Interactions Between Rising CO, Concentration and Nitrogen Supply in Cotton. I. Growth and Leaf Nitrogen Concentration

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1 Aust. J. Plant Physiol., 1996, 23, Interactions Between Rising CO, Concentration and Nitrogen Supply in Cotton. I. Growth and Leaf Nitrogen Concentration G. S. ~ o ~ e rp. s J. ~ ~ilharn~, ~, and J. P. conroya M.-C. hi baud^^ *School of Horticulture, University of Western Sydney, Hawkesbury, Locked Bag No. 1, PO Richmond, NSW 2753, Australia. BBiological and Chemical Research Institute, NSW Agriculture, PMB I0 Rydalmere, NSW 2116, Australia. CPermanent address: Departement de Physiologie Vegetale et Ecosysternes, CEA, Centre de Cadarache, F Saint Paul Lez Durance, Cedex, France. DAuthor for correspondence, g.rogers@uws.edu.au Abstract. The influence of sink development on the response of shoot growth in cotton (Gossypium hirsutum L. cv. Siokra BT1-4) was investigated by growing plants at three levels of C02 concentration: 350 (ambient), 550 and 900 y~ L-I and six levels of nitrogen (N) supply ranging from deficient to excess (0-133 mg N kg-' soil week-'). Changes in leaf N concentration were also investigated. At 59 days after sowing, there was an average 63% increase in shoot growth at 550 ILL C02 L-' compared with ambient C02-grown plants, with no significant growth increase at 900 yl C02L-' and, this response was closely matched by sink development (flower number and stem weight). Low N supply restricted the responses of both sink development and shoot growth to high C02. At elevated C02, leaf N concentration was reduced by an average 27% at low to adequate N supply. The high C02-induced reduction in leaf N concentration, however, disappeared when the N supply was increased to a high level of 133 mg N kg-' soil week-'. These C02 effects on leaf N concentration were smaller when N was expressed per unit leaf area, apparently due to a combination of the effects of elevated C02 or high N supply reducing specific leaf area and, to an N uptake limitation at low to moderate levels of N supply. The critical foliar N concentrations (leaf N concentration at 90% of maximum shoot growth) were reduced from 42 to 38 and 36 mg g-' when C02 concentrations were increased from 350 to 550 and 900 yl L-' respectively, indicating that changes in fertiliser management may be required under changing C02 concentrations. Introduction Few CO, enrichment studies employ more than one level of CO, elevation and thus the shape of the plant growth curve in response to rising C02 concentration remains largely unknown. The photosynthetic response to rising atmospheric C02 concentration, however, has been more carefully studied. It appears that the capacity of plants to generate sinks in their shoots is crucial in determining the magnitude and shape of the photosynthetic response to increasing atmospheric CO, concentration (Webber et al. 1994; Xu et al. 1994; Krapp and Stitt 1995). In species where sink strength is limited genetically (e.g. clover or sunflower) or where sink limitations are imposed (Xu et al. 1994), down-regulation of the rate of CO, assimilation (A) may limit both the magnitude of the growth response and the range of C0, concentrations over which such a response will occur (Stitt 199 1; Morin et al. 1992; Woodrow 1994~). Conversely, in species which are more flexible in their sink capacity, e.g. wheat, which can produce large numbers of tillers (Gifford 1977; Hocking and Meyer 1991; Rogers et al. 1995), the down-regulation of photosynthesis through feedback inhibition in response to elevated CO, is less likely to occur (Woodrow 1994b). Thus in the wheat variety Hartog, where optimal nutrient conditions were maintained, the tiller number nearly doubled in the vegetative phase under conditions of elevated C0, and there was a matching linear increase in vegetative shoot dry weight as CO, concentrations were increased from 350 to 550 and then to 900 pl L-' (Rogers et al. 1995). The capacity to generate sinks is dependent not only upon species but is also a function of the soil nutrient supply (Radin and Mauney 1982; Wardlaw 1990). In wheat, both the tiller number and the capacity to respond to elevated C0, is greatly stimulated by increasing nitrogen (N) supply (Conroy and Hocking 1993; Rogers et al. 1995). Furthermore, the shape of the growth response to elevated C02 is altered by N supply, with extra shoot growth occurring between 350 and 550 pl CO, L-' but not above when N is deficient (Rogers et al. 1995).

