Planta
Planta (i 984) 162 : 450-456
9 Springer-Verlag 1984
Carbon metabolism and gas exchange in leaves of Zea mays L. Changes in CO 2 fixation, chlorophyll a fluorescence and metabolite levels during photosynthetic induction Richard C. Leegood and Robert T. Furbank Research Institute for Photosynthesis, Department of Botany, University of Sheffield, Sheffield S I 0 2TN, U K
Abstract. Changes in the rate of COg uptake, chlorophyll a fluorescence and contents of metabolites were measured during illumination and darkening of maize leaves. Upon illumination, the contents of aspartate and alanine declined rapidly and there were rapid increases in the contents of 3-phosphoglycerate and triose phosphates. The amounts of pyruvate and phosphoenolpyruvate increased much more slowly. Upon darkening, the levels of 3-phosphoglycerate, phosphoenolpyruvate and triose phosphates fell sharply, while the amount of pyruvate increased. It is proposed that metabolite gradients in C a photosynthesis are built-up during induction through interchange of carbon between amino acids, metabolites of the C a pathway and 3-phosphoglycerate and triose phosphates, since CO 2 fixation during the first 5 min of photosynthesis was insufficient to account for the observed build-up of intermediates. Changes in the rates of CO 2 uptake and chlorophyll a fluorescence quenching are discussed in the light of the changes in metabolites. Key words: C a photosynthesis - CO 2 fixation Chlorophyll a fluorescence - Photosynthesis (induction, metabolites) - Zea (C metabolism, gas exchange).
Introduction Cellular differentiation into mesophyll and bundlesheath compartments lies at the heart of the C 4 mechanism. The energy-driven CO 2 pump of C 4 photosynthesis involves complex intercellular interaction, requiring transport of C a acids manufactured in the mesophyll cells to the bundle sheath
and the return of a C 3 compound. In maize, which is photosystem-II-deficient in the bundle sheath, additional co-operation between the two cell types is required because up to half the 3-phosphoglycerate generated in the Calvin cycle must be exported to the mesophyll for reduction to triose phosphates. Unlike the Calvin cycle, the C 4 cycle is not autocatalytic and does not catalyse a net fixation of CO 2. During photosynthetic induction, a simple increase in the rate of turnover of the C4 cycle would be sufficient to increase the rate of CO 2 transfer into the bundle sheath. However, it is believed that the transport of C 4 acids and of 3phosphoglycerate and triose phosphates between the mesophyll and the bundle sheath occurs by diffusion. To achieve sufficiently high rates of diffusion-driven flux, it is necessary for the leaf to develop appropriately large pools of these metabolites during steady-state photosynthesis, since concentration gradients of the order of 10 mM are required (Hatch and Osmond 1979) and it is unlikely that such gradients would persist in the dark. Evidence for substantial increases in pools of phosphoenolpyruvate, oxaloacetate and 3-phosphoglycerate during illumination has been obtained using leaves of Chloris gayana (a phosphoenolpyruvatecarboxykinase-type species) (Hatch 1979), but in general little is known about how metabolite pools change during the onset of CO z fixation in C 4 plants and by what mechanisms metabolite buildup occurs in the C 4 cycle. In an attempt to shed light on these questions we have investigated how some photosynthetic metabolites change during CO 2 fixation and how these changes relate to the pattern of chlorophyll a fluorescence quenching and the rate of CO 2 assimilation.
