Plant Physiol. (1998) 118: 573-580

Does a Low Nitrogen Supply Necessarily Lead to Acclimation of Photosynthesis to Elevated CO₂?¹

Peter K. Farage, Ian F. McKee, and Steve P. Long^*

Department of Biological Sciences, John Tabor Laboratories, University of Essex, Wivenhoe Park, Colchester, Essex CO4 3SQ, United Kingdom

	ABSTRACT

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Long-term exposure of plants to elevated partial pressures of CO₂ (pCO₂) often depresses photosynthetic capacity. The mechanistic basis for this photosynthetic acclimation may involve accumulation of carbohydrate and may be promoted by nutrient limitation. However, our current knowledge is inadequate for making reliable predictions concerning the onset and extent of acclimation. Many studies have sought to investigate the effects of N supply but the methodologies used generally do not allow separation of the direct effects of limited N availability from those caused by a N dilution effect due to accelerated growth at elevated pCO₂. To dissociate these interactions, wheat (Triticum aestivum L.) was grown hydroponically and N was added in direct proportion to plant growth. Photosynthesis did not acclimate to elevated pCO₂ even when growth was restricted by a low-N relative addition rate. Ribulose-1, 5-bisphosphate carboxylase/oxygenase activity and quantity were maintained, there was no evidence for triose phosphate limitation of photosynthesis, and tissue N content remained within the range recorded for healthy wheat plants. In contrast, wheat grown in sand culture with N supplied at a fixed concentration suffered photosynthetic acclimation at elevated pCO₂ in a low-N treatment. This was accompanied by a significant reduction in the quantity of active ribulose-1, 5-bisphosphate carboxylase/oxygenase and leaf N content.

	INTRODUCTION

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Growth at elevated pCO₂ frequently brings about change in plant physiology that is commonly interpreted as acclimation (Drake et al., 1997). Photosynthesis is inextricably involved because CO₂ is the substrate in C₃ species that is limiting at the current atmospheric pCO₂. However, results from investigations on the effects of elevated pCO₂ on photosynthesis have been inconsistent. The stimulatory response brought about when pCO₂ is suddenly increased (Long, 1991) has often been found to decline with increasing duration of exposure (for review, see Gunderson and Wullschleger, 1994; Sage, 1994; Drake et al., 1997), but some experiments have failed to find any long-term effect, either in controlled environments (Radoglou and Jarvis, 1990; Wong, 1990) or in the field (Arp and Drake, 1991; Jones et al., 1995; Pinter et al., 1996). Why, then, is the acclimatory response so varied? Species differences can no doubt account for some of the variability, but often the same species in apparently similar conditions can yield different results with different investigators (Sage, 1994). This fact in itself suggests that there may be some uncontrolled factor(s) in the experimental design that may be crucial to the acclimatory response of photosynthesis.

Evidence that additional factors may be interacting with the CO₂ response was brought to prominence by Arp (1991), who, after reviewing the data from several investigations using a variety of experimental designs, suggested that root restriction by pot size had a significant effect on the acclimatory response. Limited rooting volume was suggested to create an imbalance in the supply and demand for carbohydrates and, consequently, would lead to carbohydrate feedback inhibition of photosynthesis (Stitt, 1991). However, further investigation has suggested that pot size and root restriction may only be involved partially in determining the degree of acclimation that occurs at elevated pCO₂; the supply of nutrients is also crucial (Pettersson et al., 1993). In particular, evidence has accumulated that N supply is of primary importance. This thesis is particularly attractive because by far the largest proportion of soluble N in the leaf is incorporated in Rubisco (Woodrow and Berry, 1988). At elevated pCO₂ carboxylation efficiency increases, enabling the photosynthetic rate to be maintained with less active Rubisco per unit leaf area. Release of N from excess Rubisco would then be advantageous if growth was limited by N supply. A number of experimental results now suggest that acclimation can be significantly slowed by high-N application (Webber et al., 1994; Drake et al., 1997).

