Effect of Temperature on the Radial Exchange of Labelled Water in Maize Roots

The temperature dependence of the efflux kinetics of labelledwater in isolated maize roots has been studied. The purposeof these experiments was to obtain the energy of activation,E (kcal/mole), of the rate-limiting step in this radial exchangeprocess under various experimental conditions. Estimates ofE were obtained from linear relations between ln{D'_w} and thereciprocal of the absolute temperature; values of the apparentdiffusion coefficient, D'_w, of labelled water in the root werefound from an analytical treatment of the efflux data in termsof a cylindrical diffusion model. The energy of activation forlabelled-water exchange in normal roots was 14.9 kcal/mole.The corresponding value for ‘dead’ (boiled) rootswas 3.9 kcal/mole. These values of E substantiate the view thatin normal roots the penetration of water across the membranesof the root cells constitutes the rate-determining step in theefflux whereas in ‘dead’ roots extracellular diffusionof water is the source of rate-control. Similar temperature dependence studies were performed on theefflux kinetics from normal and ‘dead’ roots treatedwith 10^–5 M phenylmercuric acetate (PMA). The energiesof activation for labelled-water exchange in normal and ‘dead’roots under these conditions were respectively 15.5 and 5.3kcal/mole. Moreover, the results of the efflux experiments onPMA-treated roots were considered to indicate that this inhibitorproduces an alteration in some structural aspect of the rate-controlling‘membranes’.     

Effects of Inhibitory Concentrations of 3-Indolylacetic Acid and 3-Indolylacetonitrile on Cell Division and Tissue 2

Inhibitory concentrations of 3-indolylacetonitrile (IAN) cause,in cultured excised tomato roots, a marked decrease in the rateof cell division at the apical meristem but only a slight reductionin the lengths of mature exodermal and cortical cells. The reducedrate of cell division is associated with a decrease in the.number of meristematic cells at the root apex. By contrast,3-indolylacetic acid (IAA) causes marked reduction in the lengthsof mature cortical cells but does not markedly reduce cell-divisionrate at the apical meristem. Various lines of evidence indicate that both IAA and IAN causea relative increase in the number of longitudinal and a decreasein the number of transverse division walls in the meristematiczone of the root apex. Partial inhibition of the linear growth of excised tomato rootsby IAA and IAN is accompanied by increases in root and stelardiameters. These increases result from radial enlargement ofthe cortical cells and increase in the number of stelar cellsin the transverse section. The enlarged steles contain an increasednumber of lignified xylem elements, but only with the most inhibitoryconcentration of IAN (10^–4g./ml.) is there evidence ofthe development of secondary xylem. Both auxins increase significantlythe xylem vessel unit length.     

Effects of External Ca2+ on K+ Permeability of the Elongating Region of Mung Bean Roots under High KCl Stress

Simultaneous measurements of the extracellular potential andthe K⁺(⁸⁶Rb) efflux, and of the intracellular and extracellularpotentials of the cortical cells were used to study the effectsof external Ca²⁺ on the plasma membrane K⁺(⁸⁶Rb) permeabilityin two-day-old mung bean (Vigna mungo L. Hepper, ‘Blackmatpe’) roots under high KCl stress. The K⁺ efflux wasenhanced by a high KCl solution (>7.5 mM), and addition of0.5 mM Ca²⁺ could suppress this efflux. The removal of membrane-associatedCa⁺ from the root surface with EDTA led to a recovery of theK⁺ efflux along with a marked decrease in the extracellularpotential. (Received November 19, 1986; Accepted March 6, 1987)     

Extrusion of Sodium Ions by Barley Roots: II. Localization of the Extrusion Mechanism and its Relation to Long-distance Sodium Ion Transport

An active sodium effux hasbeen demonstrated in excised barleyroots. Isolated cortical and stelar tissues from the same materialdo not show any evidence of such an efflux mechanism. When intactbarley plantswere allowed to absorb ²²Na active sodium extrusionwas not observed, although the percentage of ²²Na transportedto the shoot was stimulated 20 to 40 percent by ouabain at 5x 10^–4 M. Absorption and long-distance transport of phosphatewas unaffected by ouabain at this concentration. Interpretationof these data lead to the conclusion.that the sodium-effluxpump in barley roots is located at or near the endodermis externalto the Casparian band. This pump thus provides a mechanism wherebysodium can be partially excluded from the shoots of barley plants.     

