Journal of Experimental Botany, Vol. 53, No. 374, pp. 1635-1642,
July 1, 2002
© 2002 Oxford University Press
The contribution of roots and shoots to whole plant nitrate reduction in fast- and slow-growing grass species
Received 10 December 2001; Accepted 13 March 2002
1 Department of Plant Ecophysiology, Utrecht University, PO Box 80084, 3508 TB Utrecht, The Netherlands
2 Zoology Department, PO Box 56, University of Otago, Dunedin, New Zealand
3 Plant Sciences, Faculty of Agriculture, The University of Western Australia, Crawley WA 6009, Australia
4 Department of Biology, University of York, PO Box 373, York YO1 5YW, UK
Abbreviations: cu, specific respiratory costs for ion transport; cg, specific respiratory costs for growth; NRA, nitrate reductase activity; NRAroot,max, maximum rate of root NO3– reduction; NNUR, net NO3– uptake rate; Proot, proportion of total plant NO3– reduction that occurs in roots; RGR, relative growth rate; RMR, root mass ratio; rt,CO2, total rate of root CO2 evolution with or without NO3– reduction being performed in the roots; rm, specific respiratory costs for cellular maintenance.
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Abstract |
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The hypothesis was tested that slow-growing grass species perform a greater proportion of total plant NO3– reduction in their roots than do fast-growing grasses. Eight grass species were selected that differed in maximum relative growth rate (RGR) and net NO3– uptake rate (NNUR). Plants were grown with free access to nutrients in hydroponics under controlled-environment conditions. The site of in vivo NO3– reduction was assessed by combining in vivo NO3– reductase activity (NRA) assays with biomass allocation data, and by analysing the NO3– to amino acid ratio of xylem sap. In vivo NRA of roots and shoots increased significantly with increasing NNUR and RGR. The proportion of total plant NO3– reduction that occurs in roots was found to be independent of RGR and NNUR, with the shoot being the predominant site of NO3– reduction in all species. The theoretical maximum proportion of whole plant nitrogen assimilation that could take place in the roots was calculated using information on root respiration rates, RGR, NNUR, and specific respiratory costs associated with growth, maintenance and ion uptake. The calculated maximum proportion that the roots can contribute to total plant NO3– reduction was 0.37 and 0.23 for the fast-growing Dactylis glomerata L. and the slow-growing Festuca ovina L., respectively. These results indicate that slow-growing grass species perform a similar proportion of total plant NO3– reduction in their roots to that exhibited by fast-growing grasses. Shoots appear to be the predominant site of whole plant NO3– reduction in both fast- and slow-growing grasses when plants are grown with free access to nutrients.
Key words: Key words: Nitrate uptake, nitrate reductase, nitrogen, nitrogen assimilation, relative growth rate.
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Introduction |
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Nitrate is a major form of nitrogen available to plants in many environments (e.g. warm temperate soils). Before the NO3– that is taken up by the plant can be assimilated into organic nitrogen, it must first be reduced to NO2– and then to NH4+ The reduction of NO3– to NO2– is catalysed by nitrate reductase (NR). Nitrite reductase (NiR) catalyses the reduction of NO2– to NH4+. The assimilation of NO3– into organic nitrogen requires the input of reductant, with two electrons being needed for the reduction of NO3– to NO2– by NR, and a further six for the NiR-catalysed reduction of NO2– to NH4+. ATP is required for the assimilation of NH4+ into amino acids and proteins.
The site of NO3– reduction (i.e. the reduction of NO3– to NH4+ in roots versus that in shoots) may have a substantial impact on the carbon demands of NO3– assimilation in a particular plant. For example, species that reduce NO3– predominantly in their shoot may have the advantage of being able to use excess reductant produced in photosynthesis (Pate, 1983). By contrast, species that reduce NO3– mainly in their roots must obtain their reductant from glycolysis and the oxidative pentose phosphate pathway (Oaks and Hirel, 1986; Bowsher et al., 1989), coupled to the release of CO2 and increasing the respiratory quotient. The site of NO3– reduction therefore has impacts on the plant’s carbon budget.
