Simulating the Mineral Environment of Aluminium Toxic Soils in Plant Cell Culture

The development of a medium for studying aluminium toxicityin plant cell cultures is described. To prevent the precipitationof Al added to the standard cell culture medium, it was necessaryto lower the phosphate concentration from 1250 mmol m^–3to10 mmol m^–3, and the pH from 5.8 to 4-0. Two additionalmodifications were the use of unchelated iron and a reductionin the calcium concentration from 3.0 mol m^–3 to 0.1 molm^–3. Since the gelling properties of agar are inhibitedat pH 4.0, cells were cultured on filter paper supported bypolyurethane foam sturated with liquid medium. The only limitationto the growth of plated Nicotiana plumbaginifolia Viv. cellson the modified medium was the reduced phosphate concentration.This was partly overcome by ‘preloading’ the cellswith phosphate prior to each experiment. In addition, the filterpaper with adhering cells was transferred to fresh medium everysecond day to replenish phosphate, and to re-establish the initialpH of4.0 (which otherwise drifts upward). With the modifiedmedium, Al toxicity was observed in plated N. plumbaginifoliacells at both 200 mmol m^–3 and 400 mmol m^–3 Al.There was no toxicity at these Al concentrations when the normalphosphate concentration or pH were restored to the modifiedmedium. Partial alleviation of Al toxicity occurred with restorationof the normal calcium concentration or chelated iron. Chelationof Al with citrate or EDTA also mitigated Al toxicity. In additonto Al toxicity, the modified medium should also prove usefulfor studying other metal toxicities in plant cell culture. Key words: Al toxicity, Cell culture, Nicotiana plumbaginifolia     

Isolation, Culture, and Plant Regeneration of Protoplasts of Two Shrubby Oxalis Species from South America

Protoplasts were successfully isolated from internodal callustissues of both Oxalis glaucifolia and O. rhombeo-ovata whenthey were digested in a solution containing 0.1% (w/v) MacerozymeR-10, 0.5% (w/v) cellulase Onozuka R-10 and 0.3 mmol m^–3sucrose. Protoplasts proliferated to give cell colonies on Gamborget al.'s B5 medium supplemented with 0.3 mmol m^–3 mannitol,0.5 mg dm^–32, 4-D, and 2.0 mg dm^–3 kinetin. Calluswas produced upon transfer of cell colonies to Murashige andSkoog medium containing 2.0 mg dm^–3 l-naphthaleneaceticacid (NAA) and 0.1 mg dm^–3 kinetin for O. glaucifolia,or with 5.0 mg dm^–3 NAA and 0.5 mg dm^–3 6-benzylaminopurine,for O. rhombeo-ovata. Plants were regenerated from O. glaucifoliaprotoplasts on a medium containing 0.1 mg dm–3 NAA, 1.0mg dm^–3 kinetin and 1.0 mg dm^–3 gibberellic acid,but only vascular nodules were differentiated by O. rhombeo-ovataprotoplast-derived calli. Key words: Tissue culture, protoplasts, plant regeneration, Oxalis spp     

Microelectrophoretic and 9-Aminoacridine Fluorescence Study of the Surface Electrical Properties of Suspension Cultured Catharanthus roseus Cells and Isolated Protoplasts: Effects of Abscisic Acid Treatment

The effects of abscisic acid (ABA) treatments on the surfaceelectrical properties of cells and isolated protoplasts fromCatharanthus roseus cell suspension cultures were studied byelectrophoretic mobility and 9-aminoacridine (9AA) fluorescencemeasurements. The surface charge densities of the cells andprotoplasts estimated from electrokinetic data were –0.064Cm^–2and –0.048 C m^–2 respectively. These values wereclose to that estimated by 9AA fluorescence technique i.e.,–0.053 Cm^–2 for the cells and –0.041 Cm^–2for the isolated protoplasts accordingly. The net negative surfacecharge density decreased after application of 10 µM and50 µM ABA in both cells and protoplats, the more pronouncedeffect being observed at 10 µM ABA. When 100 µMABA was supplemented to the cell suspension culture the oppositeeffect was observed. The average charge density increased to–0.074 C m^–2 for the cells, and to –0.055C m^–2 for protoplasts, as revealed from the 9AA measurements.The results are discussed in terms of specific concentrationdependent ABA-induced alterations of the electrostatic propertiesof cell and protoplast membranes. (Received December 12, 1994; Accepted April 3, 1995)     

No Light Activation and High Malate Sensitivity of Phosphoenolpyruvate Carboxylase in Guard Cell Protoplasts of Commelina communis L.

