Arnold J. Bloom, Department of Vegetable Crops, University of California, Davis

August, 2010

Abstract

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

Introduction

Atmospheric CO₂ concentrations have increased from about 280 to 370 μmol mol^–1 since 1800 (Etheridge et al. 1996; Whorf and Keeling, 1998) and may reach between 500 and 900 μmol mol^–1 by the end of the century (Joos et al. 1999). Several responses of higher plants to such changes were not anticipated (Bazzaz 1990). 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 (Curtis 1996). This general phenomenon, known as CO₂ acclimation, is correlated with a decline in the activity of Rubisco and other enzymes in the Calvin cycle (Bowes 1993; Moore et al. 1998). The change in Calvin cycle enzyme activities is not necessarily selective; rather, it often follows a decline in overall shoot protein and N contents (Makino and Mae 1999). Shoot N contents diminish by an average of 14% with a doubling of CO₂ (Cotrufo et al. 1998), a difference that exceeds what would be expected if a given amount of N were diluted by additional biomass (Makino and Mae 1999).

The degree of CO₂ acclimation varies with N supply (Adam et al. 2000; Farage et al. 1998). Wheat shoots accumulate free NO₃^– under elevated CO₂ (Smart et al. 1998), and shoot protein declines (Fangmeier et al. 1999; Kimball et al. 2001) despite little change in total N (Sinclair et al. 2000; Smart et al. 1998). Here, we present several lines of evidence that elevated CO₂ concentrations inhibit NO₃^– assimilation in shoots and suggest three physiological mechanisms for this phenomenon.

Gas Exchange Measurements

We evaluated canopy photosynthesis, both CO₂ consumption and O₂ evolution, in seedlings of wheat (Bloom et al. 2002), barley (Bloom et al. 1989), and tomatoes (Searles and Bloom, unpublished data) as a function of either photosynthetic photon flux density at plant height (the PFD response) or internal CO₂ concentration (the A/C_i response). Plants that were grown or measured at ambient or elevated CO₂ concentrations (360 or 700 μmol mol^-1 ), received NH₄⁺ and NO₃^– as a sole nitrogen source during measurements. For wheat shoots grown at ambient CO₂, net CO₂ consumption at any given PFD was higher at elevated CO₂ than ambient CO₂ (Figure 1A). Net O₂ evolution was also higher at elevated CO₂ than ambient CO₂ under NH₄⁺, but was insensitive to CO₂ concentration under NO₃^– (Figure 1B). The response of net CO₂ consumption versus C_i (shoot internal CO₂ concentration) was similar among all treatments (Figure 2), as is usually observed for C₃ plants (Sage 1994). Net O₂ evolution, by contrast, was lower under NH₄⁺ than NO₃^– for the wheat grown under ambient CO₂ and measured at the two lowest C_i’s(Figure 2).

Figure 1   Net CO₂ consumption (A) and O₂ evolution (B) by the shoot of a wheat seedling as a function of photosynthetic photon flux density (PFD) at plant height. The plants were grown in controlled environment chambers at 360 μmol mol^–1 CO₂ and measured at 360 (red or gold symbols) or 700 (blue or cyan symbols) μmol mol^–1 CO₂ . They received either NH₄⁺ (circles) or NO₃^– (triangles) as a sole N source during measurements. The leaves in the gas-exchange cuvette were at their natural orientation. Shown are mean ± SE for 5 to 8 replicate plants.

Figure 2   Net CO₂ consumption (A) and O₂ evolution (B) by the shoot of a wheat seedling as a function of internal CO₂ concentration (C_i), estimated from changes in CO₂ and water vapor concentrations. The plants were grown in controlled environment chambers at 360 (red or gold symbols) or 700 (blue or cyan symbols) μmol mol^–1 CO₂ . During measurements, the plants were exposed to NH₄⁺ (circles) and then to NO₃^– (triangles). The PFD at plant height was 1200 μmol quanta m^–2 s^–1 . Shown are mean ± SE for 6 replicate plants.