R.C. Leegood and R.T. Furbank: Changes in
CO 2
fixation during photosynthetic induction
Materials and methods Materials. Zea mays L. (Pioneer Hybrid 3780) was grown in a greenhouse with supplemental lighting during May and June in soil with added fertiliser. Substrates, cofactors and enzymes came from Boehringer, Mannheim, FRG. Acid-washed, activated charcoal came from BDH, Poole, Dorset, UK. Chlorophyll. Chlorophyll was measure by the method of Arnon (1949). Phaeophytin was extracted from perchloric-acid residues into 80% acetone using a glass-in-glass homogeniser and measured by the method of Vernon (1960). Treatment of leaves and gas analysis. Plants were placed in darkness for 15 h before the leaf samples were taken. Leaf samples were cut from about two-thirds along the secondary or tertiary leaves of three-week-old plants. Particular care was taken to ensure uniform sampling; to this end, several leaf samples were taken for each determination of metabolites fi'om the same tray of uniformly growing plants. For metabolite measurements, leaf samples were illuminated in a stream of humidified air. The irradiance was 250 W m -2 and was provided by a 250-W quartz-lamp projector filtered through a heat-reflecting mirror (Oriel Corp., Stamford, Conn., USA) and a Corning blue-green 4-96 filter (Coming Glass, Coming, N.Y., USA). Leaves were treated similarly in the gas-analysis system which was as described previously (Furbank and Walker ] 984). Measurement of substrates. Samples of leaves (approx. 0.25 g) were frozen in the light or in darkness, as appropriate, in liquid N z and pulverised in a pre-cooled pestle and mortar. A pellet of 1 ml frozen 1 M HC10 4 was added to the powder and gently pulverised with it (this procedure allowed more efficient mixing of the HC104 with the sample than if liquid HC104 was added). The mixture was allowed to thaw slowly and was transferred to a tube kept at 4 ~ C. The mortar was rinsed twice with 0.5 ml ice-cold 0.1 M HC104. The extract was left for 30 rain at 4 ~ C and centrifuged at 2000 g for 2 rain. The supernatant was retained and the pellet was washed with 0.5 ml water and centrifuged at 2000 g for 2 rain. The pellet was reserved for measurement of phaeophytin and the supernatant fractions were combined and neutralised to approx pH 6.0 with 5 M K2CO3; 10 mg of charcoal were added as a suspension in 100 gl water. The extract was centrifuged at 2000 g for 2 rain. This procedure resulted in the removal of a bright yellow pigment which interfered with spectrophotometric assays. The supernatant was immediately used for the determination of metabolites by the methods of Lowry and Passonneau (1972) in a spectrophotometer (SP 1800; Pye Unicam, Cambridge, UK). The sum of the amounts of diyhdroxyacetone phosphate and 3-phosphoglyceraldehyde was determined by Method II from a single measurement made in the presence of triose-phosphate isomerase and 3-phosphoglyceraldehyde dehydrogenase. Malate did not interfere in this assay. 3-Phosphoglycerate was determined by Method I. Glutamate and aspartate were measured according to Bernt and Bergmeyer (1974) and Bergmeyer et al. (1974), respectively. 2-Oxoglutarate was measured in a mixture containing 50raM imidazole-HC1 (pH 7.0), 2 mM aspartate, 160 gM N A D H , 1.8 units glutamate-oxaloacetate aminotransferase and 1.4 units malate dehydrogenase. Separate measurement of fructose 1,6-bisphosphate and sedoheptulose 1,7-bisphosphate (Leegood and Walker 1980) showed that the latter was always below the level of accurate detection in these leaves and did not contribute appreciably to the estimate of the content of fructose 1,6-bisphosphate. For recovery experiments, leaf samples were weighed and divided into two along the midrib. Measured amounts of inter-
451
mediates, comparable to the content of the tissue, were added to the pestle and mortar prior to one of the samples of leaf tissue and pulverised with it.
Results and discussion
The recoveries of all the compounds added to frozen leaves show that there were no serious losses during extraction and analysis (Table 1). For ease of manipulation, freeze-clamping was not routinely employed to stop leaf metabolism, but comparisons of metabolite contents in freeze-clamped leaves and in leaves plunged into liquid N 2 showed that there were no significant differences in metabolite contents between the two treatments. The amount of oxaloacetate in the leaves was always below levels which permitted accurate detection, despite precautions to preserve it in the extract (Lowry and Passonneau 1972).