Guard cell protoplasts (GCP) were prepared from leaves of Commelinacommunis L. and phosphoenolpyruvate carboxylase (PEPc) activityrecorded after injection of the protoplasts directly into theassay medium. The GCP were lysed immediately by the presenceof Triton X-100 and a lowered osmotic concentration in the assaycuvette enabling PEPc activity to be measured with ‘nascent’enzyme. There was no light activation of the enzyme with K_mPEP (about 3.7 mol m^–3) and Vmax being similar in light-ordark-treated protoplasts. Illumination of the GCP in the presenceof CO₂-free air and KCI, a treatment which is known to swellGCP, did not change the kinetics. PEPc activity at saturating PEP was very sensitive to malateinhibition, 20 mmol m^–3 (the I₅₀ value) inhibiting activityby about 50%. Inhibition was similar in light- or dark-treatedprotoplasts. Malate inhibition was, however, much less (I₅₀= 500 mmol m^–3) if the enzyme source was a protoplastextract kept in the absence of glycerol. Inclusion of 20% glycerolin the extraction medium maintained the enzyme in the malate-sensitiveform as occurred in the in vivo assays. The high apparent K_mPEP and the high sensitivity to malate inhibition of GCP PEPcare features unlike those observed with PEPc from leaf tissuesof C₄ and CAM plants and from GCP extracts. PEPc activity increased slightly in the presence of KCI in theassay medium up to about 10 mol m^–3 and thereafter activityslowly declined as KCI concentrations increased further. Key words: Guard cell protoplasts, phosphoenolpyruvate carboxylase     

Effects of Aluminium on Honeylocust (Gleditsia triacanthos L.) Seedlings in Solution Culture

Honeylocust (Gleditsia triacanthos L.) seedlings were grownin solution culture at pH 4.0) with 50, 150, 600 and 1500 mmolm^–3 aluminium. All levels of aiuminium reduced the sizeand weight of roots, shoots and leaves with the exception ofroot elongation at 50 mmol m^–3 Al. Aluminium content ofroots was 50 to 100 times that of shoots. With increasing concentrationof aluminium, aluminium content of leaves and roots increasedexponentially while a linear increase was observed for stems.The nutrient content of seedlings was improved in 50 mmol m^–3where increases in shoot calcium, magnesium, and phosphorusconcentrations were observed. Aluminium concentrations greaterthan 50 mmol m^–3 reduced shoot nutrient content. Presenceof aluminium increased the root phosphorus and calcium levelsbut had no effect on potassium and magnesium concentrations.Results show that honeylocust is an aluminium sensitive treespecies whose growth may be reduced by high soil Al levels. Key words: Aluminium toxicity, Gleditsia triacanthos, nutrient solution     

The Generation of Non-Spherical Oat Protoplasts Indicates that Hyperpolarization is both Necessary and Sufficient for Stimulating Membrane Incorporation into the Plasma Membrane

We have devised conditions which produced isolated protoplastsof non-spherical shape and which, therefore, affected the mechanismsthat control the exchange of membrane material between the plasmamembrane and an intracellular membrane reservoir. Non-sphericalprotoplasts of Avena sativa were obtained if protoplasts weretreated with hypertonic shock in the presence of 1.0 mol m^–3LaCl₃ at pH 8.3. This indicated that their ability to removeplasma membrane material via endocytotic vesiculation was suppressed.Non-spherical protoplasts were obtained under isotonic conditionsif protoplasts were incubated with 1.0 mol m^-3 LaCl₃ at pH 8.3and the proton carrier CCCP (12 mmol m^–3) was added. Thenon-spherical protoplasts had intact membranes as judged bystaining with fluorescein diacetate. The loss of the sphericalshape was reversible. On addition of EDTA protoplasts resphericulatedimmediately. Incubation in isotonic solution at pH 8.3 containingeither only 1.0 mol m^–3 LaCl₃ or only CCCP did not influencethe protoplast shape. We conclude that the membrane hyperpolarizationinduced by CCCP at high pH acted to stimulate the incorporationof membrane material into the plasma membrane and, subsequently,produced nonspherical protoplasts if the removal of membranematerial was simultaneously suppressed. This demonstrates thatmembrane incorporation and removal are two largely independentprocesses.     

