Plasmalemma Transport of OH- in Chara corallina: II. FURTHER ANALYSIS OF THE DIFFUSION SYSTEM ASSOCIATED WITH- OH EFFLUX

The dynamic and steady state aspects of the pH and electricpotential () profiles, that develop in the experimental mediumin association with the photosynthetic assimilation of exogenousHCO^–₃ by internodal cells of Chara corallina, were investigated.A theoretical treatment is presented which explains the originof the phenomenon. This theory was tested by comparing thepH and values generated by a numerical analysis model (whichsimulated the experimental system) against experimental data.Verification of our model indicates that the steady state ionicfluxes, associated with HCO^–3 assimilation (HCO^–₃,OH^–, and CO^23–, are not significantly influencedby the electric potential gradients. The main driving forcecausing the observed fluxes is the diffusion gradient associatedwith the respective ion. By simultaneous measurement of and pH, at the centre of analkaline band, a direct correlation was established betweenlight-activation and dark-deactivation of the OH^– transportsystem and the light-mediated changes in at the cell surface.In addition, under steady state conditions, an almost perfectcorrelation was observed between alkaline band pH centres andthe negative electric potential maxima. These data offer strongsupport for the hypothesis that OH^–efflux, in this system,is an electrogenic process. Based on our present analysis, the profile along the cell indicatesthat, in terms of the spatial aspect of HCO3^– transport,the rate of HCO^–₃ influx varies quite dramatically alongthe length of an internodal cell. This aspect is discussed interms of the cellular integration of OH^– and HCO^–₃transport in this species.     

Circulating Electric Currents Between Acid and Alkaline Zones Associated with Assimilation in Chara

Some charophyte species, like some other freshwater plants,have been shown to take up for photosynthetic fixation. Such plants show localized deposits of CaCO₃ associatedwith localized ‘OH^– excretion’. Chara corallinaproduces acid and alkaline regions in unbuffered external media,in the light, even when CaCO₃ is not being precipitated. Theseregions are associated with uptake and ‘OH^–efflux’ respectively. We have found that large currentscirculate, in the light, between acid and alkaline zones ofChara internodes, by (i) direct measurement of the short-circuitcurrent and (ii) measurement of potential differences in thesolution around the cell. Both methods suggest currents of 0.5–1µA per zone, with local current densities of about 75mA m^–2, or local univalent fluxes of 0.75 µmol m^–2s^–1. These circulating currents produce mean peak-to-peakpotential differences of 7 mV in the external medium. The fluxesare believed to be of and H⁺ or OH^–.While active transport of is strongly indicated, passive uniport of H⁺ or OH^– appears to be sufficient.Control systems for both fluxes would be required.     

Pharmacological and biophysical isolation of K+ currents encoded by ether-à-go-go-related genes in murine hepatic portal vein smooth muscle cells

The functional properties of the Saccharomyces cerevisiae bicarbonate transporter homolog Bor1p (YNL275wp) were characterized by measuring boron (H₃BO₃), Na⁺, and Cl^– fluxes. Neither Na⁺ nor Cl^– appears to be a transported substrate for Bor1p. Uphill efflux of boron mediated by Bor1p was demonstrated directly by loading cells with boron and resuspending in a low-boron medium. Cells with intact BOR1, but not the deletant strain, transport boron outward until the intracellular concentration is sevenfold lower than that in the medium. Boron efflux through Bor1p is a saturable function of intracellular boron (apparent K_m 1–2 mM). The extracellular pH dependences of boron distribution and efflux indicate that uphill efflux is driven by the inward H⁺ gradient. Addition of 30 mM HCO₃^– does not affect boron extrusion by Bor1p, indicating that HCO₃^– does not participate in Bor1p function. Functional Bor1p is present in cells grown in medium with no added boron, and overnight growth in 10 mM H₃BO₃ causes only a small increase in the levels of functional Bor1p and in BOR1 mRNA. The fact that Bor1p is expressed when there is no need for boron extrusion and is not strongly induced in the presence of growth-inhibitory boron concentrations is surprising if the main physiological function of yeast Bor1p is boron efflux. A possible role in vacuolar dynamics for Bor1p was recently reported by Decker and Wickner (10). Under the conditions used presently, there appears to be mildly abnormal vacuolar morphology in the deletant strain. boron; SLC4; YNL275w     

The Influence of Light Intensity on the Activation and Operation of the Hydroxyl Efflux System of Chara corallina

