Annals of Botany 89: 715-722, 2002
© 2002 Annals of Botany Company
Short-term Changes in Xylem N Compounds in Lolium perenne Following Defoliation
1Macaulay Land Use Research Institute, Craigiebuckler, Aberdeen AB15 8QH, UK and 2Institute of Grassland and Environmental Research, Aberystwyth Research Centre, Aberystwyth, Dyfed SY23 3EB, UK
* For correspondence. Fax +44 (0)1224 498206, e-mail b.thornton{at}macaulay.ac.uk
Received: 8 October 2001; Returned for revision: 11 January 2002; Accepted: 1 March 2002.
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ABSTRACT |
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Previous studies have indicated that an increased asparagine to glutamine ratio (Asn : Gln) occurs in the xylem fluid of Lolium perenne 24 h after defoliation. However, the absolute changes in Asn and Gln leading to the increased Asn : Gln ratio are unknown. The present study tested the hypotheses that: (1) defoliation-induced changes in xylem amino acid composition occur in L. perenne within the first 24 h following defoliation, irrespective of phasing with respect to the diurnal light/dark cycle; and (2) the increase in Asn : Gln ratio in the xylem fluid of L. perenne following defoliation is due to an increase in Asn content. Plants of L. perenne L. ‘Aurora’ were grown in flowing solution culture for 40 d. Plants were then either left intact, defoliated at the end of the light period or defoliated at the end of the dark period. 15N-labelled NO3– was supplied following defoliation to discriminate between the recovery of N absorbed prior to, and following, defoliation. Xylem samples were collected over the subsequent 24 h period with amino acids speciated by GC-MS. There was support for the first hypothesis: increased Asn : Gln ratios occurred within the first 24 h, irrespective of the phasing of defoliation with respect to light/dark cycles. The second hypothesis was not supported: the concentration of all amino acids in the xylem exudate declined after defoliation, and the increased Asn : Gln ratio was accounted for by a disproportionately large reduction in Gln levels. Low concentrations of amino acids in the xylem of defoliated plants precluded accurate discrimination of their nitrogen content into pre- and post-defoliation sources.
Key words: Amino acids, defoliation, diurnal variation, flowing solution culture, Lolium perenne, nitrate uptake, perennial ryegrass, xylem exudate.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSLITERATURE CITED |
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Bigot et al. (1991) observed an increase in the asparagine to glutamine ratio (Asn : Gln) in xylem exudate collected from Lolium perenne 1 d after defoliation. The ratio peaked 3 d after defoliation, after which it gradually returned to an undefoliated value (Bigot et al., 1991). These authors interpreted the increased Asn : Gln ratio in terms of a switch in the source of xylem N from root uptake to mobilization of proteins, although they also suggested that perturbation in the normal root–shoot–root cycling of amino acids (Cooper and Clarkson, 1989) could be a possible cause. Bigot et al. (1991) based their interpretation on relative concentrations of amino acids in xylem sap, because high variability prevented the use of absolute changes in concentrations of individual amino acids in the exudate. Hence it was unclear as to what the absolute changes were in Asn and Gln leading to the increased Asn : Gln ratio.
In defoliated grass plants, both the remaining shoot material and roots supply nitrogen for the growth of younger leaves (Ourry et al., 1988, 1990; Thornton and Millard, 1993). Whilst nitrogen metabolism in senescing leaves of Triticum aestivum was directed towards Gln (Peeters and Van Laere, 1994), in senescing leaves of Lolium temulentum it was directed towards formation of Asn (Thomas, 1978). Up-regulation of gene expression for asparagine synthetase (Chevalier et al., 1996) and increased Asn concentrations (Brouquisse et al., 1992, 1998) have also been observed in sugar-starved roots. Hence changes in metabolism of senescing leaves and roots appear to be consistent with the increased post-defoliation Asn : Gln ratio in xylem fluid observed by Bigot et al. (1991) being the result of increased nitrogen mobilization from senescing tissues. If the changes in xylem amino acids of defoliated grasses are related to mobilization of nitrogen, they may ultimately offer an indirect method for estimating post-defoliation N mobilization in grasses under field conditions. Measurements of post-defoliation N mobilization in grasses growing under field conditions have been made (Louahlia et al., 2000). However, 15N tracer techniques used in the laboratory to measure N mobilization directly are difficult to apply in the field due to problems in uniformly labelling the many different soil N pools that exist. Changes in the xylem amino acid composition of grasses following defoliation may be inferred from analysis of xylem exudate collected from recently cut tissues, subject to the limitations associated with this approach (Jackson et al., 1995; Schurr, 1998). In an analogous manner, changes in the xylem amino acid composition of Betula pendula trees following bud burst in spring were shown to be due to mobilization of the over-wintering N stores (Millard et al., 1998).
