A Photorespiratory Mutant of Chiamydomonas reinhardtii - NCBI

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(data not shown). The rapid recovery suggested that inhibition of photosynthesis in 1 8-7F by preillumination was not caused by irreversible photoinhibition or ...
Plant Physiol. (1990) 93, 231-237 0032-0889/90/93/0231 /07/$01 .00/0

Received for publication September 29, 1989 and in revised form December 21, 1989

A Photorespiratory Mutant of Chiamydomonas reinhardtii' Kensaku Suzuki2, Laura Fredrick Marek, and Martin H. Spalding* Department of Botany, Iowa State University, Ames, Iowa 50011 ABSTRACT

limiting CO2 concentrations excrete copious amounts of glycolate, indicating substantial flow of carbon into the photorespiratory pathway (4, 8). Glycolate excretion cannot be detected in air-adapted cells, however, except under very low levels of external CO2 (4, 8), or in the presence of metabolic inhibitors (14, 15). This is evidence that carbon flow into the pathway is substantially decreased and balanced with the metabolic capacity to process glycolate in air-adapted cells (14, 15). Because air-adapted wild-type cells can be forced to excrete detectable amounts of glycolate by the addition of an inhibitor of glycolate metabolism ( 14), some photorespiratory carbon flow apparently continues in the presence of a fully operational CO2 concentrating mechanism. Mutants which require an elevated CO2 concentration for photoautrophic growth have been isolated in C. reinhardtii. All high-CO2 requiring mutants characterized so far have apparent defects in the CO2 concentrating mechanism or in its regulation (12, 13, 21, 22, 26). We have reported that some high-CO2 requiring mutants of Chlamydomonas have a photosynthetic affinity for CO2 comparable to that of wild type (25). It is unlikely that this characteristic would be associated with a mutation involving the CO2 concentrating mechanism. In this paper, we demonstrate that one of these mutants, 187F, is apparently deficient in phosphoglycolate phosphatase, the first enzyme in the photorespiratory metabolic pathway.

A mutant strain of Chiamydomonas reinhardtii, designated 187F, has been isolated and characterized. 18-7F requires a high CO2 concentration for photoautrophic growth in spite of the apparent induction of a functional CO2 concentrating mechanism in air-adapted cells. In 2% 02 the photosynthetic characteristics of 18-7F and wild type are similar. In 21% 02, photosynthetic 02 evolution is severely inhibited in the mutant by preillumination in limiting C02, although the apparent photosynthetic affinity for inorganic carbon is similar in preilluminated cells and in cells incubated in the dark prior to 02 evolution measurements. Net CO2 uptake is also inhibited when the cells are exposed to air (21% 02, 0.035% C02, balance N2) for longer than a few minutes. [14C]Phosphoglycolate accumulates within 5 minutes of photosynthetic 14CO2 fixation in cells of 18-7F. Phosphoglycolate does not accumulate in wild type. Phosphoglycolate phosphatase activity in extracts from air-adapted cells of 18-7F is 10 to 20% of that in wild-type Chiamydomonas. The activity of phosphoglycolate phosphatase in heterozygous diploids is intermediate between that of homozygous mutant and wild-type diploids. It was concluded that the high-CO2 requiring phenotype in 18-7F results from a phosphoglycolate phosphatase deficiency. Genetic analyses indicated that this deficiency results from a single-gene, nuclear mutation. We have named the locus pgp-1.

MATERIALS AND METHODS

The unicellular green alga Chiamydomonas reinhardtii shows almost no oxygen inhibition of photosynthesis and near zero CO2 compensation concentration when grown under air levels of CO2 and 02 (9, 21). Air-adapted wild-type cells also show a much greater apparent affinity for CO2 in photosynthesis than do cells grown under elevated CO2 concentrations (21). These characteristics have been explained as resulting from the operation of a CO2 concentrating mechanism in air-adapted cells (3). This mechanism is thought to involve active import and accumulation of Ci3 which results in an increased CO2 concentration at the site of Rubisco. The increased CO2 to 02 ratio inhibits oxygenase activity of Rubisco and presumably decreases the flow of carbon into the photorespiratory pathway (3). Wild-type cells adapting to

