Thermal Kinetics of Glutathione Reductase and their Relation to Thermotolerance in Diverse Cultivars of Maize

The characteristics of glutathione reductase from a number ofmaize cultivars with contrasting thermotolerance have been investigated.The apparent K_m (Michaelis constant) for oxidised glutathione(GSSG) was measured between 10 and 45°C at constant pH.The enzyme from highland cultivars adapted to cool environmentshad a slightly lower apparent K_m for GSSG than that from lowlandtropical cultivars at low assay temperatures. Similarly theenzyme from lowland tropical cultivars had a lower apparentK_m for GSSG at high assay temperatures. However these effectswere small and regression lines plotted through the data werenot significantly different in slope or intercept. There wasa strong correlation (r = 0·939) between apparent K_mand V_max (maximum initial velocity) as assay temperature wasvaried. The interpretation of apparent K_m/temperature relationshipsis discussed with hypothetical examples of the effects of temperatureon enzyme activity/substrate concentration plots. It is demonstratedthat an increase in apparent K_m at higher assay temperaturesneed not necessarily reflect any temperature-dependent impairmentof enzyme function. The apparent K_m for GSSG of glutathionereductase from maize increased over four-fold when the temperaturewas raised from 10 to 45°C, but it is concluded that invivo rates of reaction are likely to be increased rather thandecreased by this same change in temperature. Glutathione reductasewould thus appear to be equally well adapted to function atall these temperatures. This suggests that the potential ofenzyme thermal kinetics to predict thermotolerance may be limited.Copyright1994, 1999 Academic Press Zea mays L., maize, glutathione reductase, thermal kinetics, thermotolerance     

Dynamics of Competition in Populations of Carrot (Daucus carota)

Populations of carrot (Daucus carota) were raised over a widerange of densities (79–5763 plants m^-2) to examine thedynamics of competition in terms of yield–density relationshipsand size variability, and to investigate the effects of nutrientsupply on competition. While the relationship between shootyield and density was asymptotic, the relationship between rootand total yield and density tended to be parabolic. For a giventime and density series the relationship between yield per unitarea and density could best be described by the model: y=w_mD_(1+aD)^b wherey is the yield per unit area,D is density,w_m, a andb arefitted parameters. The parametersw_m anda increased over timebut nutrient availability affected onlyw_m. An extension of thebasic yield-density model is proposed to describe the dynamicsof the yield–density relationship over time: y=kD_{[1+cexp(-rt)]{1+}     

Thermal Dependence of the Apparent Kmof Glutathione Reductase from Three Wetland Grasses and Maize

The thermal dependence of enzyme kinetic parameters has beenpresented as an indicator of species’ thermal optima andtolerance limits. Previous studies suggest the relationshipbetween temperature and the apparent Michaelis–Mentenconstant (K_m) of an enzyme system can be used to predict wholeplant success at specific temperatures. The apparent K_mfor glutathionereductase (EC 1.6.4.2; GR) (oxidized glutathione as substrate)extracted from leaves of American sloughgrass (Beckmannia syzigachneSteud.), tufted hairgrass (Deschampsia caespitosa L.), tallfescue (Festuca arundinaceae Schreb. ‘Titan’), andmaize (Zea mays L.), was determined over a range of temperatures(1–40 °C). For all species, minimum apparent K_mforGR was observed at 1 °C, and K_mvalues increased as temperatureincreased. The apparent K_mvalues differed among all speciesat the lower temperatures (1–15 °C), but were similarat higher temperatures. The enzyme from tufted hairgrass hadthe lowest apparent K_mat low temperatures (<15 °C), followedin increasing order by American sloughgrass, tall fescue andmaize. Our experimental system failed to reproduce thermal kineticwindow profiles similar to those reported elsewhere. With respectto the enzyme systems reported here, results suggest that thesecool-season grasses can be ranked as more to less eurythermicwithin the temperature range from 1 to 15 °C. Copyright0000 American sloughgrass, Beckmannia syzigachne Steud., tufted hairgrass, Deschampsia caespitosa L., tall fescue, Festuca arundinaceae Schreb. ‘Titan’, Zea mays L., plant competition, temperature stress, kinetics, Michaelis–Menten constant (K_m), glutathione reductase     

