shown in Figure 2.1 and indicate that Alhad a minimal effect on the half-time for K efflux for the slow,intermediate and rapidly exchanging components (thought toapproximately represent the fluxes from the vacuole, cytoplasmand the cell wall respectively) in Al treated and control roots(Table 2.1).The influx of K ( 86Rb) was increasingly inhibited by higherAl concentrations (Table 2.2). The K influx was reduced by 68%when treated with 10 gM Al for 5 days, but by only 17% whentreated with 1 pM Al. The K content of roots treated for 5 dayswith 5 pM Al was reduced 30% compared to control plants (data notshown).When plants which were grown in 5 pM Al for 5 days weretransferred to Al-free growth medium, the rate of K ( 86Rb) influximmediately returned to control levels (plants transferreddirectly from Al growth medium to the labelled influx mediumwithout Al). The influx values were 3.03 + 0.1 SE for thecontrol and 2.91 + 0.14 SE for the Al-pretreated plants (notsignificantly different at the 95% confidence level).42Short term Al treatment (from 30 sec up to 10 min) Al induced K ( 86Rb) influx inhibition was very rapid.Influx was reduced by 28% within 30 seconds by 50 pM Al (Table2.3). The inhibition did not persist in the absence of Al. Kinflux was the same as the controls when roots were pretreatedwith 50 pM Al and then placed in Al free medium during the 10minute 86Rb influx period. The influxes for Al-pretreated andthe control plants were 4.18 + 0.12 and 4.27 + 0.17 respectively(not significantly different at the 95% confidence level).The effects of Al (50 AM) upon kinetic parameters (V max andKm) for K ( 86Rb) influx were also determined (Fig 2.2a and b,Table 2.4). The presence of Al in the uptake medium increasedthe Km (although not statistically significant) for K influx buthad no effect on Vmax (Table 2.4). The Lineweaver-Burke plot(Fig 2.2b) shows that well defined regression lines describe thedata for both control and Al treated plants. The regressionlines converge at the y-axis (Fig 2.2b) which might beinterpreted as competitive kinetics.DiscussionThis study measured the effect of A1 3+ on ion influx overthe whole root. The influencing of fluxes over most of the rootsurface by A1 34- probably are unimportant in the development ofAl 3+-toxicity symptoms such as root growth. Ryan et al. (Inpress), showed that only the meristem is sensitive to Al 3+ . AlsoMiyasaka et al. (1991) found that there was a net K + efflux and+ .H influx at the root apex (first 1 cm) while in the rest of the43root, these fluxes were reversed. Thus the fluxes might not beconsistent or even in the same direction over the whole root. Inthis study, the K influx measured was probably the averagebetween the K influx in the mature root minus the K efflux at theroot apex. The Al-induced inhibition of K influx described herethen probably do not contribute to Al toxicity but still servesas a useful model to assess the effects of Al on ion fluxes.The half-times for potassium ( 86Rb) efflux were unaffectedby aluminum (Al) treatment (Table 2.1). Pettersson and Strid(1989) found that Al had no effect on the efflux of K from wheatroots. Other researchers have found that Al increased K effluxonly when the growth medium did not contain ammonium (Lee et al.1984) or the growth medium contained low concentrations ofphosphate (McCain and Davies 1984). The latter also found that Kefflux was reduced by Al when the same plants were grown in highphosphate media. It appears then that K efflux is affected onlyin some species and that low concentrations of Al in barley hadno effect on K efflux when grown under the conditions used inthis study.Potassium ( 86Rb) influx was reduced by Al concentrations aslow as 2 AM in plants treated for 5 days (Table 2.2). These Alconcentrations are much lower than those used by Clarkson andSanderson (1971, 100 AM) and Pettersson and Strid (1989, 50 AM).The inhibition of K influx by 50 AM Al was instantaneous. Theinflux rate was reduced by 28% within thirty seconds of Altreatment. Miyasaka et al. (1989) also found rapid inhibition ofK influx in wheat. This is in total contrast with the results ofPettersson and Strid (1989), who found that the reduction in K44influx by Al (50 gM) was affected only after 2 days ofpretreatment with Al. If Al-tolerance is related to the plants'ability to resist influx inhibition by Al (Miyasaka et al. 1989),then our results suggest that the barley cultivar Kearney is muchmore sensitive to Al than either of the two wheat cultivarstested by Pettersson and Strid (1989).The data in Table 2.3 suggests that the influx is higherwhen the influx period is short. The influx when plants weretreated with Al for only 30 seconds was over 8 pmol g -1 hr-1compared to 4.39 pmol g-1 hr-1 when the treatment lasted for 10minutes. This is probably due to a small component of the 86Rbcounts being bound to cell wall sites and not located inside thesymplast (Siddiqi et al. 1989). The roots were washed in 86Rbfree medium for 5 minutes (approximately 3 times the cell wallhalf time) which removed almost 90% of the cell wall bound 86Rb.Since determination of specific uptake rates (pmol g-1 hr-1 )involve multiplying by 120, 60 or 6 (for 30 sec, 1 min and 10 minuptake periods, respectively) error associated with remainingfree space would progressively overestimate influx withdecreasing uptake periods.A detailed kinetic analysis revealed that Al affected theinhibition of K influx by causing a decrease in the affinity ofthe K transporter for the K. Al had little effect on V max for Kinflux (Fig 2.2, Table 2.4)(see also Pettersson and Strid 1989).This finding might have practical implications. The adverseeffects of Al in acid soils, particularly on the uptake andaccumulation of K may be related in part to the soil K level andmay be perhaps minimized by correcting the soil K status.45The Lineweaver-Burke plot of these data suggests that Alcompetitively inhibits K influx. Baliger et al. (1979) haveshown that rubidium (Rb) and cesium (Cs) compete for access tothe K transporter with K. However, these ions are chemicallyvery similar to potassium (all carry a single positive charge,are similar in size and hydrated radius), whereas the Al-ion isnot at all like the K-ion since it carries three positive chargesinstead of one and is of a different size. How then could theAl-ion interact competitively with K-ions? Haug (1984), hassuggested that Al-ions can induce changes in the conformation ofmembrane proteins which might influence ion-protein interactions,such as the binding of K-ions to the K transporter. At presentone can only speculate regarding the mechanism of inhibition atthe molecular level. If Al could bind progressively to membraneproteins (perhaps displacing Ca in the process) such astransporter proteins, it is possible that transporter functioncould be gradually impeded. This could in turn result in theloss of binding affinity to K-ions, hence resembling competitiveinhibition (See also discussion in Chapter 3). Also, Al couldreduce the negative surface charge on the plasmalemma, whichwould reduce the concentration of K attracted to this negativesurface. Aluminum then could inhibit K influx by decreasing theK concentration at the transport site (Kinraide et al. 1992).When plants were transferred from both long and short termtreatment with Al to Al free medium, K influx returned to controlrates immediately. This suggests that no permanent change hasoccurred in the K transporter. It also indicates that the maintarget of Al-induced inhibition of the K transporter is limitedto its extracellular domains.4647Table 2.1. Half-times for efflux for the slow (vacuole),intermediate (cytoplasm) and rapid (cell wall) components ofefflux from roots treated with or without 5 gM Al for 5days. Each value is the mean of 2 experiments ± SE. Halftimes for Al treated roots were not significantly differentfrom the corresponding control (95% confidence level).t1/2 slow t1/2 intermediate ti/2 rapid-Al^21 + 2.0 hr^31 + 15.0 min^2.1 + 0.1 min+Al^21 + 0.4 hr^32 + 11.0 min^1.8 + 0.3 min48Table 2.2. The effect of Al on the influx of K ( 86Rb) inplants grown in Al for 5 days (influx measured in thepresence of the corresponding Al concentration). The influxwas measured over a 10 minute period in growth mediumlabelled with 86Rb at pH 4.2. Each value is the mean of 3replicates^SE. *^.^.significantly different from thecontrol (0.001 < P < 0.05). [K] = 82 gM.[Al](ihm)influx,^1 hr-1gmol K( 86Rb)(g FWroot) -0 4.55 ± 0.051 3.78 + 0.372 3.27 + 0.11 *5 2.69 + 0.09 *10 1.47 + 0.05 *49Table 2.3. The effect of a brief treatment with 50 AM Al on theinflux of K (86R„0) in plants grown without Al (Al was onlypresent during the specified influx period). Each value isthe mean of 3 replicates ± SE. * significantly differentfrom the corresponding control (0.001 < P < 0.05).influx period (min)^-Al^+Al0.5 8.05 ± 0.36 5.76 + 0.22 *1.0 6.05 ± 0.27 5.27 + 0.07 *10.0 4.39 ± 0.28 3.23 + 0.18 *50Table 2.4. Parameters Km and Vmax calculated from K isotherms(Figure 2). The Km from the Al treated curve was found tobe not significantly different at the 95% confidence level(0.05 < P < 0.1).Km ( µM)^Vmax (AM gFW-1 hr-1 )control 60.5 + 2.5 9.8 + 1.3+Al 106.0 + 14.5 9.8 ± 1.351Figure 2.1. Linear regression on semi-log plot of K (86Rb)tracer content vs time in roots of barley, var. Kearney,after elution to unlabeled solution. Total efflux from thewhole root in the absence (a) and presence (b) of 5 AM Al.Insets show the relationship after the slowest exchangingphase has been subtracted.LOG REMAINING COUNTS/g57)^P-P,^cn0 0 0 00LOG REMAINING COUNTS /gLOG REMAINING COUNTS/gFigure 2.2. a) K ( 86Rb) influx from labelled solutionscontaining K concentrations from 10 to 500 pMwith and without 50 pM Al. b) Lineweaver-Burke plotof the data shown in "a".53•08-7-6-0o•00 Control• Al0 Control• Aloi-., 1.0Y6 0.8E....