Aug 4, 1983 - ABSTRACT. Mesophyll chloroplasts of three C4 sub types, Panicum miliaccum. (NAD-malic enzyme), Panicum maximum (PCK), and Zea ...
Plant Physiol. (1983) 73, 1038-1041 0032-0889/83/73/1038/04/$00.50/0
Photoreduction of Oxygen in Mesophyll Chloroplasts of C4 Plants A MODEL SYSTEM FOR STUDYING AN IN VIVO MEHLER REACTION Received for publication June 1, 1983 and in revised form August 4, 1983
ROBERT T. FURBANK', MURRAY R. BADGER, AND C. B. OSMOND Department of Environmental Biology, Research School of Biological Sciences, Australian National University, P.O. Box 475, Canberra, A.C. T. 2601, Australia ABSTRACT Mesophyll chloroplasts of three C4 sub types, Panicum miliaccum (NAD-malic enzyme), Panicum maximum (PCK), and Zea mays (NADPmalic enzyme), were prepared from protoplast extracts and used to study the photoreduction of 02. The processes of 02 uptake and evolution in these preparations, which lack ribulose 1,5-bisphosphate carboxylase/ oxygenase, were studied simultaneously using stable isotopes of 02 and mass spectrometry. The responses of 02 uptake to 02 tension and addition of various substrates (3-phosphoglycerate, pyruvate, and oxaloacetate) were studied in detail. The addition of photosynthetic substrates differing in ATP to NADPH demands indicated that photoreduction of 02 in these chloroplast preparations is linked to ATP production and strongly regulated by NADP' levels. The results clearly indicate that photoreduction of 02 could be of physiological relevance in balancing the ATP to NADPH requirements of C. mesophyll chloroplasts.
Light-dependent 02 uptake in higher plants has previously been studied in a variety of systems: isolated thylakoids (11), intact chloroplasts and cells (5, 9, 10, 16, 21), and in the intact leaf (3, 6, 12, 22, 24). Measurements using these systems indicate that photoreduction of 02 (Mehler reaction) may be a significant component of photosynthetic 02 consumption. Photoreduction of 02 may be physiologically important in the production of ATP without concurrent NADP+ reduction (pseudocyclic photophosphorylation). The interpretation of results obtained using intact photosynthetic systems is, however, complicated by the presence of photorespiratory 02 uptake (via RuP22-oxygenase) at all but saturating CO2 concentrations. In the C4 plant, RuP2 carboxylase-oxygenase is exclusively located in bundle sheath chloroplasts (14), and hence mesophyll chloroplasts are incapable of 02 uptake by this enzyme. Because of this, the isolated C4 mesophyll chloroplast is an ideal system for the characterization of an in vivo Mehler reaction and for the study of its regulation. Photoreduction of 02 in a thylakoid system appears to be strongly inhibited by NADP+ (11). Thus, the rate of Mehler reaction (and pseudocyclic photophosphorylation) in vivo is pre-
sumably dependent upon the utilization of NADPH relative to the ATP demand as has been proposed for some time (15). Few measurements have been made of chloroplast pyridine nucleotide levels (23) and manipulation of these levels in an intact system is difficult. Hence, there is little evidence of such a regulation in vivo. Although there is substantial physiological evidence for light and ATP-dependent ion transport associated with photoreduction of 02 ( 13), there is little direct evidence of the capacity of the Mehler reaction to generate ATP for photosynthetic carbon assimilation in vivo. In this study, 02 uptake and evolution in chloroplasts in mesophyll protoplast extracts from Zea mavs (NADP malic enzyme), Panicum maximum (PEP carboxykinase), and Panicum miliaceuim (NAD malic enzyme) are examined using 1802 and a mass spectrometer. The photoreduction of 02 in this system is characterized and its response to the addition of various substrates is examined in order to study the regulation of the process and its ability to produce ATP.