2 120 G. S. Rogers et al. It appears therefore that sink strength, mediated at least partly through N supply, is crucial in determining the growth response of sink-flexible species such as wheat to elevated CO,. This raises two questions: first, what effect would alteying N supply have on the sink development of a more sink-limited species such as cotton (Benedict 1984); and second, how would the growth response to increasing atmospheric CO, concentration be affected. Growth of cotton may increase at elevated CO, concentrations but there is also an accumulation of nonstructural carbohydrate, mainly as starch (Wong 1990). Consequently, specific leaf area (SLA) declines at high CO,, and low N supply is likely to accentuate this effect (Radin and Eidenbock 1986; Paul and Stitt 1993). Given that high C02 influences the dry weight allocation between structural and non-structural pools (Wong 1990), the manner in which the N concentration is expressed is important. The question of whether differences in leaf N concentrations at high CO, involve changes in N metabolism or uptake, or if they are merely reflections of changes in the mode of expression has broad implications. An understanding of the mechanisms underlying changes in nutrient concentrations is crucial for modelling long-term growth responses to high CO,, e.g. in forest ecosystems where N limits productivity (Kirschbaum et al. 1994). There are also practical implications for the interpretation of foliar analyses which are used in the management of fertiliser applications in agricultural crop production. This is particularly important for cotton because timing and amount of N addition can affect disease reactions and the rate of boll development (Constable 1988). This study investigates differences in the shoot growth and leaf N concentration of cotton grown at N supplies which vary from deficient to excess at three CO, concentrations of 350 (ambient), 550 and 900 pl L-l, and attempts to explain how the reduction in foliar N concentration occurs. Materials and Methods Plant Culture Batches (5.8 kg) of soil, the properties of which are described by Rogers et al. (1993), were mixed with lime and dolomite (7 g kg-' CaCO, and 1.8 g kg-' MgC03) to adjust the ph from 4.3 to 6.5 (1:5 w/v in 0.01 M CaCI,). Basal nutrients were also mixed with the soil (mg kg-' dry soil): P (1800), K (90+360), B (5), Cu (3, Zn (lo), Mo (0.1), Mn (50), and Fe (50) as: CaHPO,, K2S04 + K,CO,, H3B03, CuS04, ZnSO,, Na2Mo0,, MnS04, and FeS0, respectively, prior to placing it into pots 0.4 m tall and 0.15 m in diameter (7 L). Cotton seeds (Gossypium hirsutum L. cv. Siokra BT1-4) were germinated on gaper towel at room temperature, ambient CO,. Eight germinated seeds at a similar developmental stage were placed on the surface of the soil. A further 0.2 kg of similarly amended soil was then added to each pot. The seedlings were culled 2 weeks later to four uniform plants per pot and, at 34 days after sowing (DAS), three further plants were culled leaving a single plant per pot. The plants were grown in three, naturally-lit, temperaturecontrolled glasshouses. The maximum photosynthetic photon flux density (PPFD) was 1000 pmol m-* s-' and temperatures in the glasshouses were C day and 21 rt 2 C night on a 12/12 h daylnight cycle. Relative humidity was not controlled but remained at approximately 80% during the day. CO, was supplied as compressed, food grade CO, from BOC (Australia) and passed through a Purafil column to remove ethylene contamination. CO, concentrations were maintained at 350 c 10 (ambient); 550 c 20 and 900 rt 20 yl L-I using an infrared gas analyser linked to solenoid valves that controlled the flow of COT Nitrogen treatments were applied at weekly rates of 0,8, 17,33, 67 and 133 mg N kg-' soil from a solution comprising KN03:Ca(N03)2:Mg(N03)2:(NH4)2S04:NH4N0, in the ratio (by weight) 1 : 2: 1 : 1 : 1. The N applications for the first 2 