Planta
Planta (1983) 159 : 505-511
9 Springer-Verlag 1983
The isolation and characterisation of a catalase-deficient mutant of barley (Hordeum vulgate L.) Alan C. Kendall, Alfred J. Keys, Janice C. Turner, Peter J. Lea and Benjamin J. Miflin Department of Biochemistry, Rothamsted Experimental Station, Harpenden, Hefts. AL5 2JQ, UK
Abstract. A mutant line of barley, R(othamsted)Pr 79/4, has been isolated which grows poorly in natural air, but normally in air enriched to 0.2% CO 2 . Analysis of the products of 14CO2 fixation showed that there was no major block in photosynthetic or photorespiratory carbon metabolism in the mutant and that rates of CO 2 fixation were only slightly lower than those measured in the wild type (c.v. Maris Mink). Leaves of the mutant line contained only 10% of the catalase (EC 1.11.1.6) activity found in the wild type; and the two major bands of catalase activity detected after starch-gel electrophoresis of extracts of normal leaves were missing from similar extracts of RPr 79/4. Peroxisomes isolated from mutant leaves contained negligible catalase activity, but normal levels of other enzymes involved in photorespiration. Genetic analysis has shown that the mutation is recessive and that both air-sensitivity and catalase-deficiency segregate together in F 2 plants derived from a cross between the mutant and the cultivar Golden Promise. [1-14C]Glycollate was not converted to 14CO2 faster in the mutant leaves than in the normal leaves. Thus there was no evidence that photorespiratory CO 2 may be obtained by the chemical action of H20 2 on glyoxylate or hydroxypyruvate.
Key words: Catalase deficiency - Hordeum (mutant) - Mutant (barley) - Photorespiration.
Introduction
Photorespiration, the oxygen and light-dependent release of CO 2 from the leaves of Ca plants, appears at present to be a wasteful process that could severely limit crop productivity (Ogren 1976;
Whittingham 1981). The pathway of metabolism leading t o C O 2 and ammonia release has been determined over many years by tracer experiments with t4C (Tolbert 1979; Zelitch 1979), by enzymological studies (Tolbert 1979; Beevers 197!9) and subsequently by tracer experiments with ~SN (Keys et al. 1978; Woo et al. 1978, 1982) and with 180 2 (Berry et al. 1978). More recently the excellent studies of Somerville and Ogren (1982) have begun to provide the ultimate proof of the pathway by isolating seven mutants of Arabidopsis thaliana (L.) Heynh., each defective in a different enzyme of photorespiratory metabolism. The mutants were selected from seedlings of the M 2 generation of chemically mutagenised seed by their failure to grow in air but having normal growth in CO2-enriched air. A. thaliana was chosen for the selection of photorespiration mutants, as it is a very small plant producing abundant seed in a short life cycle (Somerville and Ogren 1982). However, as Arabidopsis has little value in agriculture, we have carried out similar selection procedures on M 2 barley plants derived originally from seed treated with azide in a manner already known to produce a wide range of mutants with altered amino-acid metabolism (Bright et al. 1979, 1982; Kueh and Bright 1981). In this paper we describe a mutant line of barley, R(othamsted)-Pr 79/4, which is unable to grow satisfactorily in normal air but thrives in 0.2% CO2. The evidence presented indicates that the mutation, which is recessive, leads to a 16ss of 90% of the catalase (EC 1.11.1.6) activity of the leaf. Materials and methods Mutant selection. The mutant-selection procedure was based on that originally devised by Sommerville and Ogren (1979) for Arabidopsis. Barley seed (cv. Marls Mink) was treated with the mutagen, sodium azide, and grown to produce the selfed
506 M z seed population which was bulk harvested (Kleinhofs et al. 1978; Kueh and Bright 1981). Seeds (Mz) were germinated and grown for 21 d in an atmosphere of >0.2% CO 2, weak and unhealthy plants were discarded. The remaining plants were then allowed to grow at normal CO2 levels of 0.03% for 7 d. Plants showing a serious discolouration of the leaves were placed back in the atmosphere of 0.2% COz, any plants that recovered to a normal healthy condition were maintained.
