The Nitrogen Use Efficiency of C3 and C4 Plants - NCBI

Plant Physiol. (1987) 85, 355-359 0032-0889/87/85/0355/05/$0 1.00/0

The Nitrogen Use Efficiency of C3 and C4 Plants' III. LEAF NITROGEN EFFECTS ON THE ACTIVITY OF CARBOXYLATING ENZYMES IN CHENOPODIUM ALBUM (L.) AND AMARANTHUS RETROFLEXUS (L.) Received for publication February 23, 1987 and in revised form June 4, 1987

ROWAN F. SAGE*2, ROBERT W. PEARCY, AND JEFFREY R. SEEMANN Department of Botany, University of California, Davis, California 95616 (R.F.S., R.W.P.), Biological Sciences Center, Desert Research Institute, P.O. Box 60220, Reno, Nevada 89506 (J.R.S.), and Department of Biochemistry, University of Nevada, Reno, Nevada 89557 (J.R.S.) ABSTRACT The relationships between leaf nitrogen content per unit area (N.) and (a) the initial slope of the photosynthetic CO2 response curve, (b) activity and amount of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and phosphoenolpyruvate carboxylase (PEPC), and (c) chlorophyll content were studied in the ecologically similar weeds Chenopodium album (C3) and Amaranthus retroflexus (C4). In both species, all parameters were linearly dependent upon leaf N.. The dependence of the initial slope of the CO2 response of photosynthesis on N. was four times greater in A. retroflexus than in C. album. At equivalent leaf N. contents, C. album had 1.5 to 2.6 times more CO2 saturated Rubisco activity than A. retroflexus. At equal assimilation capacities, C. album had four times the Rubisco activity as A. retroflexus. In A. retroflexus, a one to one ratio between Rubisco activity and photosynthesis was observed, whereas in C. album, the CO2 saturated Rubisco activity was three to four times the corresponding photosynthetic rate. The ratio of PEPC to Rubisco activity in A. retroftexus ranged from four at low N. to seven at high N.. The fraction of organic N invested in carboxylation enzymes increased with increased N. in both species. The fraction of N invested in Rubisco ranged from 10 to 27% in C. album. In A. retroflexus, the fraction of Na invested in Rubisco ranged from 5 to 9% and the fraction invested in PEPC ranged from 2 to 5%.

activity. Moreover, Rubisco specific activities may be two times higher for C4 as compared to C3 plants (21). Consequently, far less Rubisco should be required to achieve high rates of photosynthesis in C4 than C3 plants. Since Rubisco is a large, N expensive enzyme accounting for typically 20 to 30% of total leaf N in C3 plants (7, 13, 19), the N savings in C4 plants may be substantial. However, this savings could be partially offset by the N cost of the C4 concentrating mechanism. A few studies have focused on the N investment in carboxylating enzymes (6, 13, 18, 19, 22), but none have compared the responses of ecologically similar C3 and C4 plants to a range of N availability. Consequently, it is unclear how much more Rubisco is present in C3 than C4 leaves of similar N content, or alternatively, leaves with identical photosynthetic capacities. In this study, we compared leaf Rubisco activity and content in the ecologically similar weeds, Chenopodium album (C3) and Amaranthus retroflexus (C4) grown on different concentrations of N. In addition, we have determined the dependence of PEPC activity, the initial carboxylation enzyme in C4 plants, on Na in A. retroflexus. We have also determined the relationship of Chl content to leaf N for both species. Since Rubisco activity and content are correlated with the initial slope of the CO2 response of photosynthesis in C3 plants (20, 26), we investigated the relationship between leaf N and the initial slope of the CO2 response curve in both species.