weeks were split over three separate days to avoid potential damage to young plants. The soil was watered to field capacity daily from sowing until harvest. The plants and CO, treatments were transferred between glasshouses and re-randimised within each glasshouse weekly to minimise variation due to glasshouse and positional effects. The six N treatments were arranged in a Randomised Complete Block Design with four replicates per treatment. Harvest At 59 DAS, the plants were cut off at soil level. The two youngest fully expanded leaves (YFEL) were removed from each plant, their areas measured on a LI-COR leaf area meter, frozen in liquid N,, freeze-dried and milled to pass through a 0.5 mm screen. The plants were then separated into stems, cotyledons, first true leaves, remaining leaves and flower buds. The leaf areas were measured, and together with the flower buds and stems, dried at 80 C for 48 h, weighed and milled as for the YFELs. Leaf and Stem Analyses The N concentrations of leaf, stem and flower bud samples were determined by thermal conductivity after combustion using a Leco Nitrogen Analyser model, FP-428 (USA). The concentration of other nutrients in the leaves of plants grown at high N supply were determined by ICP-AES after digestion in H,SO,/H,O,. Soluble carbohydrates were extracted by heating 50 mg samples of freeze-dried leaf in 5 ml water at 90 C for 30 min in polypropylene tubes, centrifuging (3000 g for 5 min) and collecting the supernatant. The residue was then re-extracted twice, the supernatants were pooled and the carbohydrate concentration determined by the anthrone method (Yemm and Willis 1954). The starch concentration was measured enzymatically (McCleary et al. 1994) on separate 50 mg freeze-dried leaf samples. Structural leaf weight was calculated as total dry weight less starch and watersoluble carbohydrate. Critical Nitrogen Concentrations Shoot dry weight was expressed as a percentage of maximum for each N and CO, treatment combination and plotted against the concentration of N in the respective YFEL at 59 DAS. At the

3 Growth and Foliar N Responses to C02 and N Supply highest levels of N supply, shoot dry weight was slightly depressed at all levels of atmospheric CO,. In order to simplify curve-fitting, these points were ignored. Polynomial equations (2") were fitted to the remaining points with? values of 0.97, 0.99 and 0.98 corresponding to CO, concentrations of 350, 550 and 900 pl L-l, respectively. Shoot Growth Results Shoot growth was significantly (P < 0.01) greater (mean 63%) in plants grown at a C0, concentration of 550 pl L-I compared to those grown at 350 pl L-' except for those at the lowest level of N supply (Fig. 1). There was no significant (P > 0.05) increase in growth when the CO, concentration was increased to 900 FL L-'. There was a significant (P < 0.01) interaction between N supply and CO, concentration. The data point at 550 pl CO, L-', 67 mg N kg-' soil week-', is difficult to explain and, given the position of surrounding points at 550 pl C02 L-', we chose to ignore it when fitting the growth response curve (Fig. 1). Increasing N supply generally resulted in greater shoot growth up to about 67 mg N kg-' soil week-' at all levels of C02. Nutrients other than N did not limit growth (data not shown). For simplicity, data for only two of the N treatments, 8 (low) and 133 (high) mg N kg-' soil week-', are presented for the shoot components (Fig. 2). Results for the other treatments are consistent with the results presented. Stem and leaf dry weight followed a similar pattern to shoot dry weight, increasing with C02 enrichment only between 350 and 550 pl L-' and was more responsive at high than low N supply (Fig. 2). The number of flower buds per plant was N supply (mg N kg-' soilweek-'' I Pig. I. Shoot dry weight of 59-day-old cotton supplied with six levels of N at either 350 (o), 550 (0) or 900 (a) p,l C02 L-'. The