The assimilatory quotient (AQ), the ratio of net CO₂ consumption to net O₂ evolution, highlights these differences (Figures 3 and 4). The AQ was verified as a non-destructive measure of in planta NO₃^– assimilation over fifty years ago for algae (Myers 1949) and over a decade ago for higher plants using barley mutants deficient in NO₃^– reductase activity (Bloom et al. 1989). Transfer of electrons to NO₃^– and NO₂^– during photoassimilation increases O₂ evolution from the light-dependent reactions of photosynthesis, while CO₂ consumption by the light-independent reactions continues at similar rates. Therefore, plants that are photoassimilating NO₃^– exhibit a lower AQ, and the difference in the AQ with a shift from NH₄⁺ to NO₃^– nutrition (ΔAQ) is proportional to NO₃^– photoassimilation. The AQ may respond to other shoot processes—principally, photorespiration and the Mehler-peroxidase reaction—but these processes probably would not differ with N form in the root medium and, thus, would not influence ΔAQ.

Figure 3   In wheat seedlings, the change in assimilatory quotient (AQ = CO₂ consumed/O₂ evolved) with a shift in N source from NO₃^– to NH₄⁺ as a function of either photosynthetic flux density (A) or internal CO₂ concentration (B), based on the data presented in Figures 1 and 2. The plants were measured (A) or grown (B) in controlled environment chambers at 360 (red symbols) or 700 (blue symbols) μmol mol^–1 CO₂ . Shown are mean ± SE for 5 to 8 replicate wheat plants. Asterisks indicate the means that were significantly different from zero (P < 0.05, a student’s t -test).

Figure 4   In barley seedlings, the change in assimilatory quotient (AQ = CO₂ consumed/O₂ evolved) with a shift in N source from NO₃^– to NH₄⁺ as a function of either photosynthetic flux density (A) or internal CO₂ concentration (B). Wild-type plants (red symbols) or a nitrate reductase deficient mutant (nar 1a:nar 7w; blue symbol) were measured and grown in controlled environment chambers at 360 µmol mol^–1 CO₂ . Shown are mean ± SE for 3 replicate barley plants. Asterisks indicate the means that were significantly different from zero (P < 0.05, a student's t -test).

Here, the ΔAQs measured at elevated CO₂ concentrations did not differ significantly from zero over a range of light levels, indicating little NO₃^– photoassimilation (Figure 3A). This is consistent with a tight coupling between the light-dependent and light-independent reactions of photosynthesis. Net O₂ evolution under NO₃^– remained high at both CO₂ levels (Figure 1B), suggesting that the rate of photosynthetic electron transport and the amount of photosynthetic reductant generated were independent of the CO₂ level. In contrast, the ΔAQs measured at ambient CO₂ increased with PFD (Figures 3A and 4A), indicating that NO₃^– photoassimilation increased with light intensity. Thus, the shoots appeared to conduct NO₃^– photoassimilation only to the extent that carbon fixation was CO₂ -limited and surplus photosynthetic reductant became available. Giving priority to carbon fixation seems an appropriate strategy given that plants can store moderate levels of NO₃^– with little difficulty until reductant becomes available, but cannot directly store significant amounts of CO₂.

The response of ΔAQ as a function of internal CO₂ concentration (C_i) supports this interpretation; wheat grown at ambient CO₂ and measured at lower C_is, exhibited ΔAQs greater than zero (Figures 3B and 4B). Therefore, exposure to elevated CO₂ concentrations, either short term (hours) or long term (weeks), diminished NO₃^– photoassimilation. The same mechanism could account for both responses: short-term inhibition of NO₃^– assimilation caused a specific down-regulation of shoot NO₃^– and NO₂^– reductase activities (see Figure 7) and, thereby, a long-term decline in the capacity of the shoot to assimilate NO₃^– even under ambient CO₂ conditions.

