Is Partitioning of Dry Weight and Leaf Area Within Dactylis glomerata A ected by N and CO 2

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1 Annals of Botany 8: 833±839, doi:1.1/anbo..13, available online at on Is Partitioning of Dry Weight and Leaf Area Within Dactylis glomerata A ected by N and CO Enrichment? H. HARMENS*{}, C.M.STIRLING{{, C. MARSHALL} and J. F. FARRAR} {Centre for Ecology and Hydrology Bangor, University of Wales, Bangor, Gwynedd LL57 UP, UK and }School of Biological Sciences, University of Wales, Bangor, Gwynedd LL57 UW, UK Received: 5 April Returned for revision: 15 May Accepted: June Published electronically: 1 August We examined changes in dry weight and leaf area within Dactylis glomerata L. plants using allometric analysis to determine whether observed patterns were truly a ected by [CO ] and N supply or merely re ect ontogenetic drift. Plants were grown hydroponically at four concentrations of NO 3 in controlled environment cabinets at ambient (3 ml l 1 ) or elevated (8 ml l 1 ) atmospheric [CO ]. Both CO and N enrichment stimulated net dry matter production. Allometric analyses revealed that [CO ] did not a ect partitioning of dry matter between shoot and root at high N supply. However, at low N supply there was a transient increase in dry matter partitioning into the shoot at elevated compared to ambient [CO ] during early stages of growth, which is inconsistent with predictions based on optimal partitioning theory. In contrast, dry matter partitioning was a ected by N supply throughout ontogeny, such that at low N supply dry matter was preferentially allocated to roots, which is in agreement with optimal partitioning theory. Independent of N supply, atmospheric CO enrichment resulted in a reduction in leaf area ratio (LAR), solely due to a decrease in speci c leaf area (SLA), when plants of the same age were compared. However, [CO ] did not a ect allometric coe cients relating dry weight and leaf area, and e ects of elevated [CO ] on LAR and SLA were the result of an early, transient stimulation of whole plant and leaf dry weight, compared to leaf area production. We conclude that elevated [CO ], in contrast to N supply, changes allocation patterns only transiently during early stages of growth, if at all. # Annals of Botany Company Key words: Allometric growth, carbon dioxide enrichment, Cocksfoot, Dactylis glomerata L., dry weight partitioning, leaf area ratio, nitrogen supply, shoot: root ratio, speci c leaf area. INTRODUCTION Doubling atmospheric [CO ] stimulates the growth of C 3 species by an average of 1 % (Poorter, 1993). The magnitude of this increase varies with the individual species (Hunt et al., 1991; Poorter, 1993), experimental duration and the availability of other resources such as N (Bazzaz, 199). When N limits growth, root dry weight increases relatively more than shoot dry weight, perhaps maintaining a functional equilibrium that results in the balanced acquisition of carbon and nitrogen (Davidson, 199; Reynolds and Thornley, 198; Brouwer, 1983; Wilson, 1988). Atmospheric [CO ] might also a ect the allocation of dry matter between shoots and roots, although contrasting results have been reported for the shoot to root ratio (S : R ratio, de ned as the dry weight of shoot divided by the dry weight of root): CO enrichment may either increase, decrease or not a ect S:R ratio (Hunt et al., 1991; Stulen and Den Hertog, 1993; Rogers et al., 199). Although [CO ] and N supply would be expected to interact regarding dry matter production and partitioning (i.e. the distribution of dry weight within the plant), the outcome cannot be predicted (Lloyd and Farquhar, 199). * For correspondence. Fax () , hh@ceh.ac. uk { Present address: School of Agricultural and Forest Sciences, University of Wales, Bangor, Gwynedd LL57 UW, UK. Comparisons of the e ects of treatments on plant growth are often based on ratios such as S : R with plants of the same age. However, patterns of allocation between plant parts change during growth and development independent of resource availability (Pearsall, 197; Bowler and Press, 