Immunofluorescence Microscopy of Microtubule Arrangement in Root Cells of Pisum sativum L. var Alaska

The arrangement of wall microtubules (MTs) in Pisum sativumroots was viewed immunofluorescently using cryosectioning. Mostcells in the tip region of pea roots (0–2 mm from tip)had wall MTs arranged transversely to the root axis. In theregion elongating at a higher rate (2–4 mm), wall MTsof epidermal, cortical and stelar cells were all transverselyarranged. In the region of about 5 mm from the tip, in whichcell elongation had already ceased, wall MTs in cortical cellschanged from a transverse to an oblique arrangement in relationto the root axis. Some cells had a crossed arrangement of wallMTs, which was interpreted as representing two sets of unidirectional,oblique wall MTs in opposite cell cortices of a single cell.This change was completed within a region of 1-mm width. Sinceroots elongated at a rate of 0.6 mm h^–1, it means thatthe arrangement of wall MTs changed within 2 h. An oblique arrangementof wall MTs was also observed in stelar cells. As the cellsaged, the oblique arrangement tended to change to a steeperor even a longitudinal one. (Received January 24, 1986; Accepted May 15, 1986)     

Pattern of Respiration of a Perennial Ryegrass Crop in the Field

‘Dark’ respiratory losses of CO₂ were measured ona one year old sward of S24 perennial ryegrass (Lolium perenneL.) at intervals during a 74 day reproductive growth period,between April and June, and a 21 day vegetative growth period,in July and August. Part of the sward was shaded for one weekbefore measure ments commenced. Measurements of ‘dark’respiration continued for 46 hand it was possible to distinguishtwo components which are designated ‘maintenance’and ‘synthetic’ ‘Maintenance’ respiration was taken to be the meanrate of CO₂ efflux after 40–46 h darkness. When calculatedon a plant d. wt basis at 15°C it ranged between 6 to 32mgCO₂ g^-1 day^-1 during reproductive growth and 10–14 mgCO₂ g^-1 day^-1 during vegetative growth. During reproductivegrowth, sward protein content ranged between 7–23 percent and when maintenance respiration was recalculated on thebasis of protein content it changed relatively little throughoutthe growth period (90–140 mg CO₂ g pro tein^-1 day^-1);the value for vegetative growth ranged between 70–100mgCO₂ g protein-day^-1. Total ‘synthetic’ CO₂ flux was determined duringreproductive growth and a rate of ‘synthetic’ CO₂flux was determined during both reproductive and vegetativegrowth. Between 15 and 35 per cent of the CO₂ fixed in the previousphotoperiod was lost in ‘synthetic’ respirationof above-ground material in reproductive swards. Previous shadingincreased the proportion of ‘synthetic’ CO₂ lossfrom above ground. The rate of ‘synthetic’ CO₂ outputduring the first hours of darkness increased with amount ofCO₂ fixed in the previous photoperiod, although it was not proportionalto it. There is some evidence that assimilate is ‘carried-over’from one photoperiod to the next.     

