Photosynthetic Characteristics of Spinach Leaves Grown with Different Nitrogen Treatments

Transcription

1 Plant Cell Physiol. 29(1): (1988) JSPP 1988 Photosynthetic Characteristics of Spinach Leaves Grown with Different Nitrogen Treatments John R. Evans 12 and Ichiro Terashima 3 ' Division of Plant Industry, CSIRO, G.P.O. Box 1600 Canberra, A.C.T. 2601, Australia 3 Plant Environmental Biology Group, Research School of Biological Sciences, The Australian National University, G.P.O. Box 475, Canberra, A.C.T. 2601, Australia Spinach plants (Spinacia oleracea L.) were grown hydroponically with different concentrations of nitrate nitrogen, ranging from 0.5 to 12 mm, in a glasshouse under full sunlight. Using an open gas exchange system, the rate of CO 2 assimilation, A, was determined as a function of intercellular partial pressure of CO 2, PJ, with a constant amount of absorbed light per unit Chi. When expressed on a leaf area basis, A measured at high irradiance and at p, = 5O0fjbaT, was proportional to the in vitro rate of uncoupled whole-chain electron transport as well as to Chi content. There was a curvilinear relationship between the mesophyll conductance (the slope of the A : Pi curve near the CO 2 compensation point) and the in vitro RuBP carboxylase activity. The curvature did not appear to be due to enzyme inactivation in vivo in leaves with high nitrogen contents. The curvature suggested the presence of a CO 2 transfer resistance between the intercellular spaces and the site of carboxylation of 2.2 m 2 s bar mol" 1 CO 2, which is similar to that previously observed in wheat. This implied that, while nitrogen deficiency increased the ratio of in vitro activity of electron transport to that of RuBP carboxylase, the two activities remained balanced in vivo. Irradiance response curves were determined by both net CO 2 and O 2 exchange. The two methods gave reasonable agreement at light saturation. The quantum yield measured by O 2 evolution was 0.090±0.003 mol0 2 mol~' absorbed quanta, whereas after correcting for pj = 500//bar, the quantum yield for CO 2 assimilation was only 82% of that measured by oxygen evolution. Key words: CO 2 assimilation CO 2 transfer resistance Electron transport Nitrogen deficiency RuBP carboxylase Spinacia oleracea. While nitrogen deficiency decreases the chlorophyll but did not alter their properties. Consequently, under content per unit leaf area, the rate of electron transport conditions where electron transport is rate-limiting (such and amounts of thylakoid components, each expressed per as at high intercellular p(co ), there should be a linear unit of chlorophyll, are unaffected (A triplex grown under relationship between the rate of CO 2 assimilation and high irradiance, Medina 1971, Spinacia, Evans and chlorophyll content, both expressed per unit leaf area, for Terashima 1987, Terashima and Evans 1988). The rela- leaves absorbing the same irradiance per unit of chlorophyll, tionship between the rate of oxygen evolution per unit of There should also be a linear relationship between the rate chlorophyll and absorbed irradiance per unit of chloro- of CO 2 assimilation and in vitro electron transport, phyll was independent of nitrogen treatment (Evans and The response of the rate of CO 2 assimilation, A, to in- Terashima 1987, Terashima and Evans 1988). This suggested tercellular CO 2 partial pressure, Pi, has been successfully that nitrogen deficiency decreased the amount of thylakoids modelled by assuming that at low p it the rate is limited by the RuBP carboxylase activity, whereas at high pj, the rate 2 Present address: Plant Environmental Biology Group, Re- is limited by the rate at which RuBP can be regenerated search School of Biological Sciences, The Australian National (Farquhar and Caemmerer 1982). RuBP regeneration is University, G.P.O. Box 475, Canberra, A.C.T. 2601, Australia. generally thought to reflect the rate at which electron 157