Characterization of the vacuolar-type H+-ATPase from guard cell protoplasts of Commelina

Characteristics of the vacuolar-type (V-type) H⁺-ATPase fromguard cell protoplasts of Commelina communis L. were investigatedusing a linked enzyme assay and nitrate inhibition as a diagnosticindicator of the enzyme activity. ATPase activity was completelyinhibited by about 50 mol m^–3 nitrate and activity wasoptimal near pH 8.0. The temperature optimum for activity wasabout 37 C and an Arrhenius plot indicated changes in activationenergy for the ATPase at 15C and possibly at about 30 C. Theenzyme was stimulated by Cl^– while Ca²⁺ inhibited activity(l₅₀ = 1.5 mol m^–3). The apparent K_m (MgATP) was 0.62mol m^–3. Incubation of guard cell protoplasts for up to 5 h in 50 µMabscisic acid (ABA) or 25µM fusicoccin (FC) did not affectsubsequent ATPase activity. In vitro assays with FC or ABA alsodid not affect enzyme activity. Activity was not affected bylight or potassium ferricyanide, two factors which are knownto influence stomatal activity. Beticoline was a potent inhibitorof activity (l₅₀ = 50 µM) while DCCD was less effective(l₅₀ = 90µM). On chlorophyll, protein and protoplast bases, V-type ATPaseactivity was greater in guard cell protoplasts than mesophyllcell protoplasts by 66, 13.9 and 1.9, respectively. On atonoplast surface area basis the enzyme activity was 5.6 timeshigher in guard cell protoplasts than in mesophyll cell protoplasts Thus, although the characteristics of the V-type, H ⁺-ATPaseof GCP are very similar to those found in other cell types,rates of activity and probably tonoplast enzyme density aremuch greater in guard cell protoplasts than mesophyll cell protoplastsof C. communis which corresponds with the large and rapid ionfluxes across the tonoplast associated with stomatal movements Key words: Guard cell protoplasts, stomata, V-type H ⁺-ATPase     

Electroporation Stimulates Plant Regeneration from Protoplasts of the Woody Medicinal Species Solarium dulcamara L.

Exposure of cell suspension protoplasts of the woody medicinalplant Solatium dulcamara L. to voltages of 250 to 1250 V cm^–1for three successive pulses, each of 10–50 us duration,stimulated growth of protoplast-derived tissues. Such tissuesexhibited increased morphogenesis and required a shorter periodin culture to exhibit this effect than tissues from untreatedprotoplasts. Regenerated shoots also rooted more readily anddeveloped more prolific root systems than shoots from untreatedprotoplasts. These observations have important implicationsfor plant genetic manipulation and may have application in therecovery and rooting of shoots from tissues of woody species,normally considered recalcitrant in culture. Key words: Electroporation, protoplasts, shoot regeneration, Solanum dulcamara (woody nightshade, bittersweet)     

Carrier-Mediated ABA Uptake by Suspension-Cultured Phaseolus coccineus L. Cells: Stereospecificity and Inhibition by Ionones and ABA Esters