CHLOROPHYLL AMOUNT AS AN INDICATOR OF MATTER PRODUCTIVITY IN BIO-COMMUNITIES

The data of chlorophyll amounts in diverse bio-communities arecompiled and discussed with reference to matter production. The chlorophyll amount in euphotic zone of lakes and oceanswas less than 1 g/m², mostly less than 0.1 g/m². In phytoplanktonblooms it was ca. 0.1–1 g/m². Large values of 5–20g/m² were obtained in the outdoor mass cultures of Chlorella,in which the high population density and chlorophyll contentof the alga were observed. In terrestrial higher plant communitiesthe chlorophyll amount (ca. 1–10 g/m²) was usually higherthan in aquatic phytoplankton communities. The largest (13.3g/m²) was obtained with an evergreen gallery forest in Thailand.The chlorophyll amount of desert bio-community could be expectedto be as high as that in water blooms. The maximum chlorophyll amount in bio-communities seems to attainup to 20 g/m² when the conditions are favorable. ¹ Dedicated to Prof. H. TAMIYA on the occasion of his 60th birthday. (Received December 25, 1962; )     

Partitioning of Transfer and Carboxylation Components of Intracellular Resistance to Photosynthetic CO2 Fixation: A Critical Analysis of the Methods Used

The partitioning of intracellular resistance to CO₂ transferin two components: mesophyll (r_m.) and carboxylation (r_x) resistances,is based upon the combination of two sub-models: one encompassingtransfer processes and the second, the carboxylation system.All the determinations derived from this method yield a highr_m/r_x ratio. it is demonstrated in low oxygen conditions, whateverthe model used, that this conclusion is highly-dependent uponthe form of the equation used at the carboxylation level. Thepossible influence of O₂ concentration on the r_m/r_x ratio isdiscussed. Starting from a Rabinowitch model (rectangular hyperbola), someof the conditions necessary to yield lower r_m/r_x ratio are considered.It is shown that the most relevant factor when modelling theCalvin cycle is the number of limiting pools (enzymes or cofactors)rather than the number of limiting reactions.     

Distribution of Calanus species in Kongsfjorden, a glacial fjord in Svalbard

The distribution of Calanus species was investigated in Kongsfjordenin summer of 1996 and 1997. In both years Calanus finmarchicusand Calanus glacialis dominated, although the boreal C. finmarchicuswas more abundant than the Arctic C. glacialis in 1997. Thiscoincided with a 2°C higher water temperature at 50 m in1997, indicating stronger influence of Atlantic origin waterthat year. Advected Calanus finmarchicus occurred in deep andsubsurface layers of the outer fjord in 1996 (200 ind. m^-3,mainly CIII). A less abundant local population aggregated insurface layers of the inner fjord (100 ind. m^-3). Similarly,advected C. finmarchicus occurred in subsurface layers in 1997(446 ind. m^-3, mainly CIII and CIV) and a local population insurface layers (183 ind. m^-3, mainly CI). Calanus glacialisin 1996 aggregated as CII and CIII in the deep layers of theouter fjord (272 ind. m^-3), whereas CIII–CV were abundant(216 ind. m^-3) in cold surface waters of the inner fjord. In1997 C. glacialis (mostly CIII–CV) was more abundant inthe outer than in the inner part of the fjord (40 and 192 ind.m^-3, respectively). Within Kongsfjorden, Calanus finmarchicusneeds one year to complete its life cycle, whereas Calanus glacialisneeds two. Calanus hyperboreus seems to be an expatriate inthe fjord system.     

