INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The green alga Chlamydomonas reinhardtii is a facultative "acetate flagellate" capable of growing heterotrophically on acetate, but not on Glc or other related carbon sources (Harris, 1989). Many studies of light stress and light regulation of photosynthetic gene expression have been carried out with acetate-grown cells (e.g. Ohad et al., 1990; Danon and Mayfield, 1991; Drapier et al., 1992). Therefore, we were interested in the potential effects acetate may have on photosynthesis and related processes. Acetate is metabolized to triose following ATP-dependent entry into the glyoxylate cycle, whereas inorganic carbon is reduced to triose during photosynthesis. Acetate metabolism may exert opposing influences on utilization of inorganic carbon. Previous studies have reported that acetate transiently inhibits photosynthesis (Endo and Asada, 1996) and stimulates respiration in light-grown cells of C. reinhardtii bubbled with air (Fett and Coleman, 1994; Endo and Asada, 1996), possibly via increased alternative oxidase activity (Weger et al., 1990a, 1990b). The ability of acetate to induce isocitrate lyase, the key glyoxylate cycle enzyme necessary for its utilization, is attenuated in the presence of light and inorganic carbon (Martinez-Rívas and Vega, 1993). Conversely, acetate represses expression of nuclear-encoded chloroplast proteins involved in light harvesting and inorganic carbon fixation (Goldschmidt-Clermont, 1986; Kindle, 1987). Thus, mixotrophically grown cells of C. reinhardtii may respond differently to light stress than photoautotrophically grown cells, potentially confounding interpretation of responses to genetic and environmental manipulations.

A particularly appropriate tool for studying biomass partitioning is stable isotope ratio mass spectrometry. Estep and Hoering (1980, 1981) determined the fraction of reduced biomass resulting from photosynthesis during mixotrophic growth of Chlorella sorokiniana on Glc or acetate medium in the presence of 1% CO₂ using stable hydrogen isotope analysis. Differences in ¹³C have also been used to determine the time of onset of autotrophy in developing seedlings (Deléens et al., 1984; Maillard et al., 1994a, 1994b). Dual isotope methods have been applied to assess carbon and nitrogen allocation during maize stem elongation (Cliquet et al., 1990) and biomass derived from translocated Suc and photosynthesis in the partially photosynthetic hypsophylls (husks) of maize (Yakir et al., 1991) and in vitro-grown potato plantlets (Wolf et al., 1998). Additionally, the effect of acetate on respiratory pathway partitioning can be assessed by on-line analysis of stable ¹⁸O₂ discrimination (Weger et al., 1990a, 1990b; Ribas-Carbo et al., 1995).

The proportions of biomass attributable to photosynthetic CO₂ assimilation and to heterotrophic respiration of a reduced carbon source in mixotrophically cultured algal cells can be estimated from stable isotope determinations using the following equation (modified after Cliquet et al., 1990), provided the isotopic signatures of the two sources of carbon are sufficiently different:

Photosynthetic fraction of carbon biomass=
This quantitative relationship prevails because: (a) uptake and respiration of reduced carbon substrates result in comparatively little discrimination (about 1) relative to the source (DeNiro and Epstein, 1976), and (b) photosynthetic CO₂ fixation is an irreversible process and therefore subsequent biochemical events have only small effects on the ¹³C value (O'Leary, 1988). Results presented in this paper show that the presence of acetate during growth in saturating light and CO₂ inhibits photosynthesis and autotrophic carbon assimilation in wild-type C. reinhardtii. This effect was exacerbated in wild-type C. reinhardtii grown under low irradiance or in air, and by site-specific chloroplast mutations that predispose C. reinhardtii to photoinhibition.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Strains

