Plant Physiol. (1 996) 1 12: 1349-1 355 Direct lnhibition of Plant Mitochondrial Respiration by Elevated CO,' Miquel A. Gonzilez-Meler*, Miquel Ribas-Carb?2, James N. Siedow, and Bert G. Drake Smithsonian Environmental Research Center, P.O. Box 28, Edgewater, Maryland 21 037 (M.A.G.-M., B.G.D.); and Developmental, Cell, and Molecular Biology, Botany Department, Duke University, P.O. Box 91 000, Durham, North Carolina 27708-1 O00 (M.R.-C., J.N.S.) Doubling the concentration of atmospheric CO, often inhibits plant respiration, but the mechanistic basis of this effect is un- known. We investigated the direct effects of increasing the concen- tration of CO, by 360 p l 1-' above ambient on O, uptake in isolated mitochondria from soybean (Glycine max L. cv Ransom) cotyledons. lncreasing the CO, concentration inhibited the oxida- tion of succinate, external NADH, and succinate and external N A D H combined. The inhibition was greater when mitochondria were preincubated for 10 min in the presence of the elevated CO, concentration prior to the measurement of O, uptake. Elevated CO, concentration inhibited the salicylhydroxamic acid-resistant cyto- chrome pathway, but had no direct effect on the cyanide-resistant alternative pathway. W e also investigated the direct effects of ele- vated CO, concentration on the activities of cytochrome c oxidase and succinate dehydrogenase (SDH) and found that the activity of both enzymes was inhibited. The kinetics of inhibition of cyto- chrome coxidase were time-dependent. The leve1 of SDH inhibition depended on the concentration of succinate in the reaction mixture. Direct inhibition of respiration by elevated CO, in plants and intact tissues may be due at least in part to the inhibition of cytochrome c oxidase and SDH. Respiration rates are often lower when plants are grown at elevated C, than when they are grown at ambient CO, levels (Wullschleger et al., 1994; Amthor, 1996). Two effects of elevated C, on apparent dark respiration in intact plants or tissues have been reported (Amthor, 1991): (a) a direct, immediate effect in which respiration is reversibly reduced by exposure to elevated C,; and (b) an acclimation effect in which respiration of plants grown in elevated C, differs from respiration of plants grown in ambient C, (when This work was supported by the U.S. Department of Agricul- ture National Research Initiative-Competitive Grants Program (grant no. 94-37306-0352), the U.S. Department of Energy, and the predoctoral programs of the Smithsonian Institution and Ministerio de Educaci?n y Ciencia (Spain) to M.A.G.-M and Ministerio-de-Educaci?n-y-Ciencia-Fulbright postdoctoral fellow- ship (Spain) to M.R.X. This paper was part of the doctoral disser- tation of M.A.G.-M. Present address: Departamento de Fisiologia Vegetal, Facultad de Ciencias, Universidad de Navarra, 31008 Pamplona, Spain. * Corresponding author; e-mail gonza1ezQserc.si.edu; fax 1-301-261-7954. measured at a common value of C,). The acclimation effect generally results in reduced respiration in plants and tis- sues grown at elevated C, (Bunce and Caulfield, 1991; Azc?n-Bieto et al., 1994), although in some plants acclima- tion leads to increased respiration (Thomas et al., 1993). Acclimation of respiration in photosynthetic tissues of plants grown at elevated C, can be related to a reduction in the maximum activity of Cyt c oxidase (Azc?n-Bieto et al., 1994; Aranda et al., 1995). The direct effect of CO, on dark respiration is reversible and is observed in most plants as a reduction in respiration within minutes of a step change in C, (Amthor, 1996). A reversible inhibition of CO, evolution by Rumex crispus leaves was observed