vertical bars represent standard errors (P < 0.05, rz = 4). - -t Shoot,& A 5 40 (b) S E 30-0, $ 20 - E 10-4/- 0 Foliage & & ,$ Stem Atmospheric CO, (pl c') Fig. 2. Influence of C02 concentration at two levels of N supply on (a) shoot, (b) foliage and (c) stem dry weight of 59-day-old cotton plants. Nitrogen was supplied at either 8 (A) or 133 (A) mg N kg-' soil week-'. The vertical bars represent standard errors (P c 0.05, n = 4). generally greater at high N supply and most responsive to C02 between 350 and 550 pl L-' (Fig. 3a). Total leaf area per plant increased in response to both C02 concentration and N supply. To aid interpretation, however, the components of leaf area (number and size) are presented. Leaf number per plant was greater at high N supply but generally unaffected by C02 concentration (Fig. 3b). The area of individual leaves was responsive to C0, from 350 to 550 pl L-' but not affected by N supply (Fig. 3c). SLA, was unaffected by CO, concentration at high N supply but decreased at both 550 and 900 pl C02 L-' when N supply was low, reaching a common minimum of about 1.8 dm2 g-' at 900 pl C02 L-' at both high and low N supply (Fig. 3d). Leaf Nitrogen Leaf N concentration per unit leaf dry weight was up to 33% lower in plants grown at C02 concentrations of 550 and 900 p,l L-I compared with those grown at ambient CO, (Fig. 4a). The C0, effect diminished as N supply was increased until, at the highest N supply (133 mg N kg-' soil week-'), there was no significant (P > 0.05) depression in leaf N concentration (Fig. 4a). The critical leaf N

4 G. S. Rogers et al. Individual leaf area (b) C - m + L O - n A t m c 0 'z 20-3 ~eaf number Specific leaf area 01 ' I I 1 I I I Atmospheric CO, (pl L-I) Fig. 3. Influence of C02 concentration at two levels of N supply on (a) flower bud number per plant, (b) leaf number per plant, (c) leaf area per leaf (all leaves) and (4 SLA (all leaves) of 59-day-old cotton plants. Symbols and error bars are as described in the Fig. 2 caption N supply (mg N kg-' soil week-') Fig. 4. The effect of C02 concentration and N supply (0-133 mg N kg-' soil week-') on the leaf N concentration of YFEL of 59-day-old cotton expressed on (a) total dry weight basis, (b) structural dry weight basis and (c) leaf area basis. Plants were grown at either 350 (o), 550 (0) or 900 (m) p,l C02 L-l, respectively. The vertical bars represent standard errors (P < 0.05, n = 4). concentrations were reduced by C02 enrichment from 42 to 38 and 36 mg N g-i at 550 and 900 p,l C02 L-' respectively (Fig. 5). The leaf N concentrations were then re-expressed on a structural dry weight basis (Fig. 4b); however, the changes in concentration in response to C02 and N supply remained similar to those expressed on a total leaf dry weight basis. Finally, when leaf N was expressed on an area basis, CO, enrichment caused an average reduction of 13% in leaf N content at low to adequate N supply (8-33 mg N kg-' soil week-') and had no significant effect (P > 0.05) when N was supplied in excess of requirement (133 mg N kg-' soil week-'). Increasing N supply caused a general increase in leaf N concentration reaching maximum concentrations of 43 mg g l, 48 mg g-' and 2.4 g mw2 expressed on a total dry weight, structural dry weight and area basis respectively (Fig. 4a-c). The N concentration of the stems was significantly (P < 0.01) lower at elevated C02 when N was supplied at 17 mg N kg-' soil week-', but unaffected by C02 concentration at low and high N supplies (0 and 133 mg N kg-' soil week-') (Table 1). Nitrogen Uptake A similar amount of N was taken up into the shoots irrespective of C02 concentration at low and moderate N supplies (0 and 17 mg N kg-' soil week-'). When N supply was increased to excess levels (133 mg N kg-' soil week-'), the high C02-grown plants (900 pl L-') were able to take up significantly (P < 0.01) more N than ambient (350 FL C02 L-I)-grown plants (Fig. 6).