of the mutant RPr 79/4. Air containing 300 gl 1- ~ 14CO2 was supplied to appropriate leaves either immediately after plants were taken from the CO2-enriched environment or 7 d later. Clip-on leaf chambers similar to that described by Shimshi (1969) were employed. The leaf was exposed to the ~4CO2 in a growth room for 10 rain at a photon fluence rate of 1,000 tamol m - 2 s- 1. The exposed leaf was imi mediately dropped into liquid nitrogen. Soluble intermediates were subsequently extracted in a mixture of methanol:chloroform: water: formic acid (12 : 5:2:1, by vol.) (RedgweU 1980) . and separated and analysed by thin-layer chromatography (TLC), autoradiography and scintillation counting as described b y Waidyanatha et al. (1975). [IJ4C]-Glycolate feeding to leaf sections was carried out as described by Waidyanatha et al. (1975). The TLC two-dimensional separation technique was modified to get a better separation of glycine, serine and glycollate. The plates were developed twice in the first direction in diethylamine: acetone: butanol : water (6: 30: 30 : 15, by vol.) and twice in the second direction in propyl acetate:formic acid: water (11 : 5: 3, by vol.). Radioactive areas were detected using a Spark Chamber (Birchover Instruments, Hitchin, Herts., UK) and counted by the method of Davies and Miflin (1978).
A.C. Kendall et al. : Catalase-deficient barley Table 1. The percent distribution of radioactivity in the products of 14CO z assimilation of mutant (catalase deficient, RPr 79/4) and wild-type (Marls Mink, MM) barley leaves. Plants were fed with 14CO z (300 gl 1-~) for 10 rain at a photon fluenee rate of 1,000 I-tmol m -z s -1. All plants were initially grown in an atmosphere enriched to 0.2% COz, but one group was transferred to normal air 7 d prior to the experiment
Analysis
Gas-exchange analysis. Leaves were detached under water and cut to 4-cm lengths (Waidyanatha et al. 1975). Batches of five leaf segments were placed with their cut bases in distilled water in 10-cm 3 water-jacketed leaf chambers and flushed at 27 1h-1 with the appropriate gas mixture from a cylinder (Arabacca 1981). The outflow gas from the chamber could be directed as required to waste or through the analytical cell of an infra red gas analyser (model 225; Analytical Development Co., Hoddesdon, Herts., UK) set in the differential mode to measure CO2 depletion.
Ribulose bisphosphate Fructose bisphosphate 6-Phosphogluconate Uridine 5'-diphosphate glucose Glucose-l-phosphate + glucose-6-phosphate Fructose-6-phosphate Unknown 1 Phosphoglyceric acid Phosphoglycolate Phosphoenolpyruvate Aspartate Glutamate Malate Maltose Sucrose Glucose Fructose Unknown z Unknown 3 Serine Glycine Glycerate Alanine Glycollate
Plants grown in air for 7 d after removal from 0.2% C02
Plants grown in 0.2% CO2
RPr 79/4
RPr
1.5 . 0.7 0.9 3.2
MM
MM
79/4 2.8
0.5
2.40
0.1 0.5
0.4
0.6
5.1
3.9
5.1
1.1 5.9
1.1 0.2 8.4
.
.
1.3 2.3 0.6 3.2 6.8 . . . 0.3 0.6 2.8 1.8 0.8 0.3 6.5 1.4 1.1 36.6 53.4 4.9 7.2 . . . 0.3 0.6 0.4 0.2 20.0 6.3 8.2 5.1 1.8 2.2 3.5 1.0 1.4 1.9
.