MATERIALS AND METHODS Growth Conditions. Chenopodium album and Amaranthus Plants possessing C4 photosynthesis have a greater photosynthetic nitrogen use efficiency (PNUE3) than C3 plants (2-4, 17, retroflexus plants were grown as previously described (17) in chambers at 27/23°C (day/night), a photon flux density 18). This is because the C4 concentrating mechanism leads to growth 600 ,mol m 2 s-' and a 16 h photoperiod. This light intensity CO2 saturation of Rubisco (14). While in C3 plants Rubisco in isofwell below the maximum that occurs in the field, but has been vivo operates at about 25% of its Vm. (26), it is believed to operate at or close to Vm. in C4 plants (14). In addition, the high found to produce plants with similar N and photosynthetic as field grown plants (17). Plants were grown in CO2 partial pressure in the bundle sheath cells nearly eliminates characteristics Rubisco oxygenase activity in C4 plants. In contrast, photores- equal volume mixtures of sand, vermiculite, and perlite, and watered daily with a 0.75 strength Johnson-Hoaglands piration is substantial in C3 plants, leading to reduced Rubisco were solution modified to contain either 12, 8, 4, 1.5, or 0.15 mM N in a 7:1 NO3- to NH4+ ratio. 'Supported in part by United States Department of Agriculture comGas Exchange Measurements. Intact leaf CO2 assimilation petitive research grant 84-CRCR-1-1474 and National Science Founda- rates were determined with an open-type gas exchange system tion grant DMB-86-08004 to J. R. S. previously described (17). The initial slope of the relationship 2 Present address: Department of Botany, University of Georgia, Ath- between photosynthetic CO2 assimilation and Ci was determined ens, GA 30602. at 270 by first allowing the leaf to equilibrate with the chamber 3 Abbreviations: PNUE, photosynthetic nitrogen use efficiency; A, net environment at a Ca of 340 ubars, light saturation, and the CO2 assimilation rate; Ca, ambient CO2 partial pressure; Ci, intercellular measurement temperature. Then, the Ca was decreased to 80 CO2 partial pressure; CABP, carboxyarabinitol bisphosphate; k,,., catalytic ,gbars. Measurements of A were made as the Ca was increased in turnover rate; N, nitrogen; Na, organic leaf nitrogen per unit area; PEPC, four to six steps back to 340 tbars. All gas exchange parameters phosphoenolpyruvate carboxylase (EC 4.1.1.31); Rubisco, ribulose-1,5- were calculated according to von Caemmerer and Farquhar (26). bisphosphate carboxylase/oxygenase (EC 4.1.1.39); V,,, substrate satuCarboxylase Assays. Leaf discs of 1.38 cm2 or 2.76 cm2 were rated enzyme activity. punched from fully expanded leaves on the main stem of 2 to 4 355

