PNAS-2002-Bloom-1730-5 | Photosynthesis | Carbon Dioxide

Nitrogen assimilation and growth of wheat under elevated carbon dioxide

Arnold J. Bloom*, David R. Smart†, Duy T. Nguyen‡, and Peter S. Searles
Department of Vegetable Crops, One Shields Avenue, University of California, Davis, CA 95616 Communicated by Emanuel Epstein, University of California, Davis, CA, November 26, 2001 (received for review September 14, 2001)

Simultaneous measurements of CO2 and O2 fluxes from wheat (Triticum aestivum) shoots indicated that short-term exposures to elevated CO2 concentrations diverted photosynthetic reductant ؊ ؊ or NO2 reduction to CO2 fixation. With longer exposures from NO3 to elevated CO2, wheat leaves showed a diminished capacity for ؊ photoassimilation at any CO2 concentration. Moreover, high NO3 ؊ translocation into chloroplasts bicarbonate levels impeded NO2 isolated from wheat or pea leaves. These results support the ؊ photoassimilation. hypothesis that elevated CO2 inhibits NO3 ؊ ؉ rather than NH4 as Accordingly, when wheat plants received NO3 a nitrogen source, CO2 enhancement of shoot growth halved and CO2 inhibition of shoot protein doubled. This result will likely have ؊ as a nitrogen major implications for the ability of wheat to use NO3 source under elevated CO2.

response (i.e., shoot photosynthesis as a function of internal CO2 concentration) experiments. The growth͞N relations experiments were also conducted at 360 or 700 ␮mol CO2 molϪ1. The CO2 added was filtered through a KMnO4 column to remove contaminating hydrocarbons such as ethylene. A combination of high-pressure sodium, metal halide, and incandescent lamps provided a photosynthetic PFD of 700 ␮mol mϪ2 sϪ1 at plant height. The light͞dark period was 16 h͞8 h at 25°C and 15°C, respectively. days old into a measurement system in which a split rubber stopper was fitted around the stem, sealing the root into an acrylic plastic and stainless steel cuvette (16) and the shoot into a gold-plated cuvette with a glass top (17). The leaves in the shoot cuvette were at their normal orientation; thus, the angle of incidence was 70–80°. Shoot gas fluxes were monitored with a commercial nondispersive infrared CO2 analyzer (Horiba VIA500R), a custom O2 analyzer, and relative humidity sensors (Vaisala, Helsinki) (17). The custom O2 analyzer contains two cells of calcia-stabilized zirconium oxide ceramic similar to those found in an Applied Electrochemistry model N-37 M. When heated to 752.00 Ϯ 0.01°C, these cells become selectively permeable to O2 and generate a 106-nV Nernst potential per ␮mol molϪ1 difference in O2 partial pressures between the cells; in practice, this analyzer can resolve O2 concentration differences to within 2 ␮mol molϪ1 on the normal background of 209,700 ␮mol molϪ1 (17). Mass flow controllers (Tylan, Torrance, CA) prepared the various gas mixtures, and a pressure transducer (Validyne, North Ridge, CA) monitored gas flows through the cuvette. In the photosynthetic PFD experiments, a 1,000-W metal halide lamp with an adjustable ballast (Wide-Lite, San Marcos, TX) and neutral density filters controlled PFD at plant height between 0–1900 ␮mol mϪ2 sϪ1. Plants exhibited photoinhibition if the PFD was increased to levels where the response became light-saturated (3000 ␮mol mϪ2 sϪ1). In the A–Ci response experiments, the PFD was 1200 ␮mol mϪ2 sϪ1. In the PFD experiments, a plant was exposed to 360 ␮mol molϪ1 CO2 and received an aerated nutrient solution of 1 mM CaSO4, 0.5 ␮M K2HPO4, and either 0.2 mM KNO3 or 0.2 mM NH4Cl for 36 h before taking any measurements. In the A–Ci response experiments, a plant received an aerated nutrient solution of 0.2 mM NH4Cl, 1 mM CaSO4, and 0.5 ␮M K2HPO4 for 16 h before taking any measurements. A plant after such a pretreatment contained no detectable NOϪ 3 in its tissues (data not shown). This plant then received a nutrient solution containing 0.2 mM KNO3, 1 mM CaSO4, and 0.5 ␮M K2HPO4 for 16 h before measurements of the A–Ci response were repeated.