Chiamydomonas reinhardtii wild-type strain 2137 (23) and a high-CO2 requiring mutant 18-7F selected as previously described (23), were grown photoautotrophically on a gyratory shaker under continuous illumination (100 umol photons m-2 s-'). The cultures were aerated with 5% CO2 (CO2 enriched cells) and adapted, when necessary, to 0.03% CO2 for 24h (air-adapted cells) as described previously (22). Strains were cultured in minimal salts medium equilibrated with 5% CO2 before innoculation. The minimal salts medium contained, per liter: K2HPO4, 0.143 g; KH2PO4, 0.073 g; NH4NO3, 0.4 g; MgSO4.7 H20, 0.1 g; CaCl2 2 H20, 0.05 g; trace elements stock (24), 1 mL; 2.0 M Mops titrated to pH 7.6 and final volume with Tris base, 10 mL. After equilibration with 5% CO2 the pH of the medium was approximately 6.9. Strains were maintained in the dark on minimal medium supplemented with 10 mM sodium acetate. Photosynthetic 02 exchange was measured at 25°C using cells suspended in 1 mL of Mops-NaOH or Mops-Tris (20 mM, pH 7.4) 5 min after washing twice with the same buffer. Before use the buffers were purged for at least 2 h with C02free air that had passed through an activated carbon filter column, a solution of concentrated NaOH, and a soda-lime

'Supported by grant No. CRCR-1-1591 from the Competitive Research Grants Office of the U.S. Department of Agriculture. 2 Present address: Institute of Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki 305, Japan. 3Abbreviations: C,, dissolved inorganic carbon; Rubisco, ribulose-

bisphosphate-carboxylase/oxygenase; NPP, p-nitrophenylphosphate; RuBP, ribulose bisphosphate; K0.5Cj, concentration of dissolved inorganic carbon required for half-maximal response of photosynthetic 02 evolution. 231

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indicating column. These treatments reduced the C1 in the buffers to a minimum. Buffers treated in this manner are hereafter referred to as 'CO2-free.' In dark-to-light ('D-L') experiments, cells suspended in C02-free buffer were incubated in the dark for about 5 min prior to adding NaHCO3, and the reaction started by turning on the light (500 ,umol photons m-2 s-'). In light-to-light ('L-L') experiments, cells suspended in C02-free buffer were incubated in the light until 02 evolution had stopped, an indication that internal C, was depleted, and the reaction started by adding NaHCO3. CO2 exchange was monitored in an open system as described previously (26). Cells were suspended in 6 mL of minimal medium (pH 7.0) in a water-jacketed glass vessel (25°C). Carbonic anhydrase (1200 Wilbur-Anderson units) from bovine erythrocytes (Sigma C7500) was added to insure rapid equilibration between CO2 and HCO3. Carbonic anhydrase assays were performed by monitoring pH change at 2°C in 25 mm barbital-buffered solution (19). Enzyme units were calculated from the equation: U = tblts 1, where tb and t, represent the time (s) measured for the pH change (8.3-7.3) with buffer alone (tb) and with sample (ta). P-glycolate phosphatase activity was determined by measuring P-glycolate dependent Pi release (7). The cells were washed twice and then sonicated in 20 mM Mes-KOH (pH 6.3) containing 5 mM MgCl2. The assay mixture included 20 mM Mes-KOH (pH 6.3), 5 mM MgCl2 and 4 mM P-glycolate, and the reaction was started by adding sonicated cell suspension. The reaction was terminated by adding perchloric acid (final concentration, 5%), and the precipitate was removed by centrifugation. Pi in the supernatant was measured with the Ames reagent (1). Nonspecific phosphatase activity was determined by the same method (7) using NPP as the sub-

strate.