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week old C. album and A. retroflexus plants following a 30 min or more exposure to high light. Immediately upon sampling, leaf discs were frozen in liquid N2 and ground with a Ten-Brock homogenizer in 2.5 to 5 ml of an extraction buffer of 100 mM HEPES (pH 7.1), 5 mm DTT, 1 mg/ml BSA, and 1% polyvinylpolypyrolidone. The homogenate was then spun for 45 s in a 1.5 ml microfuge tube. Rubisco activity was determined using a modification of the procedure of SEEMANN et al. (21) for C4 plants. Immediately following centrifugation, 1 ml of the supernatant was added to 4 ml of activation buffer (100 mM Bicine at pH 8.4, 10 mm NaHCO3, and 20 ,uM 6-P-gluconate) and allowed to activate at room temperature for 10 to 15 min. Ribulose 1,5bisP was generated in situ from ribose 5-P using yeast ribose 5-P isomerase (Sigma) and spinach ribulose 5-P kinase purified by us. Rubisco assays were performed at 25°C by injecting 100 dl of the activated sample mixture into 400 ,l of reaction buffer which initially contained 100 mM Bicine at pH 8.2, 30 mM MgCl2, 1 mM EDTA, 1,5 mM ribose 5-P, 2 mm ATP, 2 units ribose 5-P isomerase, 2 units ribulose 5-P kinase, 5 mM DTT, and either 15 mm NaH'4CO3 (for C. album) or 28 mm NaH'4CO3 (for A. retroflexus). The ribose 5-P was assumed to be fully converted to ribulose 1,5-bisP. The specific activity of all added NaH'4CO3 in this study was about 29.6 Bq nmol-'. After 60 s, the reaction was stopped by injecting 100 ,u of 4 N HCI. Acid stable radioactivity was determined by liquid scintillation counting. Rubisco amounts were measured using a technique described by Evans and Seemann (7) employing the radiolabeled transition state analog CABP. An aliquot of 100 1I of the crude extract was placed in 0.5 ml tubes containing 100 Al rabbit anti-Rubisco sera, 5 nmol CABP, and 100 ,l of Bicine (100 mm, pH 8.2) containing 30 mm MgCl2 and 1 mm EDTA. This mixture was allowed to incubate 18 h at 37°C, and was then filtered through a 0.45 ,m Gelman (Ann Arbor, MI) metricel membrane filter. The precipitated Rubisco-'4C-CABP complex was collected on the filter, which was washed free of unbound CABP by extensive rinsing with 0.85% NaCl, 10 mM MgCl2, and 10 mM Na2HPO4 at pH 7.6. Washed filters were transferred to scintillation vials and the amount of bound '4C-CABP determined by scintillation counting. PEPC activity was measured at 25°C by adding 20 1l of the above centrifuged, but unactivated supernatant to 480 Al of reaction buffer containing 50 mM Bicine (pH 8.2), 10 mM DTT, 25 mM PEP, 0.2 mM NADH, 2 units of malic dehydrogenase (Sigma), and 5.28 mm NaH'4C03. After 60 s, the reaction was terminated with 100 ,l of 6 N HCI. The acid stable radioactivity was then determined by scintillation counting. All of the above operations were conducted at 0 to 4°C except the centrifugation or as otherwise noted. Chlorophyll and Leaf Nitrogen Determinations. Leaf Chl content was measured by determining the absorbance of 80% acetone leaf extracts at 645 and 663 nm. Total leaf N and N03 were determined using a Kjeldahl procedure previously described (16). Leaf NO3 was subtracted from total N to yield organic leaf N. RESULTS Photosynthesis and Leaf Nitrogen. The initial slopes of the A versus Ci curves were linearly dependent on Na in both species (Fig. 1). At 27°C, the dependence of the initial slope on Na was over three times greater in A. retroflexus than C. album. At the same Na, the initial slope measured at 27°C was consistently three times greater in A. retroflexus than C. album. However, at equal assimilation capacities, the initial slope in A. retroflexus was only twice as great as in C. album. The dependence of A on Na in C. album and A. retroflexus grown under the same conditions as used here has been previously reported by Sage and Pearcy (17). CO2 assimilation was

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of photosynthesis (I.S.) and organic leaf nitrogen in A. retroflexus (A) and C. album (0) at 27C. The regression equations are: y 4.4 x 10-' -0.28 (R2 = 0.88) for A. retroflexus and y = 1.3 x 10- -0.08 (R2 = 0.87) for C. album. =

found to be linearly dependent on Na at 27°C in both A. retroflexus and C. album. A. retroflexus had a greater slope of A versus Na and a higher A per unit Na than C. album, indicating it had a greater PNUE. C. album, however, allocated more N per unit of leaf area than A. retroflexus at a given applied N level. Therefore, at equal applied N, C. album had a similar A as A. retroflexus. Because A and Rubisco activity were typically not measured on the same leaves, Rubisco activity and A were compared using predicted values from the respective regression equations for A versus Na and Rubisco activity versus Na. Because of this, the regression coefficients for A versus Na at 27°C from Sage and Pearcy (17) have been included in Table I. Rubisco Activity and Content. Rubisco activity increased with Na (Fig. 2) with a slope that was three times greater in C. album than A. retroflexus (Table I). At equal Na, C. album had from 1.5 to 2.6 times the Rubisco activity of A. retroflexus. At the respective Na values where assimilation rates were equal for the two species (as determined from the regressions of the respective A versus Na responses in Table I), C. album had a four times greater Rubisco activity than A. retroflexus. In C. album, Rubisco activity was estimated to be three to four times the photosynthetic rate at the same Na. By contrast, in A. retroflexus, a 1:1 relationship existed between A and Rubisco activity. Changes in the initial slope of the A versus C1 curve were correlated with changes in Rubisco activity in C. album. For example, from 120 to 240 mmol m-2 Na, both Rubisco activity and the initial slope increased 3.0 times. This is consistent with the results of von Caemmerer and Farquhar (26) and Seemann and Berry (20), showing that the initial slope of the A versus C1 curve in C3 plants is a function of Rubisco activity. In A. retroflexus, the initial slope increased 500% from 80 to 150 mmol m-2 Na. Rubisco activity increased by 260%, and PEPC activity increased by 340% over the same range of Na in A. retroflexus. This indicates that the initial slope of the A versus Ci curve in A. retroflexus is not solely dependent on either Rubisco or PEPC activity. The mean catalytic turnover rate at 25°C (kj) of Rubisco equaled 22.1 ± 2.6 mol CO2 molF' Rubisco s-' in C. album and 20.5 ± 0.8 mol CO2 mol -' Rubisco s-' in A. retroflexus. These differences were not significant (P < 0.1). Assuming 8 mol catalytic sites per mol Rubisco and a mol wt of 550,000, the calculated specific activity of Rubisco was 2.41 ± 0.28 umol min-' mg-' Rubisco protein in C. album and 2.24 ± 0.08 gmol min-' mg-' in A. retroflexus.