Investigating the role that N supply has on photosynthesis at elevated pCO₂ is not easy. When plants are grown in pots and irrigated with a solution containing a fixed concentration of nutrients, the available N-to-plant mass ratio will decline with experimental duration. This occurs because there is a finite limit to the quantity of nutrient solution that can be applied to the pot and also because of the likely spatial constraint that the roots will progressively encounter within the container. If elevated pCO₂ increases growth, then the available N-to-plant mass ratio will decline more rapidly, with the danger of confounding the CO₂ treatment with earlier N deficiency (Pettersson and McDonald, 1994). The RAR method of Ingestad and Lund (1986) eliminates this problem by supplying N in direct proportion to the plant growth rate using a hydroponics-culture technique.

To test the hypothesis that acclimation to elevated pCO₂ is primarily a response to N availability, wheat (Triticum aestivum L.) was grown hydroponically at the current atmospheric [CO₂] or at 650 µmol mol¹ and with either free access to N or at a relatively low-N RAR. The effects on photosynthesis were subsequently analyzed and the results were compared with those of a previous experiment in which wheat had been grown in sand culture at elevated pCO₂ and the two N treatments were applied in the traditional way by irrigating with solutions containing a fixed high and low N concentration.

	MATERIALS AND METHODS

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Plant Material

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Hydroponics System

Nutrient Solutions

1

1

Sand Culture Experiment

Growth Conditions

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1

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Wet Weight, Dry Weight, and Leaf-Area Measurement

At the end of the growing period (ligule emergence of the sixth leaf) plants were divided into their component parts (roots, pseudo stems, leaf laminae, and tillers). Leaf area was measured with a leaf-area meter (Delta T Devices, Burwell, UK). The plant tissue was dried to constant mass in a fan-assisted oven at 80°C before weighing on an analytical balance (model 2006 MP, Sartorius, Göttingen, Germany), which was self-calibrating and cross-checked annually by the manufacturer.

Gas-Exchange Measurements

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Tissue Analysis of Rubisco and N

Total leaf N content was measured by GC using an elemental analyzer (model PE 2400 series II CHNS/O Analyser, Perkin-Elmer Cetus). Samples were first ground to a fine powder and the instrument was calibrated with acetanilide standards (Perkin-Elmer Cetus).

Statistical Analysis

	RESULTS

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The two N treatments used in the hydroponics-culture technique produced wheat plants with significantly different rates of growth (P > 0.01), but pCO₂ had no significant effect on growth rate (P > 0.05; Fig. 1a). The elevatedpCO₂-grown plants did, however, exhibit an initial slight growth advantage and so were always bigger than their ambient-pCO₂-grown counterparts. Growth was exponential at 0.18 and 0.20 d¹ for the control and elevated-pCO₂-grown plants with free access to N and was 0.09 and 0.10 d¹ for the control and elevated-pCO₂ plants grown at the restricted rate of N supply (Fig. 1b). There was no significant effect of CO₂ treatment (F = 1.609, P <0.2) or N supply (F = 0.021, P <0.8) on A_sat, so there was no evidence that photosynthesis was down-regulated by elevated pCO₂ (Fig. 2). An identical pattern of results was obtained when the measurements were made at 650 µmol mol¹ CO₂ (data not shown), which is commensurate with this apparent lack of acclimation. The A_sat of the elevated pCO₂-grown plants measured at their growth pCO₂ at the sixth-leaf stage was significantly higher (56%) than the rate obtained for plants grown and measured at 360 µmol mol¹ (P <0.01).

View larger version (15K): [in this window] [in a new window]   Figure 1. The effects of pCO2 and N supply on the increase in total wet weight (a) and ln wet weight (b) of wheat. Plants were grown hydroponically with day/night temperatures of 20°C/15°C and a photosynthetically active photon flux density at leaf height of approximately 750 µmol m2 s1. Treatments were: , 650 µmol mol1 CO2, free access to N; , 360 µmol mol1 CO2, free access to N; , 650 µmol mol1 CO2, RAR of 0.07 mol N mol1 N d1; and , 360 µmol mol1 CO2, RAR of 0.07 mol N mol1 N d1. Vertical bars represent SE; n = 10.