2 158 J. R. Evans and I. Terashima transport regenerates NADPH and/or photophosphorylation regenerates ATP. However, in some cases RuBP regeneration can be limited by the availability of inorganic phosphate in the chloroplasts (Farquhar and Caemmerer 1982, Sharkey 1985, Sharkey et al. 1986, Stitt 1986). The CO 2 response of A when RuBP carboxylase activity is limiting can be predicted from the kinetic constants for the enzyme. When the substrate partial pressure of CO 2 is taken as equal to the intercellular ptcoj, Pi, there should be a linear relationship between the RuBP carboxylase activity and the mesophyll conductance (the slope of the A : Pi curve near the CO 2 compensation point). The linear relationship ignores the possible resistance to CO 2 diffusion from intercellular spaces to the sites of carboxylation within the chloroplast. For wheat, this resistance was found to be important (Evans 1983a, Evans et al. 1986). In contrast to the independence of the ratio of thyloakoid components to chlorophyll with changing nitrogen nutrition, the ratio of RuBP carboxylase to chlorophyll declines with nitrogen deficiency in A triplex (Medina 1971), Gossypium (Wong 1979), Solarium (Ferrar and Osmond 1986), Phaseolus (Seemann et al. 1987) and Spinacia (Evans and Terashima 1987), though not in Triticum (Evans 1983, 1985a). In spinach, the increase in the ratio of electron transport to RuBP carboxylase activities with nitrogen deficiency, is therefore expected to alter the relationship between the rate of CO 2 assimilation and pi if there is little resistance to CO 2 transfer from the intercellular spaces to the sites of carboxylation. The independence of electron transport capacity per unit of chlorophyll with nitrogen treatment implies that the different activities per unit leaf area induced by nitrogen deficiency can be removed by expressing CO 2 assimilation on a chlorophyll basis. For leaves absorbing the same irradiance per unit of chlorophyll, the rate of CO 2 assimilation per unit Chi at high p ( would be independent of nitrogen treatment, whereas nitrogen deficient leaves should have a lower mesophyll conductance. Nitrogen deficiency would therefore increase the Pi where CO 2 assimilation changes from an RuBP carboxylase limitation to an electron transport limitation (Evans and Terashima 1987). This expectation based on the in vitro biochemical analysis contrasts with observations based on gas exchange analysis, which showed that potential electron ransport activity approximately matched the RuBP carboxylase activity, irrespective of nitrogen treatment in Phaseolus (Caemmerer and Farquhar 1981), leaf age in Triticum (Evans 1986) and phosphorus treatment in Spinacia (Brooks 1985). The aim of the experiments described here was to compare the in vivo photosynthetic performance of the leaf with its underlying biochemistry. We have also compared net CO 2 assimilation with net O 2 evolution as a function of irradiance in order to facilitate comparisons between measurements of CO 2 or O 2 exchange. Materials and Methods Plant material Spinach plants {Spinacia oleracea L. cv. Henderson's hybrid 102) were grown hydroponically in a glasshouse under full sunlight, with a 9 h photoperiod and 18/13 C day/night temperature. Four nitrate treatments were imposed, 0.5, 1, 4, and 12 mm. Measurements were made on the plants after about 50 days growth. The chlorophyll content of the leaves was estimated from leaf discs by the method of Arnon (1949). The proportion of white light absorbed by the leaf was measured using an integrating sphere to determine both leaf transmittance and reflectance. The leaf opposite that used for gas exchange analysis was used for the leaf disc oxygen electrode measurements and subsequently for thylakoid preparations as described previously (Evans and Terashima 1987). Other samples were taken from the remaining parts of the leaves for soluble protein/rubp carboxylase assays and nitrogen determinations. The thylakoids were used to assay the whole chain electron transport activity at light saturation in the presence of an uncoupler (Terashima and Evans 1987). RuBP carboxylase (EC ) activity was assayed in crude extracts following preincubation in the presence of CO 2 and Mg 2 * as previously described (Evans and Terashima 1987). Assays were made at 30 C and the activity at 25 C was taken to be 65% of that at 30 C, using the Q 10 of 2.2 (see Evans 1986). To measure in vivo RuBP carboxylase activity, leaf discs collected in situ under 1,600 //mol quanta m~ 2 s~' were rapidly frozen in liquid nitrogen. A rapid extraction was made in CO 2 -free