Cultures of wild-type (CC-125, 137C mt⁺), a non-photosynthetic psbA deletion mutant (CC-744, ac-u- mt⁺), and a respiration-deficient mutant of Chlamydomonas reinhardtii lacking cytochrome oxidase activity (CC-314, dk-97 mt⁾ described by Harris (1989) were obtained from the Chlamydomonas Genetics Center (Duke University, Durham, NC). The 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-resistant transformant dr (CC-2827) originated from biolistic bombardment of CC-125 with a cloned 10-kb BamHI-BglII fragment of chloroplast DNA containing the psbA gene from the herbicide-resistant DCMU-4 mutant (Erickson et al., 1984) bearing a Ser-264 to Ala change in the D1 protein of photosystem II (PSII). The spr/sr strain (CC-2811) is impaired in chloroplast protein synthesis as a consequence of two single antibiotic-resistance point mutations (A₄₇₄-> C, A₁₁₂₃-> G) in the chloroplast-encoded 16S rRNA gene (Harris et al., 1989; Heifetz et al., 1997). This strain was obtained by biolistic transformation of CC-125 with a cloned 7.0-kb BamHI chloroplast DNA fragment containing the mutant 16S rRNA gene and most of the 23S rRNA gene proximal to the intron near the 3' end of this gene.

Growth Conditions

Cells were grown in liquid cultures shaken and bubbled with 5% (v/v) CO₂-enriched air at 25°C under continuous illumination from cool-white fluorescent lamps under low (<25 µmol m² s¹), moderate (350 µmol m² s¹), or high (600 µmol m² s¹) photosynthetically active radiation (400-700 nm). High-salt minimal medium (HS) was used for photoautotrophic experiments, whereas mixotrophic and heterotrophic growth were carried out in either high-salt acetate medium (HSHA) containing 29.4 mM sodium acetate or in Tris-acetate phosphate (TAP) buffer containing 17.5 mM acetate (Harris, 1989). Liquid cultures were maintained in the early- to mid-exponential growth phase by periodic dilution for several days to ensure acclimation to the growth environments. Aliquots of these cultures were used to inoculate 250- to 300-mL liquid cultures into 500-mL baffled shake flasks (Bellco, Vineland, NJ) at 2 × 10⁵ cells mL¹, or were spread onto 1.5% agar plates of the same medium for analysis. The pH of liquid cultures in HS, HSHA, and TAP medium bubbled with 5% CO₂ remained within the range 6.6 to 7.4. Cultures on agar plates supplemented with 5% CO₂ were placed inside a closed plexiglass chamber and supplied with mixed gas at a flow rate of approximately 500 cm³ min¹, while those at ambient CO₂ levels (in air) were maintained on lighted shelves at 25°C.

Measurement of Photosynthesis, Respiration, and Growth Rates

Cells for photosynthesis measurements were grown under high light and bubbled with 5% CO₂ in cultures of HS supplemented with 0, 3.7. 7.4, 14.7, and 29.4 mM sodium acetate to the early exponential phase (A₇₅₀ = approximately 0.1), gently pelleted, and resuspended (A₇₅₀ = 0.175) in fresh growth medium with 10 mM NaHCO₃. Respiration, maximum rate of net photosynthetic O₂ evolution, and chlorophyll fluorescence quenching were measured at growth temperature under 300 and 600 µmol m² s¹ red actinic light, as described by Heifetz et al. (1997).

The incorporation of ¹⁴CO₂ into acid-stable products was measured under high light in 1.5-mL aliquots of cells in 40-mL centrifuge tubes (Corex, Corning, NY) containing 0.5 mL of a bicarbonate reaction mixture (0.2 M Tris, pH 8.0, 40 mM NaHCO₃, 4 µCi NaH¹⁴CO₃ [6.6 Ci/mol, NEN Life Science Products, Boston]) and a 1-cm magnetic stir bar. Cells were stirred continuously, and 0.5-mL aliquots were removed after 6, 12, and 18 min and placed in scintillation vials with 500 µL of 1 N HCl to drive off the unincorporated ¹⁴C. Duplicate 100-µL aliquots from each sample were counted in 10 mL of EcoLume (ICN, Costa Mesa, CA) scintillation fluid. Rates of ¹⁴C incorporation into acid-stable products were linear for all samples over the 18-min assay period.

Chlorophyll content and exponential growth rates (cell/biomass doubling times) were calculated as described previously (Lers et al., 1992; Förster et al., 1997).