__. 0.6> 1.2-",1..__ca)540 50 100 150 200 250 300 350 400 450 500 550 600[K] (NM)b)0.00^0.02^0.04^0.06^0.08^0.101/[11 (PM)55CHAPTER 3The effects of aluminum on the influx of calcium, potassium,ammonium, nitrate and phosphate in an aluminum-sensitive cultivarof barleyIntroductionAluminum (Al) has been found to inhibit the net uptake ofcalcium, magnesium and nitrogen into plant roots while phosphateappeared to be unaffected (Huang et al. 1992a,b, Godbold 1991,Gomes et al. 1985, Clarkson 1966). Aluminum was found toinhibited calcium (Ca) uptake very strongly and to a greaterextent than other ions (Huang et al. 1992a,b). In contrast theuptake of phosphate into roots was found to remain unaffected byAl treatment (Clarkson, 1966). There are conflicting reports asto the effects of Al on the net uptake of potassium ions.Mugwira et al. (1980) found increased potassium concentrations inroots and shoots of six cultivars of wheat treated with 231 gMAl. Clarkson and Sanderson (1971) reported severe inhibition(83% at pH 4.2) of net potassium ( 8612b) uptake in barley plantsgrown in the presence of 100 gM Al. More direct measurements ofthe effects of Al on potassium transport revealed that inhibitoryeffects on net uptake were the result of inhibition of potassiuminflux rather than changes in efflux (Pettersson and Strid, 1989;Nichol et al., 1991). Long-term studies of the effects of Al onnitrate and ammonium uptake have shown that nitrate uptake was56inhibited (Fleming, 1983; Jarvis and Hatch, 1986), while ammoniumuptake remained unaffected (Fleming, 1983).The mechanism(s) whereby Al inhibits the transport of theabove ions is not known. Three mechanisms however are likely:1) Al 3+ binds to the phospholipid heads of the cell plasmamembrane, alters the lipid-protein interaction and modifies theactivity of the transporter (Suhayda and Haug, 1986), 2) A1 3+binds directly to the transport proteins, impairing theirfunction (Schroeder 1988) and 3) Al 3+ reduces the negative chargeassociated with the plasma membrane phospholipids and proteins bybinding to these charged groups or shielding the surfacepotential (Kinraide et al. 1992).Most of the studies on the effects of Al on mineralnutrition have been long-term studies, lasting for a period of 1or more days (Taylor, 1988). In the present study, in order toensure that the inhibition of ion influx was the result of theimpairment of membrane ion transporters rather than due toimpairment of the root function through general Al-inducedtoxicity, plants were grown in the absence of Al and measurementsof the effects of Al were carried out over a time period oftwenty minutes or less. These conditions allow us:(1) to examine the effects of aluminum on short-term cation(calcium, potassium, ammonium) and anion (nitrate, phosphate)influx, in order to reveal any possible patterns ofdiscrimination between cations and anions transport and(2) to critically evaluate the proposed basis of Al inhibition ofion uptake.57Materials and MethodsSeeds of Hordeum vulgare L. emend. Lam. var. Kearney, analuminum-sensitive cultivar, were surface-sterilized for 10 minin 1% NaOC1, rinsed 5 times in distilled water and then soaked inaerated distilled water for 3-5 h. The seeds were spread ontoplastic mesh supported by plexiglas rings which were firmlybedded into moist sand, covered with aluminum foil and allowed togerminate in the dark for 3 days at 20 °C. The seedlings werethen washed to remove adhering sand particles and placed in 3.7liter hydroponic pots. The aerated hydroponic solution contained(in AM), 127 Ca, 27 Mg, 82 K, 32 NH4, 370 NO3, 13 PO4, 13 SO4,1.8 FeEDTA, 3.6 Na, 1 Cl, 1.3 B03, 0.5 Mn, 0.1 Zn, 0.03 Cu and0.02 Mo. The plants were grown for 5 days prior to use inexperiments.In ion influx experiments, the uptake solutions were labeledwith 45Ca, 8612b, 13NH4, 13NO3 or 32PO4 (approx. 200 MBq. 1 -1 ).Plants were preincubated in fresh unlabeled growth medium asdescribed above for 15 minutes. The pH of the solutions used inthis and subsequent experiments was adjusted to 4.0 with HC1.The plants were then placed in isotope-labelled growth mediumwith or without Al2(SO4)3 for 5 min at the concentrations shownin the text. 45Ca influx, was carried out over a 20 min period.This was followed by a 5 min wash in unlabeled solution, exceptin the case of 45Ca experiments where the wash period was 30minutes.In 45Ca experiments, possible error in the flux measurementsdue to incomplete desorption of Ca from the cell wall was58compensated for by treating some plant roots at 70 °C for 15minutes, before handling them as described above. This hightemperature pretreatment destroyed the root cell membranes andthus counts represented only cell wall binding. These countswere subtracted from the counts in the live experimental roots togive 45Ca plasma membrane influx.The binding of the isotopes to the root cell wall was alsomeasured for all of the above ions. Roots were grown as aboveand were placed in growth medium at 70 °C for 15 minutes todestroy the root cell membranes. The roots were then placed inthe appropriate isotopes as described above for 5 minutes. Aftera 15 second rinse to remove surface-bound isotope, the cell wall-bound isotope was measured as described above.The roots were excised, weighed and ashed at 400 °C for 16-20hours in all but the 13N experiments. Distilled water (10 ml)was added to the ashed samples in 86Rb experiments before theradioactivity was measured by Cerenkov counting in a Beckman LS60000C liquid scintillation counter. In 45Ca and 32 Pexperiments, the ashed roots were dissolved in 1 ml of distilledwater to which 9 ml scintillation cocktail was added (Ecolume,ICN Biomedicals, Inc.) before counting took place (Beckman LS60001C liquid scintillation counter). In 13N experiments, rootradioactivity was counted in fresh plant material with a gammacounter programmed to correct for isotope decay (Minaxi 5000series gamma counter, Packard). All experiments were carried outin duplicate.59ResultsTable 3.1 shows the effects of aluminum (Al) on the influxof calcium, ammonium, potassium, nitrate and phosphate. Aluminuminhibited the influx of calcium, potassium ( 86Rb) and ammoniumbut enhanced the influx of nitrate and phosphate ions. Among thecations, calcium influx was the most strongly inhibited by Al.The inhibition increased from 59% to 69% as the Al concentrationincreased from 50 to 100 M. Ammonium influx was only slightlyinhibited (10%) at 50 AM Al and strongly inhibited (40%) at 100AM. Potassium ( 86Rb) was inhibited to an extent which wascomparable to the effect on ammonium at 50 gM Al (12%) but failedto show any further inhibition at 100 pM Al (13%). In contrast,the influxes of nitrate and phosphate were stimulated by Al. Theinflux of nitrate increased by 16% with 50 pM Al and furtherincreased to 44% when treated with 100 pM Al. The increase inphosphate influx remained the same (approximately 20% higher thanthe control) with both 50 pM and 100 pM Al.Table 3.2 shows the effect of aluminum on the binding ofcalcium, ammonium, potassium, nitrate and phosphate to cell wallpreparations. Cell wall-bound calcium, ammonium, potassium andnitrate declined with Al treatments while phosphate bindingincreased. 100 pM Al reduced the binding of calcium, ammoniumpotassium and nitrate to the cell wall by 70%, 25% 4% and 13%respectively. Cell wall-bound phosphate increased by 1487% with100 pM Al treatment.60DiscussionThe toxic aluminum species is generally thought to be Al 3+since there is no evidence that other species are toxic (Kinraideet al. 1992). Therefore only A1 3+ will be considered to be toxicin this discussion. The primary factors responsible for observedaluminum toxicity symptoms in plant roots are not well understood(Taylor 1988, Kochian et al. 1991, Ryan et al. 1992a). Recentreports have suggested that Al 3+-induced inhibitions of ionfluxes, particularly calcium (Ca 2+ ), could play an important rolein mechanisms of Al 3+-toxicity in higher plants (Huang et al.1992a,b). Nevertheless the mechanism(s) by which Al influencesion fluxes are not known (Huang et al. 1992b). In the presentstudy we report that A1 34- inhibits divalent cation influx morethan those of monovalent cations. In contrast, anion influx wasstimulated. Possible models which might explain the differentialeffects on cations and anions are that A1 3+ binds to thenegatively charged phospholipids and/or acidic amino acidresidues of proteins in the plasma membrane or shields thesecharges in a manner similar to that described by Kinraide et al.(1992). Both binding and shielding would reduce theelectrostatic attraction of cations and repulsion of anions,reducing the cation activity and increasing the anion activityclose to the membrane. In turn this could inhibit the influx ofcations and stimulate the influx of anions. Kinraide et al.(1992) has reported that cations ameliorate A1 3+ toxicity in the2-F3+> C > cfollowing order, H+ approx.