. 0.4 0.5 2.9 1.3 0.7 0.3 6.7 2.9 0.40 0.2 52.40 52.9 5.8 6.6 . 0.8 7.9 5.6 2.3 1.8 0.6
0.2 0.7 4.6 6.8 2.6 1.3 0.8
Enzyme analysis. Glutamine synthetase (biosynthetic hydroxa-
Results
mate formation), ferredoxin-dependent glutamate synthase and both N A D - and NADP-dependent glutamate dehydrogenase were determined as described by Wallsgrove et al. (1979). Glutamate: glyoxylate and serine: glyoxylate transaminases were assayed by the method of Cossins and Sinha (1966). Ascorbate oxidase and glutathione reductase were assayed as described by Foyer and Halliwell (1976). Hydroxypyruvate reductase was assayed by the method of Tolbert et al. (1970). Usually the catalase assay described by Luck (1965) was used, but for one set of experiments when parent, F1 and mutant F 2 lines were examined, hydrogen-peroxide-dependent oxygen evolution in an oxygen electrode was measured. Similar results were obtained with both methods. Catalase activity separated by electrophoresis on starch gels at pH 8.5 was visualised using the KI method of Scandalios (1969). Protoplasts were isolated from barley leaves, ruptured and the organelles separated on sucrose density gradients as described by Wallsgrove et al. (1980). Molecular-weight determinations were carried out on Sephacryl 300 (Pharmacia, Uppsala, Sweden) as described by Siegel and Monty (1966). Protein was assayed by the method of Bradford (1976) and chlorophyll by the method of Hiscox and Israelstam 0979).
Mutant selection. Out of 18.176 seeds initially screened, nine mutant plants were selected, of which seven have survived (RPr 79/1-7). After 24 h in normal air, brown lesions appeared on the leaves of one plant, ultimately the leaves turned white, shrivelled up, and died. New leaves were, however, formed; these were green initially and the plant grew slowly in normal air. The initial mutant plant was maintained in a chamber at > 0.2% CO2, and reproduced vegetatively through tillers. This plant was designated Rothamsted photorespiration mutant 79/4 (RPr 79/4); this designation is also used for all homozygous mutants derived from this plant.
Analysis of products of 1~C02 assimilation. The products of 1r assimilation in wild-type (Maris Mink) and RPr 79/4 leaves were determined either
A.C. Kendall et al. : Catalase-deficient barley
507
2. The metabolism of [1-14C]-glycollate by illuminated leaf segments of Maris Mink and mutant RPr 79/4 barley incubated for 60 rain in either normal air, or air from which CO 2 had been completely removed. Data expressed as % of metabolised 14C-glycollate
Table
Metabolite
CO2 Serine Glycine Glycerate Sucrose Sugar phosphates
Normal air
Zero CO 2
Marls Mink
RPr 79/4
Marls Mink
RPr 79/4
25.8 7.4 5.6 4.5 53.9 2.8
30.1 7.3 3.8 5.7 48.6 3.5
65.3 7.7 4.7 6.7 10.7 4.9
51.8 16.4 6.1 7.9 12.4 5.4
in plants immediately after removal from elevated CO 2 levels or in other plants maintained for 7 d in normal air (Table i). There was no evidence of a major block in the photosynthetic or photorespiratory pathways of the mutants. However there was an indication of accumulation of 14C in serine and also in 6-phosphogluconate (this value increased to 2.9% in other experiments) in RPr 79/4. Likewise the metabolism of [1-~4C]glyeollate by detached leaves gave no indication of a major block in the photorespiratory pathway in either normal or CO2-free air (Table 2), although again there was an indication of accumulation in serine in RPr 79/4. The data shown are for plants taken directly from the CO2-enriched cabinet in which they were grown, but essentially similar results were obtained from plants grown in normal air for up to 48 h before [l~C]glycollate feeding.
Gas-exchange analysis. Leaves from plants grown at high CO 2 levels assimilated CO 2 rapidly at 1,000 vol. per million CO 2 with a photon flux of 1,000 gmol m - 2 s- 1 (Experiment I, Table 3). After 1 h, the plants were transferred to normal air when there was the expected drop in the rate of assimila-
tion followed by a slow increase, probably due to the further opening of stomata. Subsequently, after 1 h in normal air, there was a slow decline in the rate of CO 2 assimilation. Neither a total of 10 min in 60% 0 2 nor a period of 30 min in CO2-free air hastened the decline. The general trend in CO2 uptake over the course of the experiment was similar for leaves of the mutant and parent plants but, throughout, RPr 79/4 had slower rates (Table 3). A similar experiment (II, Table 3) in which leaves were fixing CO 2 initially in 1% 0 2 , 350 ~tl 1-1 CO 2 followed by a period of 3 h in normal air, indicated again that, although the rates of COz fixation of RPr 79/4 were lower than the parent, there was no appreciable fall-off in the rate in air during the course of the experiment.