when C, was increased stepwise through the range of O to 1000 pL L-' (Amthor et al., 1992). Inhibition of respiration by increasing C, has also been reported in whole plants (Bunce, 1990; Ryle et al., 1992), leaves (Reuveni and Gale, 1985; Bunce, 1990; El Kohen et al., 1991; Amthor et al., 1992; Byrd et al., 1992; Thomas and Griffin, 1994; Ziska and Bunce, 1994), roots (Reuveni and Gale, 1985; Palta and Nobel, 1989; Qi et al., 1994), micro- organisms (Koizumi et al., 1991), and animal tissues (Palet et al., 1991). Respiration can also be unaffected or even increased when C, increases (Palet et al., 1991; Ryle et al., 1992). Although direct effects of C, on apparent respiration in plant tissues have long been reported (e.g. Kidd, 1916), these effects have recently been reevaluated in the context of the rising C,, which is expected to reach a value of twice the preindustrial concentration during the second half of the next century. A reduction of dark respiration in aerial plant tissues by elevated C, would have important conse- quences for the carbon balance in terrestrial ecosystems. The site of action of the direct, short-term inhibition of respiration by CO, is unknown. Levels of C, 5% or higher may inhibit some enzymes of the glycolytic pathway (Kerbel et al., 1988, 1990), as well as mitochondrial O, uptake (Shipway and Bramlage, 1973; Palet et al., 1992). Abbreviations: C,, concentration of CO, in the air; DIC, dis- solved inorganic carbon; SDH, succinate dehydrogenase; SHAM, salicylhydroxamic acid; Vcyt.KCN, SHAM-resistant O, uptake, the activity of the Cyt pathway in the presence of SHAM; Va,t.SHAM, cyanide-resistant O, uptake, the activity of the alternative pathway in the presence of KCN. 1349 1350 Gonzilez-Meler et al. Plant Physiol. Vol. 1 1 2, 1996 This includes the activities of mitochondrial enzymes such as SDH (Zeylamaker et al., 1970) and Cyt c oxidase (Miller and Evans, 1956; Palet et al., 1991, 1992). Al- though the dissolved CO, concentration can reach up to 1.7 mM (equivalent to 0.5% C,) in nongreen tissues (Raven and Newman, 1994), there are no reports show- ing that elevated C, inhibits enzyme activity associated with respiration of photosynthetic tissues at more phys- iological levels of C, (up to 1000 pL L-?). It has also been suggested that extramitochondrial factors such as dark CO, fixation or measurement artifacts (Reuveni et al., 1993; Wullschleger et al., 1994; Amthor, 1996) contribute to the apparent inhibition of respiration by elevated C,. The goal of this work was to determine whether the direct effects of CO, reported in tissues can be seen in isolated plant mitochondria and, if so, to study the sites of action of any such inhibition. To accomplish this we iso- lated mitochondria from soybean (Glycine max L.) cotyle- dons and exposed them to an increase in DIC equivalent to 360 pL L-? above the current ambient level of C,. MATERIALS A N D M E T H O D S Seeds of soybean (Glycine max L. cv Ransom) were planted in a 1:l mixture of sand and perlite. Plants were grown in growth chambers in the Duke University Phy- totron at 25?C under a 13-h/11-h (light/dark) photoperiod at 1000 pmol photons m-?s-?. In late spring of 1994 seeds were also planted and grown in the greenhouse with no control of incident light or temperature. In both cases plants were watered at least once a day and cotyledons were collected between 7 and 10 d after sowing. Mitochondrial lsolation and Assay Cotyledon mitochondria were isolated using a Perco11 gradient as described by Day et al. (1985) with minor modifications (Umbach and Siedow, 1993). Isolated mitochondria were assayed at 25?C in 10 mM Tes-buffered medium, pH 7.2, containing 