= c i+.It was suggestedthat the amelioration was due to these cations binding to or61screening the negative charges on the plasma membrane surface,thus reducing the activity of A1 31- close to the cell membranesurface.It has been suggested that Al could influence ion transportby displacing ions from the cell wall (Godbold 1992). Howeverthere is no evidence that binding to the cell wall is a necessaryprerequisite for transport into the cell. Maize roots andprotoplasts from these roots were shown to have identicalkinetics (Kochian and Lucas 1983). Rengel and Elliott (1992a)have also shown that a cell wall does not need to be present todemonstrate Al3+ effects on ion fluxes since A1 31- inhibits netuptake of Ca2+ into protoplasts of Amaranthus. A1 3+ , however,could act directly on the transport protein. A1 3+ has been shownto block K+ channels in higher plants (Schroeder 1988). Theliterature suggests that A1 3+ inhibits the net uptake of Ca 2+more than any other flux (Huang et al. 1992a), and thisinhibition of Ca2+ was suggested to be due to the blocking ofCa2+ channels (Huang et al 1992b, Rengel and Elliott 1992b).A13+ appeared to compete directly with Ca2+ at the Ca2+ channels(Haung et al. 1992b). It seems unlikely that the resultspresented here solely result from the direct binding of A1 31- totransport proteins since this would be expected to inhibit anionfluxes as well as cation influxes. Nevertheless, since thetransport proteins for different ions are quite different innature (Ca2+ influx via a passive channel compared to K + influxvia an active transporter), Al might directly inhibit the Ca 2+and K+ transport proteins while having only a small or negligibleeffect on the anion transporters. The results reported here then62could possibly represent a mixture of a reduction in the negativesurface charge and direct inhibition of certain transportproteins, likely only the Ca 2+ and K+ transporters to anysignificant extent.This study measured the effect of A13+ on ion influx overthe whole root. However, it has recently been proposed that thetoxic effects of A1 31- are highly localized. Ryan et al. (1992b),showed that only the meristem was sensitive to A1 3+ . AlsoMiyasaka et al. (1991) found that there was a net e efflux and+ •H influx at the root apex (first 1 cm) while in the rest of theroot, these fluxes were reversed. Thus, fluxes measured over theentire root may not represent directions or the extent of fluxesin specific regions of the root. There is growing evidence thatthe inhibition of Ca 2+ fluxes at the root apex might be animportant factor in the development of Al 3+ toxicity in higherplants (Huang et al. 1992a). It was found that net Ca 2+ influxoccurs over the whole root although the net influx was muchgreater at the root apex. In this study, Ca 2+ influx wasinhibited more strongly than other cations and within 20 minutesof Al 3+ treatment. This is before growth inhibition is observed,normally within 1 to 3 hours of A1 3+ treatment (Ryan et al.1992a) and could be a major cause of the growth inhibion seen.Although only a small component of this Ca 2+ flux is in the tipregion, which must be exposed to A1 3+ in order to induce rootgrowth inhibition (Ryan et al. 1992b), the overall flux and roottip flux appear to be net influxes so these results do supportthe idea that Ca2+ flux inhibition could result in the observed3+ •Al -induced growth inhibition. In the cultivar of barley used63in this study treated with 50 AM Al 3+ , root growth inhibition isdue to inhibition of cell elongation and not cell division(Nichol and Oliveira in preparation). Cells in the elongationzone not only were reduced in length but appeared to increaseslightly in width, suggesting a disorientation of the cellelongation process. Ca2+ is known to be a regulator ofmicrotubule assembly (Snyder and McIntosh 1976, Kirschner 1978)and exocytosis (Steer 1988), processes important in cellelongation. It is then possible that an A1 3+-induced charge incell Ca2+ homeostasis could disrupt cell and root elongation andthe A1 3+-sensitive root tip region.A13+ was found to influence binding of ions to the cell wallroughly in the same order as it affected the influx of these ions(nitrate being an exception). It should be noted that thesemeasurements were made by treating roots at 70 ° C in growthmedium. This treatment was thought to destroy the cell membranesand provide an estimate of cell wall binding. However, thistreatment may have exposed previously inaccessible intracellularmembrane sites. The values reported may therefore represent anoverestimate of the binding expected for the cell wall alone, andcould explain the dramatic stimulation of phosphate bindingstimulated by Al3+ , although this could also be due toprecipitation with A1 3+ (Clarkson, 1966).In conclusion, A1 3+ inhibited cation influx and stimulatedanion influx probably by reducing the surface negetive charge atthe plasma membrane, with a possible additional inhibition due todirect inhibition of the cation transport proteins. The stronginhibition of Ca2+ influx could disturb cellular Ca 2+ homeostasisin the root tip cells and contribute to the A1 3+-inducedinhibition of root elongation observed.6465Table 3.1. Rates of ion influx with 50 and 100 AM Al. Percentageinhibition (in brackets) or percentage enhancement("+" in brackets) are shown. * represent significantlydifferent at the 95% confidence level.Ion gmol. g(fresh) =1^hr=1Control 50 AM Al 100 gM AlCa2+ 0.162 + 0.027 0.067 * + 0.011(59) 0.050* + 0.010(69)NH4+ 3.27 + 0.28 2.94 * ± 0.09(10) 1.95 * ± 0.19(40)K+ 4.78 + 0.133 4.18 * + 0.224(12) 4.16 * ± 0.263(13)NO3 4.77 + 0.057 5.53 + 0.088^(+16) 6.85 * ± 0.030(+44)PO4 3- 0.754 ± 0.041 0.931 * + 0.054^(+23) 0.883 ± 0.095(+17)66Table 3.2. Aluminum-induced changes in ion binding to the cellwall. The numbers in brackets represent percentagereductions or enhancement ("+") of binding. * Representssignificantly different at the 95% confidence level.Ion gmol. g(fresh)=1Control 100 gM AlCa2+ 2.176 ± 0.11 0.658 * ± 0.04(70)NH4+ 0.736 + 0.03 0.551 * + 0.01(25)K+ 0.254 + 0.02 0.240 + 0.01^(4)NO3 0.219 + 0.02 0.191 + 0.01^(13)PO4 3 0.564 + 0.03 8.95 * + 0.25^(+1487)67CHAPTER 4The effects of aluminum on cellular calcium distribution asmonitored by Fluo-3 and Chlorotetracycline in root hair cells anddifferent regions of the roots of Hordeum vulgare variety KearneyIntroductionCalcium deficiency and aluminum (Al, predominantly A1 31- )toxicity have often been found to produce similar symptoms (Foyet al. 1969, Edwards et al. 1976). Aluminum has been found toinhibit the net uptake of calcium (Ca, in the form Ca2+) ions(Johnson and Jackson 1964, Lance and Pearson 1969, Clarkson andSanderson 1971, Godbold 1991, Huang et al. 1992a,b). Aluminumwas shown also to interfere with the transport of Ca to theshoots in several plants (Taylor 1988).Recently, it was suggested that some of these Al-inducedeffects might be due to Al-induced changes in Ca transport and adisruption of calcium homeostasis (Huang et al. 1992a,b). Haunget al. (1992a) and Rengel and Elliot (1992a) have shown that Alinhibited Ca uptake in wheat roots and protoplasts of Amaranthusrespectively. These observations then suggest that calciumhomeostasis might be disturbed by an inhibition of Ca influx andthis possibly could be a major contributing factor to theobserved Al toxicity symptoms.One of the most striking symptoms of aluminum toxicity inplants is the shortening of roots (Foy et al. 1978). This isthought to be due to the inhibition of cell division and/or cell68elongation (Taylor 1988). Calcium is important in mitoticregulation mainly through the control of microtubule assembly anddisassembly (Kirschner 1978, Snyder and McIntosh 1976).Therefore disruption of cellular assembly and disassembly ofmicrotubules by Al-induced changes in calcium could result in theinhibition of cell division.There is a good correlation between the orientation ofcortical microtubules and the deposition of extracellularcellulose microfibrils in the newly synthesized cell wall (Hepler1985). Reorganization of the microtubule apparatus caused by anAl-induced disruption of calcium homeostasis could disrupt cellwall synthesis and therefore cell elongation. Calcium is alsoimportant for the process of exocytosis (Steer 1988). Ifcellular Ca homeostasis is disrupted by Al treatment and resultsin changes in Ca distribution close to the plasma membrane, thenthe exocytosis of cell wall materials, essential for cell wallgrowth, could also be adversely affected.Aluminum has the potential to interfere with other calcium-dependent cellular processes. The calcium-dependent regulatoryprotein calmodulin was found to be structurally changed by itsinteraction with Al in vitro. When the Al ion to calmodulinratio was four to one, the activity of Ca and calmodulin-dependent cyclic nucleotide phosphodiesterase was completelyinhibited (Siegel and Haug 1983). It is possible then that toxiceffects due to Al arise, at least in part, from the disruption ofcalcium/calmodulin-dependent processes.Since there is growing circumstantial evidence suggestingthat Al interference with Ca fluxes (resulting in a possible69disruption of calcium homeostasis) contributes to Al toxicitysymptoms (Huang et al. 1992a,b), the major aim of this study isto investigate whether or not Al toxicity is associated withchanges in cellular Ca concentration and distribution.Fluorescence studies on cytoplasmic calcium distribution havebeen restricted in the past, due to the unavailability ofindicators for localizing cytoplasmic free Ca capable ofpenetrating plant cells (Brownlee and Wood 1986, Cork 1986), tothe use of chlorotetracycline (CTC). Chlorotetracycline is areliable indicator of membrane bound calcium (Caswell 1979). Wehave found that Fluo-3 (Kao et al. 1989) penetrates readily intothe root cells of barley when dissolved in 1% DMS0 solution andit enables studies on the distribution and relative concentrationof free cytoplasmic calcium to be carried