Following defoliation of grasses such as Lolium perenne L. there is a rapid decline in NO3– uptake over the subsequent 12–24 h, followed by a period of negligible or very low net uptake rates lasting several days, provided the plants are not N deficient (Clement et al., 1978b). If, as suggested by Bigot et al. (1991), the increased xylem Asn : Gln ratio following defoliation results from a switch in the N supply from uptake to mobilization of stores, some change in xylem Asn : Gln ratio may be expected within the first 24 h following defoliation as root nitrogen uptake declines. This may even occur prior to mobilization of nitrogen induced by defoliation per se as nitrogen mobilization is an ongoing process in undefoliated grasses with nitrogen moving from older senescing leaves to younger growing leaves (Schulte auf’m Erley et al., 2000; Bausenwein et al., 2001).
Nitrate uptake and the subsequent translocation of N to the shoot exhibit substantial diurnal fluctuations in many grasses (Clement et al., 1978b; Ourry et al., 1996; Macduff et al., 1997). The magnitude of this variation depends on a number of environmental factors, most notably the duration of the photoperiod and the daily integral of photosynthetically active radiation (PAR). Delhon et al. (1995a, b) showed that accumulation of NO3– and Asn in the roots of Glycine max L. Merr. during darkness is causally associated with the dark-induced decrease in transpiration, but is unrelated to the decline in NO3– uptake. Their work also implied some degree of diurnal variation in the composition, concentration and flux of amino acids in the xylem. Whether this diurnal variation in xylem amino acids can be extrapolated to grasses is unclear.
The objectives of the present study were to test the hypotheses that: (1) defoliation-induced changes in xylem amino acid composition occur in L. perenne within the first 24 h following defoliation, irrespective of phasing with respect to the diurnal light/dark cycle; and (2) the increase in Asn : Gln ratio in the xylem fluid of L. perenne previously observed 1 d after defoliation is due to an increase in Asn content. Plants were grown in flowing solution culture for 40 d, and the experiment was performed on day 41, when plants were either left intact, defoliated at the end of the light period or defoliated at the end of the dark period. 15N-labelled NO3– was supplied following defoliation to discriminate between the recovery of N absorbed prior to, and following, defoliation. Xylem samples were collected over the subsequent 24 h period with amino acids speciated by GC-MS.
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MATERIALS AND METHODS |
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Plant material and culture conditions
Seeds of Lolium perenne L. ‘Aurora’ were sown in a glass house in a flowing solution culture system (Clement et al., 1974; Hatch et al., 1986). Seedlings were thinned on emer gence to give 12 plants in each of 24 x 1 l culture vessels in each of three culture units containing 200 l nutrient solution. Air temperature was controlled at 19 ± 1 °C day/night, with solution temperatures between 19–20 °C throughout the experiment.
Plants were grown under natural light until 14 d after sowing when supplementary light was introduced (1600–2400 h), provided by a single 400 W HPI/T lamp (Philips) suspended 1·5 m above the surface of each culture unit (300 ± 25 µmol m–2 s–1 PAR at canopy height). From day 34 onwards, natural light was excluded and plants were subject to artificial illumination over a 10 h photoperiod (0800–1800 h) provided by paired 400 W HPI/T and SON-T lamps (Philips) above each unit (650 ± 50 µmol m–2 s–1 PAR at canopy height). On day 37, the light period was displaced to run between 2300–0900 h for two of the three culture units, to assist timetabling of measurements.