View larger version (36K): [in this window] [in a new window]   Figure 2. Rates of Asat and Vc,max of the sixth leaf at ligule emergence of wheat grown hydroponically (hy), and the Vc,max of the sixth leaf at ligule emergence of wheat grown in sand culture (sd). Measurements were made with a leaf temperature of 23°C with a photosynthetically active photon flux density of 1400 µmol m2 s1 and pO2 of 210 mmol mol1. The Asat was measured at 360 µmol mol1 CO2, and Vc,max was obtained from the initial slope of the CO2 response curve. Treatments consisted of the following: Hydroponics: , 650 µmol mol1 CO2, free access to N; , 360 µmol mol1 CO2, free access to N; , 650 µmol mol1 CO2, N RAR of 0.07 mol N mol1 N d1; , 360 µmol mol1 CO2, N RAR of 0.07 mol N mol1 N d1. Sand culture: , 650 µmol mol1 CO2, 10 mmol nitrate; , 360 µmol mol1 CO2, 10 mmol nitrate; , 650 µmol mol1 CO2, 4.5 mmol nitrate; , 360 µmol mol1 CO2, 4.5 mmol nitrate. Vertical bars represent SE; n = 4.

Analysis of tissue N showed that the low-N hydroponically grown plants exhibited a significant reduction (P <0.001) in the N content of each plant organ (roots, stems, and leaves) compared with controls, although the leaves suffered the smallest decrease (Table I). This change in N content was accompanied by a significant increase in the C-to-N ratio of each organ (Table I). However, when N content was expressed on a leaf-area basis, the effect of N treatment was greatly reduced at the sixth-leaf stage and was only significantly different between N treatments of the elevated pCO₂ leaves (Table I; F = 14.037; P > 0.01). This result is apparently due to the LAR, which showed the greatest change in response to CO₂ treatment (Table II), brought about by a decrease in SLA (P <0.001) rather than a decrease in LWR (P = 0.1).

View this table: [in this window] [in a new window]   Table I. Tissue N and C-to-N ratio of hydroponically grown wheat Tissue N and C-to-N ratio of hydroponically grown wheat in 360 µmol mol1 CO2 (LC) or 650 µmol mol1 CO2 (HC) with either free access to N (HN) or at a low rate of N supply (LN). Plants were harvested when the sixth leaf had fully expanded. Values shown are the means ± SE per unit dry weight; n = 4 to 5.

View this table: [in this window] [in a new window]   Table II. Leaf growth analysis of hydroponically grown wheat LAR, SLA, and LWR of hydroponically grown wheat in 360 µmol mol1 CO2 (LC) or 650 µmol mol1 CO2 (HC) with either free access to N (HN) or at a low rate of N supply (LN). Plants were harvested when the sixth leaf had fully expanded. Values are the means ± SE.

A comparison with results from the sand-culture experiment shows that leaf N content on a leaf-weight basis was not substantially lower than that of the hydroponically grown plants, but only when plants were irrigated with the high-N treatment (Table III). Those plants given the low-N solution suffered a large, significant reduction in leaf N content even when expressed on a leaf-area basis (Table III; F = 130.503; P > 0.001). Elevated pCO₂ exacerbated this reduction in [N] (F = 13.650; P > 0.001). This result occurred in spite of the low-N sand-culture plants receiving a higher total N dose over the course of the experiment than their hydroponically grown counterparts. Thus, growing wheat with fixed [N] dramatically reduced leaf N content; an effect that was augmented by elevated pCO₂, whereas growth in hydroponic culture had very little effect on leaf N. 

View this table: [in this window] [in a new window]   Table III. Leaf tissue N content of sand-grown wheat Leaf tissue N content of sand-culture-grown wheat at 360 µmol mol1 CO2 (LC) or 650 µmol mol1 CO2 (HC) with either 10 mmol of nitrate (HN) or 4.5 mmol of nitrate (LN). Analyses are for the third leaf at ligule emergence. Values shown are the mean ± SE per unit dry weight; n = 10.

