Journal of Experimental Botany, Vol. 51, No. 352, pp. 1879-1892,
November 1, 2000
© 2000 Oxford University Press
Original Papers |
The nitrogen handling characteristics of white clover (Trifolium repens L.) cultivars and a perennial ryegrass (Lolium perenne L.) cultivar
Institute of Grassland and Environmental Research, Plas Gogerddan, Aberystwyth, Ceredigion SY23 3EB, UK
Received 12 January 2000; Accepted 15 June 2000
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Abstract |
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Ryegrass (Lolium perenne L.) and white clover (Trifolium repens L.) have contrasting responses to soil mineral N availability and clover has the ability to fix atmospheric N2 symbiotically. It has been hypothesized that these differences are the key to understanding grass–clover coexistence and vegetative dynamics in pastures. However, the whole plant response of clover and ryegrass to mineral N availability has not been fully characterized and inter-cultivar variability in the N-handling dynamics of clover has not been assessed. A detailed experimental study to address these issues was undertaken. For all clover cultivars and ryegrass, mass specific mineral N uptake rates (of whole plants) were similar saturating functions of mineral N availability. For all clover cultivars total N assimilation rates, whole plant C : N ratios and root : shoot ratios were independent of mineral N availability. Clover growth rates were also independent of mineral N availability except for a slight (<10%) reduction at very low N availability levels. Specific N2 fixation rate (whole plant) was precisely controlled to ensure fixation balanced the deficit between mineral N uptake and the total N assimilation required to maintain constant whole plant C : N ratio. There was always a deficit between N uptake and the total N assimilation required to maintain C : N ratio. Consequently, some N2 fixation remained engaged even at high mineral N availability levels. All inter-cultivar variation in N2 fixation dynamics could be attributed to variations in growth rate. Clover mass specific growth rate declined as plant size increased. Ryegrass specific growth rate, whole plant C : N ratio and root : shoot ratio were dependent on N availability. Increased N availability led to increased growth rate and decreased C : N and root : shoot ratios. Specific growth rate was also dependent on plant size, growth rate declining as plant size increased. It is concluded that clover inter-cultivar variation in field performance is unlikely to be a consequence of variation in N-handling characteristics. Inter-cultivar differences in growth rate are likely to be a much more important source of variation.
Key words: C : N ratios, growth rates, Lolium perenne, nitrogen fixation, nitrogen uptake, plant size, Trifolium repens.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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White clover (Trifolium repens L.) is an important herbage legume in low input sustainable pastures in temperate regions of the world (Sprent and Mannetje't, 1996
). It is often grown in association with perennial ryegrass (Lolium perenne L.). Perennial ryegrass (and other grass species) and white clover have contrasting responses to soil mineral N availability. Ryegrass acquires most of its N from mineral N in the soil whereas clover can supplement uptake from the soil with N acquired by fixation. It has been hypothesized that this difference is the key to understanding grass–clover coexistence and vegetative dynamics in low input agricultural swards (Schwinning and Parsons, 1996a).
Recent field-scale dynamic growth models emphasize the importance of grass–clover–soil mineral N interactions (Thornley et al., 1995; Schwinning and Parsons, 1996b). In these models, the yields, grass–clover balance and soil mineral N levels of swards are ultimately determined by the physiological characteristics of the vegetation. The competitive ability, N2 fixation efficiency (rates of N2 fixation per unit energy consumed) and N-handling characteristics of the clover can all have an impact on sward dynamics. N-handling characteristics may be a useful trait to alter by breeding to produce clover cultivars that are more abundant in swards (Chapman et al., 1996).
There have been several studies of the N-handling characteristics of ryegrass and white clover (Høgh-Jensen et al., 1997; Macduff and Jackson, 1992; Macduff et al., 1989, 1996), but most of these have been carried out far from field conditions in nutrient solution systems. These physiological studies have tended to concentrate on characterizing the response of root mass specific N uptake and nodule mass specific N2 fixation rates to mineral N availability rather than the whole plant response. Furthermore, these studies have tended to concentrate on one or two cultivars and so the issue of inter-cultivar variability has not been addressed. In addition, although several studies have shown that there is in white clover an inverse relationship between N2 fixation and nitrate uptake (Hartwig and Nösberger, 1994, 1996; Parsons et al., 1993; Heim et al., 1993) this response has not been quantitatively characterized. Such characterization is required in order to progress the understanding of the control mechanisms of N2 fixation in white clover and other species.