out with reliabilityand relative ease. Therefore, when used in conjunction with CTC,these studies provide the basis for a comprehensive analysis ofhow cellular Ca distribution and concentration is affected by Alapplied at environmentally and agriculturally realisticconcentrations (Haug and Caldwell 1985).Materials and MethodsGrowth conditions Seeds of Hordeum vulgare L. emend. Lam. var. Kearney weresurfaced-sterilized for 10 minutes in 1% NaOC1, rinsed 5 times indistilled water and then soaked in aerated distilled water for 24hours. The seeds were then spread onto plastic mesh supported byplastic rings and placed on 3.7 liter hydroponic pots filled to70the top with a hydroponic solution so as to contact the seeds.The aerated hydroponic solution contained (in AM), 127 Ca, 27 Mg,82 K, 32 NH4, 370 NO3, 13 PO4, 13 SO4, 1.8 FeEDTA, 3.6 Na, 1 Cl,1.3 B03, 0.5 Mn, 0.1 Zn, 0.03 Cu and 0.02 Mo. Plants were grownfor 3 days at pH 4 - 4.5, with 16 hour photoperiod, at a lightintensity of 180 AE.m -2 .sec-1 and 20-22 °C prior to their use inexperiments. For experimental purpose, roots were treated withand without 50 AM Al2(SO4)2, within the range commonly found inacid soils (Haug 1984). The pH of the experimental solutions wasmaintained between 4.0 to 4.2 by the addition of 1M HC1. Altreatment lasted for 4 hours in all experiments.Fluo-3 studies Excised roots from control and Al-treated plants were loadedwith 44 AM Fluo-3/AM for 10 minutes at room temperature in thedark. The loading solution of Fluo-3/AM was obtained by dilutingan 8 mM stock solution of Fluo-3/AM prepared in DMSO withnutrient medium. Al2(SO4)2 (50 AM) was added to the loadingsolution when required. The final concentration of DMSO was 1%and the pH 4.0. This concentration of DMSO and pH value did notproduce any observable change in the cell which appeared to beboth structurally and physiologically healthy (e.g. accumulatedneutral red was in the vacuole in the same manner as controls).Control experiments with 1% DMSO added to the nutrient mediumwere conducted whenever appropriate. Loading was terminated bywashing the roots three times with Al-free nutrient medium.Epifluorescence microscopy was conducted with a Leitz Dialuxmicroscope. Confocal laser scanning microscopy was carried outwith a MRC-500 Scanning Confocal System attached to a Zeiss71Axioplan microscope. The preparations were excited by an argonion laser beam passing through a BHS filter block with a 488 nMexcitation filter. Emission fluorescence passed through a 515 nmbarrier filter. The signal was processed by a computer equippedwith Biorad Confocal software.Semi-quantitative estimate of the calcium concentration weremade by line scanning through root apices and root hair cells.The relative intensity of the fluorescence signal was assessed ateach point along the line. This line plot was repeated at leastthree times.Photographic exposures and printing conditions were keptstandard so comparative studies of the actual fluorescenceintensity could be made.Chlorotetracycline studies Excised roots were incubated in darkness for 5 minutes with100 AM chlorotetracyline (CTC) prepared in nutrient medium, at20-22 °C and pH 4. The duration of the treatment never exceeded 5min to minimize the artifactual binding of Ca 2+ to membranes byCTC (Reiss et al. 1983). The roots were then washed 3 times withdistilled water to remove the loading medium and root apices androot hair cells were subsequently observed by epifluorescencemicroscopy with a Leitz Dialux microscope. CTC-treated materialwas excited with a filter system ( "A" filter block) passing 340-380 nm uv light, while the emission fluorescence was directedthrough a filter passing light frequencies above 430 nm. BecauseCTC is known to fluoresce in the presence of Mg as well as Ca,Oxytetracycline (OTC) was used in parallel experiments as acontrol. OTC is an analog of CTC which is specific for Mg72(Wolniak et al., 1980).^The OTC preparation and experimentalprocedure were identical to those used for CTC.Microscopic observations Root hairs of intact plants grown as described above wereobserved with differential contrast (Nomarski) optics todetermine the general structure of the cells. Some roots werestained with neutral red, 0.05% (v/v) in nutrient medium at pH 4,to visualize the vacuole and observed by bright field microscopy.Root hair growth was measured over a period of 24 hours withand without 50 gM Al at pH 4.0. The same root hairs weremeasured on a marked section of the root using the same scale barmounted in the ocular piece.ResultsGeneral observations with neutral red staining and differential contrast microscopyRoot hair growth was found to be inhibited by approximately60% of the control after 20 hours treatment with 50 gM Al. Roothairs stained with neutral red accumulated the dye in thevacuole. The distribution of neutral red did not change with Altreatment (Figs. 4.1 and 4.2) or 1% DMSO without or incombination with aluminum (Al) (not shown). When roots wereheated to 70°C for 10 minutes, neutral red failed to accumulatein the vacuole (not shown). Root hairs of Al treated rootsobserved with differential contrast microscopy appearmorphologically healthy in both control and Al-treatedpreparations (Figs. 4.3 and 4.4). However, a large accumulation73of optically dense materials occurred in the apical region of theAl-treated root hairs. This was not observed in root hairstreated with DMSO alone (not shown).Epifluorescence Microscopy Studies of Root TipsRoot tips in controls showed a uniform distribution offluorescence when stained with CTC (Fig. 4.5). Al treatmentsignificantly increased the CTC fluorescence, while thedistribution remained unchanged (Fig. 4.6). OTC staining wasevenly distributed in controls throughout the root tip (Fig.4.7). This fluorescence pattern did not change with Al treatmentbut the intensity of staining increased, although not as much aswith CTC staining (compare Fig. 4.8 with Fig. 4.6). Fluo-3staining in control roots showed almost no fluorescencethroughout the root tip (Fig. 4.9). Al increased thefluorescence intensity although not uniformly. The root cap andparticularly the meristematic region displayed lower fluorescencethan in the elongation zone (Fig. 4.10).Confocal Laser Scanning Microscopy Studies of Root Tips Longitudinal line plot studies of fluo-3 fluorescenceintensity distribution in the root tip showed that thefluorescence intensity was greater in the zone of elongation thanin the meristem and root cap in both control (Fig.4.11) and Altreated roots (Figs.4.12). However, overall the fluorescenceintensity was enhanced in the Al-treated roots. Figures 4.15through 4.18 are higher magnifications of Fluo-3 fluorescence inboth root cap and meristem cells. Intracellular fluorescence canbe seen to be localized to the cytoplasm and not in vacuoles.Fluorescence intensity is higher in Al-treated (Figs. 4.16 and744.18) compared to control cells (Figs. 4.15 and 4.17) of both theroot cap and root meristem regions respectively. Transverse lineplots, passing through the elongation zone, showed that theepidermal cells displayed more intense fluorescence than thecortex and stele regions in both control (Fig.4.13) and Al-treated cells (Fig.4.14), with Al treatment increasingfluorescence intensity.Epifluorescence Microscopy Studies of Root Hairs Figure 4.19 shows the distribution of calcium (Ca), asindicated by CTC staining, in root hairs of control plants.Calcium was found exclusively associated with the cell peripherycorresponding to the region of the plasma membrane, cytoplasmimmediately adjacent to the plasma membrane and cell wall. InAl-treated root hairs the CTC-dependent fluorescence was morewidespread and it appeared to extend throughout most of thecytoplasm. The fluorescence appeared to be more intense in theapical and subapical compared to the basal regions of the roothair cell (Fig. 4.20).The OTC fluorescence in control root hairs was very dim(Fig. 4.21). Aluminum treatment increased the root hairfluorescence throughout the cell body, but in contrast to the CTCsignal, the highest OTC fluorescence was located in a subapicalband (Fig. 4.22). Overall, the OTC fluorescence was brighterthan that produced by CTC.Calcium distribution in control root hairs, as determined byFluo-3 fluorescence was very dim and largely restricted to thecell periphery (Fig. 4.23). Fluo-3 staining of root hairstreated for 4 hours with 50 gM Al showed an increase in75fluorescence, although the overall fluorescence remained dim(Fig. 4.24). Manganese (0.1 mM) did not change the distributionof Fluo-3 fluorescence (Fig.4.26) in comparison with the control(Fig.4.25).Confocal Laser Scanning Microscopy in Root Hairs The Fluo-3 fluorescence in control root hairs wasdistributed throughout the cytoplasm, with highest levels ofintensity observed in the cortical cytoplasm immediately adjacentto the surface membrane (Fig.4.27). With Al-treatment the Fluo-3fluorescence increased in the apical cytoplasm, while remaininguniformly distributed, albeit at a lower level of intensity,throughout the remainder of the cytoplasm (Fig.4.28).Semi-quantitative estimates of cytosolic free Caconcentration were obtained from confocal longitudinal line plotsof Fluo-3 studies. Line plots indicated higher levels of freecytoplasmic Ca in the peripheral region, corresponding to theplasma membrane and adjacent cytoplasm of control root hairs,while within the cytoplasm the calcium concentration was more orless uniform throughout the length of the cell (Fig. 4.29). Thelarge peak to the right, corresponding to the bright spot at thecell base, indicates the position of the cell nucleus (see alsoFig. 