MATERIALS AND METHODS Preparation of C4 Mesophyll Protoplasts Extracts. Mesophyll protoplasts were isolated from the C4 species Panicutm miliaceum, Zea mays, and Panicum maximum, essentially by the methods described in Day et al. (7) and Edwards et al. (8). Basically, the same method was applied for each species; however, the age of leaves used and the digestion times were varied depending upon the material chosen. Plant material was glasshouse grown under full sunlight and night/day temperatures of 20°C and 30°C, in 30-cm-diameter pots, with approximately 20 plants per pot. Maize leaves were used 10 to 14 d after planting, other species required 21 to 28 d for expansion to a suitable size. Approximately 10 g of leaves were harvested for each preparation. The leaves were sliced into approximately 1-mm strips with a razor blade and digested at 30°C for 2 to 2.5 h, with illumination (1 50-w flood lamp), in 50 ml of 0.5 M sorbitol, 0.2 mM CaCI2, I mM MgC92, 0.2 mM KH2PO4, 10 mM Mes, 0.2% BSA, 2% cellulase (Onozuka S-3), and 0.2% pectinase (Macerozyme R-10) at pH 5.5 with HCI. Leafstrips were examined under a microscope each hour to determine the correct digestion time. When gentle agitation of the tissue resulted in mesophyll protoplast release, the incubation medium was carefully decanted and the leaf strips washed in a plastic tea strainer with 50 ml of 0.6 M sorbitol, 5 mM Hepes, 1 mM MgCl2, 0.2 mM CaCl2, 0.2 mM KH2PO4, and 0.1 % BSA, pH 7.8. The filtrate was passed through two nylon nets (250 ,um net and then an 80 ,um) and the filtrate was centrifuged at 350g for 3 min. The pelletted protoplasts were gently resuspended in 20 ml of the solution previously described but with 0.6 M sucrose substituted for sorbitol. The suspension
'Current address: A.R.C. Research Group on Photosynthesis, Dept. of Biology, University of Sheffield, Sheffield SIO 2TN England. 2 Abbreviations: RuP2, ribulose 1,5-bisphosphate; PEP, phosphoenolpyruvate; OAA, oxaloacetate; 3-PGA, 3-phosphoglycerate; ME, malic enzyme; CK, carboxykinase. 1038
02 PHOTOREDUCTION IN C4 MESOPHYLL CHLOROPLASTS divided into two 15-ml COREX tubes, each tube overlayed with 2 ml of the sorbitol medium, and the gradient centrifuged in a swing-out head rotor for 5 min at 350g. Protoplasts were collected at the sorbitol/sucrose interface and stored on ice until required. All procedures were carried out at room temperature up to this point. Protoplasts were pelletted at 350g for 3 min and resuspended in 0.33 M sorbitol, 10 mm EDTA, 50 mm Tricine at pH 7.8 (KOH). Chloroplasts were then isolated by twice passing protoplasts through a 20-um mesh over the end of a 1-ml syringe and collection by centrifugation. The chloroplasts were resuspended and subsequently assayed in the medium used for protoplast lysis. Chloroplasts isolated by this method were routinely greater than 80% intact as measured by ferricyanide exclusion (17). Mass Spectrometric 02 Exchange. Measurements of 02 uptake and evolution in isolated chloroplasts were made using 1802 + 602 and a Varian MAT GD150 magnetic sector mass-spectrometer, as previously described (1 1). Illumination was provided by a quartz projector lamp giving a photon fluence rate of 1500 ME m 2 s-' at the cuvette surface. Chl concentration was estimated by the method of Arnon (1). Catalase (1000 units/ml) was included in all assays. Chemicals were obtained from the Sigma Chemical Co. with the exception of cellulase and pectinase which were obtained from the Kinki Yakult Biochemical Co. (Japan). was
rate was significantly suppressed by the addition of OAA, particularly in Z. mays. The presence of 3-PGA did not perturb 02-
uptake from the basal level even though it stimulated 02 evolution. Table I shows that 02 uptake in C4 mesophyll chloroplasts responds dramatically to the ATP/NADPH requirements of the substrate metabolized. This supports previous reports that pseudocyclic photophosphorylation may contribute substantially to ATP production in these organelles (18). Pyruvate, requiring 2 ATP for the conversion to PEP resulted in high rates of 02 uptake, presumably to generate the required ATP when no NADPH sink was present. Oxaloacetate suppressed this uptake by providing a source of NADP+ for reduction by the electron transport chain in lieu of 02. The reduction of 3-PGA also resulted in lowered 02 uptake, due to the requirement of only 1 ATP/NADPH. Suppression of O2 Uptake by OAA. In Table I, OAA was seen to suppress pyruvate-dependent 02 reduction, presumably due to a competition for electrons between the NADP+ produced in OAA reduction and 02. Figure 1 