'4C-labeling of photosynthetic products was done as described previously (21) with some modifications. Cells were washed once with C02-free Mops-KOH (50 mM [pH 7.0]), resuspended in 1 mL of the same buffer and equilibrated in a Rank 02 electrode in the dark. NaH'4CO3 was added (100 ,uM or 2.5 mm initial concentration) and photosynthesis initiated by turning on the light (500 ,umol photons m-2 s-'). Soluble cell extracts were fractionated into a cation fraction and a neutrals plus anions fraction on a cation exchange column of Bio-Rad AG 50W-8X, H+. Separation of the neutrals plus anions fraction was by anion exchange HPLC using a Spheresorb 5/25 SAX column from Phasesep. Neutral compounds eluted in the void. The anions were eluted with a KH2PO4 gradient based on the method developed by Giersch (5). Using 200 mM KH2PO4 as B (pH 2.8, adjusted with H3PO4) and 10 mm KH2PO4 as A (mixed as 5% B in deionized H20, no additional pH adjustment), all 14C-labeled compounds were eluted with a linear gradient from 0 to 60% B (1.09% min-', flow rate 1 mL min-'). The column was regenerated by increasing the solvent strength to 100% B (2.66% min-'), washing with 100% B for 10 min, decreasing solvent strength to 100% A (3.33% min-'), and washing with 100% A for 40 min before injecting another sample. Radioactivity was detected with a flowthrough heterogeneous scintillation counter. Standard ['4C]PGA was synthesized from RuBP using Rub-

isco and NaH'4CO3. Standard P-glycolate was synthesized from [U-'4C]RuBP using Rubisco. Standard [U-'4C]-labelled glucose-6-P, 6-P-gluconate, ribulose-5-P, and RuBP were synthesized from [U-'4C]glucose in sequential reactions using hexokinase, glucose-6-P dehydrogenase, 6-P-gluconate dehydrogenase, and phosphoribulokinase. [U-'4C]RuBP was purified by anion exchange chromatography (Dowex 1 x 8, Clcolumn eluted with a gradient of 0-0.3 M LiCl in 1 mM HCI), desalted (Sephadex G- 10-120) and the volume reduced before being used to synthesize standard ['4C]P-glycolate. All enzymes and chemicals were from Sigma except that [U-_4C] glucose was from NEN and Rubisco was isolated from soybean leaves using the method of Paech and Dybing (16). Genetic analyses were performed as previously described (26, 28). 1 8-7F was crossed with wild-type CC80 1 (spr- 1) for tetrad analysis. 1 8-7F/arginine requiring double mutants were constructed by crossing 18-7F with strains carrying the arg-2 or arg-7 markers. Diploids were constructed as described by Moroney et al. (23) using 18-7F/arginine requiring double mutants and wild-type strains CC1930 (arg-2) and 186A81 (arg-7). Complementation analyses were done as described by Winder and Spalding (28) based on the low frequency of recombination of the arg-2 and arg-7 loci (10). Phenotypes of tetrads and diploids were determined with spot tests on agar plates (23). Chl was determined after extraction into 96% (v/v) ethanol (29). RESULTS CO2 Concentrating Mechanism An important physiological characteristic of induction of the CO2 concentrating mechanism is a large increase in the apparent affinity of photosynthesis for CO2, most easily observed as a decrease in the K0.5CQ of photosynthetic 02 evolution. The Ko.5CQ for photosynthesis in air-adapted cells of 1 8-7F was less than that of CO2 enriched cells (Table I). The decrease in Ko.5Ci after adaptation to air is similar to that seen in wild type. Although the absolute value of the Ko.5Ci for photosynthesis is greater in the mutant than in wild type, the relative magnitude of the decrease is almost identical. At the pH ofthe measurements used in this study, this still represents a Ko.5CO2 of much less than that expected for Rubisco (4). An increase in carbonic anhydrase activity is another char-

Table I. Photosynthetic characteristics of C. reinhardtii Wild-Type 2137 and the High-CO2 Requiring Mutant 18-7F Air-adapted cells were exposed for 24 h to air after being grown at 5% Co2. CO2 enriched cells were grown at 5% Co2. CA Activityb Cell Type Strain Ko 5C,a units mg-' Chi /.M 70.4 25 24 h air-adapted 2137 12.3 200 CO2 enriched 24.9 55 24 h air-adapted 1 8-7F 5.3 450 C02 enriched a carbon Concentration of dissolved inorganic giving half-maximal b Carbonic response of photosynthetic 02 evolution at 21% 02anhydrase activity determined with intact cells.