4.27). The peak of the Fluo-3 signal showed that thecalcium level doubled in the tip region after Al treatment.Otherwise the line plot reveals lower intensity values throughoutthe subapical and basal regions of Al-treated root hairs(Fig.4.30). Table 4.1 shows relative measurements of Fluo-3intensity expressed as pixels in root hairs of both control andAl-treated plants.76DiscussionRoot hairs were stained with Neutral Red to ensure that theyremained viable after Al and DMSO treatment. It was found thatcontrol cells did accumulate Neutral Red in the vacuole, whilecells heated up to 70 °C did not. Treatment with aluminum (Al)and/or 1% DMSO did not interfere with the normal uptake ofNeutral Red into the cell vacuole. This suggested that the cellvacuole was intact and the root hair cells of Al and DMSO-treatedroots were physiologically stable. Root hair cell structure wasalso observed by differential contrast microscopy (DIC) andappeared to be healthy after Al treatment compared to controls.At the concentration used DMSO did not affect the cells viabilityor structure. However, the apical region of the root hairappeared morphologically altered (an accumulation of opticallydense material) in DIC images after Al-treatment. This changecould be related to Al-induced reduction in exocytosis andsubsequently in root hair growth, since it resembles alterationsobserved in other cells with treatments affecting calcium (Ca)homeostasis (Herth 1978, Harold and Harold 1986).Most of the studies on the distribution of cellular Ca inplant cells have been restricted to membrane-bound Ca, using theCa indicator CTC (Reiss and Herth 1979, Oliveira and Fitch 1988).This is due to the fact that most attempts to apply cytosolic Ca-indicators to plant cells have been unsuccessful. Failure todetect intracellular fluorescence with these fluorescent dyes hasbeen attributed to extracellular hydrolases of the77acetoxymethylester, the permeant form of the dyes (Brownlee andWood 1986, Cork 1986). In order to obviate these difficulties,microinjection (Rathore et al. 1991), electroporation(Scheuerlein et al. 1991) and permeation with the use ofchemicals such as digitonin (Timmers et al. 1991) have been usedto load these tetracarboxylate dyes into plant cells. Fluo-3 isa new tetracarboxylate intracellular Ca-indicator (Minta et al.1989, Kao et al. 1989). Fluo-3 has several potential advantageswhen compared to other commonly used tetracarboxylate indicatorssuch as Quin-2, Fura-2 and Indo-1 in that (i) the excitation andemission maxima (i.e. 506 and 526 nm, respectively) are in thevisible range, which results in lower autofluorescence when usingconfocal microscopy; (ii) the binding of Ca does not cause ashift in either the excitation or emission maxima which makes thecalibration of dye loading for measurements of [Ca]i lesscomplex, and (iii) its fluorescence intensity increases 40-foldupon binding to Ca, which permits the detection of smallerchanges in [Ca]i (Hagar and Spitzer 1992). In addition it hasbeen demonstrated that Fluo-3/AM when dissolved in DMSO readilyand efficiently penetrates plant cells and can be used as areliable indicator of cytosolic Ca (Williams et al. 1990). Theadvantage of our technique in relation to that developed by theprevious authors is that our concentration of DMSO was kept low(1% v/v) as not to affect our material. The reason why this lowconcentration of DMSO works in the present situation cannot beattributed exclusively to the low pH, a factor known tofacilitate tetracarboxylate dye loading in other plant cells(Reiss et al. 1991, Hahm and Saunders, 1991). Indeed, similar78loading efficiency was observed with the brackish water algaVaucheria longicaulis at higher pH values (Oliveira, unpublishedresults). Another advantage of this Fluo-3/DMSO assisted loadingis the fact that Fluo-3 remains in the cytoplasm for extendedperiods of time without being compartmentalization intoorganelles, particularly vacuoles. A problem frequentlyencountered with other tetracarboxylic dyes (Hodick et al. 1991).Heske€h (1983) reported that 0.1 mM manganese quenchesapoplastic tetracarboxylate dye fluorescence. This concentrationof Mn did not change the distribution of the root hairfluorescence confirming that the fluo-3 fluorescence wascytoplasmic in origin. When the free cytoplasmic and membranebound calcium was monitored by Fluo-3 and CTC, respectively, overa period of time after exposure to 50 AM Al, the fluorescenceintensity was found to gradually increase and peak at 4 hours.This peak fluorescence was then found to be subsequently stablefor many hours.It is generally accepted that Al reduces net uptake ofcalcium into roots (Clarkson and Sanderson 1971, Godbold 1991).Huang et al. (1992a) found the net calcium flux at the root apex,measured with a Ca-selective microelectrode, was stronglyinhibited by Al in the aluminum-sensitive cultivar of wheat Scout66. The same Al exposure, which inhibited root elongation inScout, had no effect on Ca influx or growth in an Al-tolerantwheat cultivar, Atlas. Aluminum was also found to affect Ca fluxat the root apex more than the fluxes of other important ions(Kochian, pers. comm.). In addition Ca influx was found to bereduced by Al in the present study (Chapter 3), while the efflux79of Ca from roots grown in up to 5 gM Al was the same as incontrol plants (data not shown). The inhibition of influx seenwith Al treatment could effect the distribution and concentrationof Ca in the cytoplasm of the root cells treated with Al.The results show that despite a decrease in Ca influx intoroot hair cells (Haung et al. 1992a, Nichol et al. inpreparation), cytoplasmic calcium as indicated by Fluo-3 and CTCfluorescence increases in the apical region of root hairs treatedwith Al compared to controls. A possible explanation is that thereduction in net uptake of Ca from the apoplasmic space resultsin the release of Ca from intracellular storage. Calcium isknown to be stored in different intracellular sites including thevacuole (the major storage site), mitochondria and endoplasmicreticulum (Clarkson and Hanson 1980). A possible speculativeexplanation involves the possibility that Al turns on thephosphatidylinositol calcium signalling system in both the roothair tip region, and root apical cells by interacting with aplasma membrane G protein (Kochian pers. comm.). Candura et al.(1991) have shown that Al interacts with phosphoinositolmetabolism in rat cerebral cortical membranes and stimulatescerebral phosphoinositide metabolism. This could then result inthe release of inositol 1,4,5-trisphophate (InsP3) into thecytoplasm which would in turn result in the release of calciumfrom the vacuole and possibly also the endoplasmic reticulum.These are the major intracellular compartments shown in plantcells to be InsP3-sensitive (Schumaker and Sze 1987; Ranjeva etal. 1988; Allen and Schumm 1990, Obermeyer and Weisenseel 1991).It might be expected that plasma membrane and ER Ca-ATPase pumps8 0(Marine, 1985) would restore Ca homeostasis and counteract therelease of Ca from intracellular storage. This then suggeststhat either the processes maintaining homeostasis are inoperativeor the rate of Ca release is so great that it overwhelms theseprocesses.Regardless of how the increase in cytoplasmic calciumoccurs, it can result in the disruption of several cellprocesses. Since root hair cells exhibit apical growth, the cellapex is a very active region where numerous calcium-dependentprocesses related to cell elongation take place (Hepler 1985).Aluminum is known to inhibit root elongation very rapidly. Thegrowth of Limnobium root hairs was reduced from 80 pm hr -1 to 16gm hr-1 by 10 pM Al in 40 minutes when grown in 0.4 mM calcium,while 20 pM Al arrested growth after 5 minutes (Ryan and Kochian,pers. comm.). Root hair growth inhibition was confirmed by ourown observations. The mechanism causing this very rapidinhibition is not known. However such an inhibition isconsistent with the results of a rapid disruption of cellularcalcium homeostasis. Intracellular calcium concentration in theroot hair cells, as indicated by Fluo-3 fluorescence, increasedshortly after and peaks within 4 hours of the start of aluminumtreatment. This is within the same time frame as the inhibitionof root elongation.Calcium concentrations as low as 10 pM were found to inhibitmicrotubule polymerization (Keith et al., 1983). Fewermicrotubules were seen in root hair cells of the wheat varietyScout treated with 5-10 pM Al in the presence of 0.2 mM CaC12than in controls (Kochian, pers. comm.). Microtubules are also81thought to be important in the transport of secretory vesiclessuch as those carrying cell wall materials necessary for cellwall elongation (Quader and Robinson, 1979). Hepler (1985) hasshown that the orientation of cortical microtubules is normallycorrelated with that of cellulose microfibrils which run parallelin the cell wall. These observations suggest that the corticalmicrotubules act as a template for new cell wall formation. Thefact that Fluo-3 studies show higher levels of Ca in theelongation zone of Al-treated compared to controls supports thehypothesis that inhibition of cell elongation may result frominterference with microtubule function.Increased cytoplasmic Ca could also interfere with celldivision through disruption of microtubules involved in theformation of the spindle apparatus. Al treatment is thought tomainly affect growing areas such as the root meristem where celldivision is very active (Huang et al., 1992a). Thisinterpretation