Essay 13.1 Elevated CO2 and Nitrogen Photoassimilation

Essay 13.1 Elevated CO2 and Nitrogen Photoassimilation

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

August, 2010


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.


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/Ci 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 Ci (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 Ci’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 (Ci), 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 (Ci) supports this interpretation; wheat grown at ambient CO2 and measured at lower Cis, 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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