Abbreviations: PFD, photon flux density; AQ, assimilatory quotient. *To whom reprint requests should be addressed. E-mail: ajbloom@ucdavis.edu.
†Present

Gas-Exchange Measurements. We transferred a seedling about 14

tmospheric CO2 concentrations have increased from about 280 to 370 ␮mol molϪ1 since 1800 (1, 2) and may reach 500–900 ␮mol molϪ1 by the end of the century (3). Several responses of higher plants to such changes were not anticipated (4). For example, a doubling of CO2 level initially accelerates carbon fixation in C3 plants by about 30%, yet after days to weeks of exposure to high CO2 concentrations, depending on species, carbon fixation declines until it stabilizes at a rate that averages 12% above ambient controls (5). This general phenomenon, known as CO2 acclimation, is correlated with a decline in the activity of Rubisco and other enzymes in the Calvin cycle (6, 7). The change in Calvin cycle enzyme activities is not necessarily selective; rather, it often follows a decline in overall shoot protein and N contents (8). Shoot N contents diminish by an average of 14% with a doubling of CO2 (9), a difference that exceeds what would be expected if a given amount of N were diluted by additional biomass (8). In wheat, CO2 acclimation varies with N supply (10, 11). Wheat shoots accumulate free NOϪ 3 under elevated CO2 (12), and shoot protein declines (13) despite little change in total N (12, 14). Here, we present several lines of evidence that elevated CO2 concentrations inhibit NOϪ 3 assimilation in wheat shoots and suggest two physiological mechanisms for this phenomenon. Materials and Methods We surface-sterilized wheat (Triticum aestivum cv. Veery 10) seeds for 1 min in 2.6% NaClO, washed them thoroughly with water, and germinated them for several days on thick paper toweling (germination paper) saturated with 1 mM CaSO4. Twenty seedlings were transplanted to 19-liter opaque plastic containers filled with an aerated nutrient solution containing 0.2 mM NH4NO3, 1 mM CaSO4, 0.5 mM K2HPO4, 0.5 mM KH2PO4, 1 mM MgSO4, 0.2 g literϪ1 Fe-NaEDTA, and micronutrients (15). The nutrient solution was replenished every 3 days. The containers were placed in a controlled environment chamber [Conviron (Winnipeg, MB, Canada) PGV-36] equipped with a nondispersive infrared CO2 analyzer (Horiba, Kyoto, no. APBA-250E) and a Conviron process controller that added CO2 to maintain the chamber concentration at 360 ␮mol molϪ1 for the photon flux density (PFD) response experiments (i.e., shoot photosynthesis as a function of photosynthetic PFD at plant height) and either 360 or 700 ␮mol molϪ1 for the A–Ci
1730 –1735 ͉ PNAS ͉ February 5, 2002 ͉ vol. 99 ͉ no. 3

A

address: Department of Viticulture and Enology, University of California, One Shields Avenue, Davis, CA 95616-8749. address: Genentech, Inc., 1000 New Horizons Way, Vacaville, CA 95688.

‡Present

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.022627299

They ϩ Ϫ received either NH4 (circles) or NO3 (triangles) as a sole N source during measurements. Wheat and pea (Pisum sativum cv.0).1-ml aliquot into 0. Net CO2 consumption (A) and O2 evolution (B) by the shoot of a wheat seedling as a function of photosynthetic PFD at plant height. To test for the intactness of the chloroplasts.5 g sulfanilamide in 150 ml of 15% (vol͞vol) CH3COOH]. 1 mM KH2PO4. A solution system consisted of a 100-liter main reservoir. 2 mM CaCl2.0. Eight to 10 plants were dried in a forced-air oven at 70°C. and 0. hydrogen. the intact chloroplasts formed a band near the bottom of the Percoll gradient. For wheat shoots grown at ambient CO2. After 2 weeks.5 ml of a sulfanilamide solution [0. The leaves in the gas-exchange cuvette were at their natural orientation.14 ϫ (A645 Ϫ A720) (19). Shown are mean Ϯ SE for 5– 8 replicate plants.5 ml of a NED solution [0. a distribution manifold with 6 4-liter hourϪ1 drip irrigation emitters.We followed standard protocols to assess the response of photosynthesis to PFD or intercellular CO2 (18). To minimize nitrification and denitrification. transferred them to two controlled environmental chambers. containing about 1 g literϪ1 chlorophyll in 50 mM K-Tricine (pH 8. Ϫ the plants received 0. The response of net CO2 consumption vs.0 mM KHCO3 at about 22°C and at a PFD of 650 ␮mol quanta mϪ2 sϪ1. where Chl (mg literϪ1) ϭ 4.05 M K-Hepes. whereas intact chloroplasts passed through the silicone oil phase and formed a pellet at the bottom of the tube. indicating that N availability was not limiting growth. SAS Institute.33 M sorbitol͞1 mM MgCl2͞1 mM MnCl2͞2 mM Na2EDTA͞0. 