buffer and the initial activity compared with that following incubation on ice for 45 min in the presence of CO 2 and Mg 2+ (Brooks 1986). Gas exchange The system used is described in detail elsewhere (Evans 1983a), except that absolute p(co 2 ) was calculated from the outputs of two mass flow controllers (Tylan FC260, Torrance, CA) which mixed CO 2 -free air of known humidity with 1 or 10% CO 2 in air and the chamber enclosed the whole leaf. The large leaves produced under 4 and 12 mm nitrate were trimmed to reduce their area to about 20 cm 2 just prior to placing them in the chamber. The leaf boundary layer conductance in the chamber was 2.5molm~ 2 s~'. Measurements were made with the leaf temperature at 25 C and leaf-to-air vapour pressure difference of 15 mbar. After 45 min equilibration at 340//bar p(co2) and an irradiance giving 2.3 mmol absorbed quanta mol"' Chi s~' (the irradiance per unit area of leaf ranging from 800 to 1,500//mol quanta m~ 2 s" 1 to compensate for the different chlorophyll contents which had been estimated beforehand) rates of CO 2 assimilation were measured with PJ ranging from the compensation point to about 500//bar. Rate of CO 2 assimilation was then measured as a function of irradiance with p ( maintained near 500//bar. Rates of gas exchange and related bio-

3 chemical parameters were calculated according to Caemmerer and Farquhar (1981). The rate of oxygen evolution was determined as a function of irradiance with a leaf-disc oxygen electrode (Delieu and Walker 1981: Hansatech, King's Lynn, U.K.). The leaf temperature was 25 C and the gas phase was ~16% O 2 and ~5% CO 2. Photosynthetic characteristics of spinach leaves 159 Results The rate of CO 2 assimilation, with Pj = 500/ibar and a constant absorbed irradiance per unit of chlorophyll was directly related to the in vitro rate of electron transport from H 2 O to methylviologen (Fig. 1). According to the model of Caemmerer and Farquhar (1981), the CO 2 assimilation rate at Pj = 500//bar that can be sustained by the electron transport rate, J, is A 500 = J(p c r*)/(4.5c r*) = 0.69J/4, where p c is the ptcoj at the site of carboxylation and /"* is the CO 2 compensation point in the absence of non-photorespiratory CO 2 evolution in the light (40/^bar, Brooks and Farquhar 1985). The rate of oxygen evolution or Hill activity, H = J/4, since there are 4 electrons per oxygen. While the slope of 0.69 is close to that observed in Fig. 1, the rate of CO 2 assimilation was not measured at light saturation. Allowing for this, the maximum rate of electron transport predicted from the rate of CO 2 assimilation would be 120 mmol O 2 mol" 1 Oils" 1. The average rate from 24 preparations measured in vitro was 103 mmol O 2 mol" 1 Chi s~'. The in vitro rate of electron transport per unit of chlorophyll was independent of the nitrogen treatment (Table 1). A strong relationship existed between A 500 and chlorophyll: A 500 (^molm" 2 s~') = 82.9xChl(mmolm" 2 ) -3.45, 1^ = Responses of photosynthesis to irradiance, measured at Pi = 500/ibar (high enough to ensure that the photosynin vitro Rate of electron transport (//mol 0 2 m" 2 s~ Fig. 1 Rate of CO 2 assimilation versus in vitro rate of electron transport. CO 2 assimilation was measured at an intercellular p(co 2 )=500//bar and an irradiance of 2.3 mmol absorbed quanta mol~' Chi s"'. Electron transport was measured from H 2 O to methylviologen at saturating irradiances. O 12, A 4, D 1, o 0.5 rrim nitrate treatments. Solid points represent the curves in Fig. 2. y= x,r 2 =0.85. thetic rate was limited by electron transport), are shown on both a unit leaf area basis and a unit chlorophyll basis (Fig. 2). Photosynthetic capacity per unit leaf area, chlorophyll content and leaf absorptance were all reduced by nitrogen deficiency (Table 1). The responses of assimilation to irradiance expressed on a chlorophyll basis were independent of nitrogen treatment, due to the proportionality between the rate of CO 2 assimilation and chlorophyll content (Fig. 2B). Table 1 The contents of nitrogen and chlorophyll, the electron transport and RuBP carboxylase activities on a chlorophyll basis and the absorptances of leaves from plants grown with different nitrate concentrations, under full sunlight Nitrogen (mmol m~ 2 ) Chlorophyll (mmol m~ 2 ) Absorptance (400 nm 700 nm) Whole chain electron transport H 2 O -* MV (mmol O 2 mol" 1 Chi s" 1 ) RuBP carboxylase activity (mmol CO 2 mol"' Chi s"') Initial RuBP carboxylase activity {%) Initial RuBP carboxylase activity expressed as a percentage of fully activated activity (n = 3). Values are means±s.e. " Dark control 62 ± ±3 0.27± ± ±8 117 ± Nitrate concentration (HIM) ± ± ± ± ± ± ± ± ± 0. 89± a 12