Enzyme analysis. Initially a number of enzymes involved in nitrogen metabolism were examined in the mutant barley plant in order to attempt to explain its inability to grow in normal air. The activities of glutamine synthetase, glutamate synthase, glutamate dehydrogenase (NAD(P)H), glutamate:glyoxylate and serine :glyoxylate aminotransferase were similar in mutant and wild-type plants. Similarly, the activities of two enzymes involved in hydrogen-peroxide breakdown in chloroplasts, ascorbate oxidase and glutathione reductase, were not appreciably different. However, the catalase activity of RPr 79/4 leaves was less than 10% of the wild-type leaves (Table 4). Mixing experiments gave no evidence of a catalase inhibitor being present in crude extracts of the mutant plant. Genetic analysis. As the original RPr 79/4 plant did not set selfed seed, the mutant line was backcrossed with cv. Golden Promise. All o f the F t progeny grew satisfactorily in normal air, although the catalase activities were intermediate between those of the two parents (Table 4). TheJ F1 plants were self-fertilised and the F 2 plants Were tested for catalase activity in the leaves. Out o f 724 F 2
Table 3. Rate of CO 2 assimilation by leaf segments of mutant and wild-type barley lines at various levels of C O 2 and 0 2 Treatment
Expt. I (Rate in gmol h - 1 rag- t chlorophyll) 60 min 1,000 #11-1 CO2, 21% O z 20 rain normal air 6 h later following 30 min CO2-free air Expt. II (Rate in mg h - 1 dm - 2) 60 min 360 gl 1-1 COz, 1% 02 60 min normal air 60rain 360 gl 1-1 COz, 1% 0 2
CO z assimilation MM
GP
F1 (RPr 79/4 x GP)
RPr 79/4
243 151 112
261 133 105
242 125 90.6
168 ~90.2 64
19.5 16.8 18.9
--
16.6 12.2
-
15.8
508
A.C. Kendall et al. : Catalase-deficient barley
Table 4. Catalase activity in parental and mutant lines of barley. Values are the means of five separate determinations Barley line
Catalase activity (mkat rag- 1 protein)
5
t-o
Lu
Mit.
3.5
Planta (1983) 159 : 505-511
9 Springer-Verlag 1983
The isolation and characterisation of a catalase-deficient mutant of barley (Hordeum vulgate L.) Alan C. Kendall, Alfred J. Keys, Janice C. Turner, Peter J. Lea and Benjamin J. Miflin Department of Biochemistry, Rothamsted Experimental Station, Harpenden, Hefts. AL5 2JQ, UK
Abstract. A mutant line of barley, R(othamsted)Pr 79/4, has been isolated which grows poorly in natural air, but normally in air enriched to 0.2% CO 2 . Analysis of the products of 14CO2 fixation showed that there was no major block in photosynthetic or photorespiratory carbon metabolism in the mutant and that rates of CO 2 fixation were only slightly lower than those measured in the wild type (c.v. Maris Mink). Leaves of the mutant line contained only 10% of the catalase (EC 1.11.1.6) activity found in the wild type; and the two major bands of catalase activity detected after starch-gel electrophoresis of extracts of normal leaves were missing from similar extracts of RPr 79/4. Peroxisomes isolated from mutant leaves contained negligible catalase activity, but normal levels of other enzymes involved in photorespiration. Genetic analysis has shown that the mutation is recessive and that both air-sensitivity and catalase-deficiency segregate together in F 2 plants derived from a cross between the mutant and the cultivar Golden Promise. [1-14C]Glycollate was not converted to 14CO2 faster in the mutant leaves than in the normal leaves. Thus there was no evidence that photorespiratory CO 2 may be obtained by the chemical action of H20 2 on glyoxylate or hydroxypyruvate.
Key words: Catalase deficiency - Hordeum (mutant) - Mutant (barley) - Photorespiration.