The Interaction of Genetic, Environmental, and Hormonal Factors in Stem Elongation and Floral Development of Cucumber Plants

Hypocotyl length was found to vary between cucumber plants carryingdifferent genes controlling sex expression. Among lines havingonly unisexual flowers (genotype M/M), the homozygous monoeciousplants (st⁺/st⁺) had significantly longer hypocotyls than theirgynoecious counterparts (st/st), heterozygous gynoecious plants(st+/st) being intermediate. Similarly, hypocotyls of plantsof an andromonoecious line (st⁺/st⁺ m/m) were significantlylonger than in their hermaphrodite counterparts (st/st m/m).Differences in intemode length were also significant and inthe same direction. Since stem and particularly hypocotyl elongationin cucumber is known to be very sensitive to applied gibberellin,these findings suggest the existence of differences in the effectivelevels of endogenous gibberellins in the different sex types,higher levels being correlated with stronger male tendency.This conclusion is in good agreement with the known effect ofapplied gibberellin on sex expression (enhancement of the maletendency) in cucumber. Application of gibberellin (GA₄+GA₇) and exposure to ‘summer’conditions (long days and relatively high temperature) inhibitedthe development of pistillate flowers while ‘winter’conditions (short days, lower temperature) had a similar effecton staminate flowers. The effect was in either case specific,that is, limited to flower development. It is concluded thatexogenous and endogenous gibberellins affect not only the initiation,but also the further development, of flowers in the cucumber.     

Summer coastal zooplankton biomass and copepod community structure near the Italian Terra Nova Base (Terra Nova Bay, Ross Sea, Antarctica)

The structure of the zooplankton biotic community and of copepodpopulation in the coastal area of Terra Nova Bay (Ross Sea,Antarctica) was investigated during the 10th Italian AntarcticExpedition (1994/1995). Zooplankton biotic community consistedmainly of pteropods (Limacina helicina and Clione antarctica),Cyclopoid (Oithona similis), Poecilostomatoid (Oncaea curvata)and Calanoid (Ctenocalanus vanus, Paraeuchaeta antarctica, Metridiagerlachei and Stephos longipes) copepods, ostracods, larvalpolychaetes and larval euphausiids. Zooplankton abundance rangedfrom 48.1 ind m^–3 to 5968.9 ind m^–3, and copepodabundance ranged from 45.2 ind m^–3 to 3965.3 ind m^–3.The highest peak of zooplankton abundance was observed between25 m and the surface and was mainly due to the contributionof O. similis, O. curvata and C. vanus. Zooplankton biomassranged from 5.28 mg m^–3 to 13.04 mg m^–3 dry weight;the maximum value was observed between 25 m and the surface.Total lipid content varied from 216.44 to 460.73 mg g^–1dry weight.     

Enhancement of Peroxidase-Dependent Oxidation of Sinapyl Alcohol by an Apoplastic Component, 4-Coumaric Acid Ester Isolated from Epicotyls of Vigna angularis L.

A water-soluble component that enhanced the peroxidase-dependent(POX-dependent) oxidation of sinapyl alcohol was isolated fromepicotyls of Vigna angularis. This compound was an ester of4-coumaric acid and a hexose, and it was found in both the apoplastand the symplast. The ester was oxidized by a basic POX isozyme(K_m, about 20 µM) and by an acidic POX isozyme (K_m, about40 µM) that had been partially purified from the apoplasticfraction of epicotyls of V. angularis. These POX isozymes oxidizedsinapyl alcohol at only a very low rate, but a 15-fold enhancementwas observed upon addition of the ester. The concentrationsof the ester required for the half-maximal enhancement weresimilar to the K_m values of the ester for its oxidation by therespective isozymes. The apoplastic concentration of the esterwas higher than 130 µM, suggesting that this ester mightact as a donor of electrons to the apoplastic POX isozymes insitu. Coniferyl alcohol also enhanced the POX-catalyzed oxidationof sinapyl alcohol. The concentrations of coniferyl alcoholrequired for half-maximal enhancement of the oxidation of sinapylalcohol were about 23 and 250 µM when reactions were catalyzedby the basic and acidic POXs, respectively. These values weresimilar to the K_m values of coniferyl alcohol for its oxidationby the respective isozymes. These results suggest that 4-coumaricacid ester and coniferyl alcohol, if it is present in the apoplast,can enhance the POX-dependent oxidation of sinapyl alcohol inthe apoplast of epicotyls of V. angularis. (Received July 1, 1996; Accepted February 5, 1997)     