4 160 J. R. Evans and I. Terashima " Irradiance (/tmol quanta m" 2 s"') (mol quanta mol" 1 Chi s" 1 ) Fig. 2 Rate of CO 2 assimilation versus absorbed irradiance. CO 2 assimilation was measured at an intercellular pccoj of 500//bar. Expressed on a leaf area basis (A). Both axes expressed on a chlorophyll basis (B). Symbols as in Fig. 1. The responses of rate of CO 2 assimilation to p, are shown in Fig. 3. As expected, for A expressed on a chlorophyll basis, no effect of nitrogen treatment was evident for pj>300/ibar (the range in which rate of electron transport should be limiting). The decreasing RuBP carboxylase/chlorophyll ratio with decreasing N-status (Table 1) was expected to result in a smaller mesophyll conductance per unit of chlorophyll for the lower nitrogen treatments. Contrary to this expectation, there were also no differences evident in the A: p; curves expressed per unit of chlorophyll for pj<200//bar. The reason for this similarity is evident from the relationship between mesophyll conductace and fully activated in vitro RuBP carboxylase activity (Fig. 4). The curvature was not due to enzyme inactivation in the high nitrate treatments (Table 1). Following rapid extraction in the absence of CO 2, the initial activities suggested that all the extracted RuBP carboxylase was catalytically active under 1,600//mol quanta m~ 2 s~' irrespective of the N-treatments. In the dark, this declined to ~60%. The curvilinear relationship has been attributed to a resistance to CO 2 transfer from the intercellular spaces to the sites of carboxylation within the chloroplast. The resistance can be obtained from the double reciprocal plot of mesophyll conductance against RuBP carboxylase activity i.e. mesophyll resistance against carboxylation resistance. Mesophyll resistance can be regarded as the sum of carboxylation resistance and the CO 2 transfer resistance. Therefore, the y-intercept where the Intercellular p(co 2) (jubar) Fig. 3 Rate of CO 2 assimilation versus intercellular p(cc>2). CO 2 assimilation was measured at an irradiance of 2.3 mmol absorbed quanta mol" 1 Chi s~'. (A) is expressed on an area basis, (B) CO 2 assimilation is expressed per unit of chlorophyll with the solid line predicted from the RuBP carboxylase limited rate. Symbols as in Fig. 1, but represent different leaves from Fig. 2. The irradiances used were 1,490, 1,350, 1,090 and 900//mol quanta m" 2 s"' for the 12, 4, 1 and 0.5 mm curves.

5 Photosynthetic characteristics of spinach leaves RuBP carboxylase activity (ju.mol m~ 2 s" 1 1/RuBP carboxylase activity (m 2 s/xmol"') Fig. 4 Mesophyll conductance versus in vitro RuBP carboxylase activity. (A) The line (i) through the data is the regression from B. Line (ii) is predited from the kinetic constants for the enzyme used by Evans and Terashima (1987) with the presence of the CO 2 transfer resistance, r w, estimated in B. Mesophyll conductance, g m, is expressed as g m =k/(l+k x r w ), where k=v c (r*+k c (l+o/ic o ))/(Pc + K c (l +O/K O )) 2, r*-40^bar, K c =296^bar, K O =212 mbar, 0=200 mbar and p c =75 ^bar. Dashed line (iii) predicted with r w =0. Double reciprocal plot of A to obtain the CO 2 transfer resistance (B). r w = 2.24±0.05 m 2 s bar mol"' CO 2. For detailed methods for calculations, see Evans (1986). carboxylation resistance is zero, equals the CO 2 transfer resistance (2.2m 2 sbarmon' CO 2, Fig. 4B). The response of rate of CO 2 assimilation per unit of chlorophyll to P changed little with nitrogen deficiency (Fig. 3). The relative capacities for electron transport/ photophosphorylation (this being proportional to the rate of CO 2, assimilation when Pj = 500/ibar, A 500, Fig. I) and RuBP carboxylation are examined in more detail at a constant irradiance per unit leaf area (Fig. 5). Lines have been calculated for the means of the ratio of calculated Hill