The initial concentrations of nutrients in the flowing solutions were (µM): NO3–, 250; K+, 250; Ca2+, 344; SO42–, 424; Mg2+, 100; H2PO4–, 50; Fe2+, 5·4; with micronutrients as described by Clement et al. (1978a). These concentrations were replaced weekly until day 39. Automatic measurements of NO3– uptake began on day 39, after all culture units were drained and refilled with nutrient solution of the same initial composition, except for NO3– (20 µM) and K+ (10 µM). Thereafter, the concentration of NO3– was measured every 14 min and maintained automatically at 20 ± 5 µM by addition of Ca(NO3)2, and K+ maintained at 10 ± 2 µM by addition of K2SO4. Net uptake of NO3– and of K+ was calculated on an hourly basis from the quantities of these ions delivered to culture units to maintain the set-point concentrations. All other nutrients were supplied automatically to culture units in fixed ratios (mol : mol) to the supply of K+ (1·0 K : 0·4 S : 0·16 Mg : 0·13 P : 0·002 Fe). The pH of the solution was maintained at 6·0 ± 0·1 in all units by automatic delivery of H2SO4 or Ca(OH)2.
Defoliation treatments and collection of xylem exudate
Each of the three culture units was subject to a different treatment (A, B or C) with respect to defoliation and its timing during the diurnal cycle on day 41. Treatment A was an undefoliated ‘control’ and is described below. In treatment B, all plants were defoliated 30 min prior to the end of the light period, and at the same time the supply of NO3– was 15N-labelled (4·49 atom % 15N excess) and maintained thereafter at 20 ± 5 µM. Xylem exudate was sampled over the subsequent 24 h from the ‘cut ends’ of these plants. In treatment C, all plants were defoliated 30 min prior to the end of the dark period, switched to a 15N-labelled NO3– supply and xylem exudate sampled as in treatment B.
The procedures for defoliation, exudate collection and harvesting of plants in treatments B and C were as follows. Defoliation was carried out in situ by making a horizontal cut with a scalpel at a shoot height of 4 cm. The ‘cut ends’ of the remaining stubble (all remaining shoot material comprising elongating leaf bases, elongating leaf blades and remaining blades) were blotted dry immediately with a paper tissue, and xylem exudate collected continuously from all plants for 20 min in pre-weighed microcapillary tubes. The exudate collected from different plants was bulked, weighed, transferred into Eppendorf tubes and freeze-dried prior to analysis. The cut ends of all plants were then re-blotted, and as further droplets of exudate formed they were collected with pasteur pipettes, more or less continuously over the next 2 h, and bulked across several plants. Two replicate samples of each treatment were collected at each time interval. At any given time interval, the exudate collected from individual plants never contributed to more than one replicate. The cut ends were then re-blotted and the exudate collection procedure repeated every 2 h until 24 h after defoliation. An upturned 125 cm3 glass beaker with a moistened paper tissue was placed over each culture vessel of plants to minimize evaporation of the exudate.
Two culture vessels of plants (12 plants each) in treatments B and C were harvested immediately following defoliation and thereafter at intervals of 2 h. On each occasion, the 12 bulked plants were separated into shoot and root material and their fresh weights recorded.
In treatment A (undefoliated ‘control’), plants were switched to a 15N-labelled NO3– supply (4·49 atom % 15N excess) from the start of the dark period (end of light period). Harvesting of these plants had begun 30 min earlier, with plants in a pair of culture vessels (12 plants in each) being defoliated in situ as described for treatments B and C, their cut ends blotted dry and xylem exudate allowed to accumulate for 20 min before collection. Afterwards, the plants were harvested as described above. Thereafter, two vessels were defoliated every 2 h (until 24 h after switching to 15N-labelled NO3–) and exudate was collected over each 20 min period prior to harvesting the plants.
Before the introduction of 15N-labelled NO3– to the flowing solutions in each treatment, plants were allowed to deplete the concentration of NO3– to <1 µM to minimize subsequent isotopic dilution of the 15N-labelled NO3 in the solutions. The 15N-enrichment of NO3– in the nutrient solutions was determined on a regular basis thereafter using a TracerMAT continuous flow mass spectrometer (Finnigan MAT, Hemel Hempstead, UK).
Evapotranspiration by plants in all treatments was determined at 2 h intervals during the experimental period by removing pairs of culture vessels to plastic beakers containing 2 l of aerated nutrient solution and measuring weight loss after 30 min. Values were corrected for weight loss from identical beakers without plants. Leaf extension during the first 21 h following defoliation was measured to the nearest mm on ten plants in both treatments B and C (B = 30 mm, C = 23 mm; P < 0·001).