330 mM sorbitol. The wheat received the nutrient solution described above. 0. Results and Discussion Gas-Exchange Measurements. the relative depletion of HCOϪ 3 by carbon fixation during the incubation period was prohibitive. and a manifold that returned the overflow from the 6 containers to the Bloom et al. Net O2 evolution.900 ϫ g for 5 min. 2 mM MgSO4. net CO2 consumption at any given PFD was higher at elevated CO2 than ambient CO2 (Fig.000 ϫ g). ؉ ؊ Growth and Nitrogen Parameters Under NH4 or NO3 .0 ͞0. NC) to perform Tukey’s Studentized Range and Bonferroni t tests on the ambient vs. 0. Every 2 days. After centrifugation of the overlayered gradients at 8. seeds as described above and. To analyze NOϪ 2 in the supernatant. weighed.000 ϫ g for 3 min. Ci (shoot internal CO2 concentration) was similar among all treatments (Fig. The calculation of fluxes included an adjustment for changes in leaf area during the measurements.3 ͞0. In another study. 1 paper. The wheat (30 –50 g) or pea (100 –130 g) shoots were blended for 10 seconds in 0. 99 ͉ no. whereas the pea was in vermiculite that was watered daily. We analyzed two plants for total protein by using the Coomassie dye binding protein assay (Bio-Rad Bradford Ϫ Protein Assay) and for in vitro activities of NOϪ 3 and NO2 reductases based on the appearance or disappearance. Here. 1. We used a general linear model (GLM procedure. added after 5 min 0. one maintained at 360 ␮mol molϪ1 CO2 and the other at 700 ␮mol molϪ1 CO2. MA).2 g literϪ1 Fe-NaEDTA. pH 8.1 M K-Tricine. To determine the amount of chlorophyll in the isolated chloroplasts. we measured and adjusted the concentration of NHϩ 4 (22) or NOϪ 3 (23) in the solution systems. The pellet was resuspended in 3 ml of a homogenization buffer (0. A concentration of 0.2 mM KNO3. 0. 6 opaque 4-liter polyethylene containers. 1B). The following procedures were conducted at 0 – 4°C. when they were 6 days old. The plants had been grown in controlled environment chambers at 360 ␮mol molϪ1 CO2 and measured at 360 (light symbols) or 700 (dark symbols) ␮mol molϪ1 CO2. After centrifugation at maximum speed for 15 seconds (about 50. Cary.1% BSA). pH 8. These were washed twice with 45 ml of the homogenization buffer and pelleted at 1.1 to 1.2 mM NHϩ 4 or NO3 . and micronutrients (15).000 ϫ g for 10 min. 2). Cheshire. We fitted a cubic spline curve to the data for NOϪ 2 concentration as a function of time and derived net NOϪ 2 uptake from the slope of the cubic spline (MATHCAD. we pipetted a 0. ANCASL). and 645 nm. and shoot N contents under all treatments were high at around 5%. 15 ␮l of chloroplast material were layered on top of 100 ␮l of silicone oil that floated above 100 ␮l of a buffer solution (0. and centrifuged them at 3. We took samples of the incubation mixture between 15 and 60 min.000 ϫ g for 30 min. broken chloroplasts floated on top of silicone oil.0 mM had little effect on wheat biomass production at 360 or 1000 ␮mol molϪ1 CO2 (12). we added ampicillin (20 mg literϪ1) and cefotaxime (10 mg literϪ1) (21). The nutrient solutions were composed of 0. elevated CO2 treatments under NHϩ 4 or NOϪ 3 nutrition and designated probabilities of less than 5% as significant. Progress 9) were grown for about 2 weeks at ambient CO2.05 M K-Tricine. Chloroplast Isolation.3. Each container held two plants.2 liters of a grinding buffer (0. The homogenate was squeezed through two layers of miracloth and centrifuged at 2. and nitrogen (CHN) elemental analyzer (PDZ Europa. Each chamber had a continuous-flow nutrient solution system that supplied NHϩ 4 as the sole N source and one that supplied NOϪ 3 as the sole source. but was insensitive to CO2 concentration under NOϪ 3 (Fig. the absorbance was read at 720. of NOϪ 2 using a colorimetric assay (24).33 M sorbitol) and layered onto a 30-ml Percoll gradient that was generated by centrifugation of 50% (vol͞vol) Percoll in an equal volume of the grinding buffer at 37. as is usually observed for C3 plants (25). MathSoft. and analyzed the extract for free NOϪ 3 by means of HPLC (23). We conducted four replicate experiments in which we switched the ambient and elevated Ϫ chambers and rotated the positions of the NHϩ 4 and NO3 treatments in a chamber. Cambridge. 