1993; Farrar and Gunn, 199). Treatments may a ect growth and development and alter S : R ratio and morphological characteristics compared to the control either because of true treatment e ects or ontogenetic drift, i.e. phenotypic traits of plants change over the course of plant growth and development (Evans, 197; Coleman et al., 199; Farrar and Gunn, 1998; McConnaughay and Coleman, 1999). Di erentiation between true e ects of treatment and of ontogenetic drift is possible using allometry, i.e. the study of the growth and development of one part of the plant in relation to another (Pearsall, 197; Troughton, 1955). A few studies have made allometric comparisons between CO treatments, usually restricted to the allometric coe cient relating the net dry matter production of shoot and root (Bowler and Press, 1993; Baxter et al., 199; Farrar and Gunn, 199; Hibberd et al., 199). More recently, some studies have included the allometric relationship of dry matter and leaf area (Farnsworth et al., 199; Gebauer et al., 199; Stirling et al., 1998; Gunn et al., 1999). In general, allometric coe cients were not a ected by [CO ]. In contrast, allocation of dry matter to roots decreased with increasing N supply (Gebauer et al., 199; McConnaughay and Coleman, 1999) // $35./ # Annals of Botany Company

2 83 Harmens et al.ðpartitioning at Elevated CO and N We suspect that many reports claiming that [CO ] alters net dry matter partitioning might be confounding e ects of [CO ] with those of uncontrolled variables, especially when plants are grown in a solid substrate. For example, limited access to nutrients due to either growth limiting concentrations or limited ux from a solid substrate to the roots, can mean that plants grown at elevated [CO ] become more nutrient de cient than controls due to increased dry weight (Stitt and Krapp, 1999), resulting in a correspondingly lower S : R ratio. Recently it has become clear that soil water content can be another confounding variable (Samarakoon and Gi ord, 1995; Knapp et al., 199), and one that is known to a ect dry matter partitioning. Plants rooted in a solid substrate with a nite supply of water may have more favourable water status at elevated [CO ] due to a lower stomatal conductance for water vapour at elevated than ambient [CO ](Drake et al., 1997). Accordingly, good control of nutrient and water supply is needed to distinguish between direct e ects of [CO ] and indirect e ects through nutrient and water status of soils. Here we examine the e ects of elevated [CO ] and the interaction with N availability on growth, partitioning of dry weight, and dry weight±leaf area relationships in Dactylis glomerata L. (Cocksfoot). Plants were grown in controlled environments at either ambient (3 ml l 1 )or elevated (8 ml l 1 )[CO ] and four N concentrations (.15,., 1.5 and. mm NO 3 ), ranging from growth limiting to optimal. Confounding e ects of soil water status were minimized by growing plants in hydroponics. Comparisons between treatments were made both as a function of plant age and by applying allometry. It was hypothesized that elevated [CO ] a ects net allocation of dry matter and distribution of dry matter and leaf area only through accelerated growth, but that N supply has a direct e ect independent of changes in ontogeny. MATERIALS AND METHODS Plant growth Seeds of Dactylis glomerata L., `Sylvan', were sown in propagators on moistened lter paper in controlled environment cabinets (Sanyo Gallenkamp, model SGC/C/HQI, Loughborough, UK) at either ambient (3 ml l 1 ) or elevated (8 ml l 1 )CO concentrations. Nine days after sowing, uniform-sized seedlings with one leaf were transferred to 3.5 l troughs, containing half-strength Long Ashton solution (Hewitt, 19) with 1 mg l 1 of sodium metasilicate. Supply of NO 3 was modi ed to give four concentrations:.15,., 1.5 and. mm. At the smaller N supplies, the potassium and calcium concentrations were made up by adding K SO and CaCl. The ph of the aerated solutions was controlled between 5.