Introduction
Photorespiration, the oxygen and light-dependent release of CO 2 from the leaves of Ca plants, appears at present to be a wasteful process that could severely limit crop productivity (Ogren 1976;
Whittingham 1981). The pathway of metabolism leading t o C O 2 and ammonia release has been determined over many years by tracer experiments with t4C (Tolbert 1979; Zelitch 1979), by enzymological studies (Tolbert 1979; Beevers 197!9) and subsequently by tracer experiments with ~SN (Keys et al. 1978; Woo et al. 1978, 1982) and with 180 2 (Berry et al. 1978). More recently the excellent studies of Somerville and Ogren (1982) have begun to provide the ultimate proof of the pathway by isolating seven mutants of Arabidopsis thaliana (L.) Heynh., each defective in a different enzyme of photorespiratory metabolism. The mutants were selected from seedlings of the M 2 generation of chemically mutagenised seed by their failure to grow in air but having normal growth in CO2-enriched air. A. thaliana was chosen for the selection of photorespiration mutants, as it is a very small plant producing abundant seed in a short life cycle (Somerville and Ogren 1982). However, as Arabidopsis has little value in agriculture, we have carried out similar selection procedures on M 2 barley plants derived originally from seed treated with azide in a manner already known to produce a wide range of mutants with altered amino-acid metabolism (Bright et al. 1979, 1982; Kueh and Bright 1981). In this paper we describe a mutant line of barley, R(othamsted)-Pr 79/4, which is unable to grow satisfactorily in normal air but thrives in 0.2% CO2. The evidence presented indicates that the mutation, which is recessive, leads to a 16ss of 90% of the catalase (EC 1.11.1.6) activity of the leaf. Materials and methods Mutant selection. The mutant-selection procedure was based on that originally devised by Sommerville and Ogren (1979) for Arabidopsis. Barley seed (cv. Marls Mink) was treated with the mutagen, sodium azide, and grown to produce the selfed
506 M z seed population which was bulk harvested (Kleinhofs et al. 1978; Kueh and Bright 1981). Seeds (Mz) were germinated and grown for 21 d in an atmosphere of >0.2% CO 2, weak and unhealthy plants were discarded. The remaining plants were then allowed to grow at normal CO2 levels of 0.03% for 7 d. Plants showing a serious discolouration of the leaves were placed back in the atmosphere of 0.2% COz, any plants that recovered to a normal healthy condition were maintained.
of the mutant RPr 79/4. Air containing 300 gl 1- ~ 14CO2 was supplied to appropriate leaves either immediately after plants were taken from the CO2-enriched environment or 7 d later. Clip-on leaf chambers similar to that described by Shimshi (1969) were employed. The leaf was exposed to the ~4CO2 in a growth room for 10 rain at a photon fluence rate of 1,000 tamol m - 2 s- 1. The exposed leaf was imi mediately dropped into liquid nitrogen. Soluble intermediates were subsequently extracted in a mixture of methanol:chloroform: water: formic acid (12 : 5:2:1, by vol.) (RedgweU 1980) . and separated and analysed by thin-layer chromatography (TLC), autoradiography and scintillation counting as described b y Waidyanatha et al. (1975). [IJ4C]-Glycolate feeding to leaf sections was carried out as described by Waidyanatha et al. (1975). The TLC two-dimensional separation technique was modified to get a better separation of glycine, serine and glycollate. The plates were developed twice in the first direction in diethylamine: acetone: butanol : water (6: 30: 30 : 15, by vol.) and twice in the second direction in propyl acetate:formic acid: water (11 : 5: 3, by vol.). Radioactive areas were detected using a Spark Chamber (Birchover Instruments, Hitchin, Herts., UK) and counted by the method of Davies and Miflin (1978).
A.C. Kendall et al. : Catalase-deficient barley Table 1. The percent distribution of radioactivity in the products of 14CO z assimilation of mutant (catalase deficient, RPr 79/4) and wild-type (Marls Mink, MM) barley leaves. Plants were fed with 14CO z (300 gl 1-~) for 10 rain at a photon fluenee rate of 1,000 I-tmol m -z s -1. All plants were initially grown in an atmosphere enriched to 0.2% COz, but one group was transferred to normal air 7 d prior to the experiment
Analysis
Gas-exchange analysis. Leaves were detached under water and cut to 4-cm lengths (Waidyanatha et al. 1975). Batches of five leaf segments were placed with their cut bases in distilled water in 10-cm 3 water-jacketed leaf chambers and flushed at 27 1h-1 with the appropriate gas mixture from a cylinder (Arabacca 1981). The outflow gas from the chamber could be directed as required to waste or through the analytical cell of an infra red gas analyser (model 225; Analytical Development Co., Hoddesdon, Herts., UK) set in the differential mode to measure CO2 depletion.