0.3 M SUC, 5 mM KH,PO,, 10 mM NaC1, 2 mM MgSO,, and 0.1% BSA. Oxy- gen uptake was measured polarographically using a Clark- type O, electrode (Rank Brothers, Cambridge, UK) and initiated by the addition of different substrates. When the reaction of mitochondrial oxidases with oxygen was initi- ated with 5 mM succinate, mitochondria were preincubated with 0.15 mM ATP, and SDH was further activated by a single state 3/state 4 transition. Oxidation of 2 mM NADH (30 p~ Ca2+) was carried out in the presence and absence of pyruvate (5 mM). A11 experiments described here were carried out under state 3 conditions, in which ADP was present in excess, to avoid ATP control of mitochondrial O, uptake. Vcyt.SHAM was assessed in the presence of 2 mM SHAM, an inhibitor of the alternative oxidase. Va,t.KCN was as- sessed in the presence of 1 mM KCN, an inhibitor of the Cyt pathway . Mitochondria were preincubated in a closed cuvette for 10 min in the presence or absence of elevated C, concen- tration (see ?CO, Treatments?) because preliminary trials showed that this was necessary to obtain stable rates of O, uptake after the step change in C,. Respiratory control (state 3-to-state 4 ratio) and ADP-to-O ratios of the mito- chondria in the cuvette remained constant for at least 30 min at room temperature, after which they started to decline. Cyt c Oxidase Assay Plant Cyt c oxidase activity was measured in mitochon- dria broken by osmotic shock. Commercial beef-heart Cyt c oxidase was from Sigma. Both enzymes were assayed polarographically at 25?C as the rate of azide (2.5 mM)- sensitive O, uptake in the reaction medium used for mitochondria (pH 7.2), with the addition of 1 mM lauryl maltoside, 8 mM ascorbate, 1 mM N,N,N?N?-tetramethyl-p- phenylenediamine dihydrochloride, and 30 p~ Cyt c. SDH Assay SDH activity was determined by measuring SDH-phena- zine methosulfate reductase activity (Burke et al., 1985). Mitochondria were placed in a medium containing 30 mM Tricine-NaOH (pH 7.5), 0.25 to 8 mM succinate, and 1 mM phenazine methosulfate. Malonate (5 mM)-sensitive O, up- take was then measured at 25?C. CO, Treatments The free CO, concentration dissolved in aqueous solu- tion is linearly proportional to the C, in the gas phase in equilibrium at constant temperature. In ambient atmo- spheric conditions, the free CO, dissolved in water is near 12 p~ at 25?C. However, the quantity of DIC is a function of the pH, determined by the Henderson-Hasselbalch equation. A stock solution of DIC was prepared fresh daily in the mitochondrial reaction medium using either potas- sium or sodium bicarbonate and was kept in containers with no gas phase at 25?C (pH 7.2), at which level equilib- rium between the dissolved chemical species was estab- lished. For the elevated C, treatment, 0.1 mM DIC (12.2 p~ CO, and 88 p~ HC0,-, pK, = 6.35) was added to the closed cuvette to emulate increasing ambient C, by 363 PL L-? in the liquid phase. In the case of the SDH-phenazine methosulfate reductase activity the reaction was carried out at pH 7.5. Therefore, 0.17 mM DIC (12.2 p~ CO, and 158 p~ HC0,-) was used for the elevated C, treatment. The reaction medium placed in the reaction cuvette was previously equilibrated with ambient air that contained a CO, concentration less than 420 pL L-? (measured with a gas analyzer [6262 IR, Li-Cor, Lincoln, NE). For the incubation experiments O or 0.1 mM DIC (10 pL from the stock solution) was added to the closed cuvette containing the reaction medium and mitochondria 10 min before the substrates (succinate or NADH) were added. Depending on the substrate used, either ATP (succinate) or Ca2+ and pyruvate (NADH) were also added to the