Carbon fixation may interfere with NO₃^– photoassimilation at several junctures. First, reduction of NO₃^– to NO₂^– occurs in the cytosol (Rufty et al. 1986; Vaughn and Campbell 1988) and requires NADH generated from malate that is shuttled from the chloroplast (Robinson 1987). The demands of carbon fixation for reductant or diminished photorespiration might limit this malate shuttle. Second, the reduction of NO₂^– to NH₄⁺, the incorporation of NH₄⁺ into amino acids, and the Calvin cycle all occur in the stroma of a chloroplast (Suess et al. 1995) and require ferredoxin that is reduced via photosynthetic electron transport (Sivasankar and Oaks 1996). Elevated CO₂ stimulates the Calvin cycle and, under light-limited conditions, can diminish the amount of reduced ferredoxin available for NO₂^– reduction or NH₄⁺ assimilation (Baysdorfer and Robinson 1985; Peirson and Elliott 1988). Our finding that ΔAQ declined to zero in low light or elevated CO₂ (Figures 3 and 4) is consistent with a diminished availability of NADH or reduced ferredoxin for NO₃^– assimilation. Third, NO₂^– transport from the cytosol into the chloroplast involves the diffusion of HNO₂ across chloroplast membranes and, therefore, requires the stroma to be more alkaline than the cytosol (Shingles et al. 1996). Carbon dioxide at elevated concentrations can dissipate this pH gradient because additional CO₂ movement into the chloroplast acidifies the stroma (Shingles et al. 1997) and because enhanced carbon fixation hydrolyzes ATP faster and requires supplementary proton exchange across the thylakoid membrane to regenerate this ATP.

To examine this last possibility, we isolated chloroplasts from wheat and pea leaves and monitored their absorption of NO₂^– from medium containing various levels of HCO₃^– (Bloom et al. 2002). The addition of 0.3, 1.0, or 3.0 mM HCO₃^– decreased chloroplast NO₂^– absorption by an average of 38, 45, or 61% in wheat and 32, 48, or 60% in pea (Figure 5 shows the 0 and 0.3 mM data). These results confirm that high CO₂ levels can interfere with NO₂^– transport into the chloroplast, and thereby, provides another mechanism through which elevated CO₂ might inhibit shoot NO₃^– assimilation.

Figure 5   Net NO₂^– uptake (μmol mg^–1 chlorophyll min^–1 ) by isolated chloroplasts as a function of NO₂^– concentration when the medium contained 0 (gold or cyan symbols) or 0.3 (red or blue symbols) mM HCO₃^– . Shown are the mean ± SE (n = 3 ) for wheat (circles) and pea (inverted triangles).

Growth under Ammonium or Nitrate

If CO₂ at elevated concentrations inhibits NO₃^– photoassimilation, then plants receiving NH₄⁺, as an N source should prove more responsive to CO₂ enrichment. To test this prediction, we grew wheat seedlings in controlled environment chambers where CO₂ was controlled at ambient or elevated levels (360 or 700 μmol mol^–1 ) and the plants received either 0.2 mM NH₄⁺ or 0.2 mM NO₃^– as the N source. The form in which N was supplied did not influence plant growth at 360 μmol mol^–1 (ambient) CO₂, but had a dramatic effect at 700 μmol mol^–1 (elevated) CO₂ (Figure 6). Leaf area in the elevated CO₂ treatment relative to the ambient CO₂ treatment increased 49% under NH₄⁺ nutrition, but only 24% under NO₃^– nutrition. Total plant biomass increased 78% under NH₄⁺ nutrition, but only 44% under NO₃^– nutrition. As a result, the plants receiving NH₄⁺ were more responsive to CO₂ enrichment than those receiving NO₃^– .

Figure 6   Biomass (g dry mass) and leaf area (cm² ) per plant of wheat seedlings grown for 14 days in controlled environment chambers at 360 or 700 μmol mol^–1 CO₂ and under NH₄⁺ or NO₃^– nutrition. Shown are mean ± SE for 4 replicate experiments, each with 8 to 10 plants per treatment. Treatments labeled with different letters differ significantly (P < 0.05).