1A). we evaluated plant growth and nitrogen parameters. extracted another subsample with 1 mM KCl adjusted to pH 2 with H2SO4. increasing the NOϪ 3 supply from 0. 663. 10 ␮l of the chloroplast suspension were diluted into 5 ml of 80% acetone and filtered through Whatman no. placed them immediately in the dark at 0 – 4°C.500 ϫ g for 5 min.66 M sorbitol) in a 4-ml Eppendorf tube. 2002 ͉ vol. England.02 ϫ (A663 Ϫ A720) ϩ 10. pH 7. or 3.3 mM HCOϪ 3 is probably higher than is present in vivo. 1. We incubated intact chloroplasts main reservoir. and ground in a ball mill. We measured total N in one subsample via a carbon.0 ͞0.3 mM KNO2. but at lower concentrations. and measured absorbance at 540 nm (20). allowed color to develop for 15 min.1 mM (NH4)2SO4 or 0. Fig. a centrifugal chemical pump. 1 mM K2HPO4. 3 ͉ 1731 PLANT BIOLOGY . We germinated Chloroplast Nitrite Absorption.2 g of N-(1-naphthyl)ethylenediamide⅐2HCl in 150 ml of 15% (vol͞vol) CH3COOH]. respectively. Net O2 evolution was also higher at elevated CO2 than ambient CO2 under NHϩ 4 . by PNAS ͉ February 5.

The plants had been grown in controlled environment chambers at 360 (light symbols) or 700 (dark symbols) ␮mol molϪ1 CO2. First. Shown are mean Ϯ SE for 6 replicate plants. or 61% in wheat and 32. 45. For example (27). 1B). highlights these differences (Fig. These results indicate that exposure to elevated CO2 concentrations. either in the short term (hours) or long term (weeks). 3. Carbon fixation may interfere with NOϪ 3 photoassimilation at Ϫ several junctures. measurements of CO2 exchange are not feasible. Our finding that ⌬AQ declined to zero in low light or elevated CO2 (Fig. the ⌬AQs measured at elevated CO2 concentrations did not differ significantly from zero over a range of light levels. Shown are means Ϯ SE for 5– 8 replicate wheat plants. The PFD at plant height was 1200 ␮mol quanta mϪ2 sϪ1.022627299 . and N source would have little effect on O2 evolution (Figs. chlorophyll fluorescence. Under these conditions. the plants were exposed to ϩ Ϫ NH4 (circles) and then to NO3 (triangles). The demands of carbon fixation for reductant might limit this malate shuttle. 3) is consistent with a diminished availability of NADH or reduced ferredoxin for NOϪ 3 assimilation. Wheat grown under ambient CO2 and measured at the lower Cis exhibited ⌬AQs greater than zero (Fig. 2).1073͞pnas. 1. indicating little NOϪ 3 photoassimilation (Fig. Net O2 evolution under NOϪ 3 remained high at both CO2 levels (Fig. plants that are photoassimilating NOϪ 3 exhibit a lower AQ and the difference in the AQ with a shift from NOϪ 3 Ϫ to NHϩ 4 nutrition (⌬AQ) is proportional to NO3 photoassimilation. requires the stroma to be more alkaline than the cytosol (36). the incorporation of NH4 into amino acids. Here. a long-term decline in the capacity of the shoot to assimilate NOϪ 3 even under ambient CO2 conditions. does not provide an accurate measure for electron transport rates of an entire wheat shoot (28). Elevated CO2 stimulates the Calvin cycle and. The AQ was verified as a nondestructive measure of in planta NOϪ 3 assimilation over 50 years ago for algae (26) and over a decade ago for higher plants by using barley mutants deficient in Ϫ NOϪ 3 reductase activity (17).0. Thus. estimated from changes in CO2 and water vapor concentrations. 48. oxygen evolution monitored with a polarographic O2 electrode at saturating CO2 did not differ between detached barley leaves given NHϩ 4 and Ϫ those given NOϪ 3 at levels similar to the xylem NO3 concentrations measured here (15. which is relatively insensitive to N source (Figs. mean Ϯ SE. Asterisks mark the means that were significantly different from zero (P Ͻ 0. 