Ribulose bisphosphate Fructose bisphosphate 6-Phosphogluconate Uridine 5'-diphosphate glucose Glucose-l-phosphate + glucose-6-phosphate Fructose-6-phosphate Unknown 1 Phosphoglyceric acid Phosphoglycolate Phosphoenolpyruvate Aspartate Glutamate Malate Maltose Sucrose Glucose Fructose Unknown z Unknown 3 Serine Glycine Glycerate Alanine Glycollate
Plants grown in air for 7 d after removal from 0.2% C02
Plants grown in 0.2% CO2
RPr 79/4
RPr
1.5 . 0.7 0.9 3.2
MM
MM
79/4 2.8
0.5
2.40
0.1 0.5
0.4
0.6
5.1
3.9
5.1
1.1 5.9
1.1 0.2 8.4
.
.
1.3 2.3 0.6 3.2 6.8 . . . 0.3 0.6 2.8 1.8 0.8 0.3 6.5 1.4 1.1 36.6 53.4 4.9 7.2 . . . 0.3 0.6 0.4 0.2 20.0 6.3 8.2 5.1 1.8 2.2 3.5 1.0 1.4 1.9
.
. 0.4 0.5 2.9 1.3 0.7 0.3 6.7 2.9 0.40 0.2 52.40 52.9 5.8 6.6 . 0.8 7.9 5.6 2.3 1.8 0.6
0.2 0.7 4.6 6.8 2.6 1.3 0.8
Enzyme analysis. Glutamine synthetase (biosynthetic hydroxa-
Results
mate formation), ferredoxin-dependent glutamate synthase and both N A D - and NADP-dependent glutamate dehydrogenase were determined as described by Wallsgrove et al. (1979). Glutamate: glyoxylate and serine: glyoxylate transaminases were assayed by the method of Cossins and Sinha (1966). Ascorbate oxidase and glutathione reductase were assayed as described by Foyer and Halliwell (1976). Hydroxypyruvate reductase was assayed by the method of Tolbert et al. (1970). Usually the catalase assay described by Luck (1965) was used, but for one set of experiments when parent, F1 and mutant F 2 lines were examined, hydrogen-peroxide-dependent oxygen evolution in an oxygen electrode was measured. Similar results were obtained with both methods. Catalase activity separated by electrophoresis on starch gels at pH 8.5 was visualised using the KI method of Scandalios (1969). Protoplasts were isolated from barley leaves, ruptured and the organelles separated on sucrose density gradients as described by Wallsgrove et al. (1980). Molecular-weight determinations were carried out on Sephacryl 300 (Pharmacia, Uppsala, Sweden) as described by Siegel and Monty (1966). Protein was assayed by the method of Bradford (1976) and chlorophyll by the method of Hiscox and Israelstam 0979).
Mutant selection. Out of 18.176 seeds initially screened, nine mutant plants were selected, of which seven have survived (RPr 79/1-7). After 24 h in normal air, brown lesions appeared on the leaves of one plant, ultimately the leaves turned white, shrivelled up, and died. New leaves were, however, formed; these were green initially and the plant grew slowly in normal air. The initial mutant plant was maintained in a chamber at > 0.2% CO2, and reproduced vegetatively through tillers. This plant was designated Rothamsted photorespiration mutant 79/4 (RPr 79/4); this designation is also used for all homozygous mutants derived from this plant.