Are there systematic differences in the site of NO3– reduction in contrasting plant species that differ in daily net carbon gain and maximum relative growth rate (RGR, the increase in dry mass per unit mass and time)? Gojon et al. (1994) proposed that, when the external NO3– supply is not limiting, slow-growing woody plant species carry out a greater proportion of total plant NO3– reduction in roots than in shoots, when compared with fast-growing herbaceous species. This proposal was based on the hypothesis that NO3– reduction in roots competes with delivery of NO3– to the xylem (for transport to the shoot), which thus determines the proportion of NO3– reduced in the roots versus that in the shoots. Significant translocation of NO3– to the shoot would occur only when the net NO3– uptake rate (NNUR) is fast enough to saturate the reduction process in the roots. In slow-growing species with a low NNUR (Garnier, 1991; Poorter et al., 1991), xylem loading of NO3– would be relatively slow when compared with fast-growing species with a high NNUR. Consequently, slow-growing species might carry out a greater proportion of total plant NO3– reduction in their roots than do fast-growing species.
Gojon et al.’s (1994) hypothesis that fast- and slow-growing species differ in the site of NO3– reduction was based on a comparison of unrelated woody and herbaceous plant species. The conclusions reached from such a comparison may, however, be confounded by differences associated with the contrasting growth form, rather than differences between the site of NO3– reduction and the inherent RGR per se. To investigate the relationship between the site of NO3– reduction and the inherent RGR more effectively, closely related plant species that differ in inherent RGR and NNUR should be compared (Poorter and Remkes, 1990; Garnier, 1992; Atkin et al., 1996a, b; 1998, 1999).
This study investigates what proportion of whole plant NO3– reduction takes place in the roots in eight grass species whose RGR values range from 102 to 255 mg g–1 d–1 (Atkin et al., 1996a; Scheurwater et al., 1998). It was hypothesized that slow-growing grass species perform a greater proportion of total plant NO3– reduction in their roots than do fast-growing grass species.The outcomes of two experimental methods were compared: the weighted distribution of in vivo NRA between root and shoot and the ratio of reduced N to total N in the xylem exudate. Moreover, the theoretical maximum proportion of whole plant nitrogen assimilation that could take place in the roots of a fast- and a slow-growing species was calculated, using information on root respiration rates, respiratory quotient, RGR, NNUR, and specific respiratory costs associated with growth, maintenance and ion uptake.
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Materials and methods |
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Plant growth
Eight monocotyledonous species were selected that differ in maximum relative growth rate (RGR): Dactylis glomerata L., Deschampsia flexuosa L., Festuca ovina L., Holcus lanatus L., Poa alpina L., P. compressa L., P. fawcettiae L., and P. trivialis L. Seeds from non-clonal sources were obtained as described in Atkin et al. (1996a) and Scheurwater et al. (1998). The RGRs of these species range from 102–255 mg g–1 d–1 (Atkin et al., 1996a; Scheurwater et al., 1998).
All species were grown under the following controlled-environment conditions: PAR: 500 µmol m–2 s–1 (using high-pressure mercury lamps, HPI-T 400W, Philips BV, Eindhoven, the Netherlands); temperature: 20 °C; relative humidity: 70%; light period: 14 h. Seeds of D. flexuosa were stratified for 6 weeks prior to germination. All seeds were germinated in the growth room in Petri dishes on moistened filter paper. Subsequently, the seedlings were transferred to sand moistened with half the strength of the following nutrient solution: 795 mmol m–3 KNO3, 602.5 mmol m–3 Ca(NO3)2, 270 mmol m–3 MgSO4, 190 mmol m–3 KH2PO4, 41 mmol m–3 Fe-EDTA, 20 mmol m–3 H3BO3, 2 mmol m–3 MnSO4, 0.85 mmol m–3 ZnSO4, 0.25 mmol m–3 Na2MoO4, and 0.15 mmol m–3 CuSO4 (Poorter and Remkes, 1990). After 7–14 d of establishment the seedlings were transferred to 32 dm3 tanks containing an aerated full-strength nutrient solution, as described above. The pH of the nutrient solution was adjusted regularly to 5.8 with H2SO4 and the nutrient solution was changed once a week.
Experimental design
After 2–5 weeks on nutrient solution, depending on the seed size and RGR of the species, the plants had reached a total fresh mass of approximately 1 g. These plants were used to determine the in vivo NO3– reduction activity (NRA) of roots and shoots and to collect xylem sap (see below), 7 h after the lights had been switched on. Three to four days before and after measuring the NRA, six to eight plants of each species were harvested to enable determination of the RGR.