The interrelationships between light intensity and the activationof OH^– bands was investigated. The lag period prior toOH^– efflux activation was longer than the photosyntheticinduction period. It was found that this lag period dependedupon the light regime employed as well as the photosyntheticcapacity of the cell. The response of the cell to low light intensities revealed thatall OH^– bands were not of equal status. Below a criticallight intensity the cell did not develop any bands even afterprolonged illumination. An hypothesis is presented to accountfor these results, interms of total cell OH^– band activationand the regulation of the HCO₃^– and OH^– transportsystems. It is proposed that the electrical properties of theChara corallina plasmalemma, observed at high pH values, canbe explained on the basis of the hypothesis presented in thispaper     

Effects of Ammonia and Methylamine on Cl- Transport and on the pH Changes and Circulating Electric Currents Associated with HCO3- Assimilation in Chara corallina

Low concentrations of ammonia and methylamine greatly increaseCl^– influx into Chara corallina. Both amines have theirmaximum effect at pH 6.5–7.5. The amine stimulation ofCl^– influx is small below about pH 5.5. Above pH 8.5 theremay be inhibition of influx by amines. Concentrations of 10–25µM ammonia are sufficient to cause the maximum stimulationof Cl^– influx; the corresponding methylamine concentrationsare 0.1–0.2 mM. It is concluded that entry of amine cations(NH₄^$ and CH₃NH₃^$), rather than unionized bases (NH₃ and CH₃NH₂),causes Cl^– transport to be increased. Increases in rates of Cl^– transport are not necessarilyaccompanied by effects on HCO₃^$ assimilation and OH^– efflux.Measurements of localized pH differences at the cell surfaceand of circulating electric currents in the bathing solutionshow that these phenomena are only significantly affected byammonia at or above 50 µM and by methylamine at or above1.0 mM. The significance of the effects of amines is assessedin relation to current ideas about transport of Cl^–, HCO₃^–,and OH^–.     

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.     

Analogue Inhibition of the Active HCO-3 Transport Site in the Characean Plasma Membrane

Competitive inhibition of the HCO^–₃ transport site, atthe plasmalemma of Chara coraUina, by the CO^2–₃ ion isdemonstrated. This CO^2–₃ inhibition was used to demonstratethat HCO^–₃ ions enter the cell by facilitated ‘diffusion’when the HCO^–₃ transport system has been inactivated bytreatment with 10 mM K⁺. Use of CO^2–₃ as a HCO^–₃analogue is limited, however, because of the necessity to employsolutions of high pH. Inhibition was not observed in the presenceof a range of organic and inorganic acid anions. These resultsdemonstrate the stereo-specific nature of the HCO^–₃ bindingsite. A variety of amino compounds were found to inhibit H¹⁴CO^–₃influx. Inhibition appeared to be competitive, being completelyrelieved at higher substrate (HCO^–₃) concentrations. Asimple correlation was not found between the degree of inhibitionand the concentration of neutral base. A combination of thepresence of neutral base and experimental pH values of at least8·0 was required to produce the reactive species thatinhibited HCO^–₃ transport. This species is consideredto be the amino carbamate. These results are discussed withrespect to further HCO^–₃ analogue experiments.     

Protein kinase C mediates the inhibitory effect of substance P on HCO3- secretion from guinea pig pancreatic ducts

The inhibitory control of pancreatic ductal HCO₃^– secretion may be physiologically important in terms of limiting the hydrostatic pressure developed within the ducts and in terms of switching off pancreatic secretion after a meal. Substance P (SP) inhibits secretin-stimulated HCO₃^– secretion by modulating a Cl^–-dependent HCO₃^– efflux step at the apical membrane of the duct cell (Hegyi P, Gray MA, and Argent BE. Am J Physiol Cell Physiol 285: C268–C276, 2003). In the present study, we have shown that SP is present in periductal nerves within the guinea pig pancreas, that PKC mediates the effect of SP, and that SP inhibits an anion exchanger on the luminal membrane of the duct cell. Secretin (10 nM) stimulated HCO₃^– secretion by sealed, nonperfused, ducts about threefold, and this effect was totally inhibited by SP (20 nM). Phorbol 12,13-dibutyrate (PDBu; 100 nM), an activator of PKC, reduced basal HCO₃^– secretion by 40% and totally blocked secretin-stimulated secretion. In addition, bisindolylmaleimide I (1 nM to 1 µM), an inhibitor of PKC, relieved the inhibitory effect of SP on secretin-stimulated HCO₃^– secretion and also reversed the inhibitory effect of PDBu. Western blot analysis revealed that guinea pig pancreatic ducts express the -, _I-, -, -, -, -, -, and µ-isoforms of PKC. In microperfused ducts, luminal H₂DIDS (0.5 mM) caused intracellular pH to alkalinize and, like SP, inhibited basal and secretin-stimulated HCO₃^– secretion. SP did not inhibit secretion further when H₂DIDS was present in the lumen, suggesting that SP and H₂DIDS both inhibit the activity of an anion exchanger on the luminal membrane of the duct cell. pancreas; Cl^–/HCO₃^– exchanger; inhibition; epithelium     

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.     