Purification and Characterization of Sucrose Synthase from Peach (Prunus persica) Fruit

Sucrose synthase (EC 2.4.1.13 [EC] ) was purified from peach fruit(Prunus persica) to a single band of protein on SDS-PAGE byammonium sulfate fractionation, DEAE-cellulose (DE-52) chromatography,Sepharose CL-6B gel filtration, PBA-60 affinity chromatographyand Sephadex G-200 gel filtration. The molecular weight wasestimated to be 360,000 by gel filtration. The enzyme was foundto be a tetramer of identical 87-kDa subunits. The maximum activityfor the synthesis and cleavage of sucrose was observed at pH8.5 and pH 7.0, respectively. The enzymatic reaction followedtypical Michaelis-Menten kinetics in both directions, with thefollowing parameters: K_m(fructose), 4.8 mmM; K_m(UDPglucose),0.033 mM; K_m(sucrose), 62.5 mM; K_m(UDP), 0.080 mM. Other properties,such as substrate specificity and the effects of divalent cations,were also investigated. The relationship between the enzymeand the accumulation of sucrose in peach fruit is discussed. Present address: Laboratory of Horticulture, Faculty of Agriculture,Nagoya University, Chikusa, Nagoya 464, Japan. (Received May 2, 1988; Accepted September 14, 1988)     

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     

The electromotive force of the Chara membrane during the hyperpolarizing response

The hyperpolarizing response of the Chara internodal cell wasstudied by applying the voltage clamp and constant current techniques.By assuming the membrane as an electromotive force (emf) inseries with a resistance r (which is the sum of a series resistancer_s and the membrane resistance r_m), it was shown that the hyperpolarizingresponse was brought about not only by the increase in membraneresistance but by the increase in membrane emf. The time dependentcurrent-voltage (I–V_m) curve obtained under the voltageclamp during the hyperpolarizing response showed a negativeresistance. The hyperpolarizing response is also an excitation,since it is a transition process of the membrane across a negativeresistance region. (Received July 22, 1974; )     

SPECIFIC RNA FROM PHOTOPERIODICALLY INDUCED COTYLEDONS OF PHARBITIS NIL

The nucleotide ratio of several RNA species from cotyledonsof Pharbitis nil subjected to a single 16 hr night with or withouta 15 min light-break, or to continuous light was investigated.RNA species examined were RNAs from nuclear, mitochondrial,microsomal, and supernatant fractions separated by differentialcentrifugation, and s-, r-, and m-RNAs fractionated by methylatedalbumin column chromatography. Of the RNAs examined, m-RNA alone was found to change its nucleotideratio with photoperiod applied. Thus as compared with m-RNAfrom non-induced cotyledons (exposed to continuous light oran interrupted night), m-RNA from cotyledons induced by an uninterruptednight contained significantly reduced guanylic and cytidylicacids on molar ratio basis. A working hypothesis was proposed that floral stimulus productionin cotyledons may be directed by gene DNA derepressed photoperiodically. (Received October 18, 1966; )     