closed cuvette during the 10-min incubation. Effects of CO, on Mitochondrial Respiration 1351 RESULTS The Direct Effect of Atmospheric CO, on Plant Mitochondria Elevated C, inhibited mitochondrial O, uptake (Fig. 1). The rates of O, uptake depended on the substrate used, but elevated C, inhibited O, uptake in a11 cases. When oxidiz- ing succinate, elevated C, inhibited mitochondrial O, up- take 16% (P < 0.001) (Fig. 1, SUCC). Elevated C, inhibited the oxidation of NADH by soybean mitochondria (9%; P = 0.056) (Fig. 1, NADH) significantly in the presence of pyru- vate (15%; P = 0.029, rank summary test) (Fig. 1, NADH+PYR). Elevated C, inhibited the oxidation of suc- cinate and NADH in the absence of pyruvate by 15% (I' < 0.001) (Fig. 1, SUCC+NADH). The Direct Effect of Atmospheric CO, on the Activity of Cyt c Oxidase and SDH Elevated C, inhibited the activity of Cyt c oxidase ob- tained from severa1 different sources (Fig. 2A). Inhibition of Cyt c oxidase activity was similar for soybean cotyledons (19%; P = 0.003) and roots (20%; P = 0.018). Slightly greater inhibition was observed for purified Cyt c oxidase from beef heart (28%; P = 0.019). The average inhibition of plant co, + 360 WL' Figure 1. The direct effect of elevated C, on soybean cotyledon mitochondrial respiration. Oxidation of either succinate (SUCC), NADH and pyruvate (NADH+PYR), NADH alone (NADH), or suc- cinate and NADH (SUCC+NADH) was measured in the presence of ADP (state 3 conditions). Values shown are for mitochondria from plants grown in the greenhouse, except SUCC and NADH+SUCC plants, which were grown in growth chambers. Mitochondria were incubated for 10 min with O (O) or 0.1 (M) mM DIC at 25?C. Respiratory controls and ADP-to-O ratios were 1.34 2 0.05 and 1.23 2 0.06, 1.49 & 0.05 and 1.54 2 0.03, and 1.42 t 0.07 and 1.66 5 0.08 for succinate, NADH, and succinate plus NADH, re- spectively. Values are means 2 SE of three to nine replicates. *, Significant difference in the mean (P < 0.05) using a Student's t test , o r a ,rank sumrw&ry.test.(see text). V, Total velocity of O, uptake of intact mitochondria. n - [7 Ambient CO, m Ambient CO, + 360 PL.L' *' 2 '""E 100 I * 1 * I Cotyledon Root Beef heart CALCULATED c, ( p ~ . ~ - l ) n 6 400 600 800 1000 1200 1400 1600 E g 100 5 90 2 E 80 b u 70 4 60 50 O H 40 o 8 I I I I 1 I I 0.0 0.1 0.2 0.3 DIC ADDED (mM) Figure 2. The direct effect of elevated C, on Cyt c oxidase activity. A, Cyt c oxidase from soybean cotyledon and root mitochondria or isolated from beef heart was incubated for 10 min with O (O) or 0.1 (M) mM DIC at p H 7.2 in a closed cuvette before the measurements were taken. 8, The effect of elevated DIC on beef-heart Cyt coxidase activity measured without preincubation time (O) and with a 1 O-min DIC preincubation (e). Values are means 2 SE of three to eight replicates. mitochondrial Cyt c oxidase, 19 to 20%, was similar to the percentage of inhibition seen with mitochondrial electron transport (Fig. 1). A titration of the beef-heart Cyt c oxidase activity showed that the effect of increasing C, on the activity of the enzyme was largest when C, was increased from normal ambient to twice ambient levels (Fig. 2B). Much less inhibition of Cyt c oxidase was observed when it was not preincubated with the elevated C, for 10 min prior to taking the measurements (Fig. 2B). The activity of SDH from mitochondria from soybean cotyledons was also inhibited by increasing C, in the reac- tion medium (Table I). The percentage of inhibition of SDH activity by elevated C, was dependent on the concentration of s?ccinate present: 20% at 0.25 mM succinate (P = 0.003), 12% at 2 mM succinate (I' = 0.022), and 7% at 8 mM 1352 