.-;

A PHOSPHOGLYCOLATE PHOSPHATASE MUTANT OF CHLAMYDOMONAS

acteristic consistently associated with induction of the CO2 concentrating mechanism (21). Air-adapted cells of 18-7F show an increase in carbonic anhydrase activity (Table I), although the magnitude of the increase is somewhat less than in wild type. 02 has little effect on photosynthesis in air-adapted, wildtype cells, presumably because of induction of the CO2 concentrating mechanism, in contrast to the inhibition it causes in CO2 enriched cells (26). Similarly, the inhibitory effect of 0° on photosynthesis in 1 8-7F cells is much less in air-adapted cells than in CO2 enriched cells (Fig. 1). These characteristics, the decreased Ko.5Cj of photosynthesis, the increase in carbonic anhydrase activity and the decreased 02 inhibition of photosynthesis suggest that a functional CO2 concentrating mechanism is induced in 18-7F cells when the cells are transferred to limiting CO2. The mutation in 1 8-7F does not appear to be directly related to the CO2 concentrating mechanism. Phosphoglycolate Phosphatase Deficiency The commonly used method for determining the effect of NaHCO3 concentration on photosynthetic 02 evolution requires pre-incubation in light to deplete internal Ci before any addition of NaHCO3. In the case of 18-7F, preillumination resulted in an inhibition of photosynthesis (L-L in Fig. 2). When cells were equilibrated in the 02 electrode without preillumination, the photosynthetic rate was much higher (D-L in Fig. 2) and less affected by 02. Although the rate of photosynthesis was inhibited by preillumination, the Ko.5Cj values were not significantly affected. If L-L cells were incubated in the dark for 10 to 15 min, and photosynthesis measured with the D-L method, photosynthetic 02 evolution recovered to almost the maximal level seen in the D-L cells (data not shown). The rapid recovery suggested that inhibition of photosynthesis in 1 8-7F by preillumination was not caused by irreversible photoinhibition or damage, but by accumulation of a photosynthetic product. Because inhibition was greatest in low NaHCO3 and 21% 02, accumulation of a photorespiratory metabolite was implied.

C'p

21

E

E 0 1c 7.E

si

Net CO2 exchange in 24h air-adapted 18-7F cells was measured in 2% and 21% 02 with and without 350 ,uL L-' CO2 through dark-light-dark transitions (Fig. 3). The CO2 exchange pattern under 2% 02, where photorespiration is suppressed, was similar to that of wild-type (data not shown, cf 27) although the maximum exchange rate was lower in 187F. In wild type there was little difference in the CO2 exchange pattern between 2% and 21% 02. In 18-7F, however, the photosynthetic rate in 21% 02 and 350 ,uL L` CO2 began to decrease after a few minutes to a steady-state rate one-third of the maximal rate in 2% 02. This steady-state rate was similar to the photosynthetic 02 evolution rate seen in L-L cells. These data support the observations based on photosynthetic 02 evolution rates that a photorespiratory metabolite was accumulating in the light and inhibiting photosynthesis. In 0 uL L' C02, the CO2 exchange pattern was the same in 21% and 2% 02 from the beginning of the first dark period to the end of light period. In 21% 02, however, C02 evolution after the light to dark transition was inhibited for about 20 min in both 0 and 350 uL L- CO2 (Fig. 3). The P-glycolate phosphatase deficient mutant strain CS 119 of Arabidopsis thaliana exhibits similar C02 exchange characteristics (1 1). Analysis of acid-stable '4C-labeled photosynthetic products from 18-7F (Table II) showed a large accumulation of label in the photorespiratory metabolite, phosphoglycolate. Seventy-seven percent of the neutrals plus anions fraction (34% of the total label incorporated) accumulated in P-glycolate after 5 min of photosynthesis in CO2 enriched 18-7F cells labelled in low bicarbonate. No glycolate was detected. In wild-type cells labeled under the same conditions, P-glycolate was barely detectable although appearance of a significant proportion of [14C] in glycolate indicated flow of carbon into the photorespiratory pathway. Glycolate was never detectable in 1 8-7F but P-glycolate accumulation was always seen. These data are evidence for a blockage in the metabolism of Pglycolate in the mutant. P-glycolate accumulation was much less in air-adapted 1 8-7F cells, and this represents biochemical evidence for induction of the CO2 concentrating mechanism. Phosphoglycolate phosphatase activity in crude extracts of

02 cells .,

0

C a

233

-------

U

(A

NaHCO3,

mM

Figure 1. Response of photosynthetic 02 evolution to NaHCO3 concentration in air-adapted (solid lines) and C02 enriched (broken lines) cells of the high-CO2 requiring mutant 18-7F of C. reinhardtii. 02 evolution was measured in 2% (0) | .and 21% (@) 02.