5 Growth and Foliar N Responses to C02 and N Supply C 2.- m 60 $ P v 40 lz 0 V) 20 0 Leaf N concentration (mg N g") Fig. 5. The effect of C02 concentration and N supply (0-133 mg N kg1 soil week-') on the critical leaf N concentration of cotton shoots grown at either 350 (o), 550 (0) or 900 (a) pl CO;, L-', respectively. Polynomial equations (2") were fitted with 1-2 values of 0.97, 0.99 and 0.98 for 350, 550 and 900 pl C02 L-I respectively. The data points corresponding to the highest level of N supply, where the shoot dry weight declined, were omitted from both the curve-fitting procedure and the figure. The numbers near the 90% yield line indicate critical leaf N concentrations. Both axes refer to measured values (n = 4) but standard error bars have been omitted for clarity. Table 1. Stem nitrogen concentration The N concentration in the main stems and lateral stems of 59-day-old cotton supplied with three levels of N (0, 17 and 133 mg N kg-' soil week-') and grown at either 350 or 900 yl C02 L-I (n = 4) N WP~Y C02 Stem N concentration (mg N kg-' soil week-') (Id- L-') (mg g-' dry weight) lsd (P < 0.05) 5.0 Discussion The hypothesis that the magnitude and shape of the growth response of cotton shoots to increasing concentration is influenced by sink strength, mediated through N supply, is supported by the results of this study. The greatest increase in shoot dry weight (83% between 350 and 550 pl C02 L-') occurred when N supply was high (Fig. I). This increase was closely matched by increases in N supply (mg N kg-' soil week-') Fig, 6. The effect of elevated C02 and N supply (0, 17 and 133 mg N kg-' soil week-') on the N uptake of whole cotton shoots grown at either (a) 350 or (b) 900 yl C02 L-l. Each vertical bar represents a standard error (P < 0.05) in one direction (either up or down, n = 4). sink strength, i.e. there were more flowers (Fig. 3a) and larger stems (Fig. 2c). When the C02 concentration was increased to 900 pl L-l, however, there was no additional shoot growth (Figs 1 and 2a) and this response was closely matched by sink strength (Figs 2c and 3a). Similarly, shoot growth of other indeterminate species such as pines and soybean showed a maximum response to C02 enrichment between 330 and 500 pl L-' (Rogers et al. 1980). In contrast, for wheat under the same experimental conditions, the greatest increase in shoot dry weight (65%) occurred between 550 and 900 pl C02 L-I with only a 35% increase between 350 and 550 pl C0, L-I (Rogers et al. 1995). The difference in C02 response between wheat and these other species when nutrient limitations are removed, may lie in a greater capacity of grasses to generate sinks in the form of tillers. In the shoots of cotton, sink-generating capacity is restricted to increasing the number of leaves (leaves are sinks initially), the number of flowers and developing fruits, and stem size. This difference between grasses and other species in the shape of shoot growth response to increasing CO, concentrations could be significant in terms of competition between species. The supply of N had a marked effect on sink generation in cotton and consequently influenced the growth response to increasing atmospheric CO, concentrations. At low N supply, flower number (Fig. 3a) and stem weight (Fig. 2c) responded little to increasing C02 concentrations and shoot growth closely matched this response (Fig. 2a). Similarly low N supply reduced tillering in wheat and therefore inhibited the capacity to respond to high C0, (Rogers et al. 1995).