Biophysical characterization of zebrafish connexin35 hemichannels

A subset of connexins can form unopposed hemichannels in expression systems, providing an opportunity for comparison of hemichannel gating properties with those of intact gap junction channels. Zebrafish connexin35 (Cx35) is a member of the Cx35/Cx36 subgroup of connexins highly expressed in the retina and brain. In the present study, we have shown that Cx35 expression in Xenopus oocytes and N2A cells produced large outward whole cell currents on cell depolarization. Using whole cell, cell-attached, and excised patch configurations, we obtained multichannel and single-channel current recordings attributable to the Cx35 hemichannels (I_hc) that were activated and increased by stepwise depolarization of membrane potential (V_m) and deactivated by hyperpolarization. The currents were not detected in untransfected N2A cells or in control oocytes injected with antisense Cx38. However, water-injected oocytes that were not treated with antisense showed activities attributable to Cx38 hemichannels that were easily distinguishable from Cx35 hemichannels by a significantly larger unitary conductance (_hc: 250–320 pS). The _hc of Cx35 hemichannels exhibited a pronounced V_m dependence; i.e., _hc increased/decreased with relative hyperpolarization/depolarization (_hc was 72 pS at V_m = –100 mV and 35 pS at V_m = 100 mV). Extrapolation to V_m = 0 mV predicted a _hc of 48 pS, suggesting a unitary conductance of intact Cx35 gap junction channels of 24 pS. Channel gating was also V_m dependent: open time declined with negative V_m and increased with positive V_m. The ability to break down the complex gating of intact intercellular channels into component hemichannels in vitro will help to evaluate putative physiological roles for hemichannels in vivo. connexin; gating; retina     

DISTRIBUTION AND PRODUCTION OF THREE HYDROBIA SPECIES (GASTROPODA: HYDROBIIDAE) IN A SHALLOW COASTAL LAGOON IN THE BAY OF CADIZ, SPAIN

The abundance, life span, growth and production of the mud snailsHydrobia minoricensis, H. ulvae and H. ventrosa in a semi-naturallagoon system were studied by taking monthly samples at threesites during 1991 and 1992. The most abundant species, H. minoricensisoccurred at mean densities of 12834 to 26264 snails m^–2(10.7 to 25.8g dry weigh m^–2), depending on the site.The least abundant species, H. ulvae, occurred at mean densitiesof 185 to 353 snails m^–2 (3.2 to 2.2g dry weight m^–2).The numerical abundance and biomass of the three Hydrobia specieswere positively related to the biomass of benthic macroalgae(P<0.01). Although H. ulvae egg capsules were recorded throughoutthe year, newly hatched snailsof this species were not observed,in contrast to the other two species. The early spring and summercohorts of H. minoricensis and H. ventrosa seemed to be themost numerous. The average life spans of these two species wereestimated to be about 18 and 13 months respectively. Annualproduction estimates for the whole lagoon system were 29.0 (6.3),5.5 (0.8) and 5.2 (1.0)g dry weight (ash-free dry weight) m^–2yr^–1 for H. minoricensis, H. ulvae and H. ventrosa respectively.The annual P/B ratio was about 2 for H. minoricensis and H.ventrosa. (Received 5 July 1994; accepted 5 October 1994)     

COMPARATIVE LIFE-CYCLE, BIOMASS AND SECONDARY PRODUCTION OF THREE SYMPATRIC FRESHWATER GASTROPOD SPECIES

The life cycles, biomass and secondary production of three sympatricfreshwater basommatophoran snails, Lymnaea palustris (MÜller),Physa fontinalis Linnaeus and Anisus rotundatus (Poiret) werestudied during two years in a freshwater ditch. L. palustrisexhibited an iteroparous life-cycle whereas the two other speciespresented a semelparous life-cycle, adults died just after oviposition.L. palustris secondary production (dry weight) value was higher(P = 11 298.4 mg 0.1 m^–1 yr^–1) than those of P.fontinalis (P = 846.3 mg 0.1 m^–2 yr^–1) and A. rotundatus(P = 1192 mg 0.1 m^–2 yr^–1). (Received 16 March 1992; accepted 30 June 1992)     

Quantity and Kinetic Properties of Ribulose 1,5-Bisphosphate Carboxylase in C3, C4, and C3-C4 Intermediate Species of Flaveria (Asteraceae)