The Formation of Alkaline and Acid Regions at the Surface of Chara corallina Cells

The magnitude of previously reported pH changes at the surfaceof Chara corallina cells has been measured with specially constructedpH electrodes. An experimental system was designed to reducethe dissipation of pH gradients by convection. When cells areilluminated their surfaces become partitioned into alkalineregions (pH 8.5–9.5) with sharp peaks and acid regions(pH about 5.5) which are more uniform. In the dark these regionsrapidly disappear and the pH of the cell surface becomes uniform(about pH 6.0). With cells initially free of any deposits ofCaCO₃, alkaline regions may appear temporarily, and may migratealong the cell surface. The presence of CaCO₃ stabilizes theregions. It is proposed that alkalinization results from HCO₃^–uptake, CO₂ fixation, and subsequent localized OH- efflux. Alkalinizationis prevented by photosynthetic inhibitors. The nature and magnitude of the HCO₃^–/OH- exchange processis discussed, and the possible role of H⁺ and OH- fluxes incontrolling the ionic and electrical properties of Characeancells is emphasized.     

ERRATA

Page 806, Preparation of Mitochondrial Fraction, line 4: The following should be inserted between ‘centrifugedat’ and ‘20 000 g for’: 3000 for 10 mm. Thesupernatant was centrifuged at The following corrections are required: Page 104, line 20: ‘2-hydroxylation’ should read ‘2-ß3-hydroxylation’ Page 106, line 11: ‘of Ga₈’ should read ‘to GA₈’ Page 113, last line:‘length 50 µm’ shouldread ‘length 150 µm’ Formula 15 should read: Formula 17 should read: y(0)– y^* = ß₁V₁+ß₂V₂ page 118: Formula 18 should read: Formula 23 should read: Formula 24 should read:      

Role of NBC1 in apical and basolateral HCO3- permeabilities and transendothelial HCO3- fluxes in bovine corneal endothelium

Corneal transparency and hydration control are dependent on HCO₃^– transport properties of the corneal endothelium. Recent work (13) suggested the presence of an apical 1Na⁺-3HCO₃^– cotransporter (NBC1) in addition to a basolateral 1Na⁺-2HCO₃^– cotransporter. We examined whether the NBC1 cotransporter contributes significantly to basolateral or apical HCO₃^– permeability and whether the cotransporter participates in transendothelial net HCO₃^– flux in cultured bovine corneal endothelium. NBC1 protein expression was reduced using small interfering RNA (siRNA). Immunoblot analysis showed that 5–15 nM siRNA decreased NBC1 expression by 80–95%, 4 days posttransfection. Apical and basolateral HCO₃^– permeabilities were determined by measuring the rate of pH_i change when HCO₃^– was removed from the bath under constant pH or constant CO₂ conditions. Using either protocol, we found that cultures treated with NBC1 siRNA had sixfold lower basolateral HCO₃^– permeability than untreated or siCONTROL siRNA-treated cells. Apical HCO₃^– permeability was unaffected by NBC1 siRNA treatment. Net non-steady-state HCO₃^– flux was 0.707 ± 0.009 mM·min^–1·cm² in the basolateral-to-apical direction and increased to 1.74 ± 0.15 when cells were stimulated with 2 µM forskolin. Treatment with 5 nM siRNA decreased basolateral-to-apical flux by 67%, whereas apical-to-basolateral flux was unaffected, significantly decreasing net HCO₃^– flux to 0.236 ± 0.002. NBC1 siRNA treatment or 100 µM ouabain also eliminated steady-state HCO₃^– flux, as measured by apical compartment alkalinization. Collectively, reduced basolateral HCO₃^– permeability, basolateral-to-apical fluxes, and net HCO₃^– flux as a result of reduced expression of NBC1 indicate that NBC1 plays a key role in transendothelial HCO₃^– flux and is functional only at the basolateral membrane. corneal endothelium; sodium bicarbonate cotransporter; small interfering RNA; bicarbonate transport     

The Apparent Induction of Nitrate Uptake by Chara corallina Cells following Pretreatment with or without Nitrate and Chlorate