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NITROGEN EFFECTS ON CARBOXYLATION ENZYMES IN C3 AND C4 PLANTS Table I. Regression Coefficients for Response of CO2 Assimilation versus Na (from Sage and Pearcy [ 171), Rubisco Activity Versus Na, Rubisco Content versus Na, PEPC Activity versus Na, and Leaf Chl versus Na in C. album and A. retroflexus All trends are significant at P = 0.01. CO2 assimilation and enzymes activities are in Mmol m-2 s-'; enzyme contents are in g m-2; chlorophyll is in Mmol m2; and leaf nitrogen is in mmol m2. Slope -Intercept R2 N CO2 assimilation versus Na C. album 0.28 -15.9 0.81 28 A. retroflexus 0.42 -20.5 0.77 31 Rubisco activity versus Na C. album 1.19 -70.5 0.76 31 A. retroflexus 0.37 -13.8 0.77 30 Rubisco content versus Na C. album 0.032 -1.97 0.83 41 A. retroflexus 0.011 -0.43 0.83 36 PEPC versus Na (A. retroflexus) Activity 2.63 -134 0.89 31 Content 0.006 -0.32 0.89 31 Chlorophyll versus Na C. album 3.72 23.3 0.54 40 A. retroflexus 5.92 -168 0.82 47

bisco in A. retroflexus, the 10-fold greater specific activity of PEPC requires that only about half as much PEPC protein as Rubisco protein be present in the leaf. PEPC activity was seven times greater than Rubisco activity at high N in A. retroflexus, but at low N, PEPC activity was only four times greater than Rubisco activity. This indicates a proportionally greater investment of N in Rubisco than PEPC at low as compared to high N availabilities. Sugiyama et al. (23) have reported similar results with maize. Nitrogen Investment in Rubisco and PEP Carboxylase. In order to calculate the N invested in Rubisco and PEPC, both enzymes were assumed to contain 16% N, a value generally accepted for most plant proteins (6). From these values, the fraction of N in Rubisco and PEPC was estimated (Fig. 4). The relationship between the percent of N in Rubisco and Na is simply the transformation of the linear equation R = dR/dNa (Na) + b (1) into the form RINa = dR/dNa + b/Na, (2) where R is the Rubisco amount and b is the y-intercept. Since 6

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FIG. 2. Relationship between Rubisco activity and organic leaf nitroin C. album (0) and A. retroflexus (A); and the relationship between PEPC activity and organic leaf nitrogen in A. retroflexus (0). All assays were conducted at 25°C. See Table I for the regression coefficients. gen

0 120 180 Leaf nitrogen, mmol m-2 FIG. 3. Amount of Rubisco as a function of organic leaf nitrogen in C. album (0) and A. retroflexus (A), and the amount of PEPC as a function of organic leaf nitrogen in A. retroflexus (0). See Table I for the regression coefficients.