The failure of elevated CO₂ to bring about a reduction of N content in hydroponically grown wheat may be a crucial factor in the ability of these plants to avoid photosynthetic acclimation as determined by A_sat. Central to this effect is likely to be the response of Rubisco. Figure 3 shows A/c_i curves for the sixth leaves once they had reached full expansion. Plants grown and measured at the current ambient pCO₂ at high and low rates of N supply had values of c_i on the initial phase of the curve, inferring Rubisco limitation. Growth at elevated pCO₂ shifted the operating point to the inflection of the curve (Fig. 3), suggesting increased control by ribulose-1,5-bisphosphate regeneration.

View larger version (23K): [in this window] [in a new window]   Figure 3. CO2-response curves for the sixth leaf at ligule emergence of wheat grown hydroponically with free access to N or with a RAR of 0.07 mol N mol1 N d1 in atmospheres of 360 or 650 µmol mol1 CO2. Symbols are data points from two representative leaves used in the calculation of Vc,max and Amax, whereas the curves are fitted by a maximum likelihood regression using the equations of Farquhar et al. (1980) to all of the leaves measured. The supply functions are indicated by dotted lines. a, 360 µmol mol1 CO2, free access to N; b, 650 µmol mol1 CO2, free access to N; c, 360 µmol mol1 CO2, RAR of 0.07 mol N mol1 N d1; and d, 650 µmol mol1 CO2; RAR of 0.07 mol N mol1 N d1. Measurement conditions are described in Figure 2.

In vivo measurements of Rubisco activity modeled from the A/c_i curves show that for the sixth leaf at ligule emergence there was no significant difference in V_c,max between the CO₂ or N treatments (Fig. 2; F = 0.170, 0.170; P > 0.65). This was confirmed by in vitro measurements, which also found no significant difference in the initial activity (Fig. 4; F = 0.032, 2.218; P > 0.1) or the activated activity of Rubisco (Fig. 4; F = 0.510, 1.449; P > 0.25). There was also no change in the quantity of this enzyme (Fig. 4; F = 1.205, 2.651; P > 0.1).

View larger version (38K): [in this window] [in a new window]   Figure 4. Vc,max of Rubisco measured in vitro together with the concentration of Rubisco protein. Activities are for the initial activity upon extraction and for the Vc,max following incubation with CO2 and Mg2+. Legend for bars is in Figure 2; vertical bars represent SE; n = 4.

When in vivo Rubisco activity was estimated for wheat grown in the sand-culture experiment, it was found that for the fourth and sixth leaves at ligule emergence there was a significant effect of both pCO₂ and N on V_c,max (Fig. 2; F = 13.326, 13.469; P <0.01). The V_c,max of the elevated pCO₂, low-N-grown plants was significantly less than for those grown at control pCO₂ and low N (Fig. 2; P <0.01), indicating a significant decrease in the amount of active Rubisco.

The operating point on the A/c_i curve of the hydroponically grown plants at elevated pCO₂ was at the inflection of the curve and so would be partially influenced by the rate of regeneration of ribulose-1,5-bisphosphate and ultimately on the rate of electron transport. The rates of A_max for the sixth leaves show that although a depression was indicated for leaves grown at elevated pCO₂ with low N, there was no significant effect of pCO₂ or N treatment (Fig. 5; F = 0.379, 4.592; P > 0.05). To verify that triose-3 phosphate export from the chloroplasts was not limiting to leaf gas exchange the pO₂ response of photosynthesis was investigated. Plants responded positively to a reduction in the pO₂ from 21 to 2.1 kPa and showed no pCO₂ or N treatment effect (Fig. 5; F = 3.333, 0.146; P > 0.05). The ability of all plants to respond similarly to the removal of photorespiration consequently supports the A_max results.

View larger version (30K): [in this window] [in a new window]   Figure 5. The rate of CO2 uptake at light and CO2 saturation (Amax) and the relative stimulation (stim.) of CO2 uptake by inhibition of photorespiration following a reduction in pO2 to 21 mmol mol1. Measurement conditions for leaf gas exchange and legends for bars are described in Figure 2; the [CO2] was 650 µmol mol1. Vertical bars represent SE; n = 4.