activity to mesophyll conductance. The means are 424 and 337mol0 2 /(molc0 2 //bar~') for plants grown with 0.5 and 12 mm nitrate, respectively. The ratio defines the transition from RuBP carboxylase to electron transport limitation, 204 and 158 ^bar, respectively. Incorporation of the CO 2 transfer resistance and deriving RuBP carboxylase kinetic constants from Fig. 4A leads to a curvilinear relationship between A 500 and RuBP carboxylase actvity (Fig. 5B). The data from all treatments fall close to a single line indicating that the capacity for electron Mesophyll conductance RuBP carboxylase activity (mol m" 2 s"' bar"') (jumol m" 2 s"') Fig. 5 Rate of CO 2 assimilation versus mesophyll conductance. CO 2 assimilation was measured at an intercellular p(co 2 ) of 500//bar and an absorbed irradiance of l,800/*mol quanta m~ 2 s" 1 (A). The two lines are predicted for transition points at (i) 204 and (ii) 158 //bar CO 2. Solid points represent the curves in Fig. 3. Rate of CO 2 assimilation versus in vitro RuBP carboxylase activity (B). Line is calculated for a transition at 185 //bar CO 2 due to a ratio of Hill activity to mesophyll conductance of 385 mol mop

6 162 J. R. Evans and I. Terashima " RuBP carboxylase activity (//mol m~ 2 s" 1 ) Fig. 6 Hill activity versus in vitro RuBP carboxylase activity. Hill activity measured with a leaf disc oxygen electrode at 2,000^mol quantam~ 2 s~', 1% CO 2. Plants were grown under full sunlight A 30% sunlight and D 15% sunlight. Lines are calculated for the mean ratios of Hill activity to mesophyll conductance for 100% sunlight (385 mol O 2 //bar mol" 1 CO 2 ) and 30, 15% (273) (data from Terashima and Evans 1988). transport remains well balanced by the RuBP carboxylase activity across the nitrate treatment. A similar analysis is shown for plants grown with different nitrate treatments and growth irradiances (data from Terashima and Evans 1988) (Fig. 6). Theoretical lines are drawn through the mean ratios of Hill activity to mesophyll conductance (calculated from the in vitro RuBP carboxylase activity) for the 100%, and for the 30 and 15% growth irradiances. The mean ratio for full sunlight was the same as that observed in the independent data set of Fig. 5B. Also, the mean ratio for the high and low nitrogen treatments of 337 and 453 mol O 2 /(moi CO 2 //bar" 1 ) respectively, was similar to that in Fig. 5A. Growth under reduced irradiance lowered the expected ratio of Hill activity to mesophyll conductance. Responses of net CO 2 assimilation and net O 2 evolution to changes in irradiance were determined on adjacent leaves (Fig. 7). All 11 comparisons were consistent with a trend that was independent of nitrogen treatment. The light saturated rates obtained with the two methods were in close agreement. The ratio of the rates calculated from CO 2 assimilation and O 2 evolution were 0.90±0.05 (mean ±S.E., n=ll) which compares with the expected ratio of 0.92 calculated on the basis that the CO 2 assimilation was measured with pj = 500^bar, while the O 2 evolution was measured at 5% CO 2 which should virtually suppress photorespiration. The average quantum yields were 0.090±0.003 mol O 2 mol" 1 absorbed quanta and ± mol CO 2 mol" 1 absorbed quanta. On average, the quantum yield measured by CO 2 assimilation (allowing for photorespiration) was only 82% of that measured by O 2 evolution, which results in a curvilinear relationship between the two rates when cmpared at different irradiances (Fig. 7B). Discussion The objectives of these experiments were to compare the in vivo photosynthetic performance of leaves with vary- 50 B A /f.0 / Absorbed irradiance (jumol quanta m~ 2 s~') 1 I Rate of oxygen evolution (//.mol m" 2 o-n Fig. 7 Rate of CO 2 assimilation or oxygen evolution versus absorbed irradiance. Open symbols and dashed lines are the rate of CO 2 assimilation with P!=500//bar, solid symbols and continuous lines were measured with the leaf disc oxygen electrode with 5% CO 2 O, 12 mm and o, 1 nim nitrate. Rate of CO 2 assimilation versus the rate of oxygen evolution (B). 12 mm, o 1 DIM. The solid line represents 1 : 1 agreement, corrected by the factor of (1 FJC), where /i=40//bar and C=500//bar. The dashed line represents the average relationship between the quanum yields (n = ll).