Nitrate reductase activity
A modified in vivo NO3– reductase assay (Jaworski, 1971) that involved anaerobic conditions (N2 flushing for 2 min) was used to estimate the activity of NO3– reductase (NR) in intact, detached roots and shoots that had been sliced into 2–4 mm sections. Six to eight replicates were used per tissue type and per species. Fresh tissue (up to 300 mg) was placed into 5 cm3 of 100 mol m–3 KH2PO4 buffer (pH 7.2), containing propanol. The optimal propanol concentration for the roots and shoots of each species was determined previously using a separate batch of plants, grown under similar conditions. The following concentrations of propanol (v/v) were used for the roots and shoots, respectively: D. glomerata 3%, 2%; D. flexuosa 2.5%, 3%; F. ovina 2.5%, 2%; H. lanatus 3%, 3%; P. alpina 2%, 2%; P. compressa 3%, 3%; P. fawcettiae 3.5%, 3.5%, and P. trivialis 3%, 2%. Incubation took place in the dark at 20 °C and a subsample was taken from the medium 15 min and 35 min after the start of the incubation in order to measure the NO2– production (Jaworski, 1971). The assay was carried out both in the absence and presence of 100 mol m–3 KNO3 in the medium. The degree to which endogenous NO3– is liberated during tissue preparation can vary between samples. The addition of KNO3 reduces the possibility of inter-assay variability. Only the results obtained via the assay with 100 mol m–3 KNO3 are presented as no systematic difference was found in NRA between the assay with, and the assay without, NO3– in the medium (data not shown).
Collection of xylem sap
Roots were severed just below the shoot junction. The cut ends of individual roots were placed in 10 mm3 glass capillaries, which were sealed with lanolin. The roots remained suspended in aerated nutrient solution of the same composition as that in which they had been grown. After approximately 3 h, xylem exudate was collected from the capillaries, pooled for the two to three plants that were used per species and immediately stored at –20 °C.
Nitrogen analyses
The total N-concentration of the xylem sap and of the freeze-dried (Unitop 600SL and Freezemobile 12SL, The Virtis Company, Inc. Gardiner, New York, USA) root and shoot samples from both RGR harvests was determined with a C-H-N analyser (Carlo-Erba, model 1106, Milan, Italy) using combustion gas chromatography (Pella and Colombo, 1973). NO3– was determined in water extracts of the freeze-dried root and shoot samples and the xylem exudates, using a modified salicylic acid method (Cataldo et al., 1975) that enabled determination of the concentration in 10 mm3 of water extract or xylem exudate.
The reduced N concentration of roots, shoots and xylem sap was calculated subtracting the NO3– concentration from the total N concentration.
Statistics and calculations
Significance of regressions was tested using the SAS statistical package (SAS, 1988). The RGR of each species was calculated from two harvests with six to eight replicates each. The first and second harvest took place 3–4 d before and after measuring the NO3– reductase activity, respectively. RGR was determined as the slope of the natural logarithm of total plant dry mass versus time (Hunt, 1982). The NNUR was calculated as described in White (1972).
The proportion of total plant NO3– reduction that occurs in the roots (Proot) was estimated using two experimental approaches and one modelling approach. The results of the in vivo NR activity assays were used in the first experimental approach (Proot,1) according to:
where RMR and SMR are the root and shoot mass ratio, respectively, and NRAroot, NRAshoot and NRAplant equal the NO3– reductase activity of roots, shoots and whole plants, respectively (µmol NO2– g–1 DM h–1).
The ratio of reduced N to total N in the xylem sap was used as the second experimental method (Proot,2) to estimate the proportion of total plant NO3– reduction that took place in the roots:
where reduced Nxylem and total Nxylem are the concentrations of reduced N and total N of the xylem sap (mol m–3), respectively.
Estimation of the maximum root contribution
A modelling approach was used to calculate the theoretical maximum root contribution to total plant NO3– reduction (see equation 1) in the roots of the fast-growing D. glomerata and the slow-growing F. ovina. To do this, it was necessary to estimate total plant NRA and the potential root NRA. Total plant NRA was estimated by multiplying the RGR by the reduced N concentration of the plants (Table 1). To estimate the potential rate of root NO3– reduction, information on gas exchange, RGR, NNUR, and specific respiratory costs was used. Furthermore, use was made of the information that the reduction of 1 mol of NO3– to NH4+ produces 2 mol of CO2 and uses 1 mol of NADH from glycolysis and the TCA cycle to reduce NO3– to NO2– and 3 mol of NADPH from the oxidative pentose phosphate pathway to reduce NO2– to NH4+ (Blacquière, 1987). Finally, it was assumed that the difference in root CO2 evolution rates with and without NO3– being reduced in the roots could be ascribed to NO3– reduction. If this difference is known, then the maximum (potential) root NRA can be calculated.