Astle, M. and Rubery, P. 1987. Carrier-mediated ABA uptake bysuspension-cultured Phaseolus coccineus L. cells: Stereospecificityand inhibition by ionones and ABA esters.—J. exp. Bot.38: 150–163. The substrate for the abscisic acid (ABA) carrier in Phaseoluscoccineus L. suspension-cultured cells is shown to be the (S)ABAenantiomer, K_m = 1?0 mmol m^–3. The methyl (MeABA) andphenyl (PheABA) esters of ABA inhibit carrier-mediated uptakeof ABA with half-maximal inhibition achieved at about 7?0 mmolm^–3 and 10 mmol m^–3 respectively: with (S)MeABAthis value is decreased to about 2?0 mmol m^–3. There isno demethylation of radioactive MeABA by the cells during 5min incubations. Although MeABA reversibly inhibits the ABAcarrier, it is not a transport substrate: association of radioactiveMeABA with living cells is unaffected by non-radioactive MeABAor ABA and, by comparison with frozen-and-thawed cells, it isshown that the radioactivity remains extracellular. It is proposedthat MeABA binds to the carrier to form an abortive complexthat is not translocated. The terpenoid ABA analogue LAB 144143also inhibits carrier-mediated ABA uptake. At concentrationsup to about 20 mmol m^–3 - and ß-ionone specificallyinhibit the ABA carrier with the half-maximal effect at about0?6 mmol m^–3 ß-ionone. However, at higher iononeconcentrations, the uptake of ABA, indol-3-yl acetic acid andof 5,5-dimethyloxazolidine-2,4-dione (DMO) are all stimulated:this may reflect general permeabilization of the membrane toweak acids by ionone. Key words: Uptake carrier, abscisic acid, methyl and phenyl esters of ABA, ionone, Phaseolus coccineus L. suspension culture     

Vanadate Sensitive ATPase and Phosphatase Activity in Guard Cell Protoplasts of Commelina

Flicker, M. D. and Willmer, C. M. 1986. Vanadate sensitive ATPaseand phosphatase activity in guard cell protoplasts of Commelina.—J.exp. Bot. 38: 642–648. Phosphatase activity was measured in extracts of guard cellprotoplasts of Commelina communis L. using the artificial substratep-nitrophenylphosphate. A pH optimum of 5.8 to 6.3 was determined.Ammonium molybdate (Ol mol m^–3) and sodium vanadate (1–0mol m^–3) gave almost complete inhibition of phosphataseactivity at pH 60. ATPase assays were, therefore, conductedin the presence of 0–2 mol m ^–3 molybdate and vanadatewas used as a specific inhibitor of plasmamembrane ATPase activity.Vanadate sensitive ATPase activity showed a pH optimum of 6.6and activity was stimulated by KC1. These properties are characteristicof plasmamembrane proton pumping ATPases in other systems andsuggest that proton extrusion in guard cells could be mediatedby a similar enzyme. The maximum ATPase activity is sufficientto account for all the proton flux observed during the stomatalopening response. Key words: ATPase, Commelina, guard cell protoplasts, phosphatase, vanadate     

The Investigation of Stomatal Ionic Relations using Guard Cell Protoplasts: I. METHODOLOGY

Clint, G. M. 1985. The investigation of stomatal ionic relationsusing guard cell protoplasts. 1. Methodology.—J exp. Bot.36: 1726–1738. A study was made of the methodology for the production and useof guard cell protoplasts in ion transport studies, with particularemphasis placed on the effects of the composition of the externalmedium on protoplast survival and performance. Addition of externalKCl to media during the production of guard cell protoplastsfrom Commelina communis L. was found to improve viability andto increase K⁺ content and physiological competence of the isolatedprotoplasts. Addition of low levels (20 x 10^–3 mol m^–3)CaCl₂ increased protoplast yield and the maintenance of viabilityin long-term incubation. Ambiguities and uncertainties werefound in the application of methods commonly used for the assessmentof viability of isolated protoplasts. Poor yields (despite highpercentage recoveries) together with difficulties in the assessmentof viability were considered to pose major potential problemsin the use of guard cell protoplasts in ion transport studies. Key words: Guard cell protoplasts, ion transport, Commelina communis     