certainly would be consistent with our observationof higher Fluo-3 fluorescence intensity in meristematic cells inAl-treated compared to control plants. This is further supportedby the Al-induced increase in CTC fluorescence of themeristematic and elongation zones of the growing root tip,indicating that calcium homeostasis had been disturbed and couldcontribute to the inhibition of root growth (Huang et al., 1992).The results reported in Chapter 1, however, suggested that celldivision was not inhibited by Al and was probably stimulated bythe [Al] used in this study. Cell division, however, might beinhibited at higher [Al] (Clarkson 1965, Morimura et al 1978).In conclusion, aluminum-induced disturbances of cellular82calcium in the root apex and root hair cells by interfering withcalcium-dependent processes involved in cell elongation and rootelongation may play a major role in the establishment of wellknown Al-induced toxicity symptoms.83Table 4.1. Confocal laser scanning fluorescence intensity(pixels) measurements using line plots superimposed on thelongitudinal axis of controls (0 AM) and Al treated root hairs(50 MM). Values represent averages of three measurements ± SEM.Treatment ^Fluorescence Intensity ^Apical region^Basal RegionControl^ 132.5+12.5^75.7+8.1Al-treated^ 222.5+22.5^50.0+6.684Figs.4.1 -4.2. Neutral red staining of vacuoles in root hairs ofcontrol (Fig.4.1) and Al-treated (Fig.4.2) materialFigs.4.3 -4.4. Differential contrast observations of the generalstructure of root hairs in control (Fig.4.3) and Al-treated(Fig.4.4) materialScale bar for Figs.4.1-4.4 represents 10 gM8 586Figs.4.5-4.6. CTC staining of the root apex in control (Fig.4.5)and Al-treated (Fig.4.6) preparations. Al treatment increasedstaining intensityFigs.4.7-4.8. OTC staining of the root apex in control (Fig.4.7)and Al-treated (Fig.4.8) material. Al-treatment increasedstaining intensity, although less than with CTC (compare Figs.4.6and 4.8)Figs.4.9-4.10. Fluo-3 staining of root apex in control (Fig.4.9)and Al-treated (Fig.4.10) material. Increased fluorescence wasobserved after Al-treatmentScale bar in Figs.4.5-4.10 represents 50 gM8 788Figs.4.11 -4.12. Longitudinal confocal line plots of Fluo-3fluorescence, in control (Fig.4.11) and Al-treated (Fig.4.12)roots showed increased fluorescence in the elongation zonecompared to the root cap and meristem in both cases and increasedoverall fluorescence in the Al-treated materialFigs.4.13 -4.14. Transverse confocal line plots of Fluo-3fluorescence, in control (Fig.4.13) and Al-treated (Fig.4.14)roots show increased fluorescence in the epidermal cells overcortex and vascular tissue and increased overall fluorescence inthe Al-treated materialScale bar in Figs.4.11-4.14 represents 200 AMRC, root cap; MZ, meristematic zone; EZ, elongation zone8 990Figs. 4.15 -4.18. Confocal observations of Fluo-3 fluorescence inroot cap (Fig.4.15, control; Fig.4.16, Al-treated) andmeristematic cells (Fig.4.17, control; Fig. 4.18, Al-treated).Al-treatment conspicuously increased fluorescence intensityScale bar in Figs.4.15-4.18 represents 10 gM9192Figs.4.19 -4.26. Epifluorescence observations of root hair cellsFig.4.19. Control stained with CTC showing fluorescencerestricted to the cell peripheryFig.4.20. Al-treated, CTC stained shows increased fluorescence inthe cell apexFig.4.21. Control stained with OTC shows very dim fluorescenceFig.4.22. Al-treatment with OTC staining shows increasedfluorescence with maximum intensity occurring in a subapical bandFig.4.23. Control stained with Fluo-3 showing very dimfluorescenceFig.4.24. Al-treated, Fluo-3 stained showing increased overallfluorescenceFig.4.25-4.26. Fluo-3 staining of root hairs treated with 0.1 mMMn2+ (Fig.4.26) compared to the Mn 2+-free control (Fig.4.25). Nochange in fluorescence is observed in the Al-treated root hair(Fig.4.26) compared to the control (Fig.4.25)Scale bar in Figs.4.19-4.22 represents 10 gMScale bar in Figs.4.23-4.26 represents 10 gM9 394Figs.4.27 -4.30. Confocal observations of root hairs treated withFluo -3Fig.4.27. Control fluorescence is mostly restricted to the cellperipheryFig.4.28. Al treatment produces increased fluorescence at thecell apex and reduced fluorescence in subapical and basal regionsFig.4.29. Control line plot showing even fluorescence throughoutthe cell. Bright fluorescence spot at the basal regionrepresents fluorescence associated with the nucleus (see alsoFig.4.27)Fig.4.30. Line plot through Al treated cell showing increasedapical fluorescence. Fluorescence is relatively low to minimalthroughout the remainder of the cellScale bar in Figs.4.27 and 4.28 represents 10 AMScale bar in Figs.4.29 and 4.30 represents 20 AM9527 28Asst ee -* — -A_30!shoe tape. ttee t She ...yeah*thetattoo elan. the line thterhttat31Ais 4L LA J_96ConclusionsAluminum (Al) toxicity in plants has been attributed to alarge number of processes including the inhibition of ion uptake,disruption of cell membrane function and the inhibition of celldivision and elongation. However, despite intensive research thebiological (physiological and biochemical) effects of Al are notwell understood (Taylor 1988). In this study, an attempt is madeto examine Al toxicity in an Al-sensitive cultivar of barley bothat the cellular and organ (root) level.Al was found to dramatically change the root anatomy asreported by previous authors (Hutterman 1985, Foy et al.1978)(Chapter 1). Secondary branching of the root system wascompletely inhibited, while the growth of the primary root wasarrested and root hair growth was inhibited. Growth inhibitionwas found to be primarily due to an inhibition of cell elongationand not cell division. Root respiration was found to be mostlyunaffected by Al suggesting that probably Al toxicity was not dueto a lack of ATP which is required to drive cellular processessuch as ion transport and cell growth. Energy dispersive X-raymicroanaylsis indicated that potassium (K) declined dramaticallyin the root tip with Al treatment while phosphorus (P) was alsoreduced, but to a lesser extent. A decline in cellular K couldbe significant since K is an important osmotic ion in cell turgorregulation. The drop in root K could contribute to the observedAl-induced inhibition of root elongation.Potassium was found to be inhibited by long term (5 days)and short term (30 seconds) treatment with Al (50 AM)(Chapter 2).97This inhibition was found to be competitive and was completelyreversible when plants were removed from Al. A decline incellular K (chapter 1) would be consistent with the observationthat Al inhibited the influx but not the efflux of K (chapter 2).Cation influx was found to be inhibited while anion influx wasenhanced by Al treatment (Chapter 3). This suggested that Al wasbinding close to the plasma membrane and acting as a positivelycharged barrier to cation fluxes while stimulating anion fluxes.Aluminum was found to strongly inhibit calcium (Ca) influx(Chapter 3) and to increase the cytoplasmic Ca in the tip(growing) region of root hair cells whose growth was inhibited bysimilar treatment (Chapter 4). Al also increased cytoplasmic Cain the root meristem and elongation zone cells raising thepossibility that Al could by interfering with cell elongation andtherefore root elongation through its effects on Ca homeostasis.Calcium is an important regulator in many cellular processesrequired for cell elongation (Chapter 4).Root cell growth in the presence of Al was abnormal (Chapter1). Instead of cells predominantly growing in length, Al treatedroot cells appeared to grow in all directions since the cellwidth was greater than in controls. These changes in cellulargrowth seem to explain the common observation that the Al-inducedreduction in root length is accompanied by an increase inthickness. It is interesting that cell elongation was affectedby Al but cell division was not. If Al-induced disturbances inCa homeostasis are responsible for these changes, why would theCa-dependent processes controlling cell elongation be sensitiveto this [Al] while those controlling cell division are not? One98possibility might be that these processes differ in theresensitivity to Al. Some microtubules are more stable than others(Luduena 1979). Another possibility might be that Ca ion fluxesare important in establishing cell polarity (and hence directcell elongation) since it is thought that some regions on thecell appear to have a greater density of Ca channels resulting ina greater Ca influx in these regions (Polito, 1985).Various questions have arisen from the results in thisthesis which should be addressed. The results reported inChapter 1 indicate that Al-induced root shortening and thickeningis caused by a change in the root cell growth from predominantlyelongating to reduced elongation and an increase in width. Theseresults should be confirmed with a more extensive study usingother methodological approaches. This study also suggested thatAl stimulated cell division. This should also be confirmed byfurther studies where the mitotic index and other relevantparameters are estimated in both control and Al treated roots.The results reported in Chapter 3 suggested that Al inhibitscation influx while stimulating anion influx. The most likelyexplanation for this is that Al binds close to or at the plasmamembrane (probably to the negatively charged phospholipids) andforms a positively charged layer which would attract anions andrepel cations. If this interpretation is correct, other ionscarrying three positive charges should also inhibit cation influxand stimulate anion transport. Also, the degree to which cationinflux