Nitrate provision has been found to regulate the capacity forChara corallina cells to take up nitrate. When nitrate was suppliedto N sufficient cells maximum nitrate uptake was reached after8 h. Prolonged treatment of the cells in the absence of N alsoresulted in the apparent ability of these cells to take up nitrate.Chlorate was found to substitute partially for nitrate in the‘induction’ step. The effects on nitrate reductionwere separated from those on nitrate uptake by experiments usingtungstate. Tungstate pretreatment had no effect on NO^–₃uptake ‘induced’ by N starvation, but inhibitedNO^–₃ uptake associated with NO^–₃ pretreatment. Chloridepretreatment similarly had no effect on NO^–₃ uptake ‘induced’by N deprivation, but inhibited NO^–₃ uptake followingNO^–₃ pretreatment. The data suggest that there are atleast two mechanisms responsible for the ‘induction’of nitrate uptake by Chara cells, one associated with NO^–₃reduction and ‘induced’ by CIO^–₃ or NO^–₃and one associated with N deprivation. Key words: Nitrate, Chlorate, Chara corallina, Induction     

Occurrence of mycosporine-like amino acids in the red-tide dinoflagellate Alexandrium excavatum: UV-photoprotective compounds?

Water extracts of the red-tide dinoflagellate Alexandrium excavatumgrown at ‘high’ light intensity (200 µE m^–2s^–1) show a broad absorbance maximum in the UV regionof the spectrum (310–360 nm). Using TLC and reverse-phaseHPLC a series of mycosporine-like amino acids have been characterized:mycosporine-glycine (_max = 310 nm), palythine (_max = 320 nm),asterina-330 (_max = 330 nm), shinorine (_max = 334 nm), porphyra-334(_max= 334 nm), palythenic acid (_max = 337 nm) and the isomericmixture of usujirene and palythene (_max = 359 nm). From theobserved spectral changes during transference from ‘low’(20 µE m^–2 s^–1) to ‘high’ (200µE m^–2 s^–1) light intensities and vice versa,the series of compounds are supposed to be biogenically relatedto one another. The presence of these compounds in A.excavatumis discussed in relation to their possible role in the photoprotectionto deleterious UV radiation.     

Plasmalemma Transport of HCO-3 and OH- in Chara corallina: Inhibitory Effect of Ammonium Sulphate

The influence of (NH₄)₂SO₄ on ¹⁴C assimilation and cyclosisin internodal cells of Chara corallina was investigated. Severeinhibition of ¹⁴C assimilation was found at pH values above7·0, this inhibition being correlated with the exogenouslevel of NH3 rather than NH⁺₄. Cyclosis was also affected athigher concentrations of (NH₄)₂SO₄. This effect was similarlycorrelated with exogenous levels of NH₃. ¹⁴C assimilation was inhibited non-competitively by (NH₄)₂SO₄,the apparent Km being increased from 0·55 to 1·5mM. The results suggest that the site(s) of inhibition is locatedat the plasmalemma, rather than at the chloroplasts. (Evidencein support of in vivo uncoupling of photophosphorylation, bylow concentrations of (NH₄)₂SO₄, was not obtained). Significant perturbation of the OH^– efflux pattern wasobserved as the level of (NH₄)₂SO₄ was increased. Induced migrationof efflux sites indicates that NH₃ may interfere with the cellularmechanism that controls OH^– transport. Using a cell-segmentisolating chamber it was shown that (NH₄)₂SO₄ inhibited OH^–efflux rather than HCO^–₃ transport. This inhibitory effectwas readily reversible. These data are discussed in terms of a possible relationshipbetween the observe NH₄)²SO₄ stimulation of ³⁶Cl^– influxand the effect of this compound on ¹⁴C assimilation.     

Ion Fluxes in 'Isolated' Guard Cells of Commelina communis L.