Changes occurring upon illumination. Figure 1 shows the changes in metabolite contents, chlorophyll a fluorescence and rate of COz uptake during 40 min illumination of maize leaves in air. The uptake of CO 2 was biphasic in character (Furbank and Walker 1984) and a steady-state rate of carbon assimilation was reached after 20-30 min. By this time the changes in metabolites were largely complete. We draw attention to three principal features of the results of metabolite determinations. First, there was a rapid fall in the content of alanine and aspartate which was completed within the first 10 min of illumination. Part of this decrease in amino-acid content was matched by an increase in the content of glutamate, while the content of 2-oxoglutarate remained high and relatively constant. The second feature was a dramatic rise in the content of triose phosphates and 3-phosphogTable 1. Recoveries ofmetabolites from maize leaves. The recoveries are expressed as a percentage of the amount added and are the averages of three separate determinations. Recoveries were estimated as described in Material and methods section Metabolite
Recovery (% • SE)
Malate Aspartate Pyruvate Phosphoenolpyruvate Glucose 6-phsphate Fructose 6-phosphate Alanine Triose phosphates 3-phosphoglycerate 2-Oxoglutarate Fructose 1,6-bisphosphate Glutamate
95 • 3 98 _+9 84 • 5 107 • 18 102 • 8 92 • 8 92 • 4 103 • 5 97 + 3 77 • 7 96+_ 10 90_+ 9
452
R.C. Leegood and R.T. F u r b a n k : Changes in CO z fixation during photosynthetic induction
,o:k/'
Planta (i 984) 162 : 450-456
9 Springer-Verlag 1984
Carbon metabolism and gas exchange in leaves of Zea mays L. Changes in CO 2 fixation, chlorophyll a fluorescence and metabolite levels during photosynthetic induction Richard C. Leegood and Robert T. Furbank Research Institute for Photosynthesis, Department of Botany, University of Sheffield, Sheffield S I 0 2TN, U K
Abstract. Changes in the rate of COg uptake, chlorophyll a fluorescence and contents of metabolites were measured during illumination and darkening of maize leaves. Upon illumination, the contents of aspartate and alanine declined rapidly and there were rapid increases in the contents of 3-phosphoglycerate and triose phosphates. The amounts of pyruvate and phosphoenolpyruvate increased much more slowly. Upon darkening, the levels of 3-phosphoglycerate, phosphoenolpyruvate and triose phosphates fell sharply, while the amount of pyruvate increased. It is proposed that metabolite gradients in C a photosynthesis are built-up during induction through interchange of carbon between amino acids, metabolites of the C a pathway and 3-phosphoglycerate and triose phosphates, since CO 2 fixation during the first 5 min of photosynthesis was insufficient to account for the observed build-up of intermediates. Changes in the rates of CO 2 uptake and chlorophyll a fluorescence quenching are discussed in the light of the changes in metabolites. Key words: C a photosynthesis - CO 2 fixation Chlorophyll a fluorescence - Photosynthesis (induction, metabolites) - Zea (C metabolism, gas exchange).
Introduction Cellular differentiation into mesophyll and bundlesheath compartments lies at the heart of the C 4 mechanism. The energy-driven CO 2 pump of C 4 photosynthesis involves complex intercellular interaction, requiring transport of C a acids manufactured in the mesophyll cells to the bundle sheath
and the return of a C 3 compound. In maize, which is photosystem-II-deficient in the bundle sheath, additional co-operation between the two cell types is required because up to half the 3-phosphoglycerate generated in the Calvin cycle must be exported to the mesophyll for reduction to triose phosphates. Unlike the Calvin cycle, the C 4 cycle is not autocatalytic and does not catalyse a net fixation of CO 2. During photosynthetic induction, a simple increase in the rate of turnover of the C4 cycle would be sufficient to increase the rate of CO 2 transfer into the bundle sheath. However, it is believed that the transport of C 4 acids and of 3phosphoglycerate and triose phosphates between the mesophyll and the bundle sheath occurs by diffusion. To achieve sufficiently high rates of diffusion-driven flux, it is necessary for the leaf to develop appropriately large pools of these metabolites during steady-state photosynthesis, since concentration gradients of the order of 10 mM are required (Hatch and Osmond 1979) and it is unlikely that such gradients would persist in the dark. Evidence for substantial increases in pools of phosphoenolpyruvate, oxaloacetate and 3-phosphoglycerate during illumination has been obtained using leaves of Chloris gayana (a phosphoenolpyruvatecarboxykinase-type species) (Hatch 1979), but in general little is known about how metabolite pools change during the onset of CO z fixation in C 4 plants and by what mechanisms metabolite buildup occurs in the C 4 cycle. In an attempt to shed light on these questions we have investigated how some photosynthetic metabolites change during CO 2 fixation and how these changes relate to the pattern of chlorophyll a fluorescence quenching and the rate of CO 2 assimilation.