3B). In contrast. The change in AQ (AQ ϭ CO2 consumed͞O2 evolved) with a shift in Ϫ ϩ N source from NO3 to NH4 as a function of either photosynthetic flux density (A) or internal CO2 concentration (B). Chloroplast Nitrite Absorption. 30) and requires NADH generated from malate that is shuttled from the chloroplast (31).Fig. The AQ may respond to other shoot processes— principally. Our ability to monitor shoot O2 fluxes simultaneously with CO2 fluxes under normal physiological conditions provided a unique perspective. can diminish the amount of ϩ reduced ferredoxin available for NOϪ 2 reduction or NH4 assimilation (34. 3A). the ⌬AQs measured at ambient CO2 increased with PFD (Fig. The response of ⌬AQ as a function of internal CO2 concentration (Ci) supports this interpretation. Measurements of photosynthetic O2 exchange are generally conducted by using polarographic O2 electrodes at saturating CO2 concentrations. Another technique. 6) and.3. The addition of 0. 3A). Carbon dioxide at elevated concentrations can dissipate this pH gradient because additional CO2 movement into the chloroplast acidifies the stroma (37) and because enhanced carbon fixation hydrolyzes ATP faster and requires supplementary proton exchange across the thylakoid membrane to regenerate this ATP. Therefore. would not influence ⌬AQ.pnas. but cannot directly store significant amounts of CO2.05. thus. was lower under NHϩ 4 than NO3 for the wheat grown under ambient CO2 and measured at the two lowest Cis (Fig. 3). Bloom et al. NO2 transport from the cytosol Ϫ Fig. 2. 1732 ͉ www.org͞cgi͞doi͞10. based on the data presented in Figs. The assimilatory quotient (AQ). in contrast with O2 fluxes.4 Ϯ 0. the shoots seemed to conduct NOϪ 3 photoassimilation only to the extent that carbon fixation was CO2-limited and surplus photosynthetic reductant became available.7 mM. into the chloroplast involves the diffusion of HNO2 across chloroplast membranes and. or 60% in pea (Fig. Giving priority to carbon fixation seems an appropriate strategy in that plants can store moderate levels of NOϪ 3 with little difficulty until reductant becomes available. 1B and 2 B). therefore. therefore. or Ϫ 3. Ϫ contrast. 1 and 2. 1 A and 2 A). under light-limited conditions. 35). During measurements. suggesting that the rate of photosynthetic electron transport and the amount of photosynthetic reductant generated were independent of CO2 level. and the Calvin cycle all occur in the stroma of a chloroplast (32) and require ferredoxin that is reduced via photosynthetic electron transport (33). while CO2 consumption by the light-independent reactions continues at similar rates. diminished NOϪ 3 photoassimilation. The plants had been measured (A) or grown (B) in controlled environment chambers at 360 (light symbols) or 700 (dark symbols) ␮mol molϪ1 CO2. the ratio of net CO2 consumption to net O2 evolution. n ϭ 55). photorespiration and the Mehler-peroxidase reaction— but these processes probably would not differ with N form in the root medium and.0 mM HCOϪ 3 decreased chloroplast NO2 absorption by an average of 38. Net CO2 consumption (A) and O2 evolution (B) by the shoot of a wheat seedling as a function of internal CO2 concentration (Ci). indicating that NOϪ 3 photoassimilation increased with light intensity. This finding is consistent with a tight coupling between the light-dependent and light-independent reactions of photosynthesis. The same mechanism could account for both these responses: shortterm inhibition of NOϪ 3 assimilation caused a specific downϪ regulation of shoot NOϪ 3 and NO2 reductase activities (Fig. Previous studies of photosynthetic responses to PFD and Ci have monitored primarily CO2 exchange. Transfer of electrons to NO3 and NOϪ during photoassimilation increases O evolution from the 2 2 light-dependent reactions of photosynthesis. a Student’s t test). reduction of NOϪ 3 to NO2 occurs in the cytosol (29. the ϩ ϩ reduction of NOϪ 2 to NH4 . Second.