Ion fluxes have been measured in ‘isolated’ guardcells of Commelina communis L. using ⁸⁶RbCl and K⁸²Br, in epidermalstrips in which all cells other than guard cells have been killedby treatment at low pH. To avoid problems of slow free spaceexchange most fluxes have been measured at pH 3.9, at whichstomata open well in K(Rb) Cl(Br) and are stable for many hours.At pH 3.9 the intracellular ⁸⁶Rb exchanged as a single compartmentwith a half-time of 2–3 h, independent of external concentration(C_o). The influx of ⁸⁶Rb rose with concentration, to a V_maxof about 23 pmol mm^–2 h^–1. The efflux curve of ⁸²Brcould be well fitted by two exponential terms, with half-timesof 38 min (independent of C_o), and 5–35 h (falling withincreasing C_o). Bromide contents of cytoplasm and vacuole (Q_cand Q_v), and fluxes at plasmalemma and tonoplast, were calculatedfrom the efflux kinetics. Over C_o 20–60 mM, as the apertureincreased from 7 µm to 17 µm, the tonoplast flux(0.5–11.5 pmol mm^–2h^–1) was always much lessthan the plasmalemma flux (7–77 pmol mm^–2 h^–1).Q_c and Q_v both increased with aperture. The increase in Q_c of10.3 pmol mm^–2 µm^–1 is adequate to accountfor the osmotic changes required to change the aperture, aspreviously estimated. However, the change in vacuolar contentof only 5.9 pmol mm^–2 µm^–1 is much too smallto account for the osmotic changes required, or to balance thecytoplasmic changes. It appears therefore that increasing KBroutside not only increases the cytoplasmic salt content, andthe Br flux at the tonoplast, but also stimulates the vacuolaraccumulation of some other solute.     

The Mechanism of Photosynthetic Use of Bicarbonate by Hydrodictyon africanum

Hydrodictyon africanum can photosynthesize at high pH underconditions in which HCO₃^– rather than CO₂ is the carbonspecies entering the cell. A passive entry of HCO₃^– seemsunlikely; a metabolic HCO₃^– pump is proposed. It is possiblethat such a pump is related to a light-dependent reaction specificto the use of HCO₃^–. This reaction is dependent on photosystem2, but appears to be independent of ATP. These characteristicsare similar to those of active lightdependent Cl influx in H.africanum, and suggest a similar energy source for the two pumps.The HCO₃^– pump may be electrogenic.     

Effect of Temperature and External pH on the Cytoplasmic pH of Chara corallina

Cytoplasmic pH (pH_c) in Chara corallina was measured (from [¹⁴C]stribution)as a function of external pH (pH₀)and temperature. With pH₀near 7, pH_c at 25?C is 7.80; pH_cincreases by 0.005 pH units?C^–1 temperature decrease, i.e. pH_c at 5 ?C is 7.90. WithpH? near 5.5, the increase in pH_c with decreasing temperatureis 0.015 units ?C^–1 between 25 and 15?C, but 0.005 units?C^–1 between 15 and 5?C. This implies a more precise regulationof pH_c with variations in pH_o at 5 or 15 ?C compared with 25?C. The observed dp H_c/dT is generally smaller than the –0.017units ?C^–1 needed to maintain a constant H⁺/OH^–1,or a constant fractional ionization of histidine in protein,with variation in temperature. It is closer to that needed tomaintain the fractional ionization of phosphorylated compoundsor of CO₂–HCO₃^– The value of dpH_c/dT has importantimplications for several regulatory aspects of cell metabolism.These include (all as a function of temperature) the rates ofenzyme reactions, the _H+ at the plasmalemma(and hence the energy available for cotransport processes),and the mechanism for pH_c regulation by the control of bidirectionalH⁺ fluxes at the plasmalemma.     

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’.     

Respiratory Efflux in Relation to Temperature of Simulated Swards of Perennial Ryegrass with Contrasting Soluble Carbohydrate Contents

Fully light-intercepting simulated swards of S24 perennial ryegrasswere exposed to contrasting environmental conditions in a growthroom for 4 days. Half experienced 20 h days of 120 Wm^–2(400–700. nm) and 5 °C, and came to have a WSC (watersoluble carbohydrate) content of 235 mg g^–1 and half 4h days of 20 Wm^–2 and 25 °C leading to a WSC of 25mg g^–1. Their rates of CO₂ efflux were monitored at anumber of temperatures during an 8 h dark period; half experiencedincreasing (5–30 °C) and half decreasing (30–5°C) temperatures. The ‘high’ WSC swards hadrespiration rates of 3.7 mg CO₂ g^–1 (d. wt) h^–1at 15 °C, and the ‘low’ swards 0.8 mg CO₂ g^–1h^–1. The order in which the temperatures were experiencedwas immaterial. Even the ‘low’ WSC swards showedno evidence of a respiratory decline during the dark periodthat could be attributed to substrate shortage. The relationshipbetween temperature and CO₂ efflux was best represented by logisticcurves. Even so, a Q₁₀ of 2 fitted the data reasonably well,at least up to 20 °C, and has practical advantages wheninterpolating estimated between measured values of respirationin the construction of a carbon balance sheet. Lohum perenne L., ryegrass, respiration, temperature, Q₁₀, soluble carbohydrate content, simulated sward