[¹³C]Acetate Labeling

The ¹³C of acetate samples from eight different suppliers ranged from 44.1 to 19.5. Laboratory compressed air (¹³C approximately 8) was mixed with bottled CO₂ from various sources to produce 5% CO₂ in which the ¹³C varied from 44 to +4 between different experiments. The dynamic range of the isotope discrimination assay for TAP-grown cells was expanded by supplementing the naturally available ¹³C from acetic acid with 2 mg L¹ 1,2 [¹³C]acetate (Sigma-Aldrich, St. Louis, catalog no. 28,201-4). Thus, the span of ¹³C of wild-type cultured in the dark or CC-744 cultured in dim light on TAP medium ranged from 21 (photoautotrophic growth on CO₂) to >110 (heterotrophic growth on ¹³C-TAP), permitting a very accurate estimation of the photosynthetic fraction of carbon assimilated under mixotrophic conditions.

Sample Preparation for Carbon Isotope Mass Spectrometry and ¹³C Determinations

Cells were harvested from liquid cultures in the mid-exponential phase (approximately 3 × 10⁶ cells mL¹) by centrifugation at room temperature, washed three times in double-deionized H₂O, pelleted in 1.5-mL microcentrifuge tubes at 4°C, and stored at 70°C until lyophilization. Cells grown for 5 to 10 d on agar plates were transferred directly to microcentrifuge tubes with sterile inoculating loops, avoiding transfer of agar substrate (¹³C = 17 to 19). Lyophilized samples were ground finely, and aliquots (200-2,000 µg) were weighed into tin capsules and combusted in an automated elemental analyzer (NA1500, Carlo Erba, Milan) for determination of ¹³C/¹²C ratios using a stable isotope ratio mass spectrometer (VG Isogas SIRA II, Middlewich, UK) (Yakir et al., 1991). Values are reported as means ± SE of duplicate or triplicate samples as indicated in the figures and tables.

On-Line Respiratory ¹⁸O Fractionation

The on-line sample trapping and preparation system used for liquid-phase ¹⁸O₂ discrimination during respiration was that described by Ribas-Carbo et al. (1995). Aliquots of exponentially growing liquid cultures (0.04 to 0.1 A₇₅₀ = 0.5 to 2 × 10⁶ cells mL¹) were transferred to a 30-mL capacity cylindrical plexiglass chamber connected to a vacuum trapping line and fitted with an O-ring-sealed plunger to facilitate sampling without the introduction of air bubbles. Dissolved gases were sparged from 5-mL aliquots of the cell suspension sampled at 8- to 20-min intervals by bubbling with He, and the O₂ was trapped at 77°K on a molecular sieve after removal of CO₂ and H₂O in a vacuum line. Cells treated with 10 µM DCMU to inhibit photosynthetic O₂ evolution gave the same results as experiments using a darkened chamber (data not shown). Oxygen isotope discrimination was calculated as described in Ribas-Carbo et al. (1995) using an Ar/N₂ ratio of 0.0388 to account for the aqueous diffusivities of these gases. Slope SEs were adjusted for sample size (Weger et al., 1990b) and F tests of significance were used for pairwise comparisons of regression slopes.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Effect of Acetate Concentration on Photosynthesis and Growth Rate in Saturating Light and CO₂

Maximum rates of net O₂ evolution and CO₂ incorporation into acid stable products by wild-type cells of C. reinhardtii declined with increasing acetate concentration in the mixotrophic growth medium under high (saturating) light and CO₂ conditions (Table I). HSHA, which contains 29.4 mM acetate, effected a 48% reduction in the maximum rate of O₂ evolution and a 56% reduction in CO₂ fixation rate. The lowest acetate concentration tested (3.7 mM) reduced O₂ evolution and CO₂ fixation by 26% and 34%, respectively. In contrast, the growth rate, respiration, PSII efficiency, and chlorophyll content were not affected by acetate concentration.

View this table: [in this window] [in a new window]   Table I.   Effect of acetate concentration on growth rate and photosynthesis of wild-type C. reinhardtii in the early log phase when grown and analyzed under saturating light (600 mmol m2 s1) in the presence of 5% CO2 Photosynthesis values are expressed in absolute units and as a percentage of the control HS culture without acetate.