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w-

.C

E Figure 2. Effect of preillumination on response of photosynthetic 02 evolution to NaHCO3 concentration in air-adapted cells of a high-CO2 requiring mutant 18-7F of C. reinhardtii. L-L: cells incubated in the light to deplete internal Ci prior to addition of NaHCO3; D-L: cells equilibrated in the dark before addition of NaHC03.

E0 'a

0 E 'f

C 0 ._ 0

-

0

NaHCO3,

mM

0

UX

x

E Figure 3. Effect of 02 and C02 on net C02 exchange in the C. reinhardtii high-CO2; requiring mutant 18-7F during dark-light-dark transitions. A, At 350 ,aL L-1 C02; B, at 0 yL L-1 C02. Exchange rates were determined with 24 h airadapted cells.

co

E 0

min

Time,

Table II. Percent of Total Acid-Stable '4C Incorporated by C. reinhardtii Wild-Type 2137 and the High C02 Requiring Mutant 18-7F Cells were grown continuously at 5% C02 or at 5% C02 and then for 24 h with air before labeling with 100 AM or 2.5 mM NaH'4CO3 for 5 min (pH 7.0, 250C). 2.5 mM NaHCO3 1 00 M NaHCO3

Ion Exchange

5%

Fraction

Wtb

CO2a

24 h

18-7Fb

wt

24 h air

5% C02

air' 18-7F

wt

18-7F

wt

18-7F

% incorporation of total acid-stable 14C

46.6 1.3

Insoluble Neutrals Acids

Glycolate Mono-P PGA P-glycolate RuBP Basic a Cell type.

6.4 11.1 3.9 0.4 6.1

46.0 1.1

50.3 2.1

52.0 4.6

58.4 2.7

61.8 5.9

61.5 2.5

51.5 8.5

16.2

4.1

17.0 3.5

3.6 3.9 5.0

7.6

10.1

10.7 10.8 1.4 12.3

6.9 5.5 3.3 0.3 13.5

c

4.6

2.4

34.0

12.2 15.5 8.0 11.0 b Strain. c Not detectable.

19.5 1.1

10.3

A PHOSPHOGLYCOLATE PHOSPHATASE MUTANT OF CHLAMYDOMONAS

Table IlIl. Phosphoglycolate Phosphatase and p-Nitrophenylphosphate (NPP) Phosphatase Activities in C. reinhardtii Wild-Type 2137 and the High-CO2 Requiring Mutant 187F Air-adapted cells were exposed for 24 h to air after being grown at 5% C02. CO2 enriched cells were grown at 5% C02. Substrate Strain

2137 18-7F

Cell Type

24 h air-adapted C02 enriched 24 h air-adapted C02 enriched

NPP P-glycolate mg-' CHI h-1 mmol Pi 32.3 8.5 19.9 7.8 4.7 9.2 3.0 6.4

1 8-7F was 10 to 20% of that in wild type (Table III). Nonspecific phosphatase activity in the cells was estimated using NPP because it is only marginally hydrolyzed by phosphoglycolate phosphatase in C. reinhardtii (7). There was no significant difference in NPP phosphatase activity among air-adapted or CO2 enriched cells of wild type and 1 8-7F. Phosphoglycolate phosphatase activity in 18-7F was about half that of NPP phosphatase activity. It is possible that the apparent residual phosphoglycolate phosphatase activity in 1 8-7F is entirely due to nonspecific phosphatase activity. If true, it is likely that much of the nonspecific phosphatase activity is not located in the stroma and would therefore be slow acting on phosphoglycolate. The activity of phosphoglycolate phosphatase increased by about 50% in air-adapted cells of 1 8-7F and wild type (Table III). Phosphoglycolate phosphatase activity in the crude extract from wild type was not inhibited by mixing with the extract from 1 8-7F (data not shown), demonstrating that the deficiency of phosphoglycolate phosphatase activity in 187F is not caused by an inhibitor produced in 1 8-7F cells but rather by reduced enzyme synthesis or the synthesis of a defective enzyme. In 1 8-7F, the activities of phosphoglycerokinase and NADPH-glyceraldehyde-3-P dehydrogenase were similar to those in wild type (data not shown), indicating that there was not a general decrease in the activity of stromal enzymes in the mutant.