A series of experiments were set up to address these issues. The specific aims were: (1) to characterize the whole plant response, in terms of mineral N uptake and N2 fixation, to changes in mineral N availability in white clover and perennial ryegrass when plants were grown in a soil-like particulate matrix; (2) determine the amount of inter-cultivar variation in these whole plant responses in white clover; and (3) make a quantitative comparison of the differences between the whole plant N-handling characteristics of ryegrass and white clover.
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Materials and methods |
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Cultivars used in the experiments
The cultivar of perennial ryegrass used in the experiments was AberElan. Details about the white clover cultivars used in the experiments are given in Table 1
. The two self-fertile white clover cultivars were bred at IGER from Australian plants (Michaelson-Yeates et al., 1997). Seedlings and young plants of both these cultivars have slow growth rates (c. 50% that of normal non-self-fertile cultivars). In flowing nutrient cultures and at field mineral N levels (10 kg N ha-1) there was no evidence of N2 fixation by self-fertile line A (Minchin et al., 1997). In flowing nutrient culture seedlings of self-fertile line B have abnormally low rates of nitrate uptake (c. 50% that of a normal cultivars) and their N2 fixation rate is not sensitive to mineral N availability (fixation declining by c. 25% in 20 µM nitrate as compared to a c. 75% reduction for normal cultivars; Minchin et al., 1997).
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Short-term response of white clover cultivars and perennial ryegrass to mineral N availability
The experiment was carried out in a controlled environment glasshouse. Growth conditions were 14 h photoperiod with a minimum illumination level of 30 W m-2 of PAR and minimum daily and nightly temperatures of 18 and 12 °C, respectively.
Soil was collected from a field on Brynllys organic farm, near Dol-y-Bont, Aberystwyth, Wales (O.S. Ref. SN 624893). This was used to prepare a soil micro-organism inoculum (which contained Rhizobium and mycorrhizal propagules as well as propagules of other soil organisms). 2.215 kg fresh weight of soil was mixed with 2 l of reverse osmosis water and the resulting soil suspension left to stand for 5 min. It was then passed through a mesh (500 µm diameter pores) and the filtrate stored overnight at 4 °C prior to use.
Scarified clover and grass seed were sown in coir loam trays. Trays were initially watered with the previously prepared inoculum solution (100 ml tray-1). Thereafter they were kept moist with tap water. After 12 d germinated seedlings were thinned out and after 17 d the remaining seedlings were watered with a nutrient solution containing 200 ppm KNO3 (100 ml tray-1; see below for details of nutrient solution).
After 25 d single seedlings with their associated coir loam plugs (5 cm deep 1.8 cm diam.) were transferred into 1.0 l plastic pots filled with Terra-Green soil conditioner, an inert absorbent granular attapulgite clay (mesh sizing ISO 565(TBL 2) 1983 mm/microns 2.36–1.18, OIL-DRI, Bannisters Row, Wisbech, Cambs, PE13 3HZ). The highest levels of nitrate N in typical samples of Terra-Green are quoted as 138 mg l-1. Seedlings of each cultivar were assigned randomly to one of eight treatments (eight replicates per treatment per cultivar) and their position in the glasshouse randomized. Treatments 1 and 2 were control treatments, treatments 3–8 were destined for nitrate applications of 8.8, 17.5, 35.1, 52.6, 70.1, and 87.7 mg of N enriched to 5.2618 atom% 15N pot-1, respectively. These N application levels are approximately equivalent to one-off N application rates of 11.5, 23, 46, 69, 92, and 115 kg ha-1, respectively. Each pot was placed on a saucer to ensure the solutions applied to each individual plant would not flow to adjacent plants.