Salt Tolerance in the Succulent, Coastal Halophyte, Sarcocornia natalensis

The effects of 0, 50, 100, 200, 300, 400 and 500 mol m^–3NaCl on growth and ion accumulation in the succulent, coastalhalophyte Sarcocornia natalensis (Bunge ex Ung.-Sternb.) A.J. Scott were investigated. Increase in salinity from 0 to 300 mol m^–3 NaCl stimulatedproduction of fresh, dry, and organic dry mass, increased succulenceand shifted resource allocation from roots to shoots. Growthwas optimal at 300 mol m^–3 and decreased with furtherincrease in salinity. Water contributed to a large proportion of the increase in freshmass. Inorganic ions, especially Na⁺ and Cl– contributedsubstantially to the dry mass. At 300 mol m^–3 NaCl inorganicions contributed to 37% of total dry mass and NaCl concentrationin the shoots was 482 mol m^–3. Expressed sap osmotic potentialsdecreased from –2.10 to –3.95 MPa as salinity increasedfrom 0 to 300 mol m^–3 NaCl. Massive accumulation of inorganicions, especially Na⁺ and Cl^–, accounted for 86% of theosmotic adjustment at 300 mol m^–3 NaCl. Salinity treatments decreased the concentrations of K+ in shoots.Plant Na+ :K+ ratios increased steadily with salinity and reacheda maximum of 16.6 at 400 mol m3 NaCl. It is suggested that the exceptional salt tolerance of S. natalensisis achieved by massive inorganic ion accumulation which providessufficient solutes for osmoregulation, increased water fluxand turgor-induced growth. Key words: Sarcocornia natalensis, salt tolerance, halophyte     

Plant Growth in Relation to the Supply and Uptake of NO3-: A Comparison Between Relative Addition Rate and External Concentration as Driving Variables

Two approaches to quantifying relationships between nutrientsupply and plant growth were compared with respect to growth,partitioning, uptake and assimilation of NO₃^– by non-nodulatedpea (Pisum sativum L. cv. Marma). Plants grown in flowing solutionculture were supplied with NO₃^– at relative addition rates(RAR) of 0·03, 0·06, 0·12, and 0·18d^–1, or constant external concentrations ([NO₃^–)of 3, 10, 20, and 100 mmol m^–3 over 19 d. Following acclimation,relative growth rates (RGR)approached the corresponding RARbetween 0·03–0.12 d^-1, although growth was notlimited by N supply at RAR =0.18 d^-1. Growth rates showed littlechange with [NO₃–] between 10–100 mmol m^–3(RGR=0·15 –0·16 d^-1). The absence of growthlimitation over this range was suggested by high unit absorptionrates of NO₃^–, accumulation of NO₃^– in tissues andprogressive increases in shoot: root ratio. Rates of net uptakeof NO₃^– from 1 mol m^–3 solutions were assessed relativeto the growth-related requirement for NO₃^–, showing thatthe relative uptake capacity increased with RGR between 0·03–0·06d^–1 , but decreased thereafter to a theoretical minimumvalue at RGR     

Phosphate uptake and transport in Agrostis capillaris L Effects of non-toxic levels of aluminium and the significance of P and Al speciation

Investigations on the effects of low levels of Al on P adsorption,uptake and translocation in seedlings of the indigenous grassAgrostis capillaris were undertaken. Apparent uptake and transportof H₂ ³²PO₄ from nutrient solutions containing 10 or 100mmolm^–3 phosphate were characterized as functions of timeand concentration. Experiments on ³²P uptake and transport insolutions containing no Al (control) or Al ranging from 3.7to 185 mmol m^–3 at pH ranging from 4.3 to 4.6, showedthat in 10 mmol m^–3 P, effects of Al at 3.7 and 37 mmolm^–3 on the size of the initial uptake shoulder were small,but some increase in subsequent P uptake to the roots was observed,though transport to the shoots was suppressed. With 37 mmolm^–3 Al in nutrient solution containing 100 mmol m^–3P, the uptake shoulder was much increased above the control.Subsequent root uptake was stimulated but transport was unaffected.Lack of toxicity of the Al concentrations used was indicatedby a lack of significant effect on plant fresh weight. AbsorbedAl was almost totally retained in the root in all treatments.Speciation calculations showed that the major species in Alamended nutrient solution at pH 4.4 were H₂PO₄^–, AI³⁺and AIHPO₄⁺, together with substantial amounts of AISO₄⁺ andsoluble aluminium hydroxy complexes (AIOH²⁺, AI(OH)₂⁺), dependingon the relative concentrations of P and Al. The effects of Al,with 10 mmol m^–3 P, on adsorption of complexed P werepartly accounted for in terms of preferential cell wall adsorptionof Al complexes not containing P. Conclusions were drawn aboutthe P-economy of A. capillaris plants growing on soils withlow levels of P and Al. Key words: Phosphorus, aluminium, speciation, Agrostis capillahs L     

Direct Effects of Ca2+-Channel Blockers on Plasma Membrane Cation Channels of Amaranthus tricolor Protoplasts