In Vitro Morphogenesis in Nardostachys jatamansi DC.: Shoot Regeneration from Callus Derived Roots

Callus cultures of Nardostachys jatamansi DC. maintained onMurashige and Skoog's medium containing 3.0 mg 1^–1 of-naphthaleneacetic acid and 0.25 mg 1^–1 of kinetin whenshifted to medium containing 0.25–1.0 mg 1^–1 ofindole-3-acetic acid or indole-3-butync acid showed profuserhizogenesis. The callus-regenerated roots when transferredto medium containing 2.0–6.0 mg 1^–1 of kinetin producedshoot buds. The de novo shoot bud regeneration took place eitherdirectly from cortical cells or from the inner stelar region.In addition, direct, concomitant root-shoot development wasalso observed. Nardostachys jatamansi, organogenesis, root-buds     

Characterization of the Ca2+-Dependent Cl- Efflux in Perfused Chara Cells

The mechanism of the Ca²⁺-dependent Cl^– efflux was studiedin tonoplast-free cells, in which the intracellular chemicalcomposition can be freely controlled. Tonoplast-free cells wereprepared by perfusing the cell interior of internodal cellsof Chara corallina with a medium that contained EGTA. The Ca²⁺-inducedCl^– efflux was measured together with the membrane potentialduring continuous intracellular perfusion. The dependenciesof Cl^– efflux and the membrane potential on the intracellularCa²⁺ or Cl^– concentrations were analyzed. When perfusionwas started with medium that contained Ca²⁺ ions, Cl^–efflux and membrane depolarization were induced. The amountof Cl^– efflux varied considerably among individual cells.The rate of efflux decreased exponentially but a residual effluxremained detectable. The Cl^– efflux was induced at concentrationsof Ca²⁺ ions above 1 µM and reached a maximum at 1 mM.By contrast, the membrane depolarization reached a maximum atabout 10 µM Ca²⁺. The rate of Cl^– efflux increasedlinearly with logarithmic increases in the intracellular Cl^–concentrations. These findings suggest that more than two kindsof Ca²⁺-dependent Cl^– channel might be present in theplasma membrane. Addition of ATP or its removal from the perfusion medium didnot affect the Ca²⁺-dependent Cl^– efflux. Calmodulin antagonistsslightly inhibited the Ca²⁺-dependent Cl^– efflux. ¹Present address: Biological Laboratory, Hitotsubashi University,Naka 2-1, Kunitachi, Tokyo, 186 Japan.     

An Analysis of the Growth of Young Tomato Plants in Water Culture at Different Light Integrals and CO2 Concentrations: I. Physiological Aspects

Young tomato plants were grown from germination in water cultureat light-flux densities from 6 to 110 W m^-2 (400–700 nm),daylengths from 8 to 24 h and CO₂ concentrations from 0.4 to2.2 g CO² m^-3 in controlled environment cabinets. The growth rates and net assimilation rates of 14–17-day-oldplants at the highest light integrals were appreciably greaterthan most values previously recorded for tomato, and diminishedwith time. Plants in the lowest light conditions had leaf arearatios five times larger than those in the highest light, attributablemainly to a difference in leaf dry weight/area. Such flexibilityin leaf area ratio has not previously been associated with ‘sun’plants such as the tomato. Relatively normal growth was obtained in continuous light, incontrast to most other reports. This may have been due to theuse of conditions which would minimise water stress. The efficiency of the conversion of incident light energy tochemical energy by the whole plant ranged from 15 per cent inseedlings in low continuous light to about 6 per cent, tendingto be higher in young plants in long days under CO₂ enrichment.The higher values are probably overestimates because of theexclusion of reflected light from the energy receipt values.     