7 Photosynthetic characteristics of spinach leaves 163 ing degrees of nitrogen deficiency with that predicted from underlying biochemistry. It was expected that nitrogen deficiency would not alter the electron transport capacity per unit of chlorophyll but would increase the capacity of electron transport relative to that of RuBP carboxylation (Evans and Terashima 1987). According to the model of Farquhar and Caemmerer (1982), the slope of the curve relating the rate of CO 2 assimilation to P; near the CO 2 compensation point is determined by the RuBP carboxylase activity. At higher Pi (~500^bar), the rate is limited by the rate of RuBP regeneration which reflects the rate of electron transport/photophosphorylation. There is considerable evidence demonstrating the relationship between RuBP carboxylase activity and the mesophyll conductance (Caemmerer and Farquhar 1981, 1984, Caemmerer and Edmondson 1986, Evans 1983a, 1986, Evans and Seemann 1984, Brooks 1986), but very limited data concerning the electron transport limitation (Caemmerer and Farquhar 1981). The measurements made here with spinach leaves having different electron transport activities confirm the expected limitation by electron transport at high irradiance and pj = 500/ibar (Fig. 1). However, as in the previous work (Caemmerer and Farquhar 1981), the electron transport rate calculated from the gas exhcnage exceeded the measured in vitro rate by about 20%. CO 2 transfer resistance The comparison of in vivo RuBP carboxylase activity (the mesophyll conductance), with extracted activity, revealed not only a curvilinear relationship but also a greater activity than expected from the kinetic constants (contrast the data with curves (ii) and (iii) in Fig. 4A). Given that the initial activity of rapidly extracted enzyme was not improved by incubation with CO 2 and Mg 2+, the curvature in Fig. 4A did not appear to be due to inactive enzyme in vivo at the higher nitrate levels. While we cannot rule out the possibility of the presence of a loosely bound catalytic inhibitor in leaves with high nitrogen contents, the subsequent discussion will suggest that the deviation is caused by a CO 2 transfer resistance. Analysis of the curvature yielded an estimate of the CO 2 transfer resistance (2.2 m 2 s bar mol" 1 CO 2 Fig. 4B) similar to that observed for wheat (Evans 1983a). The double reciprocal plot analysis assumes that the CO 2 transfer resistance is the same for all replicate leaves. While this is probably untrue, it is difficult to predict how it would differ with leaf nitrogen content. To the extent that it decreases with increasing leaf nitrogen, the double reciprocal plot will underestimate the resistance (Evans 1983b). The existence of the CO 2 transfer resistance in wheat has also been inferred from an independent method which examined the discrimination against I3 CO 2 (2.4m 2 sbarmon' CO 2, Evans et al. 1986). A consequence of the resistance is that the in vivo RuBP carboxylase activity is reduced progressively with increasing amounts per unit leaf area due to the lowering of the p(cc>2) at carboxylation sites. So, for carboxylation to remain in balance with th electron transport capacity, the ratio of RuBP carboxylase to electron transport needs to increase with increasing leaf nitrogen content. The comparison of these two capacities estimated in vitro suggested an excess of electron transport capacity and a dramatic change in their ratio with leaf nitrogen content (Evans and Terashima 1987). Incorporation of the CO 2 transfer resistance and in vivo RuBP carboxylase activity clearly showed that the capacity for electron transport at 1,800 /rniol quantam~ 2 s~' was closely matched to the RuBP consumption rate (Fig. 5B). The observed relationship between A 500 and mesophyll conductance in spinach (Fig. 5A) is remarkably similar to that for wheat (Evans 1986). RuBP carboxylase kinetic constants for modelling The rate of CO 2 assmilation has been successfully predicted from the amount of RuBP carboxylase, given the appropriate p (. However, the kinetic constants used to obtain this agreement vary dramatically among laboratories. Three examples will be compared at 25 C; the data of Besford et al. (1985), Makino et al. (1985) and Evans (1986). In all three, reasonable agreement was claimed between the measured rates of CO 2 assimilation and the rates predicted from the RuBP carboxylase measurements. This was despite more than a two-fold variation in expected performance of the enzyme. The initial slope of the curve relating the rate of CO 2 assimilation to the p(co 2 ) can be described as follows (Farquhar and Caemmerer 1982), da/dc = V cmax /(r* + K,(l + O/K o )) where V cmax is the maximum RuBP carboxylase activity, K c and K o are the Michaelis constants for RuBP carboxylase for CO 2 and O 2 repectively, O is the p(o 2 ) and r* is the CO 2 compensation point in the absence of non-photorespiratory CO 2 evolution in the light. The