6 124 G. S. Rogers et al. We also report two significant findings in relation to the interactive effect of CO,, and N supply on leaf N concentration. First, the reduction in leaf N concentration in cotton in response to elevated C02 (Wong 1979; Huluka et al. 1994) is highly dependent on N supply. At levels of N supply which restricted growth (less than 33 mg N kg1 soil week-'), we observed an 18-33% reduction in leaf N concentration. This is comparable with the effects reported by Wong (1979) and Huluka et al. (1994), i.e. respective reductions of 50 and 32%. We found, however, that if the N supply was increased to that which allowed maximum shoot growth, the concentration of N in the leaves of elevated C02-grown plants increased while those of ambient C02- grown plants did not (Fig. 40). The result was that at N supplies of 67 and 133 mg N kg-] soil week-', there was no reduction in leaf N concentration due to C02 enrichment. Increased non-structural carbohydrate in the leaves at high C02 contributed to, but did not fully account for, the effect of elevated CO,, on leaf N concentration (Fig. 4b). The lack of CO,, effect on leaf N concentration when N is freely available may be a general response since we found a similar pattern in wheat (Rogers et al. 1995). Our second finding relates to the importance of the basis of expression of leaf N concentration. The reduction in leaf N concentration due to C02 enrichment appeared to be much greater when leaf N was expressed on a dry weight basis (Fig. 4a) compared with a leaf area basis (Fig. 4c). This discrepancy cannot be explained by additional nonstructural carbohydrate (Fig. 4b). We therefore suggest that increases in structural leaf weight at either high CO,, or high N supply are an important component of the explanation of effects of elevated CO,, and N supply on leaf N concentration. Low SLA (about 1.8 dm2 g-') at either high CO,, or high N supply support this idea (Fig. 34. At high N supply, A, will potentially be high (Wong 1979; Evans 1989). When N supply is low, A,,, will be lower but able to increase in response to elevated CO,, due to the increased carboxylation efficiency of Rubisco, provided electron transport and ribulose 13-bisphosphate regeneration capacity are not limiting (Bowes 1991). Thus, when either N or CO,, supplies are high, we suggest there may be sufficient assimilate available to permit maximum secondary cell wall thickening in the leaves to allow an increase in the thickness of the mesophyll cell layer; perhaps explaining the SLA response (Fig. 3d). The idea of a thicker mesophyll layer at elevated CO,, is supported by Rogers et al. (1981) who report a 28% increase in the thickness of expanded soybean leaves due to increased mesophyll growth at 910 pl C02 L-' compared with plants grown at ambient C02. Further evidence of an increase in the number or size of mesophyll cells is supported by the increase in individual leaf size at elevated CO,, in this experiment (Fig. 3c), and by reports of increased mesophyll cell expansion at high CO,,. See Taylor et al. (1994) for a review. In addition to the effects of elevated CO,, on nonstructural carbohydrate and on structural changes in the leaves, the other main factor responsible for the lower leaf N concentration in high C02-grown cotton in this experiment appeared to be an uptake limitation. The total amount of N in the shoot was the same at both ambient and elevated (900 pl L-') CO,, when the N supply was low or moderate (0 or 17 mg N kg-' soil week-') (Fig. 6). At these levels of N supply, leaf N concentration was significantly (P < 0.01) lower at 900 pl C02 L-', indicating that the limited N in the shoot was distributed over a greater weight of leaf (Fig. 2b) and stem tissue (Fig. 2c). When N was supplied at levels in excess of requirement (133 mg N kg-' soil week-'), the uptake limitation appeared to be overcome and the high C02-grown plants were able to take up proportionally more N than ambient C02-grown plants (Fig. 6). Consequently, under these conditions no reduction in leaf or stem N concentration occurred at high C02 (Fig. 4a and Table 1) despite increased leaf (Fig. 2b) and shoot dry weight (Figs 1 and 2a). The combined effects of elevated CO,, on shoot growth and leaf N concentration resulted in a reduction in the critical N level (leaf N concentration at 90% of maximum shoot growth) from 42 mg g-' at 350 to 36 mg g-' at 900 pl CO,, L-' (Fig. 5). The value of 42 mg g1 agrees with published values for adequate levels in field-grown cotton of a similar age (Reuter and Robinson 1986). The reductions in critical leaf N concentration at elevated C02 were similar in trend but lower in magnitude than previously reported for younger (34 DAS) cotton plants (Rogers et al. 1993). Similarly the critical N concentrations in wheat were reduced at high C02 (Rogers et al. 1995). Acknowledgments We thank Linda Payne for expert technical assistance and Alan Wheen for assistance with the manuscript. This work was supported by an Australian Research Council grant No. A , and a Rural Industries Research and Development Corporation scholarship No. UWS-5a to G. R. References Benedict, C. R. (1984). Physiology. In 'Cotton'. (Eds R. J. Kohel and C. F. Lewis.) pp (Soil Science Society of America: Madison.) Bowes, G. (1991). Growth at elevated C02: photosynthetic responses mediated through Rubisco. Plant, Cell and Environment 14, Conroy, J., and Hocking, P. (1993). Nitrogen nutrition of C, plants at elevated atmospheric C02 concentrations. Physiologia Plantarum 89, Constable, G. (1988). 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