	DISCUSSION

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Acclimation of photosynthesis to elevated pCO₂ was accentuated by low-N supply when wheat was grown in pots with a fixed [N]. However, when N was supplied in direct proportion to plant growth, elevated pCO₂ did not produce acclimation of photosynthesis regardless of whether the N supply was strongly limiting growth or optimal. Pettersson et al. (1993), who have previously used the RAR for applying N, have obtained similar findings with birch. The data support the hypothesis that acclimation of photosynthesis to elevated pCO₂ results from a greater dilution of plant N content rather than from a low availability of N. Our results suggest that if a plant commences development with a low availability of N, paralleling a plant germinating on a N-deficient soil, the major effect will be on the rate of leaf-area development rather than on leaf N content (Scott et al., 1994). As the root system expands, further N may become available, a situation that may be simulated by the RAR method. When a plant germinates within a pot with N supplied at a fixed concentration, as simulated by our sand-culture experiment, initially there is a high availability of N relative to plant mass, allowing rapid growth. However, with further growth the relative amount of N will decline and trigger acclimation of photosynthetic capacity. A corresponding situation may occur in the field, when over-winter mineralization or fertilizer application at sowing creates a flush of available N followed by a depletion of the N reserve through the season.

The dilution effect on plant N status produced by growth in elevated pCO₂ can result either from increasing dilution of a given N supply (Coleman et al., 1993) or by increased carbohydrate accumulation diluting the [N] within the plant (Wong, 1990; Kuehny et al., 1991). Complications can arise if changes in SLA are not taken into account. This is because elevated pCO₂ frequently alters leaf morphology and, consequently, effects of pCO₂ on [N] are decreased (Norby et al., 1992; Rogers et al., 1996a) or absent (Rowland-Bamford et al., 1991) when results are expressed per leaf area rather than leaf weight. Our hydroponically grown wheat did show an increase in the C-to-N ratio, but the effect was relatively small and leaf N content remained above 1.8 g m². It is well established that rates of photosynthesis are correlated to N content, but for wheat, this relationship begins to plateau at leaf concentrations above 1.75 g m² (Evans, 1989). In another investigation, when growth techniques were compared the C-to-N ratio of tobacco plants was markedly increased at elevated pCO₂ when they were grown in pots but was largely unchanged following growth in hydroponic culture (Ferrario-Méry et al., 1997).

Availability of N is also a crucial factor in sink development. Rogers et al. (1996b) have shown that the degree of N fertilization is an important contributor to sink strength, demonstrating that it can prevent acclimation at all but the lowest rates of N application. In relation to wheat, Rogers et al. (1996a) have demonstrated the requirement of N for tiller and leaf production for avoidance of acclimation. Similar conclusions were obtained by Ryle et al. (1992) and by Newbery and Wolfenden (1996). However, in our hydroponics experiment both leaf area and the number of tillers were significantly reduced by the low-N RAR at both control and elevated pCO₂, but acclimation was still avoided. This demonstrates that production of a large sink capacity may not be necessary to avoid acclimation, rather, the balance between source and sink at the whole-plant level is the key factor, as has been proposed by Pettersson and McDonald (1994). Thus, at elevated pCO₂, although our hydroponically grown, low-N wheat plants maintained their photosynthetic rates, the absolute amount of photosynthate produced was lessened because of their smaller leaf area (data not shown) compared with that of their high-N-grown counterparts. In addition, the ability of the hydroponically grown wheat to respond to a lowering of the pO₂ demonstrates that the requirement for Pi by ATP phosphorylase was not greater than the rate of sugar phosphate use (Sharkey, 1985). An interesting observation, however, was that the hydroponically grown wheat had a greater mass than those plants grown in ambient air, despite there being no significant effect of pCO₂ on relative growth rate. This difference must have been initiated from a very early and transient stimulation of growth, and consequently appears similar to observations made in other elevated-CO₂ studies where differences in biomass between pCO₂ treatments are reported to have arisen from brief alterations in relative growth rate (Poorter, 1993).