RuBP carboxylase activity in the leaf is sensitive to the effective A" m (CO 2 ) in the presence of oxygen, and to the relative activities of the oxygenase and carboxylase reactions. The effective K m (CO 2 ), K c (l+o/k o ), in the presence of 200mbar O 2 and the (/"*) for the three sets of data were 244 (24.1), 399 (27.5) and 575 (37.8) respectively. At 25 C, A has been estimated to be 43 by an in vitro technique (Jordan and Ogren 1984) and 40.7 by gas exchange (Brooks and Farquhar 1985). The two lower values used by Besford et al. (1985) and Makino et al. (1985) enhanced the carboxylase activity expected in vivo. By contrast, Evans (1986) used a high turnover rate of the enzyme (32mol CO 2 mor'enzymes" 1, Evans and Seemann 1984) which compares with 16.4 (Makino et al. 1985) and 26.5 mol CO 2 mor' enzyme s" 1 (Evans and Terashima 1987). To account for the observed CO 2 assimilation rates, its has always been necessary to assume very

8 164 J. R. Evans and I. Terashima high affinities for CO 2 and/or high specific activities. Considerable changes to the kinetic constants would be required to account for the data in Fig. 4A. Balance between electron transport and RuBP carboxylase The relative capacities for electron transport and RuBP consumption by RuBP carboxylase define a transition from an RuBP carboxylase limited to an electron transport limited rate of CO 2 assimilation. Within the transitional region, both capacities are fully expressed and therefore utilised with the greatest efficiency. Increasing the electron transport capacity raises the p s at the transition and if efficiency were to be maintained, the partial pressure of intercellular CO 2 would need to be greater. As with leaf aging in wheat (Evans 1986), nitrogen deficiency in spinach was associated with a relative increase in the electron transport capacity (Fig. 5A), although this is not readily apparent from the normalised CO 2 response curves (Fig. 3). In wheat, the Pj observed under normal external conditions increased in a manner consistent with p, following the transition point (Evans 1985). A more obvious example of change in the ratio between electron transport and RuBP carboxylase capacities is evident in acclimation to different growth irradiances. Growth under lower irradiance reduces the electron transport capacity for a given RuBP carboxylase activity (Fig. 6). Therefore, the ratio of Hill activity to mesophyll conductance, is reduced, as implied by curves relating the rate of CO 2 assimilation to Pj for Agathis (Langenheim et al. 1984), Pisum (Evans 1987a), Phaseolus (Seemann et al. 1987) and Piper (Walters and Field 1987). O 2 versus CO 2 measurement Comparison of the irradiance response curves obtained by measuring CO 2 assimilation and O 2 evolution showed a consistent difference (Fig. 7). The quantum yields measured by CO 2 assimilation were unexpectedly lower than those measured by O 2 evolution, while the light saturated rates were equal. The irradiance response curve measured by CO 2 assimilation was obtained after measuring a CO 2 response curve and with decreasing irradiance, whereas the O 2 evolution irradiance response curve was measured with increasing irradiance from darkness. No difference has been found when irradiance response curves in the leaf-disc electrode were measured using increasing or decreasing irradiance (data not shown). If the irradiance for the CO 2 assimilation measurements had been underestimated, it would result in both a low quantum yield and the need for a higher irradiance to reach light saturation. In fact, light saturation was reached at similar irradiances with both methods. Thus the cause of the curvature in Fig. 7B is uncertain. Bjorkman and Demmig (1987) also noted that quantum yields measured by CO 2 assimilation were only 83% of that measured by the leaf disc oxygen electrode. After correcting for photorespiration, the quantum yields obtained here by CO 2 assimilation were only 82% of that obtained by the leaf disc electrode. While the quantum yield of mol O 2 mol" 1 quanta agrees with the average value in the literature, no difference was seen between oxygen and CO 2 quantum yields in previous work with pea leaves, although the measurements were made on different plants at different locations (Evans 1987b). This work was carried out during the tenure of a CSIRO postdoctoral fellowship (JRE) with additional support from an ANU- CSIRO collaboration grant. H. Ficker's assistance is gratefully acknowledged. We thank Drs. S. von Caemmerer and G. D. Farquhar for helpful criticism of the manuscript. References Arnon, D. I. (1949) Copper enzymes in isolated chloroplasts. Polyphenoloxidases in Beta vulgaris. Plant Physiol. 