Pots were watered with nutrient solution which contained no N (400 ml pot-1) twice a week for the duration of the experiment. This amount of solution was enough to flood pot saucers and therefore wash through the growth medium and leach out residual N. The nutrient solution was then made up by adding 300 ml of solution A and 60 ml of solution B to 120 l of 0.3587 g l-1 CaSO4.2H2O (precipitated general purpose reagent). Solution A: 1.0 l of 275 g l-1 MgSO4.7H2O, 1.0 l of 40 g l-1 NaFeEDTA, 2.0 l of 125 g l-1 K2SO4, and 1.0 l of micronutrient stock solution. Solution B: 1 kg of K2HPO4 in 5.0 l of water. Micronutrient stock solution: 2.025 g MnSO4.4H2O, 0.08 g CuSO4.5H2O, 0.22 g ZnSO4.7H2O, 2.85 g H3BO3, 0.1 g NaMoO4.2H2O, 0.575 g CoSO4.7H2O, 0.005 g NaCl, and 0.0475 g NiSO4.7H2O in 1.0 l of water.
After 62 d (when all grass plants and non-fixing white clovers were showing classic symptoms of N deficiency) eight plants of each cultivar were sampled (control treatment 1) in order to establish the base line status of plants. Each plant was separated from the Terra-Green and coir loam medium and divided into roots and shoots. These were then washed in tap water and dried in an oven at 60 °C for 48 h. Their oven dry weights were determined and each ground to a fine powder. Samples of the powdered material were then analysed by mass spectroscopy to determine C : N and 15N : 14N ratios.
Experimental treatments 2–8 were applied to remaining plants on the following day. Appropriate amounts of N enriched to 5.2618 atom% 15N were added to each pot in the form of KNO3 dissolved in 200 ml of nutrient solution amended with 0.7978 g l-1 K2HPO4 and 0.3989 g l-1 KH2PO4. Two hundred ml of solution would not overflow the pot saucers, thus all N present in the treatment solutions was available for plant uptake.
All plants were sampled 5 d after experimental treatments had been applied. Each plant was separated from the Terra-Green and coir loam medium, washed in tap water and dried in an oven at 60 °C for 48 h. Their oven dry weights were determined and each ground to a fine powder. Samples of the powered material were then analysed as before.
The mass of 15N taken up from the enriched N pool over the 5 d experimental period (A0-5) was:
 | (1) |
, 14N : 15N ratio in the enriched N pool available for uptake; 
, 14N : 15N ratio in the unenriched N pool.
Equation 1 was derived as follows: the mass of 14N taken up from the enriched pool over the 5 d experimental period was A0-5. The total mass of 14N taken up prior to exposure to the enriched N pool was (C5-A0-5). Therefore:
 | (2) |
Equation 2 can be rearranged to give Equation 1. It was assumed that the 15N : 14N ratio of atmospheric N and any N present in the growth medium was the same and constant with respect to time. It was also assumed that there was no biological discrimination between N isotopes.
The dry weight of plants (D0) at beginning of experiment was estimated as follows:
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The specific N uptake rate (U, mass N per unit dry mass per day) was:
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The 15N : 14N ratio of plant material at the end of the 5 d experimental period (
5) was:
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 | (6) |
is the mass 14N fixed per unit mass 15N fixed. By rearranging Equation 5 we find F:
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Note that the aim was to assess the whole plant response to mineral N and that therefore these fixation and uptake rates are expressed in terms of rates per unit whole plant dry mass. These rates are not the same as the physiological rates of these processes which are usually expressed in terms of fixation per unit nodule mass and uptake per unit root mass.
Long-term response of white clover and perennial ryegrass to mineral N availability
Pots of Terra-Green containing 25-d-old seedlings of white clover cv. AberCrest or perennial ryegrass cv. AberElan were prepared in exactly the same way as described for the short-term experiment. Seedlings of each plant cultivar were assigned randomly to one of three treatments (24 replicates per treatment per cultivar) and their position in the glasshouse randomized. Treatments 1, 2 and 3 were control, low and high nitrate treatments, respectively. Each pot was placed on a saucer to ensure the solutions applied to each individual plant would not flow to adjacent plants.
Twice a week plant pots were flooded through with tap water and then 400 ml of nutrient solution (for details see above) or nutrient solution amended with either 0.14 or 1.38 g l-1 KNO3 (treatments 2 and 3, respectively) was added to each.