Evidence for Symplasmic Ion Transport in Maize Roots

Excised maize roots, placed in saturated water vapour to limitthe external ionic supply, continued to produce exudates attheir basal ends for at least 24 h. The mean rate of fluid exudationfrom roots in water vapour was about 28 per cent of the correspondingrate in ‘control’ roote placed in a solution containing0.1 mil CaCl₂ and 1 mM KC1. Moreover, the net fluxes (mean ±S.E.)of potassium and calcium ions into the exudate were reducedfrom (35.8±3.2) x 10^– and (4.37±0.39) xlO^–9 mole cm^–2 h^– for roots in solution to(10.9±0.6) x 10^–9 and (l.00±0.06)x 10^–9molecm^–2 h^–1 respectively for roots in vapour. It isconsidered that the observation of a prolonged exudation ofwater and ions from the roots placed in water vapour demonstratesthe existence of an alternative ionic supply within the roottissue itself and that this parallel route of ion transportto the exudate constitutes the cortical symplasmic pathway. Pre-treatment of the excised roots with 0.8 M mannitol beforeexudation studies in water vapour and solution led to a significantreduction in the rates of fluid and ion exudation which hadbeen observed in untreated roots under similar conditions. Itis concluded that the plas-molysis, induced by mannitol, disruptedthe symplasmic connections between root cells and that thisperturbation significantly reduced the operation of the symplasmicmode of ion transport into the exudate.     

Transport of Amino Acids in Barley Leaf Tissue: I. EVIDENCE FOR A SPECIFIC UPTAKE MECHANISM AND THE INFLUENCE OF 'AGEING' ON ACCUMULATORS CAPACITY

Amino-acid uptake by barley leaf strips was investigated withthe aid of an unnatural analogue, -aminoisobutyric acid (AIB).Negligible metabolism of this acid by the tissue was detected. Accumulatory capacity rose with ‘ageing’ of tissuein 10^–4 M CaSO₄. This effect was not attributable to bacterialcontamination. Net uptake was linear with time for at least5 h and proceeded against a strong concentration gradient. Therate constant for the loss of previously accumulated labelledAIB into water was one-tenth of that into unlabelled AIB. When influx and efflux were estimated separately, ‘ageing’was found to have reduced the rate of exit by a factor of 4.The rise in influx, though proportionately smaller, was largerin absolute value.     

The effect of CCCP on ion fluxes in the stele and cortex of maize roots

Summary The accumulation of ⁸⁶Rb labelled potassium by isolated stelar and cortical tissues from 7-day-old roots of Zea mays has been compared with the levels accumulated by these tissues in the intact root. Cortical tissues have similar uptake eapacities in these two conditions whereas stelar tissues only exhibit an uptake capacity in the intact root system. The uncoupler carbonylcyanide m-chlorophenylhydrazone caused a considerable decrease in the uptake of potassium by these tissues. In the intact root system it prevented ions from the bathing medium reaching the stelar tissues. The efflux pattern from preloaded isolated stelar and cortical tissues was considerably altered by the inhibitor, a promotion of the efflux occurring in both of these tissues.It is concluded that stelar tissues only accumulated ions when these are supplied through the root symplasm and that the stelar plasmalemma has only a limited uptake capacity per se. Stelar uptake is thus a reflection of vacuolar accumulation across the tonoplast. There is no evidence in the present study of a carrier-mediated active secretion of ions across the stelar plasmalemma. The fact that the efflux was promoted rather than depressed by the uncoupler supports the postulate that a passive leakage is the final stage in the transport of ions across the plant root.     

Respiratory Efflux of Carbon Dioxide from Mature and Meristematic Tissue of Uniculm Barley During Eighty Hours of Continuous Darkness

Young plants of uniculm barley were grown singly in pots ina growth room at 23/21 °C, and an irradiance of 655 µEm^–2s^–1 during each 12 h photoperiod. At the fifth leaf stage,they were subjected to 80 h of continuous darkness during whichthe rates of CO₂ efflux of vegetative shoot meristems, and maturefully expanded leaves, were separately monitored. Respiratoryefflux from the meristematic tissue was initially high, 12–15mg CO₂ g^–1 h^–1, equivalent to a daily loss in weightof 20–25 per cent. It remained high, or even rose slightly,during what would have been the normal dark period, but thenfell sharply. Even so, it was still three times that of themature tissue at the end of the experimental period. The rateof CO₂ efflux of the mature tissue began low, and fell evenfurther during the first 12 h of darkness. It then levelledoff at a rate of 2·0–2·5 mg CO2 g^–1h^–1, equivalent to a daily loss in weight of about 3 percent. It is suggested that the rate of ‘mature tissue’respiration, established after 12–24 h of darkness, mightbe a useful selection criterion to employ in attempts to increasethe total dry matter yield of the grass crop by breeding. Hordeum vulgare L., barley, respiration, synthetic respiration, maintenance respiration, meristem, mature tissue respiration, simulated sward     