Gonzilez-Meler et ai. Plant Physiol. Vol. 112, 1996 Table 1. The direct effect of elevated C, on the activity of SDH ~ from soybean cotyledon mitochondria SDH was preincubated for 10 min with 0 (ambient CJ, 0.1 7 (ambient C, + 360 pL LK'), or 0.51 (ambient C, + 1080 pL L- l ) mM DIC at pH 7.5 before the measurements were taken. Values are means 5 SE of three to six replicates. Letters indicate statistical differences in the degree of inhibition of an increase in ambient C, in 360 p L L-', P C 0.05 (one-way analysis of variance). For other statistical details, see text. Succinate Concentration c a 0.125 mM 0.25 ITlM 2 I l lM 8 mM nmol O, mg-' protein min-' Ambient 32 t 0.5 40 5 0.8 110 i 2.1 124 t 3.7 Ambient + 24 ? 0.7 32 i 0.9 97 -C 2.0 115 _t 4.0 Ambient + 17 5 0.6 22 2 0.7 - - 360 pL L-' 1080 p L L-' 0.75a 0.80b 0 . 8 8 ~ 0 . 9 3 ~ '-, Not determined. succinate (P = 0.143). The degree of inhibition of SDH activity by elevated C, was lessened as the concentration of succinate was increased (P < 0.05) (Table I). The Direct Effect of Atmospheric CO, on the Cyt and Alternative Pathways Elevated C, inhibited the Vcyt.SHAM with a variety of substrates (Fig. 3 ) . Elevated C, caused a greater inhibition of the Cyt pathway than O, uptake by mitochondria in the absence of any inhibitor (Fig. l), except for the oxidation of NADH alone. The percentage of inhibition of the Cyt path- way by elevated C, during oxidation of succinate was 16% 200 I 180 1 20 OI II 3 Ambmt CO, AmbientCO,+ 36OpL.L' I I Figure 3. The direct effect of elevated C, on Cyt pathway activity. Vcyt.KCN was measured in mitochondria preincubated for 10 min with 0 (O) or 0.1 (m) mM DIC in the presence of 2 mM SHAM. For other details see "Materiais and Methods" and the legend to Figure 1. (P < 0.001) (Fig. 3, SUCC); 22% when externa1 NADH and pyruvate were supplied (P = 0.030) (Fig. 3, NADH-tPYR); and 17% with both NADH and succinate (P = 0.009; plants grown in growth cabinets) (Fig. 3, SUCC+NADH). Oxida- tion of NADH alone gave the lowest inhibition (9%, P = 0.056) (Fig. 3, NADH). Increased C, had little or no effect on V,lt.KCN (Fig. 4). Elevated C, inhibited the alternative pathway only when the mitochondria were oxidizing succinate (17%; P = 0.145, rank summary test) and not when NADH, either alone or in the presence of pyruvate, was the substrate. with the oxidation of succinate, the time needed to attain maximal inhibition of O, uptake by increased C, was greater for the Vcyt.SHAM than for Valt.KCN (Fig. 5). With the Cyt pathway, maximal inhibition was achieved after 5 to 6 min, whereas only 2 to 3 min were needed to maximally inhibit the alternative pathway. DISCUSSION The results of this study show that the reported direct inhibitory effect of increasing C, on plant respiration (Amthor et al., 1992) is also seen in isolated plant mito- chondria. The effect was mediated at least in part by inhi- bition of Cyt c oxidase and SDH. Elevated C, did not inhibit the alternative oxidase. The levels of inhibition ob- tained in soybean cotyledon mitochondria matched those reported for soybean leaves and whole plants by doubling ambient levels of C, (Bunce, 1990; Byrd et al., 1992; Thomas and Griffin, 1994). Elevated C, inhibited the oxidation of succinate and NADH by mitochondria (Fig. 1). Inhibition of O, uptake o" k a 5O Ambient CO, J- = Ambient C02 + 360pL L r Figure 4. The direct effect of elevated C, on cyanide-resistant alter- native pathway activity. V,,, wd3 rnedsured in mitochondria pre- incubated for 10 min with 0 (O) or 0.1 (m) mM DIC in the presence of 1 mM KCN. For other details see "Materials and Methods" and the legend to Figure 1. Effects of CO, on Mitochondrial