respectively.04 g literϪ1 (mean Ϯ SE. In vitro shoot activities of NO3 reductase Ϫ and NO2 reductase decreased 12% and 27% from ambient to elevated CO2. Shown are means Ϯ SE for four replicate experiments. If CO2 at Fig.01 g Ϫ literϪ1 and 0. the plants receiving NHϩ were more responsive 4 to CO2 enrichment than those receiving NOϪ 3. 4 shows the 0 and 0. 5). as might be expected given the dilution by additional biomass. Total plant biomass increased Ϫ 78% under NHϩ 4 nutrition but only 44% under NO3 nutrition (Fig. but increased 62% at elevated CO2 in those Ϫ receiving NOϪ 3 (Fig. Leaf area in the elevated CO2 treatment relative to the ambient CO2 treatment increased 49% under NHϩ 4 nutrition but only 24% under NOϪ 3 nutrition (Fig. Biomass (g of dry mass) and leaf area (cm2) per plant of wheat seedlings grown for 14 days in controlled environment chambers at 360 or 700 ϩ Ϫ ␮mol molϪ1 CO2 and under NH4 or NO3 nutrition. 6). Treatments labeled with different letters differ significantly (P Ͻ 0. ؉ ؊ Growth and Nitrogen Parameters under NH4 and NO3 . respectively. Net NO2 uptake (␮mol mgϪ1 chlorophyll minϪ1) by isolated chloroϪ plasts as a function of NO2 concentration when the medium contained 0 (light Ϫ symbols) or 0. 4. Thus. shoot protein per Ϫ plant increased 73% and 32% under NHϩ 4 and NO3 . each with 8 –10 plants per treatment. n ϭ 2) for the NO3 treatment at 360 and 700 ␮mol molϪ1. indicating that N absorption per unit plant mass remained unchanged (Fig. To test this prediction.05). 5. respectively. Elevated CO2 concentrations stimulated shoot growth of the plants receiving NOϪ 3 to only half the extent of those receiving NHϩ 4 . Although the plants received the various CO2 and N treatments only from day 6 through day 20. 6) and decreased 33% and 30%. These results confirm that high CO2 levels can interfere with NOϪ 2 transport into the chloroplast and thereby provide another mechanism through which elevated CO2 might inhibit shoot NOϪ 3 assimilation.05). 2002 ͉ vol.07 g literϪ1 (mean Ϯ SE. In the elevated CO2 treatment relative to the ambient CO2 treatment. Shown are the mean Ϯ SE (n ϭ 3) for wheat (circles) and pea (inverted triangles).2 mM NHϩ 4 or 0. 99 ͉ no. 6). Shown are means Ϯ SE for four replicate experiments. PNAS ͉ February 5. The fate of N after it was absorbed. respectively. The form in which N was supplied did not influence plant growth at 360 ␮mol molϪ1 (ambient) CO2. NO3 reductase Ϫ Ϫ activity (␮mol NO2 generated mgϪ1 protein hϪ1).3 (dark symbols) mM HCO3 . and NO2 reductase activity Ϫ (␮mol NO2 consumed mgϪ1 protein hϪ1) in the shoot (Top) or root (Bottom) of wheat grown for 14 days in controlled environment chambers at 360 or 700 ϩ Ϫ ␮mol molϪ1 CO2 and under NH4 or NO3 nutrition.3 mM data).30 Ϯ 0.32 Ϯ 0. on a total protein basis (Fig. 6). Fig. 5). each with 8 –10 plants per treatment. Thus. but had a dramatic effect at 700 ␮mol molϪ1 (elevated) CO2 (Fig.Ϫ Fig.26 Ϯ 0. NOϪ 3 . Total N concentration (mg gϪ1 dry mass). NO3 concentration (mg gϪ1 fresh mass). protein concentration (mg Ϫ Ϫ gϪ1 fresh mass).34 Ϯ 0. and 0. and enzyme activities were similar under both CO2 treatments (Fig. 3 ͉ 1733 PLANT BIOLOGY elevated concentrations inhibits NOϪ 3 photoassimilation. These results support a hypothesis that elevated CO2 inhibits NOϪ 3 photoassimilation (12). but decreased 13% under NOϪ 3 nutrition despite less additional biomass (Fig. Chlorophyll concentrations were 0. 