Analysis of the Diffusion Symmetry Developed by the Alkaline and Acid Bands which form at the Surface of Chara corallina Cells

The diffusion symmetry developed by the alkaline and aeid bandsof Chara corallina was studied. The alkaline system developeda diffusion pattern which could not be fitted to the equationfor a continuous point-source efflux. However, good correlationwas obtained between experimental data and the diffusion equationfor a hollow sphere. The calculated OH- efflux values, obtainedusing the equation of a continuous spherical-surface source,were checked against the influx values of H₁₄ obtained under the same experimental conditions. IndividualOH– band efflux values ranged from 0.07 to 5.95 pmol s^–1and total cell fluxes of 25 pmol cm^–2 s^–1 for OH^-and H were obtained (in the presence of 0.5 mM NaHCO₃). The acid system developed a cylindrical diffusion pattern, butthis could not be fitted to a mathematical equation. Numericalanalysis will have to be employed to obtain values of H⁺ efflux.     

Plasmalemma Transport of HCO3- and OH- in Chara corallina: Non-antiporter Systems

An experimental system was designed to test the obligate couplingbetween HCO₃^– and OH^– fluxes (i. e. a ‘Mitchell-type’antiporter) proposed by Lucas and Smith (1973). The resultsof these experiments demonstrated categorically that the OH^–efflux process can function in the absence of exogenous HCO₃^–at the actual OH^– efflux site. Hence, the obligate couplinghypothesis is invalid. It is proposed that HCO₃^– and OH^–are transported across the plasmalemma ‘independently’,on quite distinct carriers. It is possible, therefore, thatthese fluxes contribute towards determining the electrical propertiesof this membrane when the bathing solution pH value is 6.5.It was also found that HCO₃^– can be transported acrossthe dark segment of a partly illuminated cell. The observedrates were always much less than those obtained in the illuminatedcell segment. The significance of this result is discussed.     

Effect of Inorganic Phosphate on the Biosynthesis of Purine and Pyrimidine Nucleotides in Suspension-Cultured Cells of Catharanthus roseus

The levels of purine and pyrimidine nucleotides in suspensioncultures of Catharanthus roseus were determined 24 h after stationary-phasecells were transferred to fresh complete (‘+Pi’)or phosphate-deficient (‘–Pi’) Murashige-Skoogmedium. The levels of ATP, GTP, UTP and CTP were from approx.3 to 5-fold greater in the cells grown in ‘+Pi’medium than in the cells grown in ‘–Pi’ medium.The levels of almost all other nucleotides were slightly higherin the cells in ‘+Pi’ medium. The rates of de novoand salvage biosynthesis of purine and pyrimidine nucleotideswere estimated from the rates of incorporation of radioactivityfrom [¹⁴C]formate, [2–¹⁴C]glycine, NaH¹⁴CO₃, [6–¹⁴C]orotate,[8–¹⁴C]adenine, [8–¹⁴C]adenosine, [2–¹⁴C]uraciland [2–¹⁴C]uridine. The results indicated that the activityof both the de novo and the salvage pathway was higher in thecells in ‘+Pi’ medium than in the cells in ‘–Pi’medium. The rate of degradation estimated from the rate of releaseof ¹⁴CO₂ from labelled purines and pyrimidines indicated thatdegradation of uridine was significantly reduced in the cellsin ‘+Pi’ medium, but no significant difference wasfound in the degradation of adenine, adenosine and uracil. Thepossible role of Pi in the control of the biosynthesis of nucleotidesand in the degradation of uridine is discussed. Catharanthus roseus, Madagascar periwinkle, suspension culture, inorganic phosphate, nucleotides, purines, pyrimidines, biosynthesis, degradation     