R.C. Leegood and R.T. Furbank: Changes in
CO 2
fixation during photosynthetic induction
Materials and methods Materials. Zea mays L. (Pioneer Hybrid 3780) was grown in a greenhouse with supplemental lighting during May and June in soil with added fertiliser. Substrates, cofactors and enzymes came from Boehringer, Mannheim, FRG. Acid-washed, activated charcoal came from BDH, Poole, Dorset, UK. Chlorophyll. Chlorophyll was measure by the method of Arnon (1949). Phaeophytin was extracted from perchloric-acid residues into 80% acetone using a glass-in-glass homogeniser and measured by the method of Vernon (1960). Treatment of leaves and gas analysis. Plants were placed in darkness for 15 h before the leaf samples were taken. Leaf samples were cut from about two-thirds along the secondary or tertiary leaves of three-week-old plants. Particular care was taken to ensure uniform sampling; to this end, several leaf samples were taken for each determination of metabolites fi'om the same tray of uniformly growing plants. For metabolite measurements, leaf samples were illuminated in a stream of humidified air. The irradiance was 250 W m -2 and was provided by a 250-W quartz-lamp projector filtered through a heat-reflecting mirror (Oriel Corp., Stamford, Conn., USA) and a Corning blue-green 4-96 filter (Coming Glass, Coming, N.Y., USA). Leaves were treated similarly in the gas-analysis system which was as described previously (Furbank and Walker ] 984). Measurement of substrates. Samples of leaves (approx. 0.25 g) were frozen in the light or in darkness, as appropriate, in liquid N z and pulverised in a pre-cooled pestle and mortar. A pellet of 1 ml frozen 1 M HC10 4 was added to the powder and gently pulverised with it (this procedure allowed more efficient mixing of the HC104 with the sample than if liquid HC104 was added). The mixture was allowed to thaw slowly and was transferred to a tube kept at 4 ~ C. The mortar was rinsed twice with 0.5 ml ice-cold 0.1 M HC104. The extract was left for 30 rain at 4 ~ C and centrifuged at 2000 g for 2 rain. The supernatant was retained and the pellet was washed with 0.5 ml water and centrifuged at 2000 g for 2 rain. The pellet was reserved for measurement of phaeophytin and the supernatant fractions were combined and neutralised to approx pH 6.0 with 5 M K2CO3; 10 mg of charcoal were added as a suspension in 100 gl water. The extract was centrifuged at 2000 g for 2 rain. This procedure resulted in the removal of a bright yellow pigment which interfered with spectrophotometric assays. The supernatant was immediately used for the determination of metabolites by the methods of Lowry and Passonneau (1972) in a spectrophotometer (SP 1800; Pye Unicam, Cambridge, UK). The sum of the amounts of diyhdroxyacetone phosphate and 3-phosphoglyceraldehyde was determined by Method II from a single measurement made in the presence of triose-phosphate isomerase and 3-phosphoglyceraldehyde dehydrogenase. Malate did not interfere in this assay. 3-Phosphoglycerate was determined by Method I. Glutamate and aspartate were measured according to Bernt and Bergmeyer (1974) and Bergmeyer et al. (1974), respectively. 2-Oxoglutarate was measured in a mixture containing 50raM imidazole-HC1 (pH 7.0), 2 mM aspartate, 160 gM N A D H , 1.8 units glutamate-oxaloacetate aminotransferase and 1.4 units malate dehydrogenase. Separate measurement of fructose 1,6-bisphosphate and sedoheptulose 1,7-bisphosphate (Leegood and Walker 1980) showed that the latter was always below the level of accurate detection in these leaves and did not contribute appreciably to the estimate of the content of fructose 1,6-bisphosphate. For recovery experiments, leaf samples were weighed and divided into two along the midrib. Measured amounts of inter-
451
mediates, comparable to the content of the tissue, were added to the pestle and mortar prior to one of the samples of leaf tissue and pulverised with it.
Results and discussion
The recoveries of all the compounds added to frozen leaves show that there were no serious losses during extraction and analysis (Table 1). For ease of manipulation, freeze-clamping was not routinely employed to stop leaf metabolism, but comparisons of metabolite contents in freeze-clamped leaves and in leaves plunged into liquid N 2 showed that there were no significant differences in metabolite contents between the two treatments. The amount of oxaloacetate in the leaves was always below levels which permitted accurate detection, despite precautions to preserve it in the extract (Lowry and Passonneau 1972).
Changes occurring upon illumination. Figure 1 shows the changes in metabolite contents, chlorophyll a fluorescence and rate of COz uptake during 40 min illumination of maize leaves in air. The uptake of CO 2 was biphasic in character (Furbank and Walker 1984) and a steady-state rate of carbon assimilation was reached after 20-30 min. By this time the changes in metabolites were largely complete. We draw attention to three principal features of the results of metabolite determinations. First, there was a rapid fall in the content of alanine and aspartate which was completed within the first 10 min of illumination. Part of this decrease in amino-acid content was matched by an increase in the content of glutamate, while the content of 2-oxoglutarate remained high and relatively constant. The second feature was a dramatic rise in the content of triose phosphates and 3-phosphogTable 1. Recoveries ofmetabolites from maize leaves. The recoveries are expressed as a percentage of the amount added and are the averages of three separate determinations. Recoveries were estimated as described in Material and methods section Metabolite
Recovery (% • SE)
Malate Aspartate Pyruvate Phosphoenolpyruvate Glucose 6-phsphate Fructose 6-phosphate Alanine Triose phosphates 3-phosphoglycerate 2-Oxoglutarate Fructose 1,6-bisphosphate Glutamate
95 • 3 98 _+9 84 • 5 107 • 18 102 • 8 92 • 8 92 • 4 103 • 5 97 + 3 77 • 7 96+_ 10 90_+ 9
452
R.C. Leegood and R.T. F u r b a n k : Changes in CO z fixation during photosynthetic induction
,o:k/'