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Figure 3 shows the relationships between the amount of Rubisco and Na. Of these data, 10 of 41 determinations for C. album and 6 of 36 for A. retroflexus were direct measurements. The remainder were calculated from the measured activities and the mean specific activities which were determined from the CABP binding assay. There were no apparent differences in the relationship of Rubisco and Na between the direct measurements

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0 and the estimates based on Rubisco activities. Because the spe10 At Rubisco cific activities of Rubisco were similar in the two species, the differences in Rubisco content between the species reflect the 5 _ PEPC differences in Rubisco activity. C. album had two to three times as much Rubisco as A. retroflexus at equivalent N contents, and 0, three to four times as much Rubisco when the species had similar 0 60 120 180 240 assimilation rates. Leaf nitrogen, mmol m 2 PEP Carboxylase. In A. retroflexus, PEPC activities were always greater than those of Rubisco (Fig. 2). As with Rubisco, FIG. 4. Percent of leaf nitrogen in Rubisco as a function of organic PEPC activity was linearly related to Na, but exhibited a slope leaf nitrogen in C. album (0) and A. retroflexus (A), and the percent of seven times greater (Table I). Assuming that PEPC in A. retro- leaf nitrogen in PEPC as a function of organic leaf nitrogen in A. flexus has a specific activity equal to the values found for maize retroflexus (0). The fitted curves represent the responses calculated by (24.8 ,mol min-' mg-' protein) (24), then the amount of PEPC transforming the respective linear regressions of carboxylase content increased linearly from 0.06 g m-2 to 0.7 g m-2 as Na increased versus Na into the form of equation 2, and assuming that carboxylase (Fig. 3). While PEPC had substantially more activity than Ru- protein is 16% N. 0

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the x-intercept of Rubisco amount versus Na is large in both species and the y-intercept negative, the predicted relationship between percent Rubisco-N and Na is curvilinear and approaches the value of dR/dNa at high Na. However, the scatter in the data obscures the curvilinear trend predicted by transforming the linear regression of Rubisco amount versus Na. Across a range of Na, the predicted fraction of N in Rubisco more than doubled in C. album from 10% at low Na to over 27% at high Na (Fig. 4). In A. retroflexus, the predicted fraction of N in Rubisco increased with Na from 5% at low Na to 9% at high Na. At an equal Na, C. album had 1.3 to 2.4 times as much N invested in Rubisco as A. retroflexus (Table II). Using the same analysis, PEPC was estimated to contain 3 to 5% of the leaf Na in A. retroflexus. The combined level of N in PEPC and Rubisco in A. retroflexus accounted for 15% of leaf N at high N, but only 8% at low N. The investment of N in PEPC in C. album was negligible, accounting for only 0.1% in high N leaves. Consequently, at an equal, high Na value, the investment of N in carboxylating enzymes was 1.5 times greater in C. album than A. retroflexus (Table II). Rubisco content declined more rapidly with decreasing Na in C. album than A. retroflexus so that at low Na, A. retroflexus had more N invested in PEPC and Rubisco than did C. album. However, comparisons made at equal assimilation capacities showed that C. album always had a substantially higher Na than A. retroflexus and therefore much greater Rubisco contents than the ratios at identical Na would indicate. For example, at a photosynthetic rate of 45 smol m-2 s-', which is equivalent to the maximum A (at a Ca of 340 gbars) found for C. album and A. retroflexus plants at 27°C, we estimate that C. album invests almost 4 times as much N in Rubisco alone, and 2.4 times as much N in both Rubisco and PEPC as A. retroflexus (Table II). Chlorophyll Contents. Leaf Chl content was positively correlated with Na in both C. album and A. retroflexus. Chl increased more rapidly with increasing Na in A. retroflexus, so that at Na values above 120 mmolm-2, it tended to have more Chl per unit leaf area than C. album (Fig. 5). Chl accounted for only a small fraction (1-2%) of leaf N in both species. For example, at an A of 45 smol m-2 s-' Chl was estimated to contain 1.5% of the leaf N in C. album and 1.9% of the leaf N in A. retroflexus. Evans (6) has estimated that the Chl protein complexes, the electron transport chain, and the coupling factors together contain approximately 55 mol N mol-' Chl. Assuming this is correct, then 21 and 27% of the leaf N would be associated with the above light harvesting components in C. album and A. retroflexus, respectively. By combining these estimates with the percent N in Rubisco and PEPC, it is possible to account for 45% of the leaf N in C. album and 41% of the leaf N in A. retroflexus. Carboxylation Enzymes in Field Grown Plants. Carboxylase enzyme activities and contents were measured on natural populations of C. album and A. retroflexus growing in fields near Table II. Predicted Ratios of Nitrogen Content ofRubisco, and Total Carboxylase between C. album (C3) and A. retroflexus (C4)