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To calculate the total rate of root CO2 evolution with NO3– reduction being performed in the roots (rt,CO2(+NRA), mmol CO2 g–1 DM d–1), the RGR and the NNUR as determined in this study were used in the following equation (cf. Scheurwater et al., 1998):
where rm (mmol O2 g–1 DM d–1), cu (mol O2 mol–1 NO3–) and cg (mmol O2 g–1 DM) are the specific respiratory costs for maintenance, ion transport (including ion uptake) and growth, respectively (Scheurwater et al., 1998). RQt is the respiratory quotient (mol CO2 mol–1 O2) of the roots as determined via gas-exchange measurements (Scheurwater et al., 1998). The theoretical rate of CO2 evolution without NO3– reduction being performed in the roots (rt,CO2(–NRA), mmol CO2 g–1 DM d –1) can be calculated using equation 4, the RGR and the NNUR as determined in this study, and the specific respiratory costs as determined by Scheurwater et al. (1998). It was assumed that the theoretical RQ for growth processes without NO3– reduction (RQg) was 1.25 (Penning de Vries et al., 1974) and that the RQ for maintenance (RQm) and uptake of ions (RQu) was 1:
The maximum rate of root NO3– reduction (NRAroot,max, µmol NO2– g–1 DM h–1) can then be calculated using the information that the reduction of 1 mol of NO3– to NH4+ produces 2 mol of CO2 (see above):
where 1000 converts mmol to µmol and 24 converts days to hours.
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Results |
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Relative growth rate
The RGR values of the eight grass species ranged from 77–243 mg g–1 d–1 (Table 1). The RGR values determined for the same species in previous studies (Atkin et al., 1996a; Scheurwater et al., 1998) were plotted against the RGR values of this study (Fig. 1). All data points were on or near the 1:1 line (Fig. 1). The RGR values in this study were, therefore, very similar to those reported before. RGR and NNUR were closely and positively correlated (correlation coefficient of 0.81, Table 1).
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Nitrate reductase activity
As expected, the NRA of roots and shoots increased with increasing RGR and NNUR (Fig. 2). The regression of NRA against RGR and NNUR was significant for both roots and shoots (p <0.001). The ratio of shoot NRA to root NRA was calculated to investigate whether shoot NRA increased relatively faster than root NRA with increasing RGR. This was not the case, as the linear regression of this ratio against RGR was not significant (data not shown). Data on biomass partitioning (Table 1) and the results of the in vivo NRA assays were used to calculate the NRA in whole plants. Total plant NRA increased significantly with increasing RGR (Table 1).
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The NRA of the whole plant can also be estimated by multiplying the reduced N concentration of the plant (Table 1) by the RGR. Assuming there were no losses of reduced N from the plants, this approach must yield highly accurate estimates of whole plant NRA, as the degree of error associated with the measurements of reduced N and RGR are small. The approach mentioned above was used to compare the whole plant NRA values with those based on the in vivo NR assay and RMR (Fig. 2; Table 1). The in vivo assay values underestimated total plant NRA by 60% (data not shown). Several factors may have been responsible, including incomplete cessation of NiR activity by anaerobiosis (King et al., 1992) and/or incomplete permeability following the addition of the surfactant propanol. Nevertheless, both methods clearly showed that total plant NRA increased significantly with increasing RGR.
The site of NO3– reduction in fast- and slow-growing species
In this study, two experimental methods were used to determine whether fast- and slow-growing grass species differ in their predominant site of NO3– reduction. With the first method, the proportion of total plant NO3– reduction that occurs in the roots was calculated from data on biomass partitioning and in vivo assay estimates of NRA in the roots and shoots (cf. equation 1). Figure 3 (closed symbols) shows that this proportion did not correlate with the RGR of the species, and that the shoot appears to be the main site of NO3– reduction in both fast- and slow-growing grass species
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The second experimental method estimated the proportion of total plant NO3– reduction that occurred in the roots from the ratio of reduced N to total N in the xylem sap (cf. equation 2). The higher the ratio, the greater the proportion of NO3– that was reduced in the roots. Figure 3 (open symbols) shows that the linear regression of this ratio against the RGR of the species was not significant and that the xylem sap was dominated by NO3– rather than by reduced N. Thus, the results obtained with this method indicates that fast- and slow-growing grass species have the same predominant site of NO3– reduction (i.e. in the shoot).