Shoot and root N concentrations were similar under the two CO₂ regimes, indicating that N absorption per unit plant mass remained unchanged (Figure 7). The fate of N after it was absorbed differed under ambient and elevated CO₂ as demonstrated by the balance of inorganic and organic N. In the elevated CO₂ treatment (relative to the ambient CO₂ treatment), shoot protein concentrations decreased 6% under NH₄⁺ nutrition. This might be expected given the dilution by additional biomass, but shoot protein concentrations decreased 13% under NO₃^– nutrition despite less additional biomass. Thus, shoot protein per plant increased 73% and 32% under NH₄⁺ and NO₃^– , correspondingly. Shoot NO₃^– concentrations were undetectable in plants receiving NH₄⁺ , but increased 62% at elevated CO₂ in those receiving NO₃^– . In vitro shoot activities of NO₃^– reductase and NO₂^– reductase decreased 12% and 27% from ambient to elevated CO₂ , respectively, on a total protein basis and decreased 33% and 30%, respectively, on a fresh mass basis. Root protein, NO₃^– , and enzyme activities were similar under both CO₂ treatments.

Figure 7   Total N concentration (mg g^–1 dry mass), protein concentration (mg g^–1 fresh mass), NO₃^– concentration (mg g^–1 fresh mass), NO₃^– reductase activity (μmol NO₂^– generated mg^–1 protein h^–1 ), and NO₂^– reductase activity (μmol NO₂^– consumed mg^–1 protein h^–1 ) in the shoot (top panel) or root (bottom panel) of wheat grown for 14 days in controlled environment chambers at 360 or 700 μmol mol^–1 CO₂ and under NH₄⁺ or NO₃^– nutrition. Shown are mean ± SE for 4 replicate experiments, each with 8 to 10 plants per treatment. Treatments labeled with different letters differ significantly (P < 0.05). Chlorophyll concentrations were 0.32 ± 0.02 and 0.34 ± 0.07 g liter^–1 (mean ± SE, n = 2) for the NH₄⁺ treatment at 360 and 700 μmol mol^–1 , respectively, and 0.30 ± 0.01 and 0.26 ± 0.04 g liter^–1 (mean ± SE, n = 2) for the NO₃^– treatment at 360 and 700 μmol mol^–1 , respectively.

These results support a hypothesis that elevated CO₂ inhibits NO₃^– photoassimilation (Smart et al. 1998). Although the plants received various CO₂ and N treatments from day 6 through day 20, the differences were substantial. Elevated CO₂ concentrations stimulated shoot growth of the plants receiving NO₃^– to only half the extent of those receiving NH₄⁺. Shoot protein concentrations at elevated CO₂ concentrations declined more than twice as much under NO₃^– than under NH₄⁺ (Figure 6). Shoot activities of NO₃^– assimilatory enzymes declined even more than the overall protein concentrations, suggesting that they were selectively inhibited. Studies on Plantago major (Fonseca et al. 1997), Nicotiana tabacum (Geiger et al. 1999), N. plumbaginifolia (Ferrario-Máry et al. 1997), and spinach (Kaiser et al. 2000) have also found that longer exposures (four hours to over two weeks) to elevated CO₂ inhibited shoot NO₃^– reductase activity. Selective inhibition of NO₃^– assimilation led to the accumulation of NO₃^– in the shoots of N. plumbaginifolia (Ferrario-Máry et al. 1997) and wheat (Smart et al. 1998).