examines this phenomenon further, showing O2 uptake by uncoupled, isolated Z. mays mesophyll chloroplasts in the presence of varying concentrations of OAA at low and high light. At low light (150 ME m 2 s-'), 02 uptake was suppressed 80% from the rate in the absence of substrate by the addition of less than 100 Mm OAA. At high light (1800 ME m 2 sg-), only 40% suppression occurred and 400 uM RESULTS AND DISCUSSION OAA was necessary. These data indicate that, when pyruvate Pi-dikinase is operResponse of Mesophyll Chloroplasts to Substrate ATP Dmand. The isolated mesophyll chloroplast is incapable of fixing ating at full capacity in vivo (simulated here by uncoupling), CO2 without added substrate and the cytoplasmic enzyme PEP- OAA concentration in the chloroplast may regulate the contricarboxylase (14). However, the enzyme complement of these bution of pseudocyclic photophosphorylation (particularly in the chloroplasts allows the study of the response of 02 uptake and NADP-ME types). As OAA levels in vivo appear to be very low evolution to substrates requiring either only ATP for their me- (M. D. Hatch, personal communication), high rates of pseudotabolism (e.g. pyruvate), ATP, and NADPH (3-PGA or pyruvate cyclic electron transport would be possible. Figure 1 also demonstrates the competition of NADP+ and 02 plus OAA) or NADPH alone (OAA). The response of 02 exchange by isolated intact mesophyll for photochemical energy, similar to that seen in isolated C3 chloroplasts of Z. mays, P. miliaceum, and P. maximum to thylakoids (1 1). At low light, less OAA was required to suppress different substrates is shown in Table I. All species supported 02 uptake than at high light and a greater suppression was high rates of 02 evolution with 3-PGA as substrate, but chloro- observed. This is also analogous to data obtained with isolated plasts from Z. mays alone were able to support high rates of thylakoids where more NADP+ is required to suppress 02 uptake OAA reduction, presumably due to the higher levels of NADP- at high light than at low light (1 1). Oxygen Response of 02 Uptake. The response of the in vivo malate dehydrogenase in NADP-ME type mesophyll chloroplasts Mehler reaction to 02 tension is a matter of contention. It has (14). Light-dependent 02 uptake was highest in all three types when been reported (2, 20) that in thylakoids a Mehler reaction is fully pyruvate was the sole substrate, showing considerable stimulation saturated by as little as 2% 02.02 uptake in isolated chloroplasts, over the basal uptake rate in the absence ofadded substrate. This however, requires considerably higher levels of 02 for saturation Table I. Light-Dependent 02 Exchange in Mesophyll Chloroplasts from Several C4 Plants Substrates were used at the following concentrations: 3-PGA, 2 mM; pyruvate (PYR), 2 mM; OAA, I mM. The 02 concentration was 250 AM, initially, in each experiment.
38 36 66
Mumol mg' Chl h' 31 147 38
PYR PYR + OAA
44 74 58
PYR PYR + OAA Z. mnays
3-PGA PYR PYR + OAA
23 24 65 24
58 34 181 53 80 20 259 63 108
Net 02 Exchange -7 111
-28 14 -14 137 -21 22 -3 235 -2 84
FURBANK ET AL.
Plant Physiol. Vol. 73, 1983 s
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FIG. 3. The response of 02 uptake (A) and 02 evolution (0) in Z. mays mesophyll chloroplasts to 02 concentration with pyruvate (2 mM) as substrate.
OAA Concentration (mM) FIG. 1. The response of 02 uptake (A), 02 evolution (0), and net 02 evolution (0) in Z. mays mesophyll chloroplasts to OAA concentration, at two light intensities: a, 150 ME m-2 s'; b, 1800 ME m-2 s-'. Five mm NH4Cl was included as an uncoupler.
with OAA (0.5 mM) as substrate in the presence of 5 mm NH4Cl. IOO1
under conditions where photorespiration is inhibited (5, 10). The response of 02 uptake and evolution in isolated Z. mays mesophyll chloroplasts to 02 tension with a number of different substrates is shown in Figures 2 to 4. The response of 02 exchange to 02 tension during 3-PGA reduction (Fig. 2) shows that lightdependent 02 uptake responded significantly to 02 concentration up to 125 uM with saturation occurring above this point. Linear electron flow (indicated by gross 02-evolution, or water-splitting) also decreased with low 02 (< 60Mm). In the absence of catalase, 3-PGA reduction was inhibited and 02 uptake stimulated, indicating the formation of H202 under these conditions (data not shown). With Z. mays chloroplasts in the presence of pyruvate, high even
FIG. 4. The response of 02 uptake (A), 02 evolution (0), and net 02 evolution (0) in Z. mays mesophyll chloroplasts to 02 concentration
02 Concentration (uM)