Genetic Analysis The crosses between 18-7F and wild-type resulted in 2:2 segregation of the high-CO2 requiring and wild-type phenotypes in all of the tetrads tested (18 tetrads, data not shown). The high-CO2 requiring phenotype cosegregated with reduced phosphoglycolate phosphatase activity (Table IV). On the other hand, no significant cosegregation was observed with respect to NPP phosphatase activity in tetrads (Table IV). Phenotype, phosphoglycolate phosphatase activity, and NPP phosphatase activity were compared in diploids heterozygous for the 18-7F mutation, homozygous for the 18-7F mutation and homozygous for the wild-type genotype (Table V). Activities in the homozygous diploids were similar to activities measured in haploid cells. NPP phosphatase activity in heterozygous diploids was similar to that in homozygous wild-type diploids and both were somewhat lower than in the homozygous mutant diploids. Although the heterozygous dip-

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Table IV. Phosphoglycolate Phosphatase and p-Nitrophenylphosphate (NPP) Phosphatase Activities in Tetrad Progeny from Crosses Between 18-7F and Wild Type CC801 (spr- 1) Activities were measured in 24 h air-adapted cells. Phenotypes (CO2R, high-CO2 requiring; wt, wild-type) were tested with spot tests on agar plates. Tetrad Progeny Progeny

Phenotype Phenotype

12-1 12-2 12-3 12-4 15-1 15-2 15-3 15-4 16-1 16-2 16-3 16-4 17-1 17-2 17-3 17-4

CO2R wt CO2R wt CO2R CO2R wt wt CO2R wt CO2R

Substrate P-glycolate

NPP

pumol P mg-' Chl h' 5.3 11.0 31.3 7.3 4.7 9.1 40.5 8.2 4.8 11.4 5.4 9.7 24.9 5.0 39.8 6.6 4.4 8.0 7.2 33.6 6.2 11.1 25.3 4.3 27.4 4.4 2.8 8.4 36.2 11.3 7.9 14.8

wt wt

CO2R wt

CO2R

Table V. Phosphoglycolate Phosphatase and pNitrophenylphosphate (NPP) Phosphatase Activities in Diploid Progeny of 18-7F and Wild Type Activities were measured in 24 h air-adapted cells. Phenotypes (CO2R, high-CO2 requinng; wt, wild-type) were tested with spot tests on agar plates. Substrate Diploid Strain

Homozygous wt Heterozygous mutant Homozygous mutant

Phenotype

wt wt

CO2R

NPP P-glycolate AMOI P mg-' Chl h-' 20.0 6.3 10.3 5.3 4.4 9.8

loids were phenotypically wild type, phosphoglycolate phosphatase activity was intermediate between that of the homozygous diploids. These characteristics are quite similar to those reported for the phosphoglycolate phosphatase mutant CS 1 19 of A. thaliana (17), and they suggest that a single recessive nuclear mutation is responsible for both the high-CO2 requirement for growth and the phosphoglycolate phosphatase deficiency in 1 8-7F. A wild-type phenotype was found in all the diploids produced from crosses between 18-7F and each of six other high-CO2 requiring mutants (data not shown): 152P and 18-2A, which have a photosynthetic affinity for CO2 comparable to that of wild type (25), and ca-1-12-1C, ca-11 8-6A, ca- 1-1 8-7C and pmp- 1- 16-5K, which have mutations affecting the CO2 concentrating mechanism (21, 22, 26). DISCUSSION The physiological, biochemical and genetic evidence presented in this paper indicates that the high-CO2 requiring