5). shoot protein concentrations decreased 6% under NHϩ 4 nutrition. differed under ambient and elevated CO2 as demonstrated by the balance between inorganic and organic N (Fig. 6).2 mM NO3 as the sole N source. the differences were substantial. n ϭ 2) for ϩ the NH4 treatment at 360 and 700 ␮mol molϪ1. Bloom et al. however. Shoot protein concentrations at elevated CO2 concentrations declined more than twice as . 6). Shoot and root N concentrations were similar under the two CO2 regimes. then plants receiving NHϩ 4 as a N source should prove more responsive to CO2 enrichment. Shoot NOϪ concentrations were undetectable in plants 3 receiving NHϩ 4 . Root protein. Treatments labeled with different letters differ significantly (P Ͻ 0. on a fresh mass basis. 6.02 and 0. we grew wheat seedlings in controlled environment chambers where CO2 was controlled at ambient or elevated levels (360 or 700 ␮mol molϪ1) Ϫ and the plants received either 0. respectively.

Campbell. (1998) Plant Cell Environ.. 753–764. F. Plant Physiol. 20. (1949) in Photosynthesis in Plants. 1387–1397. At this Ci. F. J. Iowa). 88. eds. Baysdorfer. and N2O release (51). Epstein. Bloom. Bowes. Shingles. R.6 ␮mol O2 mϪ2 sϪ1 with ϩ Ϫ the shift from NH4 to NO3 nutrition (Fig. Nitrogen parameters from the growth analysis were consistent with the gas-exchange measurements.. D. G. (1993) Annu. Bazzaz. 1375–1381. J. addition of such high N levels to compensate for CO2 inhibition of shoot NOϪ 3 assimilation is unlikely for both economic and environmental reasons. suggesting that they were selectively inhibited. C. J. Robert Pearcy. H. J. 28. Wall. 145–157. F. J. LaMorte. Plant Mol. 39. Adamsen. G. 83.. Plants in the high N treatment could compensate for CO2 inhibition of shoot NOϪ 3 assimilation because they received additional NHϩ 4 . L.. R. 573–580. W. Consequently. (1986) Plant Physiol. J. P. 10. Ϫ NOϪ 3 is a major N form. Langenfelds. B. & Robinson.. (1988) Plant Physiol. 19. 39. Steven Theg for his assistance in chloroplast preparation. net CO2 uptake did not differ with N source (Fig. M. W. B. VA). Despite extensive evidence on the importance of N availability for determining plant responses to CO2 enrichment (4. The Ϫ two major N forms.. Nicotiana plumbaginifolia (40). A. whereas these parameters varied only slightly with CO2 level in the high N treatment (13. ambient CO2 in the high N treatment (13).. 425– 429. G. 22. & Robinson.org͞cgi͞doi͞10.. J. 5. Ecosyst. D. 54 –54. D. 42– 45). L. C. 107. J. A. Periodic watering of pots with solutions containing various N forms may not provide adequate control because N transformations are both rapid in nonsterile cultures (47) and sensitive to atmospheric CO2 (48). S.1073͞pnas. & McCarty. Moore. & Keeling. a value consistent shoots of these plants was 1. J. 118. A. 675– 680. L. Ritchie. H. T. (1996) Plant Cell Environ. Studies on Plantago major (38). (1994) Photosynth. K. but net O2 evolution increased by 0. 79. W. B. & Huffaker.. Ritchie. 18.§ This rate was sufficient trations was about 0. D. D. 33. few other studies have considered the form of N. T. Res. & Campbell Wilbur.3 ␮mol mϪ2 sϪ1 ϫ 0. 2. & Seemann. S. J. 1734 ͉ www. K. 16.. J. and spinach (41) have also found that longer exposures (4 h to over 2 weeks) to elevated CO2 inhibited shoot NOϪ 3 reductase activity. R. P. Franck. 