Respiration 1353 /L I' O 1 2 3 4 5 6 10 Time after adding O.1mM DIC (min) Figure 5 . The time course of inhibition of the Cyt (Vcyt.SHAM) and alternative (Va,,.KCN) pathways by elevated C, in soybean cotyledon mitochondria. Concentrations used were: 5 mM succinate, 1 mM KCN, 2 mM SHAM, and 0.1 m M DIC. Values are the means of two different experiments for each parameter. should reflect the inhibition of those components of the mitochondrial electron transport chain that control the overall rate of respiration (Padovan et al., 1989; Moore, 1992). The inhibition of Cyt c oxidase activity from differ- ent sources by elevated C, (Fig. 2A) and SDH activity (Table I) supports the results obtained with the entire mi- tochondrial electron transport chain, and suggests that the direct effect of elevated C, on intact mitochondrial respi- ration mainly affects the activity of the Cyt pathway. In our study, inhibition was greater under conditions in which Padovan et al. (1989) reported that substantial control of respiration resided at the level of Cyt c oxidase (Fig. 1, The inhibition of Cyt c oxidase was greater when the enzyme was preincubated with elevated C, prior to taking the measurement. Very high concentrations of DIC (10-20 mM, equivalent to 5-10% C,) inhibited the activity of pu- rified beef-heart Cyt c oxidase 40 to 50% (Palet et al., 1991, 1992). However, Palet et al. (1991, 1992) did not preincu- bate the enzyme with elevated C, prior to measurement. Without preincubation, titration of the activity of the pu- rified beef-heart Cyt c oxidase versus C, showed no signif- icant inhibition of activity at physiological levels of C, (Fig. 2B), but showed 50% inhibition when 10 mM DIC (5% C,; data not shown) was added without preincubation (GonzA- lez-Meler, 1995). These data show that the reaction between Cyt c oxidase and any form of DIC is time-dependent. The time-dependent lag in the effect of elevated C, on the activity of Cyt c oxidase (Fig. 2B) is also observed in intact mitochondrial respiration (Fig. 5). The slow equilib- rium reaction among soluble species of inorganic carbon in the mitochondrial matrix (Forster et al., 1969; Balboni and Lehninger, 1986) may explain this time lag, at least for the alternative pathway (Fig. 5). However, the time needed to reach maximal inhibition for the Cyt pathway was consid- SUCC). erably longer than the time expected for equilibrium of DIC chemical species to be attained (Fig. 5). Other proteins (e.g. Rubisco or hemoglobin) also react slowly with CO, (Mitz, 1979; Lorimer, 1983). The fact that CO, can readily cross biological membranes (Balboni and Lehninger, 1986) and the facility with which it carbamylates proteins (Mitz, 1979) suggest that a reversible protein carbamylation may be involved in the inhibition of Cyt c oxidase. Although from this study we cannot conclude which chemical species of inorganic carbon inhibited Cyt c oxidase, Palet et al. (1991, 1992) showed that inhibition of Cyt c oxidase from carna- tion callus and pea leaf mitochondria depended on the concentration of free CO, dissolved in the reaction me- dium. Bicarbonate can also inhibit plant Cyt c oxidase competitively, but only at very high concentrations (Miller and Evans, 1956). SDH activity of soybean cotyledons was also inhibited by elevated C, (Table I). However, the relative inhibition of SDH was greater at lower concentrations of succinate. Di- carboxylic (malonate, acetoacetate, and oxaloacetate) or monocarboxylic acids (formate, glycolate, and glyoxylate) all inhibit competitively the activity of SDH (DerVartanian and Veeger, 1964). Bicarbonate is also a monocarboxylic acid and has been reported to be a competitive