is inhibited and anion influx stimulated should bedependent on the charge carried by that ion. For example, theion flux of an anion carrying three positive charges should be99stimulated more than that of an ion carrying two, and this inturn should be stimulated more than one carrying only onepositive charge. Studies along these lines would verify andstrengthen this hypothesis.Aluminum was found to increase the cytoplasmic Caconcentration in the growing apex of root hair cells and in cellsof the zone of elongation (Chapter 4). The increase incytoplasmic Ca induced by Al was counterintuitive since calciuminflux and net uptake were found to be inhibited by Al (Chapter3). It would be expected that cytoplasmic Ca would declinerather than increase. The object of future studies would then beto investigate how this increase in cytoplasmic Ca comes about.This can be tested by treating roots with calcium modulatorswhich reduce the availability of extracellular Ca (EGTA), blockCa influx (lanthanum and veripimil) or enhance it (ionophoreA23187) and block Ca release from intracellular storage (TMB-8).These modulators will help clarify the source of this Ca signal.This, in turn, will help elucidate the role(s) of the varioussources of Ca in controlling cell growth. A possible explanationfor the increase in cytoplasmic Ca is that the reduction in netuptake of Ca from the apoplastic space, results in the release ofCa from intracellular storage. Ca is known to be stored atdifferent intracellular sites including the vacuole (the majorstorage site), mitochondria and endoplasmic reticulum (Clarksonand Hanson 1980). If the source of the Ca is intracellular, TMB-8 treatment could limit the Al induced increase in Ca seen withAl treatment. From the combination of Ca modulators describedabove, it can be determined how Al affects Ca homeostasis.100Literature CitedAllen NS, Schumm JH (1990) Endoplasmic reticulum, calciosomes andtheir possible role in signal transduction. Protoplasma 154:1-12Arieff A (1985) Aluminum and the pathogenesis of dialysisencephalopathy. Am. J. Kidney Dis. 6: 317-321Baligar VC, Nielsen NE, Barber SA (1979) Kinetics of absorptionof K, Rb, and Cs from solution culture. J. Plant. Nutr.1: 25-37Bolla KI, Briefel G, Spector D, Schwartz BS, Wieler L, Herron J,Gimenez L (1992) Neurocognitive effects of aluminum. Arch.Neurol. 49: 1021-1026Botti C, Vasil IK (1984) Plastic embedding for light microscope.In: Vasil IK (ed) Cell Culture and Somatic Cell Genetics ofPlants, Vol. 1, Academic Press , New York, pp 684-688Brownlee C, Wood JW (1986) The gradient of cytoplasmic freecalcium in growing rhizoid cells of Fucus serratus.Nature 320: 624-626Campbell PGC, Stokes PM (1985) Acidification and toxicity ofmetals to aquatic biota. Can. J. Fish Aquat. Sci.42: 2034-2049Candura SM, Castoldi AF, Manzo L, Costa LG (1991) Interaction ofaluminum ions with phosphoinositide metabolism in ratcerebral cortex membranes. Life Sciences 49: 1245-1252Caswell AH (1979) Methods of measuring intracellular calcium.Int Rev Cytol 56: 145-181101Claesson A, Tornquist L (1988) The toxicity of aluminum to twoacido-tolerant green algae. Water Res. 22: 977-983Clark RB (1977) Effect of aluminum on growth and mineral elementsof Al-tolerant and Al-intolerant corn. Plant Soil 47:653-662Clark KL, LaZerte BD (1985) A laboratory study of the effects ofaluminum and pH on amphibian eggs and tadpoles. Can. J. FishAquat. Sci. 42: 1544-1551Clarkson DT (1965) The effect of aluminum and some othertrivalent metal cations on cell division in the root apicesof Allium cepa. Ann Bot 29: 309-315Clarkson DT (1966) Effect of aluminum on the uptake andmetabolism of phosphorus by barley seedlings. Plant Physiol41: 165-172Clarkson DT, Hanson JB (1980) The mineral nutrition of higherplants. Plant Physiol 31: 239-298Clarkson DT, Sanderson J (1971) Inhibition of the uptake andlong-distance transport of calcium by aluminum and otherpolyvalent cations. J Exp Bot 22: 837-851Cork RJ (1986) Problems of application of quint-am to measuringcytoplasmic free calcium in plant cells. Plant CellEnvironment 9: 157-160Driscoll CT, Baker JP, Bisogni JJ, Schofield CL (1980) Effect ofaluminum speciation on fish in dilute acidified waters.Nature 284: 161-164Edwards JH, Horton BD, Kirkpatrick HC (1976) Aluminum toxicitysymptoms in peach seedlings. J Am Soc Hort Sci 101: 139-142102Fleming AL (1983) Ammonium uptake by wheat varieties differing inAl tolerance. Agron J 75: 726-730Folsom BR, Popescue NA, Wood JM (1986) Comparative study ofaluminum and copper transport and toxicity in an acid-tolerant freshwater green alga. Envir. Sci. Technol. 20:616-620Foy CD, Burns GR, Brown JC, Fleming AL (1965) Differentialaluminum tolerance of two wheat varieties associated withplant-induced pH changes around their roots. Soil Sci. Soc.Am. Proc. 29: 64-67Foy CD, Chaney RL, White MC (1978) The physiology of metaltoxicity in plants. Ann Rev Plant Physiol 29: 511-566Foy CD, Fleming AL (1982) Aluminum tolerances of two wheatgenotypes related to nitrate reductase activities. J. Plant.Nutr. 5: 1313-1333Foy CD, Fleming AL, Armiger WH (1969) Aluminum tolerance ofsoybean varieties in relation to calcium nutrition.Agron J 61: 505-511Foy CD, Flemming AL, Gerloff GC (1972) Differential aluminumtolerance in two snapbean varieties. Agron. J. 64: 815-818Foy CD, Lee EH, Wilding SB (1987) Differential aluminumtolerances of two barley cultivars related to organic acidsin their roots. J. Plant Nutr. 10: 1089-1101Gensemer RW (1990) Role of aluminum and growth rate on changes incell size and silica content of silica-limited populationsof Asterionella ralfsii var. Americana (Bacillariophyceae).J. Phycol. 26: 250-258103Glass ADM (1989) Plant nutrition: An introduction to currentconcepts. Jones and Barlett Publishers, BostonGodbold D (1991) Aluminum decreases root growth and calcium andmagnesium uptake in Picea abies seedlings. In: Wright RJ,Baligar VC, Murrmann RP (eds) Developments in Plant and SoilSciences: Plant-Soil Interactions at Low pH, vol 45. KluwerAcademic Publisher, Dordrecht, pp 747-753Gomes MMM, Cambraia J, Sant'anna R, Estevao MM (1985) Aluminumeffects on uptake and translocation of nitrogen in sorghum(Sorghum bicolor L. Moench). J. Plant Nut. 8: 457-465Hagar AF, Spitzer JA (1992) The effect of endotoxemia onconcanavalin A induced alterations in cytoplasmic freecalcium in rat spleen cells as determined with Fluo-3. CellCalcium 13: 123-130Hahm SH, Saunders MJ (1991) Cytokinin increases intracellularCa2+ in Funaria: detection with Indo-1. Cell Calcium12: 675-681.Harold RL, Harold FM (1986) Ionophores and cytochalasinsmodulate branching in Achlya bisexualis. J Gen Microbiol132: 213-219Hartwell BL, Pember FR (1918) The presence of aluminum as areason for the difference in the effect of so-called acidsoil on barley and rye. Soil Sci 6: 259-279Haug AR (1984) Molecular aspects of aluminum toxicity. CTC CritRev Plant Sci 1: 345-373104Haug AR, Caldwell CR (1985) Aluminum toxicity in plants: the roleof the root plasma membrane and calmodulin. In: St.JohnJ.B., Berlin E., Jackson PC (eds) Frontiers of MembraneResearch in Agriculture, Rowman and Allanheld, Totowa, pp359-381Hepler PK (1985) The plant cytoskeleton. In: Robards AW (ed)Botanical Microscopy, Oxford Univ. Press, Oxford, pp 233-262Herth W (1978) Ionophore A23187 stops tip growth, but notcytoplasmic streaming in pollen tubes of Lilium longiflorumProtoplasma 96: 275-282Hesketh TR, Smith GA, Moore JP, Taylor MV, Metcalf JC (1983) Freecytoplasmic calcium concentration and mitogenic stimulationof lymphocytes. J Biol Chem 258: 4876-4882Hodick D, Gilroy S, Fricker MD, Trewavas AJ (1991) Cytosolic Ca 2+concentration and distribution in rhizoids of Chara frac:fills Desv. determined by ratio analysis of the fluorescent probeIndo-1. Bot Acta 104: 222-228Horst WJ, Wagner A, Marschner H (1983) Effects of aluminum onroot growth, cell division rate and mineral element contentin roots of Vigna unguiculata genotypes. Z Pflanz109: 95-103Huang JW, Shaff JE, Grunes DL, Kochian LV (1992a) Aluminumeffects on calcium fluxes at the root apex of aluminum-tolerant and aluminum-sensitive wheat cultivars. PlantPhysiol 98: 230-237Huang JW, Grunes DL, Kochian LV (1992b) Aluminum effectson the kinetics of calcium uptake into cells of the wheatroot apex. Planta (in press)105Hutterman A (1985) The effects of acid deposition on thephysiology of the forest ecosystem. Experienta 41: 584-590Jarvis SC, Hatch DJ (1986) The effects of low concentrations ofaluminum on the growth and uptake of nitrate-N by whiteclover. Plant Soil 95: 43-55Johnson RE, Jackson WA (1964) Calcium uptake and transport bywheat seedlings as affected by aluminum. Soil Sci Soc AmProc 28: 381-386Kao JPY, Harootunian AT, Tsien RY (1989) Photochemicallygenerated cytosolic calcium pulses and their detection byFluo-3. J Biol Chem 264: 8179-8184Keith C, DiPaola M, Maxfield FR, Shelanski ML (1983)Microinjection of Ca2+-calmodulin causes a localizeddepolymerization of microtubules. J Cell Biol 97: 1918-1924Kinraide TB (1988) Proton Extrusion by Wheat Roots ExhibitingSevere Aluminum Toxicity Symptoms. Plant Physiol. 88:418-423Kinraide TB, Ryan PR, Kochian LV (1992) Interactive effects ofAl3+ , H+ , and other cations on root elongation considered interms of cell-surface electrical potential. Plant Physiol.99: 1461-1468Kircschner MW (1978) Microtubule assembly and nucleation.Int Rev Cytol 54: 1-71Kochian LV, Lucas WJ (1983) Potassium transport in corn roots. IIThe significance of the root periphery. Plant Physiol73: 208-215106Kochian LV, Shaff JE, Ryan PR (1991) Microelectrode-basedinvestigations into the relationship between Al toxicity androot-cell membrane transport processes. Curr. Topics PlantBiochem. Physiol. 