±. using mm MES and KOH. Twenty seedlings were planted in each trough (two troughs per treatment) and after 1 week the nutrient solutions were replaced and the number of plants per trough was reduced to 15. Thereafter, nutrient solutions were replaced twice weekly and the number of plants per trough was reduced at every harvest. The conditions of growth were as described previously (Harmens et al., ). In order to reduce cabinet e ects and e ects of environmental heterogeneity within the cabinets, CO treatments were swapped between the two cabinets twice a week and troughs were randomized within each cabinet. Growth analysis Seven plants per treatment (three±four plants per trough) were harvested at 3, 8, 3 and 38 d after sowing. Plants were separated into leaf blades, leaf sheath stem, and roots. The area of leaf blades was determined using a digital leaf area meter (Delta T Ltd, Cambridge, UK) and plant parts were dried for at least 8 h at 5 8C and weighed. The following parameters were calculated: shoot : root ratio (dry weight), leaf area ratio (LAR; leaf area per plant dry weight), speci c leaf area (SLA; leaf area per leaf dry weight) and leaf weight ratio (LWR; leaf dry weight per plant dry weight). Allometric relationships were determined using means per trough for each harvest and an ordinary linear regression equation (Pearsall, 197; Troughton, 1955): ln y ˆ ln a k ln x where ln a is the y-intercept, k is the slope, and y and x are respectively: shoot and root dry weight; leaf dry weight and total plant dry weight; leaf area and total plant dry weight; and leaf area and leaf dry weight. In the majority of cases, the regression was linear as determined by linear and sequential polynomial regression. Subsequently, the slope (v) of the geometric mean regression was determined for all relationships (Ricker, 198; Farrar and Gunn, 199), as both y and x are dependent variables: v ˆ k=r where r is the correlation coe cient of the ordinary linear regression and v is the allometric coe cient calculated by geometric mean regression. Where there was no signi cant di erence between the slopes due to [CO ] a comparison of the elevations (as opposed to the y-intercepts) of the regressions (i.e. comparison of the vertical position of the lines) was carried out (Zar, 199). Regression lines with the same slope and elevation coincide, whereas regression lines with the same slope but di erent elevation are parallel. Statistical analysis Data were analysed by analysis of variance (ANOVA) of the mean values of each trough (n ˆ per treatment per harvest) using the Genstat statistical package (Lawes Agricultural Trust, Rothamsted, UK). Where indicated, data were ln-transformed prior to analysis to obtain homogeneity of variances. E ects of CO and N supply on the allometric coe cient (v) and elevations of the regression were analysed by pairwise comparison using Student's t-test with 1 degrees of freedom. Unless indicated otherwise, signi cant treatment e ects refer to P.5.

3 Shoot:root ratio Total dry wt (g) mm NO 3. mm NO mm NO 3. mm NO 3 E 3 Harmens et al.ðpartitioning at Elevated CO and N 835 F Time (d) F IG. 1. Total dry weight (A±D) and shoot: root ratio (E±H) of D. glomerata grown at 3 (ÐdÐ) or 8 (- - -s ---) ml l 1 CO and varying [NO 3 ]. Each data point represents the mean of two troughs, three±four plants per trough (+s.e.). G H RESULTS Growth and ratios at the same plant age Total plant dry weight was greater at elevated than ambient [CO ](Fig. 1A±D; Table 1) and although the magnitude of the increase varied, no signi cant interactions between [CO ] and time, or [CO ] and N supply were found. From 8 d onwards, total plant dry weight increased with increasing [NO 3 ]. Responses of shoot and root dry weight to elevated [CO ] and [NO 3 ] were similar to that of total plant dry weight (data not shown), except that N supply did not a ect root dry weight until 3 d. The S:R ratio was generally not a ected by [CO ] and increased with increasing N supply (Fig. 1E±H; Table 1). However, at TABLE 1. Summary of statistical signi cance of e ects of date of harvest, [CO ] and N supply on growth and development parameters of D. glomerata Parameter Source of