63, 82 and 97 d after sowing eight replicate plants per treatment per cultivar of AberCrest and AberElan were sampled. Each plant was separated from the Terra-Green and coir loam medium and divided into roots and shoots and analysed as before.
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Results |
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Short-term response of white clover cultivars and perennial ryegrass to mineral N availability
Details of the characteristics of white clover cultivars and ryegrass plants (prior to N application) are given in Table 2
. Ryegrass had nearly double the root : shoot ratio of clover cultivars and much higher C : N ratios of root and shoot materials.
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Mineral N availability, cultivar and interactions between these factors had significant effects on whole plant C : N ratio and specific N fixation and uptake rates (Table 3
). N availability had no effect on white clover growth over the 5 d experimental period. Significant differences in the dry weights of clover cultivars at the end of the 5 d experiment were a result of differences that existed before N-application (Table 2
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Overall the self-fertile white clover cultivars and L. perenne had approximately double the specific N uptake rates of the other white clover cultivars (Table 4
). Out of the non-self-fertile cultivars Grasslands Huia had the fastest uptake rate and AberVantage and AberCrest the slowest. For all white clover cultivars and L. perenne specific N uptake rate was a saturating function of N availability (Figs 1, 2). A first order Michaelis-Menten type relationship fitted the data reasonably well (Cornish-Bowden, 1995; Table 5). Since these experiments deal with rates expressed on a unit whole plant dry mass basis the parameters describing these saturating functions are not directly equivalent to physiological parameters Vmax and Km. Estimated maximal (saturated) uptake rates (Umax; maximum nitrogen influx rate mg N unit whole plant dry weight-1 d-1) were similar for all white clover cultivars, but estimated K values (nitrate concentration at which uptake is half Umax) were more variable. Amongst the non-self-fertile cultivars AberVantage had the highest K and Grasslands Huia and AC57 had the smallest (compare estimated K values in Table 5). Both self-fertile cultivars had much lower K than the other cultivars.
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Overall amongst the non-self-fertile cultivars Grass-lands Huia had the fastest mass specific N fixation rate and AberVantage and AberCrest the slowest, the fixation rate of Grasslands Huia being approximately twice that of the latter (Table 4
). Self-fertile lines A and B had the lowest and highest fixation rates, respectively, of all the cultivars studied.
For all of the white clover cultivars there was a clear inverse linear relationship between N uptake rate and N fixation rate (Fig. 3). For all the cultivars, with the exception of self-fertile A, the gradient of the response (m; decrease in uptake rate per unit increase in fixation rate) did not differ significantly from -1. However, the specific fixation rate when uptake was zero (intercept with x-axis) varied between cultivars being greatest for Grasslands Huia and self-fertile B and least for AberVantage.
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Total specific N assimilation rate (fixation and uptake) was approximately constant and independent of mineral N availability for all the non-self-fertile cultivars (Fig. 1
). Specific N assimilation rate was greatest in Grasslands Huia and least in AberVantage and AberCrest. For self-fertile line A specific N assimilation rate increased to a constant value at high mineral N availability. For self-fertile line B specific N assimilation rate decreased to a constant value at high mineral N availability.
Overall all non-self-fertile lines had similar C : N ratios (c. 8.5; Table 4). The mean C : N ratios of self-fertile lines A and B were slightly higher and lower, respectively. Ryegrass had a mean ratio of 16.8. The C : N ratio of non-self-fertile cultivars declined slightly (c. -0.6) with increasing N availability. For AC57, AberCrest and AberVantage F=6.15, 9.50 and 4.69, respectively (df=6, P<0.001). For Grassland Huia and Menna F=4.06 and 3.47, respectively (df=6, P<0.01). For AberHerald F=3.09 (df=6, P<0.05). There were similar but more marked declines for self-fertile lines A and B and ryegrass (-6.1, -1.8 and -14.3 respectively). For lines A and B and ryegrass F=43.33, 16.09 and 16.45, respectively (df=6, P<0.001).