Acclimation of photosynthesis to growth in elevated pCO₂ is most frequently accompanied by a reduction in carboxylation capacity (for review, see Bowes, 1991). It is commonly suggested that a decrease in Rubisco activity is ultimately responsible for acclimation of photosynthesis to elevated pCO₂ (Bowes, 1991). Estimations of V_c,max in vivo and direct measurement of Rubisco activity and content in vitro confirmed that the pCO₂ and N treatments had no significant effect on the hydroponically grown wheat, and this is the most likely reason that net photosynthesis was unaffected. The lack of effect on Rubisco most likely stems from the absence of either any major reduction in leaf N content or of any end-product inhibition of photosynthesis. Increase of leaf carbohydrate has been correlated with a decrease in V_c,max (McKee and Woodward, 1994), and it is this increase in photosynthate that is suggested to be the signal that brings about a decrease in Rubisco levels (Stitt, 1991). The underlying mechanism is believed to operate via the repression of photosynthetic gene expression (Webber et al., 1994; Koch, 1996; Drake et al., 1997). Whether the quantity of Rubisco is decreased at elevated pCO₂ in response to buildup in leaf carbohydrate or not, less active enzyme is required because it is not saturated at the current atmospheric pCO₂ (Long, 1991). Consequently, N may be reallocated to proteins of the electron-transport chain or photosynthetic carbon-reduction-cycle enzymes (Woodrow, 1994). Wheat grown under true field conditions (Free Air CO₂ Enrichment) showed no loss of Rubisco activity or quantity in unshaded leaves, and this was accompanied by a complete absence of any down-regulation of photosynthesis (Nie et al., 1995; Drake et al., 1997).

In conclusion, the results from our investigation have shown that low rates of N supply need not cause acclimation of photosynthesis to elevated pCO₂. Development of large sinks, e.g. tillers and leaves, was not necessary for the avoidance of acclimation; rather, an adjustment of plant growth rate to match the N supply appears to be the decisive factor. This agrees with the hypothesis by Pettersson and McDonald (1994) that acclimation to elevated pCO₂ is dependent upon whether the whole-plant growth response has acclimated to elevated pCO₂, together with any other resource limitations in the immediate environment. The majority of experimental methodologies used in elevated pCO₂ investigations by necessity grow plants in artificial conditions, but the outcome may be that resources do not keep pace with demand and, therefore, the plants will be prone to acclimation. The experimental approach that has least often found acclimation is the field system (Gunderson and Wullschleger, 1994; Sage, 1994; Drake et al., 1997). Plants growing in the natural environment are more likely to be adjusted to surrounding environmental influences. However, this does not preclude acclimation from occurring; natural environments are often limiting in nutrients, especially N (Eamus and Jarvis, 1989; Gifford, 1992), and, consequently, there is ample opportunity for acclimation of photosynthesis to occur. Plants can experience periods of source:sink imbalance, especially late in the growing season when reproductive sinks are developing. For example, translocation of N during grain filling was assumed to produce a decrease in Rubisco concentration of wheat exposed to elevated CO₂ in a free air CO₂ enrichment system (Nie et al., 1995), and changes in developmental state have been shown to initiate reversible acclimation in beet (Ziska et al., 1995). We might therefore expect phenology, together with specific environmental conditions, to be instrumental in determining the response of plants to elevated pCO₂ in the natural environment.

	FOOTNOTES

   Received March 2, 1998; accepted July 7, 1998.

	ABBREVIATIONS

Abbreviations: A_max, rate of CO₂ uptake at light and CO₂ saturation. A_sat, rate of CO₂ uptake at light saturation. c_i, intercellular pCO₂. LAR, leaf area ratio. LWR, leaf weight ratio. pCO₂, partial pressure of CO₂. RAR, relative addition rate. SLA, specific leaf areaV_c,. _max, maximum velocity for carboxylation.

	ACKNOWLEDGMENTS

We thank S. Corbet for technical assistance with the hydroponics culture and P. Beckwith for general technical skills. J. Bullimore's help with the Rubisco analyses is much appreciated.

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