variation Time N CO Time N W total *** *** *** *** W shoot *** *** *** *** W root *** ** *** *** W shoot :W root *** *** n.s. *** Leaf area *** *** ** *** Leaf area ratio *** ** *** *** Speci c leaf area *** *** *** *** Leaf weight ratio *** *** n.s. *** Number of f.e.l. main stem *** *** n.s. * Total number of leaves *** *** n.s. *** Total number of tillers *** *** n.s. *** Data for dry weight (W) and leaf area were ln-transformed to obtain homogeneity of variance. A general linear ANOVA model was tted to the mean value of each trough, so n ˆ per treatment at each harvest. n.s., Not signi cant, *P.5, **P.1, ***P.1. `Time CO ', `N CO ' and `Time N CO ' interactions were not signi cant; f.e.l., fully expanded leaves. [NO 3 ] above.15 mm there was a tendency towards an increase in S : R ratio due to CO enrichment. Developmental parameters such as number of tillers, leaves and fully expanded leaves on the main stem were not a ected by [CO ], but increased with increasing N availability (Table 1). LAR was signi cantly higher at ambient than elevated [CO ] at all [NO 3 ] and it decreased with time (Fig. A±D; Table 1). SLA was lower in plants exposed to elevated than ambient [CO ], independent of [NO 3 ] (Fig. E±H; Table 1). At.15 mm NO 3 the SLA increased with time, but it decreased with time at 1.5 and. mm NO 3, resulting in a higher SLA at the nal harvest at.15 mm NO 3 compared to 1.5 and. mm NO 3. LWR was not a ected by CO (Fig. I±L; Table 1), thus the decrease in LAR at elevated [CO ] was due solely to a decrease in SLA. At low N supply LWR decreased with time, whilst at high N supply it was nearly constant, resulting in a higher LWR at the nal harvest at high compared to low N availability. Clearly, most ratios changed with time and therefore with increasing plant dry weight. Allometric relationships Elevated [CO ] had no signi cant e ect on any of the allometric coe cients (v) and therefore did not a ect net dry matter partitioning and dry weight±leaf area relationships within the plants between the rst and nal harvest (Figs 3, ; Table ). However, CO enrichment had a signi cant e ect on some of the elevations of regression lines, indicating a transient change in the partitioning of dry matter and leaf area during the initial stages of growth (before the rst harvest). At low N supply the allocation of dry matter to the shoot or leaf blades was increased early on at elevated compared to ambient [CO ]. At high N supply CO enrichment decreased the production of leaf area relative to whole plant dry weight early in growth. Independent of N supply, CO enrichment reduced the

4 83 Harmens et al.ðpartitioning at Elevated CO and N LWR SLA (cm g 1 ) LAR (cm g 1 ) mm NO 3. mm NO mm NO 3. mm NO 3 E F G H I J K L Time (d) F IG.. LAR (A±D), SLA (E±H) and LWR (I±L) of D. glomerata grown at 3 (ÐdÐ) or 8 (- - -s ---)ml l 1 CO and varying [NO3 ]. Each data point represents the mean of two troughs, three±four plants per trough (+s.e.). ln DW leaf (mg) ln DW shoot (mg) 8 E. 15 mm NO 3. mm NO mm NO 3. mm NO F 8 1 ln DW root (mg) G ln DW total (mg) 8 1 H 8 1 F IG. 3. The allometric relationship between shoot and root dry weight (A±D) and leaf and plant dry weight (E±H) of D. glomerata grown at 3 (ÐdÐ) or 8 (---s---) ml l 1 CO and varying [NO 3 ]. Each data point represents the mean three±four plants per trough. production of leaf area relative to leaf dry weight early in ontogeny. In contrast with [CO ], nitrogen supply signi cantly a ected most allometric coe cients (Figs 3, ; Table ). At.15 and. mm NO 3, dry matter was preferentially partitioned to the root (v 5 1), whereas at 1.5 and. mm NO 3 it was allocated equally to shoot and root (v was not signi cantly di erent from 1). Consequently, the increase in leaf weight per unit of plant dry weight was signi cantly lower at low compared to high N supply. At