The leaf nitrogen contents 80, 120, and 160mmoI-2 were selected to

represent the range of nitrogen overlap between the species. The leaf nitrogen contents 215 and 158 mmol m-2 are the predicted leaf N levels in C. album and A. retroflexus, respectively, at an assimilation rate of 45 ismolmn2 s'. Ratio

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Davis, CA. Consistent with the results from growth chamber grown plants, field grown C. album had a substantially greater Rubisco activity and content than A. retroflexus. At 25°C, Rubisco activity was 181 ,umol m-2 s-' in C. album and 54 ,umol m-2 s-' in A. retroflexus. This corresponded to 4.5 and 1.5 gm-2 Rubisco in C. album and A. retroflexus, respectively. PEPC activity in field grown A. retroflexus was 353,umol m-2 S-", which corresponded to 0.9 g mi2 PEPC. Rubisco accounted for 29% of the leaf N in C. album and 9% of the leaf N in A. retroflexus. PEPC accounted for 5% of the leaf N in A. retroflexus.

DISCUSSION The functional role of the C4 pathway is presumably to increase the CO2 concentration in the bundle sheath cells to levels at which Rubisco is rate saturated (14). Consequently, in C4 plants Rubisco can operate at its Vmax and the in vitro C02 saturated activity should equal the in vivo activity, as indicated by the assimilation rate. This was found in A. retroflexus and is consistent with results reported from other C4 plants (15, 25). In C3 plants, the fully activated, CO2 saturated Rubisco activity should be four to five times greater than the CO2 assimilation rate (26). In C. album it was three to four times greater, indicating that the extracted Rubisco activities were not quite sufficient to account for the observed photosynthesis rates. C. album required four times more Rubisco as A. retroflexus for similar photosynthetic capacities, which is in agreement with studies on other species (11, 18). C. album contained 2 to 2.5 times more Rubisco per unit of N than A. retroflexus, which is consistent with comparisons between other C3 and C4 species (1 1, 14, 18). Rubisco generally accounts for 30 to 60% of the soluble protein in C3 plants (8, 12, 27, 28), but only 5 to 20% in C4 plants (1, 1 1, 18, 23). PEPC has been found to account for 8% of the soluble protein in maize (23). Typically, soluble protein accounts for about half of the organic leaf N. Direct measurements show that 15 to 30% of the leaf N in C3 plants is in Rubisco (7, 13, 19, 20). Ratios of Rubisco to N have rarely been directly determined in C4 plants. In maize, Rubisco accounted for 6.5% of the leaf N (18), which is comparable to the levels observed for A. retroflexus. At an assimilation rate of 45,umol m-2s-", the regression equations of A versus Na predict that C. album has an Na of 215 mmol m-2 and A. retroflexus has an Na of 158mmol m-2. At these N contents, C. album is estimated to invest 25%, or 55 mmol m-2 of its leaf N in Rubisco, while A. retroflexus invests 9%, or 14 mmolmi2 of its N in Rubisco. If Rubisco in C. album operated at Vmax in vivo, as it apparently does in A. retroflexus, then this C3 plant could save about 40 mmol Nmi2. This would increase the PNUE of C. album (using A/Na as an index of PNUE) by 23%, and would decrease the difference in PNUE