Evaluation of Isotopic Fractionation during Heterotrophic and Photoautotrophic Growth of C. reinhardtii

The assessment of the relative contributions of photosynthetic CO₂ fixation and respiration of acetate to cell metabolism during mixotrophic growth first requires baseline isotopic signatures of cells grown heterotrophically and photoautotrophically. The ¹³C value of heterotrophically grown wild-type cells (data not shown) and cells of a nonphotosynthetic psbA deletion mutant (CC-744) grown in dim light on HSHA (Fig. 1A) was strongly correlated with the ¹³C of the acetate present in the growth medium. These results demonstrate that heterotrophic metabolism of acetate by C. reinhardtii results in little or no carbon isotope discrimination. The ¹³C values of wild-type C. reinhardtii biomass grown photoautotrophically under saturating light and CO₂ remained relatively constant throughout the exponential portion of the growth curve and increased only slightly in the early stationary phase (Fig. 1B). Growth of wild-type C. reinhardtii in HS liquid cultures supplemented with 5% CO₂ at three irradiance levels (200, 350, and 600 µmol m² s¹) resulted in an average isotopic discrimination relative to the source () of 24.6 ± 0.3, which in agreement with earlier data (Sharkey and Berry, 1985). The much more negative biomass ¹³C values in Figure 1B are due to the use of CO₂ sources with different isotopic compositions. The  with respect to source CO₂ in cells grown autotrophically on agar plates with 5% CO₂ was 21.9 (19.9 to 22.6 in four separate experiments with different genotypes). In cells of wild-type grown autotrophically on agar plates in air, an even lower discrimination was observed ( = 6.3 to 10.7). Evidently, CO₂ limitations in the wet cell mass on the agar surface and/or the CO₂ concentrating mechanism were responsible for the smaller discriminations. As there was no detectable change in discrimination during heterotrophic growth on HSHA plates or liquid HSHA medium (Fig. 1A), acetate diffusion problems on the agar plates can be ruled out.

View larger version (16K): [in this window] [in a new window]   Figure 1.   Carbon isotope composition (13C) of cell biomass during photoautotrophic and heterotrophic growth of C. reinhardtii. A, Heterotrophic growth of C. reinhardtii on HSHA containing 29.4 mM acetate of varying 13C. Cells of the non-photosynthetic mutant strain CC-744 were grown in dim light (5-25 µmol m2 s1) on HSHA plates () or 250-mL HSHA shake flask cultures () formulated with sodium acetates differing in natural abundance of 13C. The stable carbon isotope compositions of the source acetates and lyophilized biomass were determined as described in the text. B, Photoautotrophic growth of wild-type C. reinhardtii in 250-mL liquid cultures bubbled with 5% CO2 under moderate light (350 µmol m2 s1) at 25°C. Samples were harvested at the indicated times for hemocytometer counts, spectrophotometric determination of biomass concentration (A750), and biomass 13C measurement. Each value and data point are the means ± SE (in parentheses) of two independent measurements. Data points were fitted to a logistic growth equation. Cell concentration at the beginning of the experiments was 2 × 105 cells mL1 (A750 = 0.01).

Estimation of Carbon Acquisition during Mixotrophic Growth of Wild-Type C. reinhardtii and Mutants with Impaired PSII Function

Isotopic composition of mixotrophically grown wild-type C. reinhardtii was determined using HSHA (29.4 mM acetate, ¹³C = 27 to 44) in liquid cultures bubbled with 5% CO₂ (¹³C = 19.5) or on agar plates exposed to ambient CO₂ in air (¹³C = 8). The photosynthetic fraction of carbon biomass (see equation) was calculated from these values, and the baseline heterotrophic and photoautotrophic isotopic composition. Consistent with the inhibitory effects of acetate on photosynthesis (Table I), marked reductions were observed in the fraction of biomass carbon assimilated photosynthetically (Table II). In HSHA liquid medium under saturating light and CO₂, the photosynthetic fraction did not exceed 55%. On plates exposed to air in high light, this fraction was only 23%. Strikingly, wild-type cells grown on HSHA plates exposed to air in moderate or low light showed little or no detectable autotrophic carbon assimilation.