5 Harmens et al.ðpartitioning at Elevated CO and N mm NO 3. mm NO mm NO 3. mm NO 3 ln leaf area (cm ) 8 1 E ln DW total (mg) F G H ln DW leaf (mg) F IG.. The allometric relationship between leaf area and plant dry weight (A±D) and leaf area and leaf dry weight (E±H) of D. glomerata grown at 3 (ÐdÐ) or 8 (---s---) ml l 1 CO and varying [NO 3 ]. Each data point represents the mean three±four plants per trough. TABLE. E ects of [CO ] and N supply on the allometric coe cient (v) relating dry weight (W) and leaf area in D. glomerata x y CO concentration (ml l 1 ) v [NO 3 concentration (mm)] W root W shoot 3.5 a (q**).79 b (q***).99 c 1.7 c 8.5 a.78 b.99 c 1.5 c W total W leaf blade 3.5 a (q**).8 b (q**).97 c.99 c 8.59 a.83 b.95 c.98 c W total Leaf area 3.7 a.77 a.85 b (Q***).9 c (Q***) 8.77 a.8 a.8 a.88 b W leaf Leaf area a (Q**).91 b (Q***).88 b (Q***).91 b (Q***) a.9 b.8 c.91 bc The natural logarithm of y was plotted against the natural logarithm of x for all harvests and an ordinary linear regression was tted. The allometric coe cient v was then calculated from the slope of the regression (k) and the correlation coe cient (r): v ˆ k=r (r ˆ.98±1.). E ects of [CO ] and N supply on v were tested by pairwise comparison using Student's t-test (degrees of freedom ˆ 1). Di erent superscripts within rows indicate signi cant di erences (P.5) between v at di erent N treatments. Although there were no signi cant e ects of CO on v, signi cant e ects of CO on the elevation of regressions were found as indicated within parentheses: Q or q indicate a signi cant higher or lower elevation at 3 compared to 8 ml l 1 CO, respectively; **P.1, ***P.1. ambient [CO ] the increase in leaf area per unit of plant dry weight was signi cantly less at.15 and. mm NO 3 than at higher [NO 3 ]. At.15 mm NO 3, leaf area increased relatively more than leaf dry weight compared to higher [NO 3 ]. DISCUSSION When growth was analysed allometrically, it was evident that elevated [CO ] in comparison with N supply had minimal e ects on the partitioning of dry matter within D. glomerata. An increase in N availability a ected partitioning throughout ontogeny, such that enhanced N supply reduced allocation of dry matter to roots. Dependent on N availability, elevated [CO ] changed the partitioning of dry weight and dry weight±leaf area relationships within D. glomerata only during the initial stages of growth. Hence, e ects of CO enrichment on S : R ratio, LAR and SLA in plants of the same age were sometimes still present when ontogenetic drift was taken into account. Ratios indicate the state of the plant at an instant in time, are the product of the plant's history and will be an insensitive measure of changes in partitioning throughout ontogeny (Farrar and Gunn, 1998). We applied allometry to identify e ects of treatment independent of ontogeny over a period of growth; allometry has a clear biological

6 838 Harmens et al.ðpartitioning at Elevated CO and N meaning and proved to be statistically more robust than comparing plants on the basis of similar total dry weights (Gunn et al., 1999). We found that dry weight and leaf area to dry weight ratios were subject to ontogenetic drift. Had we not accounted for this, we would have misjudged, in some treatments, the adjustments in allocation patterns. For example, the decrease in LAR due to CO enrichment at low N supply when plants of the same age were compared was simply the result of accelerated growth. On the other hand, a transient increase in allocation of dry matter to the shoot at elevated [CO ] and very low N supply during early stages of growth would not have been detected when only S : R ratios of plants of the same age were compared. Although other studies have shown that elevated [CO ] has little e ect on allometric coe cients (Bowler and Press, 1993; Baxter et al., 199; Farnsworth et al., 199; Gebauer et al., 199; Stirling et al., 1998; Gunn et al., 1999), little attention has been paid to changes in the elevation of regressions (Gunn et al., 1999; Marriott, 1999). Whilst previously values for ln a have been used to quantify treatment e ects on allometry early in growth, extreme care is required since the results of analyses will be highly dependent on the units used, so ln a has little biological signi cance (Baxter et al., 199; Stirling et al., 1998). This study clearly shows that both allometric