NITROGEN EFFECTS ON CARBOXYLATION ENZYMES IN C3 AND C4 PLANTS between C. album and A. retroflexus by 65%. The cost of photorespiration and the N-requirement of the photorespiratory cycle enzymes in C. album, and the N-cost of the C4 cycle enzymes in A. retroflexus may account for much of the remaining difference in PNUE. The N-cost of the C4 cycle enzymes may be substantial, since in A. retroflexus, PEPC may account for half as much of the leaf N as Rubisco. At the lowest applied N levels, C. album has a greater PNUE and whole plant NUE than A. retroflexus, which is the opposite of the case at high N (16, 17). It is at these low N levels that the relative differences between the carboxylase contents of these two species are minimal (Table II). The ecological significance of this may be limited, because in the field, sun leaves of both C. album and A. retroflexus were consistently found to have N and carboxylase contents similar to those of our high N grown plants. It is at these high N levels where the differences in PNUE and carboxylase contents between the species were found to be the greatest. The greater specific activity reported for Rubisco from C4 plants (21) should contribute to their greater PNUE. Differences in the specific activities of Rubisco in C3 dicots have been correlated with differences in PNUE (20). However, for C. album and A. retroflexus, no differences in specific activities were observed. The values for both species were comparable to those of many C3 dicots but less than those found for other C4 plants (21). We have no explanation for this discrepancy. In wheat, Evans (6) found that the proportion of N invested in Rubisco did not vary as Na varied. In contrast, the investment of N in Rubisco more than doubled across the observed range of Na for C. album. Similar results have been observed in Phaseolus vulgaris and Alocasia macrorrhiza (22). Wheat has a higher A/Na than C. album and the x-intercept of Rubisco activity versus Na is near zero (6). Consequently, the response of Rubisco/Na to Na exhibits little curvilinearity. The value of A/ Na and PNUE may depend somewhat on the x-intercept of A versus Na such that species with a large x-intercept may have a lower A/Na and PNUE than those with a small intercept. What determines the value of the x-intercept of A versus Na is unclear, but it may be influenced by the minimum N level at which Rubisco is formed, as is indicated by the similar x-intercepts of A versus Na (56 mmol N m-2) and Rubisco activity versus Na (60 mmol m-2) in C. album. In addition to its catalytic function, Rubisco has been suggested to serve as a storage protein (9, 10). This has been interpreted to mean that Rubisco is produced in excess of its catalytic capacity, with the excess serving to store excess N (5). In this study we saw no evidence of excess Rubisco capacity in

either C. album or A. retroflexus. The levels of Rubisco present in C. album appear to be less than that required to account for the measured rates of photosynthesis. This has also been found in other species (5, 7, 13, 26) and may be a result of less than 100% extraction ofthe enzyme. In A. retroflexus the close parallel between Rubisco activity and Na, and A and Na argues against a distinct storage role for Rubisco in this species as well. Rubisco does serve as a source of N during seed fill, as do most of the remaining N pools in the leaf. In both C. album and A. retroflexus for example, we found that fully senesced leaves typically contained 15 to 30 mmol m-2 N, demonstrating that up to 90% of the leaf N may be remobilized during senescence. Acknowledgments-We wish to thank Drs. T. D. Sharkey, R. C. Huffaker, and T. M. DeJong for their helpful comments on this work. We also thank the JastroShields foundation for financial support of this study. LITERATURE CITED 1. BJ6RKMAN 0 1976 Adaptive and genetic aspects of C4 photosynthesis. In RH Bunris, CC Black, eds, CO2 Metabolism and Plant Productivity. University

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Park Press, Baltimore, pp 287-309 2. BOLTON JK, RH BROWN 1980 Photosynthesis of grass species differing in carbon dioxide fixation pathways. V. Response of Panicum maximum, Panicum milioides, and tall fescue (Festuca arundinacea) to nitrogen nutrition. Plant Physiol 66: 97-100 3. BROWN RH 1978 A difference in N use efficiency in C3 and C4 plants and its implications in adaptation and evolution. Crop Sci 18: 93-98 4. BROWN RH, JR WILSON 1983 Nitrogen response of Panicum species differing in CO2 fixation pathways. II. CO2 exchange characteristics. Crop Sci 23: 1154-1159 5. EVANS JR 1983 Nitrogen and photosynthesis in the flag leaf of wheat (Triticum aestivum L.). Plant Physiol 72: 297-302 6. EVANS JR 1983 Photosynthesis and nitrogen partitioning in leaves of Triticum aeslivum and related species. PhD thesis, Australian National University, Canberra 7. 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