Long-term response of white clover and perennial ryegrass to mineral N availability
MANOVA analysis indicated species, sample time, N availability and interactions between these factors had significant (P<0.001) effects on measured variables (Table 6). Most of the variation was due to species and time differences. Ryegrass growth (cv AberElan) was directly related to mineral N availability (Fig. 4). White clover (cv AberCrest) growth was relatively independent of N availability, although growth was slightly enhanced in the presence of mineral N (c. 10% increase at both high and low mineral N levels; Fig. 4).
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Litter production by ryegrass increased markedly after 80 d and after 100 d was correlated with N availability and plant size (Fig. 4
). White clover litter production rate did not show a similar marked increase between 80 and 100 days. The C : N ratio and root : shoot ratio of white clover was independent of N availability (Fig. 4). However, for ryegrass these ratios were dependent on N availability. Both C : N and root : shoot ratios declined at high levels of N availability (Fig. 5).
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For both ryegrass and white clover at all N levels whole plant dry mass (g) was a power function of time (d). R2 values for fitted relationships ranged from 0.97–0.99 for white clover and 0.93–0.98 for ryegrass (all significant at P<0.001 level). For white clover the allometric exponents and constants at zero, medium and high N availability levels were 6.22 and 1x10-11, 6.12 and 2x10-11 and 5.62 and 2x10-10, respectively (Fig. 6
). For ryegrass the allometric exponents and constants at zero, medium and high N availability levels were 3.00 and 5x10-6, 4.00 and 1x10-7 and 6.82 and 1x10-12, respectively.
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Discussion |
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Many plants, including white clover and perennial ryegrass, have nitrate-inducible nitrate uptake systems which characteristically have high-affinity for nitrate ions and low maximum influx rates (Vmax, µmol g-1 root FW h-1; Glass and Siddiqi, 1995
; Høgh-Jensen et al., 1997). In many plants Vmax is dependent on internal N status, the uptake system being up-regulated when N status is poor and down-regulated when N status is high (Glass and Siddiqi, 1995; Oscarson et al., 1989). The Km (external nitrate concentration at which uptake is half Vmax, mmol m-3) values of these uptake systems are usually less sensitive to internal N status (Van de Dijk et al., 1982). Several studies of plants growing in nutrient solutions at room temperature have shown that ryegrass and white clover nitrate uptake systems typically have Vmax values in the ranges c. 9–11 and 7–5 and Km values in the ranges c. 86–140 and 130–156, respectively (Høgh-Jensen et al., 1997; Macduff and Jackson, 1992; Macduff et al., 1989, 1996). It is recognized that in the long-term it is probably growth rate, rather than kinetic N uptake parameters, which determines net N absorption (Clement et al., 1978).
In the present study nitrate uptake rates are expressed in terms of rates per unit whole plant dry mass and so direct comparisons with the above physiological parameter values are not possible. In addition, the kinetic parameters for nitrate uptake in this study refer to net uptake rates since, over the relatively long duration of the experiment, there may have been significant nitrate efflux from the plants (Høgh-Jensen et al., 1997). In this study the K of ryegrass was slightly greater than the K of clover plants of similar size (c. 95 compared to 80 kg N ha-1). However, ryegrass had nearly double the root : shoot ratio of white clover suggesting that the physiological Vmax of the ryegrass in this study was probably smaller than that of white clover.
In white clover and other legumes, mineral N concentration in the growth medium has an indirect impact on N2-fixing activity. It has been previously reported that legumes like white clover down-regulate N2 fixation to minimal but non-zero levels when soil N is high, fixed N still accounting for c. 15% of total plant N (Davidson and Robson, 1985, 1986a, b). Nitrate uptake and N2 fixation tend to be negatively correlated. It has been hypothesized that N2-fixing activity (nodule activity) is regulated by the internal N status of the plant (Hartwig and Nösberger, 1994).
In the present study, a clear linear inverse relationship was found between specific nitrate uptake and N2 fixation rates in white clover (Fig. 2). For all the cultivars, with the sole exception of self-fertile line A, the gradient of the response did not differ significantly from -1. This is a strong indication that N2 fixation was regulated in order to keep specific N assimilation rate constant. Thus, the results provide strong support for the hypothesis that N2-fixing activity is regulated by the internal N status of the plant.