10: 117-133Lance JC, Pearson RW (1969) Effect of low concentrations ofaluminum on growth and water and nutrient uptake by cottonroots. Soil Sci Soc Am Proc 33: 95-98LaZerte BD (1986) Metals and acidification: an overview. WaterAir and Soil Pollut. 31: 596-676Lee CR (1971) Influence of aluminum on plant growth and mineralnutrient of potatoes. Agron J 63: 604-608Lee J, Pritchard MW, Sedcole JR, Robertson MR (1984) Aluminum andammonium ion effects on the depletion of potassium fromhydroponic solutions by Trifolium repens L. cv. "GrasslandsHuia". J. Plant. Nutr. 7: 1635-1650Luduena RF (1979 Biochemistry of tubulin. In: Roberts K, Hyams JS(eds) Microtubules, Academic Press, London, pp 86-88Macdonald TL, Martin RB (1988) Aluminum ion in biologicalsystems. TIBS 13: 15-19Marme D (1985) The role of calcium in the cellular regulation ofplant metabolism. 23: 945-953Matsumoto H, Morimura S, Takahashi E (1977) Less involvement ofpectin in the precipitatiro of aluminum in pea root. PlantCell Physiol 18: 325-335107McCain S, Davies MS (1984) Effects of pretreatment withphosphate in natural populations of Agrostis capillaris. IIInteractions with aluminum on the acid phosphatase activityand potassium leakage of intact roots. New Phytol.96: 589-599.Minta A, Kao JPY, Tsien RJ (1989) Fluorescent indicators ofcytosolic calcium based on rhodamine and fluoresceinchromophores. J Biol Chem 264: 8171-8178Miyasaka SC, Kochian LV, Shaff JE, Foy CD (1989) Mechanisms ofaluminum tolerance in wheat : An investigation of genotypicdifferences in rhizosphere pH, K+ , and H+ transport, androot-cell membrane potentials. Plant Physiol. 91: 1188-1196Moore DP (1974) Physiological effects of pH on roots. In: Carson(ed) The plant root and its environment, University Press ofVirginia, Charlottesville, 135-151Morimura S, Takahashi E, Matsumoto H (1978) Association ofaluminum with nuclei and inhibition of cell division inonion (Allium cepa) roots. Z Pflanz 88: 395-401Mugwira LM, Patel SU, Fleming AL (1980) Aluminum effects ongrowth and Al,Ca,Mg,K and P levels in triticale, wheat andrye. Plant Soil 57: 467-470Nichol BE, Oliveira L, Glass ADM, Siddiqi MY (1991) The effectsof short and long term aluminum treatment on potassiumfluxes in the roots of an aluminum sensitive cultivar ofbarley. In: Wright RJ, Balingar VC, Murrmann RP (eds)Developments in Plant and Soil Sciences: Plant-SoilInteractions at Low pH, Vol. 45. Kluwer Academic Publisher,Dordrecht, pp 741-746108Niedziela G, Aniol A (1983) Subcellular distribution of aluminumin wheat roots. Acta Biochim. Polon. 30: 99-105Noggle GR, Fritz GJ (1983) Introductory Plant Physiology.Prentice-Hall, Englewood Cliffs, New JerseyObermeyer G, Weisenseel MH (1991) Calcium channel blockers andcalmodulin antagonists affect the gradient of free calciumions in lily pollen tubes. Eur J Cell Biol 56: 319-327Oliveira L, Fitch RS (1988) Visualization of Ca 2+-gradients ingerminating aplanospores of Vaucheria longicaulis var macounii Blum (Tribophyceae) with chlorotetracyclinefluorescence. J Submicrosc Cytol Pathol 20: 741-750Okazaki Y, Iwasaki N (1991) Injection of a Ca 2+ chelating agentinto the cytoplasm retards the progress of turgor regulationupon hypotonic treatment in the alga, Lamprothaminium. PlantCell Physiol 32: 185-194Okazaki Y, Tazawa M (1990) Calcium ion and turgor regulation inplant cells. J Memb Biol 114: 189-194.Parker DR, Kinraide TB, Zelazny LW (1988) Aluminum speciation andphytotoxicity in dilute hydroxy-aluminum solutions. SoilSci. Soc. Am. J. 52: 438-444Parker DR, Zelazny LW, TB Kinraide (1989) In: Lewis TE (ed)Environmental chemistry and toxicology of aluminum, LewisPublishers Inc., Michigan, pp 117-145Perl DP, Gajdusek DC, Garruto RM, Yanagihara RT, Gibbs CJ Jr(1982) Intr-neuronal aluminum accumulation in amyotropiclateral sclerosis and parkinonism-dementia of Guam. Science217: 1053109Pettersson S, Strid H (1989) Effects of aluminum on growth andkinetics of(86R,,D) uptake in two cultivars of wheat(Triticum aestivum) with different sensitivity to aluminum.Physiol Plant 76: 255-261Polito VS (1985) Intracellular calcium dynamics and plant cellfunction. In: Hidaka H, Hartshorne DJ (eds) CalmodulinAntagonists and Cellular Physiology. Academic Press, NewYork, pp 457-467Quader H, Robinson DG (1979) Structure, synthesis and orientationof microfibrils: VI The role of ions in microfibrildeposition in Oocystis solitaria. Eur J Cell Biol 20: 51-56Ranjeva R, Carrasco A, Bonlet AM (1988) Inositol trisphosphatestimulates the release of calcium from intact vacuolesisolated from Acer cells. FEBS Lett 230: 137-141Rathore KS, Cork RJ, Robinson KR (1991) A cytoplasmic gradient ofCa2+ is correlated with the growth of lily pollen tubes.Dev Biol 148: 612-619Reiss HD, Herth W (1979) Calcium gradients in tip growing plantcells visualized by chlorotetracycline fluorescence.Planta 146: 615-621Reiss HD, Herth W, Schnepf E, Nobiling R (1983) The tip to basecalcium gradient in pollen tubes of Lilium longifloriummeasured by proton-induced X-ray emission (PIXE).Protoplasma 115: 153-159Reiss U, Grolig F, Wagner G (1991) Changes of cytoplasmic free2Ca + in the green alga Mougeotia scalaris as monitored withIndo-1, and their effect on the velocity of chloroplastmovement. Planta 184: 105-112110Rengel Z, Elliott DC (1992a) Aluminum inhibits net 45Ca2+ uptakeby Amaranthus protoplasts. Biochem. Physiol. Pflanzen 188:177-186Rengel Z, Elliott DC (1992b) Mechanism of aluminum inhibition ofnet 45Ca2+ uptake by Amaranthus protoplasts. 98: 632-638Ross M (1987) The silent epidemic - a comprehensive guide toAlzheimer's disease. Hounslow Press, Willowdale, Ont.,CanadaRyan PR, Shaff JE, Kochian LV (1992a) Aluminum toxicity in roots:correlation among ionic currents, ion fluxes, and rootelongation in aluminum-sensitive and aluminum-tolerantcultivars. Plant Physiol. 99: 1193-1200.Ryan PR, DiTomaso JM, Kochian LV (1992b) Aluminum toxicity inroots: An investigation of spatial sensitivity and the roleof the root cap. J. Exp. Bot. (in press)Rygiewicz PT, Bledsoe CS, Glass ADM (1984) A comparison ofmethods for determining compartmental analysis parameters.Plant Physiol. 76: 913-917Sampson M, Clarkson D, Davies DD (1965) DNA synthesis in aluminumtreated roots of barley. Science 148: 1476-1477Scheuerlein R, Schmidt K, Poenie M, Roux SJ (1991) Determinationof cytoplasmic calcium concentration in Dryopteris spores.A developmentally non-disruptive technique for loading ofthe calcium indicator fura-2. Planta 184: 166-174Schroeder JI (1988) K + transport properties of IC+ channels in theplasma membrane of Vicia faba guard cells. J. Gen. Physiol92: 667-683111Schumaker KS, Sze H (1987) Inositol 1,4,5-trisphosphate releasesCa2+ from vacuolar membrane vesicles of oat roots. J BiolChem 262: 3944-3946Siddiqi MY, Glass ADM, Ruth TJ, Fernando M (1989) Studies of theregulation of nitrate influx by barley seedlings using13NO3. Plant Physiol. 90: 806-813Siegel N, Haug A (1983) Aluminum interaction with calmodulin:evidence for altered structure and function from optical andenzymatic studies. Biochim Biophys Acta 744: 36-45Snyder JA, McIntosh JR (1976) Biochemistry and physiology ofmicrotubules. Ann Rev Biochem 45: 699-720Spencer PS, Nunn PB, Hugan J, Ludolph AC, Rose SM, Roy DN (1987)Guam amyotrophic lateral sclerosis-parkinsonism-dementialinked to a plant excitant neurotoxin. Science 237: 237Steer MW (1988) The role of calcium in exocytosis and endocytosisin plant cells. Physiol Plant 72: 213-220Suhayda CG, Haug A (1986) Organic acids reduce aluminum toxicityin maize root membranes. Physiol Plant 68: 189-195Taylor GJ (1987) Exclusion of metals from the symplasm: apossible mechanism of metal tolerance in higher plants.J. Plant Nutr. 10: 1213-1222Taylor GJ (1988) The physiology of aluminum phytotoxicity. In:Sigel H, Sigel A (eds) Metal Ions in Biological Systems,Marcel Dekker Inc, New York, pp 123-163Taylor GJ, Foy CD (1985a) Mechanisms of aluminum tolerance inTriticum aestivum L. (wheat) I. Differential pH induced bywinter cultivars in nutrient solutions. Am. J. Bot.72: 695-701112Taylor GJ, Foy CD (1985b) Mechanisms of aluminum tolerance inTriticum aestivum L. (wheat) II. Differential pH induced byspring cultivars in nutrient solutions. Am. J. Bot.72: 702-706Timers ACJ, Reiss HD, Schel JHN (1991) Digitonin-aided loadingof Fluo-3 into embryogenic plant cells. Cell Calcium12: 515-521Tornqvist L (1989) Ultrastructural changes and alteredlocalization of acid phosphatases in Monoraphidium andStichococcus cells (Chlorophyceae) influenced by aluminum.Envir. and Exp. Bot. 29: 457-465Williams DA, Cody SH, Gehring CA, Parish RW, Harris PJ (1990)Confocal imaging of ionized calcium in living plant cells.Cell Calcium 11: 291- 297Wolniak SM, Hepler PK, Jackson WT (1980) Detection of themembrane-calcium distribution during mitosis in Haemanthus endosperm with chlorotetracycline. J Cell Biol 87: 23-32Zhang G, Taylor GJ (1988) Effect of aluminum on growth anddistribution of aluminum in tolerant and sensitive cultivarsof Triticum aestivum L.. Commun. Soil Sci Plant Anal.19: 1195-1205Zhang G, Taylor GJ (1990) Kinetics of aluminum uptake in Triticumaestivum L. Identity of the linear phase of aluminum uptakeby excised roots of aluminum-tolerant and aluminum-sensitivecultivars. Plant Physiol 94: 577-584


Citation Scheme:


Citations by CSL (citeproc-js)

Usage Statistics



Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            async >
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:


Related Items