Despite extensive evidence on the importance of N availability for determining plant responses to CO₂ enrichment (Bazzaz 1990; Drake et al. 1997; Eamus and Jarvis 1989; Geiger et al. 1999; McGuire et al. 1995; Oren et al. 2001; Sage 1994; Sinclair et al. 2000), few other studies have considered the form of N. The two major N forms, NH₄⁺ and NO₃^–, have distinct physiological effects upon plant growth and development (Bloom 1997), yet this may be the first study to examine CO₂ responses under controlled levels of NH₄⁺ versus NO₃^– as sole N sources. Periodic watering of pots with solutions containing various N forms may not provide adequate control because N transformations are both rapid in non-sterile cultures (Padgett and Leonard 1993) and sensitive to atmospheric CO₂ (Smart et al. 1997). In the present study, we compared NH₄⁺ and NO₃^– as sole N sources using a continuous flow system to maintain constant, non-limiting levels of nutrients and a cocktail of antibiotics to minimize conversion among N forms (Smart et al. 1995).

Our hypothesis is consistent with numerous studies on the responses of wheat to elevated CO₂ (Amthor 2001; Fangmeier et al. 1999; Mitchell et al. 1999). For example, in a multi-year FACE (Free Air CO₂ Enrichment) experiment conducted at Maricopa, Arizona, wheat received either moderate (about 150 kg N ha^–1 ) or high (about 500 kg N ha^–1) N as a mix of NH₄⁺ and NO₃^– and was exposed to 360 or 550 μmol mol^–1 CO₂ (Sinclair et al. 2000). Grain yields did not vary with CO₂ level in the moderate N treatment, but were 15% higher at elevated versus ambient CO₂ in the high N treatment (Kimball et al. 2001). Leaf N concentrations and grain protein declined by more than 10% at elevated versus ambient CO₂ in the moderate N treatment, whereas these parameters varied only slightly with CO₂ level in the high N treatment (Kimball et al. 2001; Sinclair et al. 2000). In the moderate N treatment, NO₃^– was the predominate N form (Sinclair et al. 2000); thus, CO₂ inhibition of NO₃^– photoassimilation might account for the decline in leaf N and grain quality at elevated CO₂ in this treatment. Plants in the high N treatment could compensate for CO₂ inhibition of shoot NO₃^– assimilation because they received additional NH₄⁺. A treatment of about 500 kg N ha^–1, however, exceeds the average fertilizer recommendations for wheat by a factor of 3 or 4 (CIMMYT 1996; FAOSTAT 2002) and would exacerbate NO₃^– leaching, NH₄⁺ volatilization, and N₂ O release (Matson et al. 1998). Consequently, addition of such high N levels to compensate for CO₂ inhibition of shoot NO₃^– assimilation is unlikely for both economic and environmental reasons.

We feel that our laboratory results have implications for the real world of crop production. Wheat is grown on over 200 million hectares worldwide (FAOSTAT 2002) and receives 18 million metric tons of N annually (CIMMYT 1996) or 20% of the world′s production (FAOSTAT 2002). In well-drained soils, generally devoted to wheat cultivation, NO₃^– is a major N form. If CO₂ inhibition of NO₃^– photoassimilation was common among wheat cultivars; rising atmospheric CO₂ levels would probably require major changes in fertilizer practices associated with wheat production.

Were CO₂ inhibition of shoot NO₃^– assimilation common to many species, it would contribute to the response of natural ecosystems to rising CO₂ levels. Plants vary in their relative dependence upon NH₄⁺ and NO₃^– as nitrogen sources (Bloom 1997) and in their balance between shoot and root NO₃^– assimilation (Andrews 1986). Our results suggest that rising atmospheric CO₂ will favor taxa that prefer NH₄⁺ as a nitrogen source or assimilate NO₃^– primarily in their roots. Clearly, a broad survey of plant species under controlled levels of NH₄⁺ and NO₃^– is warranted to determine whether CO₂ inhibition of NO₃^– photoassimilation is a general phenomenon.

We thank Teena Stockert for her technical assistance, Steven Theg for his assistance in chloroplast preparation, and Jeffrey Amthor, Werner Kaiser, Robert Pearcy, and Emanuel Epstein for their comments on the manuscript. Supported by Department of Energy under grant 95ER62128 TECO and National Science Foundation under grant IBN-99-74927.

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