The Water Permeability of Unplasmolysed Tissues2

1. The permeability of unplasmolysed cells of beetroot, v. ‘CrimsonGlobe’, was determined from the rate of water loss ofbeet slices on placing in sucrose solutions having O.P. greaterthan the suction pressure of the beet. The absolute values obtainedwere about 0?7µ³ water per µ² cell-surface per hourper atmosphere osmotic pressure difference, i.e. 0?7 µ/hr./atm.
2. The permeability of similar beet cells plasmolysed withintheircell walls was found to be about 13µ/hr./atm.
3. Thepermeability of beet cells which had been plasmolysed andallowedto recover was shown to be approximately the same asthat ofunplasmolysed cells.
4. The hypothesis is advanced that the increasein water permeabilityon plas-molysis is due to those partsof the plasma-membranewhich had formerly been pressed againstthe micelles of thecell wall becoming free and able to takepart in water transfer.
5. The energy requirement for the maintenanceof an excess hydrostaticpressure of five atmospheres withina cell by its vital activitywas shown to be about one-tenthof the total respiratory energyreleased in freshly cut beetslices.

     

Longitudinal Water Movement in the Primary Root of Zea mays

The rates of transfer of tritiated water (THO) along lengthsof excised primary roots of Zea mays have been measured undera variety of conditions. The following values of ‘apparentdiffusion coefficients’ for THO in the root tissue havebeen evaluated: 1.5±0.1x10^-5 cm² sec^-1 in roots boiledfor 3 min before use,0.5±0.03x10^-5 cm² sec^-1 in rootspoisoned with 10^-2 M NaF,0.9±0.07x10^-5 cm² sec^-1 in rootspoisoned with 10^-2 M NaN₃,and 2.1±0.2x10^-5 cm² sec^-1in normal roots. The bathing medium in all cases was 1.0 mMKCl/0.1 mM CaCl₂ with the addition of the inhibitors where appropriate.Thefourfold increase in the rate of THO transfer in normal rootscompared with poisoned ones is attributed to the existence ofa long-distance convective flow in the first case, which isterminated by the addition of inhibitors. Since experimentsshow that this convective flow must occur both acropetally andbasipetally with equal velocity, it is thought to occur in thephloem.By assuming the ‘streaming transcellular strands’model for phloem transport, the rate of movement required togive the observed transfer has been computed as approximately4.5x10^-2 cm sec^-1 (160 cm h^-1).The earlier report of the existenceof a highly impermeable barrier surrounding the xylem vesselshas been further substantiated by the experiments reported here.     

Permeation of Ca2+ and monovalent cations through an outwardly rectifying channel in maize root stelar cells

A depolarization-activated outwardly-rectifying channel (OR),most likely involved in the passive release of K⁺ from the rootsymplasm into the stelar apoplast (for subsequent transportto the shoot via the xylem vessels), has been characterizedin the plasma membrane of maize root stelar cells (Roberts andTester, 1995). In the present study, the selectivity of thischannel was further characterized using single channel current-voltagecurves generated using a voltage ramp protocol. This protocolpermitted the accurate and unambiguous measurement of the reversalpotentials of currents resulting from single channel openings.Using the voltage ramp protocol, it was shown that the OR allowsboth K⁺ efflux and Ca²⁺ influx at potentials positive of E_Kand negative of E_Ca. The OR had a P_Ca/P_K of 1.72–0.21decreasing as extracellular Ca²⁺ was increased. The permeabilityof the OR for monovalent cations other than K⁺ was also investigated.In biionic conditions, a relative permeability sequence of was determined (i.e. Eisenman sequenceIV). The physiological implications of the selectivity of theOR are discussed. Key words: Maize roots, K⁺ channel selectivity, Ca²⁺ permeation