The absence of any deviation from linearity in the response at high nitrate uptake rates suggests that white clover had the potential to down regulate fixation to zero levels. However, for this study's clover plants (which were all less than 100 d old) N2 fixation rate was never zero. This is because there was an upper limit to the specific rate of N uptake (Umax, 0.009 g N g-1 d-1) and the specific rate of N assimilation required to maintain a constant C : N ratio (see below) was always greater than Umax (Fig. 1).
At high mineral N availability the specific growth rate of white clover cv. AberCrest (the white clover cultivar which was used as an example in the long term experiment) was similar to that ryegrass cv. AberElan (Table 2; Fig. 4). For both plants specific growth rate decreased at a similar rate as whole plant dry mass increased. For white clover the observed inter-cultivar variation in the mass of plants of similar age (Table 2) indicates that these growth functions are cultivar specific. At low mineral N availability ryegrass specific growth rate decreased more rapidly than that of clover as whole plant mass increased. The size related decline in AberCrest specific growth rate was unaffected by mineral N availability (although actual growth rate was slightly lower; Fig. 6). White clover has, therefore, a clear growth advantage over ryegrass under N-limited conditions. It is important to note that under such conditions clover's advantage will be dependent on the sizes of the competing grass and clover plants, a factor that is not taken into account in existing models of grass–clover interactions.
The reasons for the decline in plant mass specific growth with increasing plant size are unclear, but similar scaling relationships are found throughout the plant and animal kingdoms (Enquist et al., 1998). In this experiment it is unlikely that these relationships arose as a consequence of increasing limitation in the availability of mineral nutrients or water, since these were always present in excess. It could be argued that the reduction in plant growth with increasing size was a response to decreasing N assimilation per unit plant mass. This might possibly be true for ryegrass, however, growth of white clover plants did not increase when mineral N availability was increased, whatever their size. It is therefore highly unlikely that white clover growth is driven by mineral N availability.
The marked effect of plant size on plant growth must have had an impact on plant N-handling rates otherwise the C : N ratio of plants would have decreased substantially as they increased in size. In the long term such decreases were never observed (Fig. 4). In white clover, and to a lesser extent ryegrass, growth and N-handling rates must therefore be co-ordinated. This fact is unaffected by the exact nature of causal relationships or interrelationships between growth and N handling. Any interpretation of the experimental data on whole plant N handling rates must therefore take effects of plant size into account.
The fact that C : N ratio of white clover was constant (independent of N availability and growth) indicates that total specific N assimilation rate in this species was proportional to growth rate. Ryegrass C : N ratio increased when N was limiting and its growth was therefore not directly proportional to N uptake. However, its C : N ratio did not decrease and therefore its specific N assimilation rate must also be some function of its specific growth rate. The increase in ryegrass shoot : root ratio at low N availability is bound to contribute to the observed reduction in its specific growth rate because it leads to a marked reduction in the proportion of the plant that is actively engaged in C assimilation.
In white clover there were substantial between-cultivar variations in the K parameter which describes the kinetics of whole plant specific nitrate uptake rate (the N availability level (kg ha-1) at which N uptake was 4.5 mg N g-1 d-1). Most of this variation can be explained by between-cultivar variation in mean whole plant mass during the experimental period (Fig. 7). Estimated K values were directly proportional to plant mass. Since Umax was independent of plant size and similar for all cultivars this suggests that specific N uptake rate was a function of specific growth rate and an inverse function of plant mass. This is exactly what would be expected if N uptake rate decreased to match the size-related decline in specific growth rate and so keep whole plant C : N ratio constant.
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Much of the between-cultivar variation in whole plant specific N fixation rate can also be explained by the combined effects of plant size and growth rate. Plant specific N fixation rate was also an inverse function of plant mass (Fig. 7
). The observed size-related reductions in specific N uptake rates in isolation would not be sufficient to keep plant C : N ratios constant. Specific fixation rates must, therefore, also decline as plants grow in order that total specific N assimilation rates remain directly proportional to growth rate. The actual N2 fixation rate of a clover plant at any given mass will depend on its specific growth and N uptake rates at that mass. Similarly sized plants with similar specific N uptake rates but different specific growth rates will have different specific fixation rates, the plant with the higher specific growth rate having the highest specific fixation rate.