TAP medium (17.5 mM acetate) is commonly used to grow wild-type and mutant strains of C. reinhardtii for photosynthetic and molecular analysis (Rochaix et al., 1998). The relative photosynthetic fraction of carbon metabolism in the wild type and in mutations affecting chloroplast protein synthesis and PSII function was determined using TAP medium supplemented with ¹³C to make the ¹³C acetate much more positive than air or the 5% CO₂ source. The biomass of wild-type cells grown mixotrophically in liquid cultures of TAP medium (¹³C = approximately +95 to +99) bubbled with 5% CO₂ (¹³C = +2.9) showed a photosynthetic fraction of only 78% under saturating irradiance and this declined to 62% at subsaturating irradiance. Thus, even under the optimal light and CO₂ conditions, nearly one quarter of the carbon in the wild type was derived heterotrophically when the cells were provided 17.5 mM acetate and 5% CO₂ as alternative carbon sources. As expected, ¹³C values for the dr mutant, with slower PSII electron transfer, showed a lower photosynthetic fraction compared with wild type grown mixotrophically under identical moderate and high light conditions (Fig. 2). The spr/sr mutant, which has defects in chloroplast protein synthesis, was even more dependent on heterotrophically assimilated carbon during mixotrophic growth. Reductions in autotrophic competence of the two mutants under high light compared with the wild type correlate well with their impaired light-saturated photosynthetic rates and growth rates (Heifetz et al., 1992, 1997).

View larger version (21K): [in this window] [in a new window]   Figure 2.   Photosynthetic fraction of carbon biomass from wild-type and mutant C. reinhardtii cells grown on 13C-TAP (17.5 mM acetate) in liquid culture. Cells of wild type (circles), spr/sr (diamonds), and dr (squares) were grown autotrophically to the early-/mid-exponential phase in HS bubbled with 5% CO2 (13C = +2.9), heterotrophically in the dark on TAP supplemented with 13C (13C = +99; ), and mixotrophically on 13C TAP bubbled with 5% CO2 at two irradiance levels, 350 µmol m2 s1 (closed symbols) and 600 µmol m2 s1 (open symbols). The fraction of carbon biomass resulting from photosynthetic carbon reduction was calculated as described in the text from the average photoautotrophic, heterotrophic, and mixotrophic biomass 13C values.

Role of the Alternative Oxidase during Mixotrophic and Autotrophic Growth

We established baseline isotopic signatures for respiratory O₂ exchange via the cytochrome oxidase and alternative (cyanide insensitive oxidase) pathways during mixotrophic and photoautotrophic growth of wild-type cells to determine if partitioning between the two respiratory pathways is influenced by acetate. For end point determinations of discrimination due to only the alternative or cytochrome oxidases, wild-type cells were pretreated for 15 min with KCN or the alternative oxidase inhibitor propyl gallate. Alternatively, photoautotrophically grown cells of the dk-97 mutant lacking cytochrome oxidase activity (Wiseman et al., 1977; Husic and Tolbert, 1987) were used to assess discrimination due to the alternative oxidase pathway. The oxygen isotope discrimination in photoautotrophically grown wild-type cells in minimal medium ( = 18.8) reveals little engagement of the alternative pathway (Table III), which is in agreement with previous work (Weger et al., 1990b). Respiratory discrimination of wild-type cells grown in the presence of acetate and 5% CO₂ (20.8) was not significantly affected by propyl gallate treatment (Table III). This demonstrates that the alternative oxidase was not engaged in the presence or absence of acetate under these conditions. These results, together with the lack of increased dark respiration in mixotrophically grown cells, indicate that the effects of acetate are on photosynthesis rather than on respiration.

View this table: [in this window] [in a new window]   Table III.   Discrimination against 18O2 during dark respiration by wild-type C. reinhardtii cells grown to the mid-exponential phase at 600 µmol m2 s1 irradiance in liquid cultures bubbled with 5% CO2 KCN results in discrimination due solely to O2 consumption via the alternative oxidase pathway. Propyl gallate results in discrimination due solely to O2 consumption via the cytochrome oxidase pathway.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Our results demonstrate that growth of wild-type C. reinhardtii in the presence of 3.7 to 29.4 mM acetate in saturating light and CO₂ inhibits photosynthesis, as measured by the maximum rates of net O₂ evolution and ¹⁴C fixation. However, neither dark respiration nor engagement of the alternative oxidase pathway were affected. Fett and Coleman (1994) reported that acetate stimulated respiration in cells grown mixotrophically in air, and Endo and Asada (1996) demonstrated a similar response immediately upon addition of acetate to autotrophically grown cells. Growth rates in our experiments were unaffected by the large reduction in photosynthesis in the presence of acetate (Table I). Moreover, analysis of stable carbon isotope composition of biomass from mixotrophically grown cells revealed a marked shift from autotrophic to heterotrophic carbon metabolism in response to both environmental and genetic manipulation of C. reinhardtii. The stable isotope data (Table II) indicate that carbon derived from acetate in the light can substitute for up to 50% of photoautotrophically acquired carbon in liquid cultures under the saturating light and CO₂ conditions optimal for photosynthetic growth of C. reinhardtii (Heifetz et al., 1997). At subsaturating irradiance and CO₂ levels in the presence of specific mutations reducing photosynthetic performance, further decreases in the contribution of photosynthetic carbon assimilation were observed under mixotrophic growth conditions.