coe cients and elevations of regressions should be analysed to determine whether changes in partitioning occur throughout ontogeny or are restricted to a de ned period in the plant's development. Whereas CO enrichment did not a ect any of the allometric coe cients (v), it did a ect elevations of the regressions in most cases, indicating a change in partitioning during the initial stages of growth of D. glomerata. These early changes in partitioning are hard to detect, mainly due to the di culties associated with measuring very small plants and the large number of replicates that would be required to pick up small changes in v, which appear to occur in a short period of time. Optimal partitioning models and theory suggest that plants respond to variation in the environment by partitioning dry weight among plant organs to optimize the capture of resources in a manner that maximizes plant growth rate (Reynolds and Thornley, 198; Brouwer, 1983; Wilson, 1988; McConnaughay and Coleman, 1999). In D. glomerata, optimal partitioning did apply to the range of N availability such that at limited N supply dry matter was preferentially allocated to roots. Whereas optimal partitioning models predict increased allocation of dry matter to roots at elevated CO, the partitioning of dry matter between shoots and roots in D. glomerata was little a ected by CO enrichment. In contrast, a transient increase in the partitioning of dry matter to the shoot (and leaves) was observed at elevated [CO ] at low N supply at an early stage of plant development. Clearly, if the basic assumptions of optimal partitioning (i.e. that plants do alter dry weight distribution in response to changes in availability of resources to maximize growth rate) are incorrect, models attempting to predict the ecological outcome of environmental changes based on the optimal partitioning theory should be re-evaluated (McConnaughay and Coleman, 1999). Reductions in SLA, and consequently in LAR, have been reported at elevated CO (Bazzaz, 199; Den Hertog et al., 1993; Poorter, 1993; Baxter et al., 199), and are often correlated with an increase in non-structural carbohydrates. Although the non-structural carbohydrate content was increased in the youngest fully expanded leaf of D. glomerata (Harmens et al., ), reduction in its SLA at elevated compared to ambient [CO ] was still evident when expressed per unit structural dry weight (Harmens, unpubl. res.). The current study indicates that decreases in SLA cannot simply be explained by accelerated growth at elevated [CO ], but are due to changes in the distribution of leaf dry weight and area during early stages of growth. In conclusion this work has shown the importance of assessing the e ects of treatment on dry weight distribution and leaf area development using the dynamic allometric approach rather than by using ratios of plant components which change as a consequence of ontogeny. There is increasing evidence that CO enrichment does not a ect allometric coe cients during ontogeny, in contrast to resources such as N and light (McConnaughay and Coleman, 1999). Therefore, sequential harvesting in combination with allometry is a useful tool to distinguish between e ects primarily due to enhanced [CO ] and confounding e ects such as water and nutrient status of soils. ACKNOWLEDGEMENTS This work was completed whilst H. H. was in receipt of a NERC Post-doctoral Fellowship. We thank Tim Sparks (ITE) for his help on statistical analyses and Ray Rafarel (ITE) for technical support. LITERATURE CITED Baxter R, Ashenden TW, Sparks TH, Farrar JF E ects of elevated carbon dioxide on three montane grass species. I. Growth and dry matter partitioning. Journal of Experimental Botany 5: 35±315. Bazzaz FA The response of natural ecosystems to the rising global CO levels. Annual Review of Ecology and Systematics 1: 17±19. Bowler JM, Press MC Growth responses of two contrasting upland grass species to elevated CO and nitrogen concentration. New Phytologist 1: 515±5. Brouwer R Functional equilibrium: sense or nonsense?. 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