The self-fertile line A cultivar was anomalous in several respects. At high N availability levels the C : N ratio of this cultivar was normal, but its fixation rate was lower than expected on the basis of its mean plant size (Fig. 7). This cultivar had an unusually low growth rate given its mean plant size (Table 2) and the plants control over fixation rate therefore appears instrumental in maintaining normal C : N ratio under these conditions. At low N availability levels, tissues of this cultivar had unusually high C : N ratios. This was probably a consequence of the fact that N fixation was not able to supply enough N to compensate for the reduced N uptake. N fixation appears to be in some way deficient. These observations suggest that in the long term the growth rate of this white clover cultivar may be influenced by N availability in much the same way, but to a less marked extent, as ryegrass growth. As in ryegrass, reductions in growth may be a result of a greater proportion of resources being channelled into root rather than shoot growth. Interestingly, the root : shoot ratio of this cultivar was greater than that of all the other clover cultivars. The N handling and growth characteristics of the other self-fertile cultivar were far less anomalous. Plants of this cultivar appear only to have a reduced growth rate.
The fact that white clover down-regulates fixation in response to increasing N uptake and plant size suggests that maximum specific growth rate is not N limited but determined by other parameters. Under many circumstances white clover's N2 fixation system may have excess capacity, since the specific fixation rates of younger (smaller) plants are greater that those of older (larger) plants. White clover plants under field conditions are subjected to varying N availability, therefore it is likely that size- and growth-related declines in N2 fixation are reversible. If this is so it is likely that the degree of root nodulation is relatively unimportant because of a high degree of effective redundancy in established nodules. Nodule development is known to be influenced by growth medium nitrate concentration (Parsons et al., 1993). Both the nitrate concentration in the nodulated root zone and the internal N status of the plant have a negative impact on nodule development (Blumenthal et al., 1997). Also white clover cultivar–Rhizobium strain interactions can have an impact on N2 fixation and nodule development (Cresswell et al., 1992). However, it remains to be seen whether these effects have any impact on whole plant growth.
Nodulated white clover plants that are actively fixing N2 respire c. 10% more fixed carbon that non-nodulated plants with an ample supply of nitrate (Ryle et al., 1979). Indeed in the long-term experiment AberCrest growth was slightly enhanced when it is less reliant on N2 fixation (Fig. 5). The slight decline in the C : N ratio of whole plants (normal self-fertile white clover cultivars) when N availability was high in the short-term experiment (Fig. 3) might also reflect this. The slight increase in levels of fixed C may not yet have been countered by a slight increase in specific growth rate.
Recent dynamic growth models of grass legume swards predict that it is advantageous for clover to down-regulate N2 fixation rapidly and compete with grass for N when soil mineral N levels start to increase above the level which is limiting for grass growth (Chapman et al., 1996; Schwinning and Parsons, 1996b). This is because metabolic costs of N2 fixation are greater than those of mineral N uptake (Ryle et al., 1979) and because growth of the grass competitor is a function of soil mineral N availability. These models, therefore, support the notion that much of the between-cultivar variability in clover performance in the field could be a result of variability in their N handling characteristics and/or N2 fixation efficiencies.
The results of this present study indicate that there is probably little inter-cultivar variation in N handling characteristics of white clover. Most of the variation encountered in the study could all be attributed to variations in plant size and cultivar growth rates. It was therefore concluded that the observed inter-cultivar variations in field performance are probably not a result of differences in N-handling characteristics. Such variations are more likely to be a result of inter-cultivar variations in N2 fixing efficiency and/or growth rate.
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Acknowledgments |
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Our thanks to Mike Collinson, Mick Fothergill, Mike Hay, Dafydd Jones, Derek Jones, Masil Khan, Mark Levey, James Macduff, Frank Minchin, Lance Mytton, Tony Parsons, Ruaraidh Sackville Hamilton, and Susanne Schwinning for helping to process ryegrass samples or providing useful advice and comments on the manuscript and data analysis. Thanks to IGER's Plant Breeding Department for providing the white clover and ryegrass cultivars. This work was funded by the BBSRC.
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Notes |
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1 To whom correspondence should be addressed. gwyn.griffith{at}bbsrc.ac.uk
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References |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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