Ca²⁺-channel blockers at concentrations greater than 1 mmolm^–3, directly affect the activity of K ⁺selective channelsin the plasma membrane of Amaranthus tricolor protoplasts. Theseeffects are not mediated by the blockade of Ca²⁺ channels. Blockers tested included 1, 4-dihydropyridines (nifedipine,nicardipine), verapamil, bepridil, Gd³⁺ and La³⁺, applied towhole-cell and detached outside-out patches of plasma membraneat concentrations from 50µmol m^–3 to 100 mmol m^–3.For certain experiments the concentration of Ca²⁺ on the cytoplasmicside of the plasma membrane ([Ca²⁺]cyt) was buffered at either50ftmol m^–3 or 500 µmol m^–3. The principal currents observed in whole-cells flowed throughcation outward rectifier (OR) channels. Each blocker causedan immediate reduction of time-dependent outward currents atdoses down to 1 mmol m^–3 and produced a different, reversible,kinetic block of the outward current, independent of the levelof [Ca²⁺]cyt. Verapamil also activated a sustained inward cationcurrent at negative p.d. The same effects were found with individualchannels in detached outside-out patches. Conductance and selectivityof the cation OR channels were unchanged by the drugs. [Ca²⁺]ex, was varied over a range from 0 to 10 mol m^–3.Progressively lower [Ca²⁺]eI, increasingly enhanced the maximumamplitude of the time-dependent currents. Time-constants fordecay of inward tail currents were increased at low [Ca²⁺]eit.These effects were rapidly reversible. Although there was noevidence that the cation ORs in plasma membrane of Amaranthustricolor were dependent on [Ca²⁺]cyl for their activation, theywere sensitive to the concentration of free Ca²⁺ in the extracellularmedium. Key words: Verapamil, blocker, cation channels, Amaranthus, protoplasts     

Determination of Volume of Dunaliella Cells by Lithium Dilution Measurements and Derivation of Internal Solute Concentrations

A method has been developed to measure the cell volume of theunicellular green alga Dunaliella parva 19/9 using Li⁺ measurementsonly. Concentrations of internal solutes can also be calculatedif they are assayed in the same samples as Li⁺. We found thatD. parva cells grown in 0.4 kmol m^–3 NaCl have an averageaqueous cell volume of 65.1 ?2.9 µm³, a K⁺ concentrationof 126?6 mol m^–3, a Na⁺ concentration of 11?11 mol m^–3and a glycerol concentration of 615?27 mol m–3 (n= 12).Algae grown in 1.5 kmol m^–3 NaCl have an average aqueouscell volume of 131 ?7.5 µm³, a K⁺ concentration of 109?4mol m^–3, a Na⁺ concentration of 10?39 mol m^–3 anda glycerol concentration of 1 425?59 mol m^–3 (n = 12).These results indicate that D. parva cells adapted to high salinitieshave larger cell volumes than those adapted to lower salinities.However, there is no evidence for a significant difference ininternal Na⁺ concentration, despite the almost 4-fold differencein the concentration of external NaCl. The intracellular glycerolconcentration alone accounts for 65% and 54%, respectively,of the osmotic balance in low and high salt grown cells. Key words: Dunaliella, cell volume, intracellular solutes     

Nitrate Inhibition of Chloride Influx in Barley: Implications for a Proposed Chloride Homeostat

Net accumulation of Cl^– by intact barley plants was virtuallyeliminated in roots and reduced by 40% in shoots when externalmedia (0.5 mol m^–3 CaSO₄ plus 0–5 mol m^–3KCI) were supplemented with 0.25 mol m^– Ca(NO₃)₂. Plasmalemma³⁶Cl^– influx (_oc) was shown to be insensitive to externalNO₃^- in plants which had previously been grown in solutionslacking ^–3, but _oc became sensitive to NO₃^-after a lagperiod of 3–6 h. Kinetic analyses revealed that the inhibitionof 36C1^– influx by external NO₃^- was complex. At 0.25mol m^–3 NO₃^- the V_max for Cl^– influx was reducedby greater than 50%, with insignificant effects upon K_m. At0.5 mol m^–3 NO₃^- there was no further effect upon V_maxbut K_m for influx increased from 38±5 mmol m^–3to 116±26 mmol m^–3. By contrast, Cl^– effluxwas found to be insensitive to external NO₃^-. A model for theregulation of Cl^– influx is proposed which involves bothnegative feedback effects from vacuolar NO₃^- +Cl^–) concentrationand (external) NO₃^- inhibition of Cl^– influx at the plasmalemma.These combined effects serve to discriminate against Cl^–accumulation, favouring NO₃^- accumulation, when the latter ionis available. Such observations are inconsistent with recentproposals for the existence of bona fide homeostats for chlorideaccumulation in higher plants. Key words: Nitrate inhibition, Chloride influx, Barley     