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phenotype in the C. reinhardtii mutant 1 8-7F results from a deficiency in the photorespiratory metabolic pathway, not from the absence of a functional CO2 concentrating mechanism. Residual amounts of phosphoglycolate phosphatase activity, large phosphoglycolate accumulation and the absence of glycolate accumulation indicate that phosphoglycolate phosphatase must be mostly lacking or nonfunctional in 187F. The phosphoglycolate phosphatase deficiency always cosegregated with the high-CO2 requiring phenotype in a pattern indicative of a single-gene, nuclear mutation. Complementation analyses established that the 1 8-7F mutation is at a locus distinct from all others previously characterized in high-CO2 requiring mutants of Chlamydomonas, and we have assigned this locus the name pgp- 1 (strain pgp- 1-1 8-7F). Phosphoglycolate phosphatase deficient mutants have been isolated in two species of higher plants; A. thaliana [line CC1 19; (17)] and barley [Hordeum vulgare, line RPr84/90; (6)]. The mutations are conditionally lethal, and both mutant lines require an elevated CO2 concentration for their growth. Arabidopsis and barley are both C3 plants. Chlamydomonas has C3 type carbon metabolism but air-adapted wild-type cells do not show physiological or biochemical evidence of photorespiration except under extreme conditions (4, 8), and the excessive glycolate excretion obvious in adapting cells is not detectable in adapted cells. It was ofinterest, therefore, to find a phosphoglycolate phosphatase deficient mutant in Chlamydomonas. It was expected that the CO2 concentrating mechanism would provide protection from the effects of a metabolic blockage in the photorespiratory pathway. Air-adapted cells of 18-7F do show some evidence that the CO2 concentrating mechanism reduces the physiological effects of the mutation: 02 inhibition of photosynthesis and accumulation of phosphoglycolate are less in air-adapted cells. However, the cells still do not grow at air levels of CO2 and they do not grow well on acetate in the light in air. We found a large accumulation of phosphoglycolate in 187F within 5 min under inducing conditions (high-CO2 grown cells labeled in low CQ). Phosphoglycolate also accumulated in the barley mutant (6), and presumably, in Arabidopsis CC 1 19. An accumulation of phosphoglycolate would be expected to inhibit triose phosphate isomerase (2) which would interfere with regeneration of RuBP, reducing the RuBP pool available to support photosynthesis. We do see less labeled RuBP in 18-7F than in wild-type cells. It takes at least eight h for complete induction of the CO2 concentrating mechanism (18) and during this time inhibitory levels of phosphoglycolate would be present. Using aminooxyacetate to inhibit glycolate metabolism, Moroney et al. (14) have recently presented evidence that detectable flow of carbon through the glycolate pathway does occur in air-adapted, wild-type cells in air. They estimate a rate of glycolate production of 5 to 10 ,umol mg-' Chl h-', which is greater than the residual phosphoglycolate phosphatase activity we measure in 18-7F. Because of the metabolic lesion in 1 8-7F, an elevated phosphoglycolate pool would be maintained in air-adapted cells which would cause a continued inhibitory effect on triose phosphate isomerase and RuBP regeneration. The inhibition of photosynthesis in air-adapted cells by preillumination and by 02 probably results from RuBP limitations to photosynthesis, and these

Plant Physiol. Vol. 93,1990

limitations may be the basis of the phenotype in 1 8-7F. Some turnover of the phosphoglycolate pool must be occurring in 1 8-7F because we see recovery in photosynthetic 02 evolution after a dark incubation. The turnover presumably is due to residual phosphoglycolate phosphatase or non-specific phosphatase activity. Dark recovery of photosynthesis was also observed in the Arabidopsis mutant. It has been suggested that the signal for induction of the CO2 concentrating mechanism in Anabaena variabilis is phosphoglycolate (11). Spalding et al. (20) have suggested that

induction in Chlamydomonas is dependent on the accumulation of a photorespiratory metabolite. Because 18-7F induces the CO2 concentrating mechanism, its characteristics argue that if a photorespiratory metabolite is involved in induction, the metabolite must be phosphoglycolate. LITERATURE CITED 1. Ames BN (1966) Assay of inorganic phosphate, total phosphate and phosphatases. Methods Enzymol 8: 115-118 2. Anderson LE (1971) Chloroplast and cytoplasmic enzymes. II Pea leaf triose phosphate isomerases. Biochim Biophys Acta 235: 237-244 3. Badger MR, Kaplan A, Berry JA (1980) Internal inorganic

4.

5.

6.

7. 8. 9.

10. 11. 12.

13. 14.

15. 16.

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