Although one might expect that the addition of a reduced carbon source would lower the proportion of biomass carbon derived from photosynthesis, the notion that acetate metabolism in saturating light and CO₂ can quantitatively substitute for photosynthetic carbon assimilation to drive growth in C. reinhardtii is probably overly simplistic. There is undoubtedly a dynamic relationship between acetate metabolism and photosynthesis that involves both mitochondria and chloroplasts. Consistent with other treatments that deplete cell ATP, Gans and Rebéillé (1990) found that the addition of acetate to autotrophically grown C. reinhardtii decreased PSII fluorescence and promoted a transition from state I to state II, presumably with attendant adjustment of the antenna architecture of the photosynthetic apparatus (Bulté et al., 1990). These observations were confirmed and extended by Endo and Asada (1996), who showed that the addition of acetate produced transient non-photochemical quenching in the light, which was sustained in the dark and associated with a reduction in PSII efficiency. Whether these primary events, thought to be mediated by chlororespiration (Bennoun, 1998), account for the long-term decline in photosynthetic O₂ evolution and carbon assimilation observed here remains to be assessed. Greater inhibition of photosynthesis by acetate at lower light intensities (Table II; Fig. 2) would be consistent with such a reduction in PSII efficiency, but this was not reflected in our dark-adapted measurements of F_v/F_m (Table I).

The first step in acetate utilization is the ATP-dependent production of acetyl coenzyme A. Therefore, in mixotrophic growth under limiting light, ATP demand for acetate assimilation may itself limit photosynthetic carbon reduction. These effects may be exacerbated if CO₂ is limited due to the induction of the carbon concentrating mechanism (Spalding, 1998). Acetate may also exert inhibitory effects on metabolism, as concentrations above 6.7 mM were reported to inhibit heterotrophic growth of wild-type C. reinhardtii (Chen and Johns, 1994). In the absence of acetate, reduced photosynthesis in several C. reinhardtii mutants with impaired D1 function (Frster et al., 1997; Lardans et al., 1998) or resistance to very high light (Förster et al., 1999) did not directly affect growth rate. These observations suggest that metabolic variables other than photosynthetic CO₂ fixation may sometimes limit growth.

The mechanisms underlying the effects of acetate on photosynthesis in our long-term growth experiments may also involve changes in gene expression. In plants and algae, carbon metabolites (including acetate) are known to down-regulate the expression of nuclear genes encoding chloroplast proteins involved in photosynthesis and in non-photosynthetic carbon metabolism (Kindle, 1987; Sheen, 1990, 1994). At the transcriptional level, acetate is a potent repressor of synthesis of enzymes involved in photosynthetic carbon reduction, as well as an inducer of the glyoxylate cycle-specific enzymes malate synthetase (Neilson and Lewin, 1974) and isocitrate lyase (Martinez-Rívas and Vega, 1993). Thus, transcriptional and translational regulation of both nuclear and chloroplast genes encoding photosynthetic components and enzymes involved in acetate metabolism would be expected to respond dynamically to the presence of acetate. These molecular processes, as well as the physiological events they influence, should therefore be compared in both mixotrophically and photoautotrophically grown cells.

In summary, we show that both photosynthetic incorporation of inorganic carbon and the maximum rate of O₂ evolution in C. reinhardtii can be significantly diminished by growth in the presence of acetate. Under some circumstances (limiting light, high acetate concentrations, growth on solid medium in air) photosynthetic carbon gain is virtually abolished. In studies involving mutants of C. reinhardtii with partial photosynthetic defects that do not cause obligate heterotrophy, very different interpretations of their metabolic consequences might be obtained depending on the presence or absence of acetate. Consequently, interpretation of the effects of environmental or other manipulations may be confounded by acetate-induced impairment of photosynthetic performance.