Analysis of products of 1~C02 assimilation. The products of 1r assimilation in wild-type (Maris Mink) and RPr 79/4 leaves were determined either
A.C. Kendall et al. : Catalase-deficient barley
507
2. The metabolism of [1-14C]-glycollate by illuminated leaf segments of Maris Mink and mutant RPr 79/4 barley incubated for 60 rain in either normal air, or air from which CO 2 had been completely removed. Data expressed as % of metabolised 14C-glycollate
Table
Metabolite
CO2 Serine Glycine Glycerate Sucrose Sugar phosphates
Normal air
Zero CO 2
Marls Mink
RPr 79/4
Marls Mink
RPr 79/4
25.8 7.4 5.6 4.5 53.9 2.8
30.1 7.3 3.8 5.7 48.6 3.5
65.3 7.7 4.7 6.7 10.7 4.9
51.8 16.4 6.1 7.9 12.4 5.4
in plants immediately after removal from elevated CO 2 levels or in other plants maintained for 7 d in normal air (Table i). There was no evidence of a major block in the photosynthetic or photorespiratory pathways of the mutants. However there was an indication of accumulation of 14C in serine and also in 6-phosphogluconate (this value increased to 2.9% in other experiments) in RPr 79/4. Likewise the metabolism of [1-~4C]glyeollate by detached leaves gave no indication of a major block in the photorespiratory pathway in either normal or CO2-free air (Table 2), although again there was an indication of accumulation in serine in RPr 79/4. The data shown are for plants taken directly from the CO2-enriched cabinet in which they were grown, but essentially similar results were obtained from plants grown in normal air for up to 48 h before [l~C]glycollate feeding.
Gas-exchange analysis. Leaves from plants grown at high CO 2 levels assimilated CO 2 rapidly at 1,000 vol. per million CO 2 with a photon flux of 1,000 gmol m - 2 s- 1 (Experiment I, Table 3). After 1 h, the plants were transferred to normal air when there was the expected drop in the rate of assimila-
tion followed by a slow increase, probably due to the further opening of stomata. Subsequently, after 1 h in normal air, there was a slow decline in the rate of CO 2 assimilation. Neither a total of 10 min in 60% 0 2 nor a period of 30 min in CO2-free air hastened the decline. The general trend in CO2 uptake over the course of the experiment was similar for leaves of the mutant and parent plants but, throughout, RPr 79/4 had slower rates (Table 3). A similar experiment (II, Table 3) in which leaves were fixing CO 2 initially in 1% 0 2 , 350 ~tl 1-1 CO 2 followed by a period of 3 h in normal air, indicated again that, although the rates of COz fixation of RPr 79/4 were lower than the parent, there was no appreciable fall-off in the rate in air during the course of the experiment.
Enzyme analysis. Initially a number of enzymes involved in nitrogen metabolism were examined in the mutant barley plant in order to attempt to explain its inability to grow in normal air. The activities of glutamine synthetase, glutamate synthase, glutamate dehydrogenase (NAD(P)H), glutamate:glyoxylate and serine :glyoxylate aminotransferase were similar in mutant and wild-type plants. Similarly, the activities of two enzymes involved in hydrogen-peroxide breakdown in chloroplasts, ascorbate oxidase and glutathione reductase, were not appreciably different. However, the catalase activity of RPr 79/4 leaves was less than 10% of the wild-type leaves (Table 4). Mixing experiments gave no evidence of a catalase inhibitor being present in crude extracts of the mutant plant. Genetic analysis. As the original RPr 79/4 plant did not set selfed seed, the mutant line was backcrossed with cv. Golden Promise. All o f the F t progeny grew satisfactorily in normal air, although the catalase activities were intermediate between those of the two parents (Table 4). TheJ F1 plants were self-fertilised and the F 2 plants Were tested for catalase activity in the leaves. Out o f 724 F 2
Table 3. Rate of CO 2 assimilation by leaf segments of mutant and wild-type barley lines at various levels of C O 2 and 0 2 Treatment
Expt. I (Rate in gmol h - 1 rag- t chlorophyll) 60 min 1,000 #11-1 CO2, 21% O z 20 rain normal air 6 h later following 30 min CO2-free air Expt. II (Rate in mg h - 1 dm - 2) 60 min 360 gl 1-1 COz, 1% 02 60 min normal air 60rain 360 gl 1-1 COz, 1% 0 2
CO z assimilation MM
GP
F1 (RPr 79/4 x GP)
RPr 79/4
243 151 112
261 133 105
242 125 90.6
168 ~90.2 64
19.5 16.8 18.9
--
16.6 12.2
-
15.8
508
A.C. Kendall et al. : Catalase-deficient barley
Table 4. Catalase activity in parental and mutant lines of barley. Values are the means of five separate determinations Barley line
Catalase activity (mkat rag- 1 protein)
5
t-o
Lu
Mit.
3.5