150. P. (1995) Plant Physiol... & Webber. eds. & Winship. & Bugbee.. Kimball. Leaf N concentrations and grain protein declined by more than 10% at elevated vs. 23. (2000) Photosynth. L. 352–356.. Farage. Remmler. 53– 60. 11. (1980) Plant Cell Environ. Cerovic. J. 147–163. J. 102. Hunsaker. Adam. F. (2000) Agric. T. Leavitt. & Brooks. O.. Biol. In the Ϫ present study. Thomas. G. B. (1996) J. plumbaginifolia (40) and wheat (12).6 ␮mol O2 mϪ2 Ϫ ϩ Ϫ Ϫ2 Ϫ1 Ϫ 1 s ϫ 4 electrons per O2)͞(8 electrons per NO3 converted to NH4 ) ϭ 0. Whorf. Sage. R. & Schmittner. 35. (1990) Annu. 21. 4115– 4128. R. O. We thank Teena Stockert for her technical assistance. Marchal. H. A. (1987) Plant Physiol. Caldwell. Rains. yet this may be the first study to examine CO2 responses under controlled levels of Ϫ NHϩ 4 vs. wheat received either moderate (about 200 kg N haϪ1) or high (about 500 kg N haϪ1) Ϫ N as a mix of NHϩ 4 and NO3 and was exposed to 360 or 550 ␮mol molϪ1 CO2 (14). Syst. 6). I. S.. C. J. C. Ineson.. and the anonymous reviewers for their comments on the manuscript. Pinter. M. Matthias. Rufty. (1985) Plant Physiol. 175–179. (1988) J. J. exceeds the average fertilizer recommendations for wheat by a factor of 3 or 4 (49. & Morgan. Cheng. (1998) Plant Physiol. Biochem. M. eds. Nicotiana tabacum (39). 6). Assuming that all of the extra O2 evolution Ϫ was associated with generating reductant for NO3 photoassimilation. 27. 371–376. Werner Kaiser.9 ␮mol NOϪ 3 m Ϫ2 s Ϫ1 with a NOϪ photoassimilation rate of 0. 14.. Kimball. P. 14). R. Adam. 14. G. 26.. F.. D. 318 –320. NOϪ 3 was the predominate N form (14).. Changes in O2 evolution Ϫ after exposure to NOϪ 3 indicated that the NO3 photoassimilation rate for plants grown and measured under ambient CO2 concenϪ2 sϪ1. J. pp. P. 40. S. & Oaks. Wall.... N. Steele. & Huffaker. 34. 1. T. R. A. Vaughn. Plant Physiol. D. J. In the well drained soils generally devoted to wheat cultivation. K. Brooks. G. Leavitt. D. NO3 as sole N sources. K. 4. L.. Thompson. R. Hunsaker.. (1999) Plant Cell Physiol. 95.. LaMorte. 464 – 467. (1997) Association of Analytical Chemists: Official Methods of Analysis of AOAC International (AOAC Int. 77. Sinclair. L. W. rising atmospheric CO2 levels would probably require major changes in fertilizer practices associated with wheat production. A. Warner. R. This work was supported by Department of Energy under Grant 95ER62128 TECO and National Science Foundation under Grant IBN-99-74927. Torrey. F. R. & Lara. LaMorte. A. 101. R. Our results may explain several responses of wheat to elevated CO2. M. De la Torre. 13. Biochem. Prokhorenko. NHϩ 4 and NO3 . (2001) New Phytol. A. Garcia. 12. 44. In a multiyear Free Air CO2 Enrichment (FACE) experiment conducted at Maricopa. 17. 609 – 620. (Iowa State College Press. Myers. J. L. Adamsen.3 ␮mol NO3 m s Ϫ would be the potential NO3 photoassimilation rate. J. 145. Finazzo. (1988) Anal.. R. Bloom. C.. (1995) J. Bloom. 295–303. Chem. Walker. J. B. J. Aslam. Morris. 309 –332. Matthias. 82. R. M. Boca Raton. (1996) Plant Physiol. J. Atmos. M. Environ.. 127–137. 31. A. S. 579 –584.. Kimball. CO2 inhibition of NOϪ 3 photoassimilation might account for the decline in leaf N and grain quality at elevated CO2 in this treatment. we compared NHϩ 4 and NO3 as sole N sources by §When plants grown under ambient CO 2 were exposed to an atmospheric concentration of 360 ␮mol molϪ1. W. B.. et al.. ambient CO2 in the moderate N treatment. A treatment of about 500 kg N haϪ1. J. 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