Estimation of the maximum contribution by roots to whole plant NO3– reduction
These maximum root NRA values (calculated using equations 3, 4 and 5) were 26.6 and 9.0 µmol NO2– g–1 DM h–1 for the fast-growing D. glomerata and slow-growing F. ovina, respectively (Table 2). The root NRA values as determined via the in vivo assay were 7.5 and 3.8 µmol NO2– g–1 DM h–1, for the two grass species, respectively (Fig. 2, open symbols). The maximum contribution of the roots to total plant NO3– reduction can now be determined using equation 1 and the outcome of the multiplication of total plant reduced N by RGR as the estimate of total plant NRA. Thus, the maximum proportion of total plant NO3– reduction that could occur in the roots of the fast-growing D. glomerata and the slow-growing F. ovina was 0.37 and 0.23, respectively (Table 2). This indicates that the shoots were the predominant site of NO3– reduction in both species.
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Discussion |
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The results of this study demonstrate that the predominant site of NO3– reduction is independent of the inherent relative growth rate and net nitrate uptake rate in several grass species grown with free access to nutrients (Fig. 3; Table 2). The shoot was the dominant site of NO3– reduction in all species (Fig. 3; Table 2). Therefore, the hypothesis was rejected that the proportion of whole plant NO3– reduction that takes place in the root is higher in species that exhibit low RGR and NNUR values, compared to faster-growing species with high NO3– uptake rates (Gojon et al., 1994).
What factor was responsible for the difference in these results and that reported by Gojon et al. (1994)? Gojon et al. (1994) calculated the ratio of shoot to root NRA from literature data for several herbaceous and woody species. On average, this ratio was more than four times higher in the herbaceous plants, which led them to their conclusion that herbaceous plants predominantly reduce NO3– in their shoot, while in woody plants the major part of total plant NO3– reduction occurs in their roots. Rates of NRA alone do not, however, determine the proportion of NO3– that is reduced in a particular tissue. Rather, the proportion of NO3– that is reduced in a particular tissue depends on the plant’s RGR, its specific rate of NRA in that tissue and the proportion of plant mass that is allocated to that tissue (cf. equation 1), as Gojon et al. (1994) state as well. The difference between these results and those of Gojon et al. (1994) may therefore be due to the fact that Gojon et al. (1994) did not take into account interspecific differences in RGR and biomass allocation.
Other studies have also suggested that a larger proportion of NO3– is reduced in the shoots than in the roots. For example, using results from in vivo NRA assays, Andrews et al. (1992) concluded that shoots are the predominant NO3– reduction site in 4 grass species grown with 5 mol m–3 NO3– (Dactylis glomerata, Lolium multiflorum and L. perenne). In fast-growing wild-type and slow-growing gibberellin-deficient Lycopersicon esculentum plants, the shoot was also the main site of NO3– reduction (Cramer et al., 1995). Furthermore, Cramer et al. (1995) suggested that the site of NO3– reduction was not related to the RGR of the plants. Both the weighted distribution of NRA and the N composition of the xylem sap did not differ between the three genotypes (Cramer et al., 1995). Recently, Cen et al. (2001) used simultaneous measurements of CO2 and O2 exchange in roots and shoots to calculate the proportion of NO3– reduction taking place in roots and shoots of white lupin (Lupinus albus). They found that the predominant site of NO3– reduction was the shoot (57–95% of whole plant NO3– reduction). Using a 15N-labelling approach, Gojon et al. (1986b) found that 73% and 80% of whole plant NO3– reduction takes place in the shoots of corn and barley, respectively, when both are grown in the presence of NO3– for extended periods.
These results show that root NRA and shoot NRA both increased with increasing RGR and NNUR (Fig. 2). Other studies have reported similar results. For example, when Hordeum vulgare plants were grown at a range of RGRs by adding NO3– at different relative addition rates, both root and shoot NRA increased with increasing RGR (Öhlén and Larsson, 1992; Agrell et al., 1997). However, the results of this study suggest that changes in NNUR and RGR are not associated with changes in the proportion of NO3– assimilated in the roots. The NNUR of the slow-growing species may already have been sufficiently high to saturate the reduction process in the roots. Such saturation of NO3– reduction with increasing NNUR has been reported in roots of Zea mays (Morgan et al., 1985).