The kinetic properties of ribulose 1,5-bisphosphate carboxylase(RuBPC) appear to have been modified during evolution of photosynthesisto adjust to changes in substrate availability. C₄ plants areconsidered to have a higher concentration of CO₂ available toRuBPC than C₃plants. In this study, the K_m(CO₂ and catalyticcapacity (k_cat) of RuBPC and the ratio of RuBPC protein to totalsoluble protein from several Flaveria species, including C₃,C₃-C₄ intermediate, and C₄ species, were determined. The C₃and intermediate species had similar K_m(CO₂) values while theC₄ species on average had higher K_m(CO₂) values. The mean ratioof K_cat/K_m for species of each group was similar, supportingthe hypothesis that changes in K_m and K_cat, are linked. Theallocation of total soluble protein to RuBPC was lowest in theC₄ Flaveria species, intermediate in the C₃-C₄ species, andhighest in the C₃ species. The results suggest that during evolutionof C₄ photosynthesis adjustments may occur in the quantity ofRuBPC prior to changes in its kinetic properties. (Received January 4, 1989; Accepted April 11, 1989)     

Effects of Osmotic Stress, Ionic Stress and IAA on the Cell-Membrane Resistance of Vigna Hypocotyls

The cell-membrane resistance (R_m) of Vigna hypocotyls was examined,and the effects of osmotic stress, ionic stress and IAA on R_mwere investigated. R_m decreased by 64 to 77% under osmotic stressin the presence of absorbable solutes (40 mM sorbitol, 15 mMKC1, 30 mM sucrose; or 40 mM sorbitol, 15 mM KC1, 30 mM sucroseplus 10^–4 M IAA) or under ionic stress (50 mM NaCl or50 mM KC1). R_m was not changed by perfusion with 10^–4M IAA. Therefore, the hyper-polarizations of the membrane potentialobserved in both cases should be ascribed totally to the activationof the electrogenic proton pump. Although R_m showed an increaseof 1.6 fold when the hypocotyls were subjected to osmotic stress(100 mM sorbitol or 100 mM sorbitol plus 10^–4 M IAA),83.6% or 92.4% of the hyperpolarization of the membrane potential(V_px was also the result of the activation of the pump. Theresults, calculated on the basis of the current source model,support the viewpoint that the hyperpolarization of the cellmembrane potential of Vigna hypocotyls under osmotic stress,ionic stress or in the presence of IAA is an expression of theactivation of the proton pump, and is not caused by an increasein R_m. ¹ Present address: Researchers and Planners of Natural Environment, Yotsugi Bldg. (2F), 1-5-4 Horinouchi, Suginami-Ku, Tokyo,166 Japan ² Present address: Graduate School of Integrated Science, YokohamaCity University, 22-2 Seto, Kanazawa-Ku, Yokohama, 236 Japan (Received February 14, 1991; Accepted July 24, 1991)     

Use of the Expolinear Growth Model to Analyse the Growth of Faba bean, Peas and Lentils at Three Densities: Fitting the Model

The expolinear equation for crop growth (Goudriaan and MonteithAnnalsof Botany66: 695–701, 1990) was fitted to measurementsof above ground dry weight made on two cultivars of each ofthree species, faba bean (Vicia fabaL.), peas (Pisum sativumL.)and lentils (Lens culinarsMedic.), each grown at three densitiesat the University of Reading, UK in 1992 and 1993. The expolinearequation fitted the data well but required frequent samplingto obtain good estimates of the parameters. The equation hasthree parameters,R_mthe maximum relative growth rate,C_ma maximumcrop growth rate, andt_bthe time at which the crop effectivelyreaches a linear phase of growth.R_mdid not differ between densities,cultivars or species but differed between years.C_mincreasedwith increased density and was lower for lentils than for fababeans or peas.t_bdecreased with increased density for faba beanbut not for the other species. Incorporating an extinction coefficientfor solar radiation and the maximum fraction of radiation interceptedenabled reasonably accurate time courses of leaf area indexto be derived, as suggested by Goudriaan (1994. In: MontiethJL, Scott RK, Unsworth MH, eds.Resource capture by crops. Nottingham:Nottingham University Press, 99–110).Copyright 1998 Annalsof Botany Company Expolinear equation, grain legumes, crop growth rate, crop density, relative growth rate, growth modelling, faba bean,Vicia fabaL., peas,Pisum sativumL., lentils,Lens culinarsMedic.     

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