Effect of Salinity on Growth, Nodulation and Nitrogen Yield of Chickpea (Cicer arietinum L.)

Chickpea cultivar ILC 482 was inoculated with salt-tolerantRhizobium strain Ch191 in solution culture with different saltconcentrations added either immediately with inoculation or5 d later. The inhibitory effect of salinity on nodulation ofchickpea occurred at 40 dS m^–1 (34.2 mol m^–3 NaCl)and nodulation was completely inhibited at 7 dS m^–1 (61.6mol m^–3 NaCl); the plants died at 8 dS m^–1 (71.8mol m^–3 NaCl). Chickpea cultivar ILC 482 inoculated with Rhizobium strain Ch191^spcstrwas grown in two pot experiments and irrigated with saline water.Salinity (NaCl equivalent to 1–4 dS m^–1) significantlydecreased shoot and root dry weight, total nodule number perplant, nodule weight and average nodule weight. The resultsindicate that Rhizobium strain Ch191 forms an infective andeffective symbiosis with chickpea under saline and non-salineconditions; this legume was more salt-sensitive compared tothe rhizobia, the roots were more sensitive than the shoots,and N₂ fixation was more sensitive to salinity than plant growth. Key words: Cicer arietinum, nodulation, N₂ fixation, Rhizobium, salinity     

Nitrate Transport into Chara corallina Cells using 36ClO-3 as an Analogue for Nitrate: I. INTERACTION BETWEEN 36ClO-3 AND NO-3 AND CHARACTERIZATION OF 36CIO-3/NO-3 INFLUX

The use of chlorate as an analogue for NO^–₃ during nitrateuptake into Chara corallina cells has been investigated. NO^–₃inhibits ³⁶C1O^–₃ influx into Chara over the concentrationrange 0–1000 mmol m^–3. Lineweaver-Burke plots ofthe data are characteristic of competitive inhibition by NO^–3in the low concentration range (0–300 mmol m^–3 ClO^–₃)and apparent K_INO3 is 140 mmol m^–3 which is of a similarorder of magnitude as apparent K_mCIO3- 180 mmol m^–3. Athigher substrate concentrations the inhibition by NO^–₃was not characteristic of competitive or uncompetitive inhibition. ³⁶C1O^–₃/NO^–₃ influx was dependent on K⁺ and Ca²⁺in the external medium and inhibited by FCCP. NO^–₃ pretreatmentor N starvation increased subsequent ³⁶C1O^–₃/NO^–₃influx into Chara. A comparison between rates of net NO^–₃uptake and ³⁶C1O^–₃/NO^–₃ influx supported the previoushypothesis that NO^–₃ efflux is an important componentin the determination of overall uptake rates. Key words: Nitrate, Chara, ³⁶CIO^–₃     

Role of Copper Binding, Absorption, and Translocation in Copper Tolerance of Agrostis gigantea Roth

The capacity of roots to accumulate and retain copper was examinedin two clones of Agrostis gigantea which differ in their toleranceto excess copper. Root elongation growth in the non-tolerantclone was completely inhibited by 16 mmol m^–3 Cu whereas40 mmol m^–3 was required for inhibition in the tolerantclone. The amount of readily exchangeable copper was greaterin roots of the tolerant clone than in the non-tolerant clone.The higher capacity for binding copper did not prevent the entryof copper into the cells of intact or excised roots of the tolerantclone. Roots of both clones contained similar amounts of copperafter removal of the readily exchangeable fraction. More copperwas translocated to the shoots of the tolerant than the non-tolerantclone. The explanation of copper tolerance in Agrostis giganteamust be sought in areas other than those of differences in grosscopper absorption and retention by roots.