In the past 30 years, several methods have been used to investigate the extent to which roots and shoots contribute to whole plant NO3– reduction (Pate, 1983; Rufty et al., 1982; Andrews, 1986; Gojon et al., 1986a, b; Wallace, 1986; Cooper and Clarkson, 1989; Touraine et al., 1994; Cen et al., 2001). Most, if not all, are subject to criticisms of one sort or another (e.g. cycling of organic nitrogen; see below). It is important, therefore, that studies such as this one do not rely on one approach. For this reason, it was decided to combine two experimental approaches and a modelling exercise to assess whether fast- and slow-growing species differ in their main site of NO3– reduction. All three of the methods used in this study (NRA assay/biomass allocation, xylem sap analyses, and modelling exercise) yielded the same result (i.e. that fast- and slow-growing species reduce a similar proportion of whole plant NO3– in their roots). Further work using other methods (e.g. 15N and/or simultaneous measurements of CO2 and O2 exchange) are needed assess whether the absolute values per se shown in Fig. 3 and Table 2 are accurate.
In addition to the NRA assay/biomass allocation, xylem sap analyses, and modelling exercise approaches described above, another way of determining the site of NO3– reduction in plants is to measure the concentration of carboxylates and reduced nitrogen in whole plants. The proportion of plant NO3– reduction that occurs in the shoots in NO3–-fed plants is taken as the ratio of carboxylates to reduced nitrogen (Touraine et al., 1994). In preliminary experiments, the carboxylate concentration was calculated by subtracting the NO3– concentration from the ash alkalinity (Poorter and Bergkotte, 1992; Jungk, 1968). No differences were found amongst the species in the proportion of total plant NO3– reduced in the roots (data not shown). This supported the conclusion reached using the three approaches listed above. However, the estimates (0.7–0.85) were much higher than shown in Fig. 3. Why was this? The most likely explanation is that the assumptions made by the carboxylate to reduced N method were not valid for the selected species. This approach assumes that a carboxylate anion is produced for each NO3– that is reduced in the shoot. Carboxylate production is necessary to regulate internal pH during shoot NO3– reduction; the pH is offset by the net release of an hydroxyl ion for each NO3– that is reduced in the shoot (the ‘biochemical pH stat’, Raven and Smith, 1976; Davies, 1986; Touraine et al., 1988). It also assumes that the carboxylates that are formed in the shoot also accumulate in the shoot cells, that no carboxylate accumulation takes place in the roots and that the carboxylate and the reduced N concentration of the plant do not change during the experiment. However, carboxylates that are formed in the shoot may not necessarily accumulate in the shoot cells (Ben Zioni et al., 1971). Rather, carboxylates may be transported from the shoot to the root and then metabolized. If so, analyses of carboxylates and organic N concentrations would result in an overestimate of the proportion of NO3– reduced in the root.
The results shown in Fig. 3 and Table 2 suggest that roots play a relatively minor role in reducing NO3– compared with the role of the shoots (typically 0.4 of whole plant NO3– reduction; Fig. 3). To what extent was this a reflection of the three methods used? More specifically, is the role of roots underestimated by the chosen assays? Although species that reduce NO3– in their roots are expected to exhibit high ratios of reduced N to total N in the xylem sap, a significant proportion of the reduced N in xylem sap can originate from the shoot (i.e. reduced N is cycled; Rufty et al., 1982; Simpson et al., 1982; Cooper and Clarkson, 1989). Cycling of reduced N causes an overestimation (i.e. not an underestimation) of the roots’ contribution to total plant NO3– reduction. Further support for the suggestion that the contribution of the roots to total plant NO3– reduction has not been underestimated comes from the fact that the maximum proportion of total plant NO3– reduction that could occur in the roots was 0.37 (Table 2). The authors feel confident, therefore, that the role of roots in total plant NO3– reduction has not been underestimated.
In conclusion, the present study indicates that the site of NO3– reduction in grass species of contrasting growth rate is independent of RGR and NNUR. It also shows that all eight grass species reduce NO3– predominantly in the shoot when grown with free access to nutrients.
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Acknowledgements |
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The authors thank David Clarkson for advice on the collection of xylem sap and Jan Kees van Amerongen and Rob Welschen for technical assistance. Seeds of Festuca ovina were kindly provided by Hendrik Poorter, Utrecht University, The Netherlands. The contribution of Ingeborg Scheurwater was financially supported by the Life Sciences Foundation (SLW), which is subsidized by the Netherlands Organization for Scientific Research (NWO). Owen Atkin acknowledges the support by an EU Postdoctoral Fellowship (Human Capital and Mobility Programme) while in Utrecht.
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