inhibitor of SDH (Zeylamaker et al., 1970). The effect of succinate con- centration on the inhibition of SDH activity by elevated C, is consistent with the competitive nature of the inhibition by bicarbonate reported by Zeylemaker et al. (1970). Inhi- bition of the activity of SDH by 1200 pL L-' C, has also been observed in root mitochondria (Reuveni et al., 1995). Oxygen uptake through the VCyt-SWAM was always in- hibited by C, independent of the substrate used, although in the case of NADH alone the inhibition was minimal(9%) (Fig. 3). During the oxidation of succinate, significantly more control of respiration resides at Cyt c oxidase than during the oxidation of NADH (Padovan et al., 1989). This means that inhibition of the Cyt c oxidase by C, (Fig. 2) will not be able to reduce the rate of oxidation of NADH alone, as is observed in oxidation of succinate (Fig. 3), in which C, also inhibited SDH activity (Table I). Inhibition of the Cyt pathway by increased C, was considerably greater during oxidation of NADH and pyruvate than with NADH alone. It should be noted that the rate of Cyt pathway activity during the oxidation of NADH alone is lower than that for the oxidation of NADH and pyruvate (Fig. 4) (see also Ribas-Carb? et al., 1995), suggesting that the sites of met- abolic control during the oxidation of NADH alone versus NADH and pyruvate are not comparable. The alternative oxidase is apparently not inhibited by elevated C,, because its pathway (Valt.KCN) was not inhib- ited when mitochondria were oxidizing NADH, when sig- nificant metabolic control at the level of the alternative oxidase would be expected (with or without pyruvate) (Fig. 4). Thus, the inhibition of the Cyt pathway shown in Figure 4 for the oxidation of NADH can be attributed to the inhibition of Cyt c oxidase. However, the alternative path- way was inhibited when mitochondria oxidized succinate. Such an inhibition is a consequence of inhibition of SDH by elevated C, (Figs. 4 and 5). 1354 GonzAlez-Meler et al. Plant Physiol. Vol. 112, 1996 O u r results suggest that a t least par t of the so-called direct effect of elevated C, on respiration reported in tis- sues (e.g. Amthor e t al., 1992) may be located a t the leve1 of the mitochondria. A n increase i n the concentration of CO, equivalent to 360 pL L-' inhibited the rate of mitochon- drial O, uptake by 10 to 15%, depending on the substrate utilized. Greater inhibition w a s found during the oxidation of succinate. This can be explained by the direct inhibition of SDH a n d Cyt c oxidase by elevated C,. There w a s no direct effect of increased C, on the alternative oxidase. Because a11 of the decarboxylations in the Krebs cycle form CO, (Balboni and Lehninger, 1986), and because bicarbon- a te concentration i n the mitochondrial matrix m a y fluctu- ate between 0.05 a n d 0.4 mM (calculated from Raven and Newman, 1994, and refs. therein), identifying which chem- ical species (free CO, or bicarbonate) inhibits mitochon- drial activity will be useful in establishing the physiologi- cal consequences of this effect. It is also noteworthy that the acclimation effect of C, on plants or tissues grown in elevated C, affects the activity and amount of Cyt c oxidase (Azc?n-Bieto e t al., 1994) a n d SDH (Frenkel and Patterson, 1973), suggesting that these enzymes are important for the control of respiration in a high-CO, world. ACKNOWLEDCMENTS The authors would like to thank Drs. Joaquim Azc?n-Bieto, Damian Barrett, Joseph Berry, Bruce Hungate, James Jacob, Roser Matamala, Josep PeAuelas, and Ann Umbach for helpful sugges- tions regarding the manuscript. 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