24: Besford, R. T., Withers, A. C. and Ludwig, L. J. (1985) Ribulose bisphosphate carboxylase activity and photosynthesis during leaf development in the tomato. /. Exp. Bot. 36: Bjorkman, O. and Demmig, B. (1987) Photon yield of O 2 evolution and chlorophyll fluorescence characteristics at 77K among vascular plants of diverse origins. Planta 170: Brooks, A. (1985) Effects of phosphorus nutrition on photosynthetic metabolism of spinach leaves. Ph. D. thesis, Australian National University. Brooks, A. (1986) Effect of phosphorus nutrition on ribulose 1-5-bisphosphate carboxylase activation, photosynthetic quantum yield and amount of some Calvin-cycle metabolites in spinach leaves. Aust. J. Plant Physiol. 13: Brooks, A. and Farquhar, G. D. 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(1983a) Nitrogen and photosynthesis in the flag leaf of wheat (Jriticum aestivum L.). Plant Physiol. 72: Evans, J. R. (1983b) Photosynthesis and nitrogen partitioning in leaves of Triticum aestivum and related species. Ph. D. thesis,

9 Photosynthetic characteristics of spinach leaves 165 Australian National University. Evans, J. R. (1985) A comparison of the photosynthetic properties of flag leaves from Triticum aestivum and T. monococcum. In Regulation of Sources and Sinks in Crop Plants, Monograph 12 Edited by Jeffcoat, B., Hawkins, A. F. and Stead, A. D. pp British Plant Growth Regulator Group, Bristol. Evans, J. R. (1986) The relationship between CO 2 -limited photosynthetic rate and RuBP carboxylase content in two nuclear-cytoplasm substitution lines of wheat and the coordination of RuBP carboxylation and electron transport capacities. Planta 167: Evans, J. R. (1987a) The relationship between electron transport components and photosynthetic capacity in pea leaves grown at different irradiances. Aust. J. Plant Physiol. 14: Evans, J. R. (1987b) The dependence of quantum yield on wavelength and growth irradiance. Aust. J. Plant Physiol. 14: Evans, J. R. and Seemann, J. R. (1984) Differences between wheat genotypes in specific activity or ribulose-l,5-bisphosphate carboxylase and the relationship to photosynthesis. Plant Physiol. 74: Evans, J. R., Sharkey, T. D., Berry, J. A. and Farquhar, G. D. (1986) Carbon isotope discrimination measured concurrently with gas exchange to investigate CO 2 diffusion in leaves of higher plants. Aust. J. Plant Physiol. 13: Evans, J. R. and Terashima, I. (1987) Effects of nitrogen nutrition on electron transport components and photosynthesis in spinach. Aust. J. Plant Physiol. 14: Farquhar, G. D. and Caemmerer, S. von (1982) Modelling of photosynthetic response to environmental conditions. In Physiological plant ecology II. Water relations and carbon assimilation Edited by Lange, O. L., Nobel, P. S., Osmond, C. B. and Ziegler, H. Encycl. Plant Physiol. New Ser. Vol. 12B, pp Springer-Verlag, Berlin. Ferrar, P. J. and Osmond, C. B. (1986) Nitrogen supply as a factor influencing photoinhibition and photosynthetic acclimation after transfer of shade-grown Solanum dulcamara to bright light. Planta 168: Jordan, D. B. and Ogren, W. L. (1984) The CO 2 /O 2 specificity of ribulose-l,5-bisphosphate carboxylase oxygenase. Planta 161: Langenheim, J. H., Osmond, C. B., Brooks, A. and Ferrar, P. J. (1984) Photosynthetic responses to light in seedlings of selected Amazonian and Australian rainforest tree species. Oecologia 63: Makino, A., Mae, T. and Ohira, K. (1985) Photosynthesis and ribulose 1,5-bisphosphate carboxylase/oxygenase in rice leaves from emergence through senescence. Quantitative analysis by carboxylation/oxygenation and regeneration of ribulose-1,5- bisphosphate. Planta 166: Medina, E. (1971) Effect of nitrogen supply and light intensity during growth on the photosynthetic capcity and carboxydimutase activity of leaves of Atriplex patula ssp. hastata. Carnegie Institution Washington Yrbk. 70: Seemann, J. R., Sharkey, T. D., Wang, J. L. and Osmond, C. B. (1987) Environmental effects on photosynthesis, nitrogen-use efficiency, and metabolite pools in leaves of sun and shade plants. Plant Physiol. 84: Sharkey, T. D. (1985) Photosynthesis in intact leaves of C 3 plants: Physics, physiology and limitations. Bot. Rev. 51: Sharkey, T. D., Stitt, M., Heineke, D., Gerhardt, R., Raschke, K. and Heldt, H. W. (1986) Limitations of photosynthesis by carbon metabolism II. O 2 -insensitive CO 2 uptake results from limitation of triose phosphate utilization. Plant Physiol. 81: Stitt, M. (1986) Limitations of photosynthesis by carbon metabolism I. Evidence for excess electron transport capacity in leaves carrying out photosynthesis in saturating light and CO 2. Plant Physiol. 81: Terashima, I. and Evans, J. R. (1988) Effects of light and nitrogen nutrition on the organization of the photosynthetic apparatus in spinach. Plant Cell Physiol. 29: Walters, M. B. and Field, C. B. (1987) Photosynthetic light acclimation in two rainforest Piper species with different ecological amplitudes. Oecologia 72: Wong, S. C. (1979) Elevated atmospheric partial pressure of CO 2 and plant growth. I. Interactions of nitrogen nutrition and photosynthetic capacity in C 3 and C 4 plants. Oecologia 44: (Received July 29, 1987; Accepted November 2, 1987)