Analysis of xylem N compounds
Determination of amino acids and their 15N enrichment in the xylem fluid followed Millard et al. (1998). Briefly, following addition of an internal standard of nor-valine, amino acids were converted to their t-butyldimethylsilyl derivatives. Analysis of the derivatives was carried out by GC-MS in single ion recording mode (Fisons series 8000 gas chromatograph linked to a VG TRIO 1 quadrupole mass spectrometer; Fisons Instruments UK, Crawley, UK). The derivatization used did not allow determination of arginine in the samples. Arginine was previously shown to contribute <2 % of the free amino acid nitrogen in root and shoot tissue of L. perenne when grown on ammonium nitrate (Thornton, 2001). Nitrate in xylem fluid was analysed by ion exchange chromatography with conductivity detection using a Dionex DX500 (Dionex UK Ltd, Camberley, UK).
Total N in the xylem fluid was defined as the sum of NO3– and amino acid N. It was assumed that NH4+-N was insignificant in the xylem exudate, as the sole source of N in the present study was NO3–. Furthermore, NH4+ comprised only 1 % of total xylem N in Triticum aestivum L. grown under NO3– nutrition (Cramer and Lewis, 1993). Peptides may contribute to the N content of xylem fluid (Williams and Miller, 2001); however, as analysis for peptides was not performed, this possibility is ignored in the current definition of the total N content of xylem fluid.
Statistical analysis and curve fitting
Data were subject to regression analysis using Genstat 5 Release 4·1 ©Lawes Agricultural Trust (IACR-Rothamsted). Fourier curves were fitted to data exhibiting diurnal variation after ensuring the curve gave a better fit (accounted for a higher percentage of the variance) than a straight line. Exponential curves or double exponential curves where these gave a better fit (accounted for a higher percentage of the variance) were fitted to data exhibiting a decline with time.
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RESULTS |
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Changes in xylem N concentrations
Concentrations of total-, NO3–- and amino acid-N in xylem exudate from the stubble of undefoliated (control) plants varied diurnally, with maximum N concentrations occurring approximately midway through the dark period (Fig. 1). Nitrate was the dominant form of N in the xylem fluid of these plants, accounting for between 75 and 92 % of the total N at all times (Fig. 1). Severe defoliation either at the end of the light period, or at the end of the dark period, resulted in a rapid change in xylem N content and in the suppression of the diurnal pattern. The data for defoliated plants indicated a two-phased decline in xylem total N over time, irrespective of the timing of defoliation relative to the light/dark cycle (Fig. 1). Phase 1 was characterized by a rapid decline in concentration during the first 2–3 h, attributable mainly to the amino acid N component. This was followed by a progressively slower decline (phase 2), associated mainly with the NO3–-N component (Fig. 1). The concentration of both glutamine and asparagine, the most abundant of the specific amino acids, declined rapidly during phase 1 after defoliation (Fig. 2).
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The swifter decline in the concentration of amino acid N compared with NO3–-N following defoliation meant that the proportional contribution of NO3–-N to the total N content of the xylem was higher in defoliated treatments than in the ‘control’. For example, NO3–-N accounted for 99 % of the total N concentration in plants 8 h after defoliation at the end of a light period, compared with 79 % in ‘control’ plants at an equivalent point during the photoperiod.
Changes in amino acid composition of xylem exudate following defoliation
Glutamine constituted >80 % of the amino acid N in xylem exudate immediately prior to defoliation (Fig. 3A). It remained the most abundant amino acid in exudate collected from ‘control’ plants (treatment A) throughout the diurnal cycle, although declining gradually over time from 90 to 60 % of the total amino acid N. The trend was very different in both of the defoliated treatments, with the proportion of Gln in the amino acid fraction declining to <20 % after 15 h (Fig. 3A). The difference between ‘control’ and defoliated plants suggests that the defoliation-induced decline in concentration was amino acid specific, at least during phase 2. The proportionately high reduction in xylem Gln concentration following defoliation was reflected in the near linear increase in the Asn : Gln concentration ratio over time (Fig. 3B). This was in marked contrast to the stability of the Asn : Gln ratio in ‘control’ plants.
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Nitrate uptake and 15N incorporation into xylem glutamine
Net uptake rates of NO3– by ‘control’ plants varied diurnally, with maximum rates occurring towards the start of the dark period and minimum rates towards the end of the dark period (Fig. 4). The diurnal variation in NO3– uptake by intact plants was out of phase with the pattern for the total N concentration in xylem exudate (Fig. 1) whose maximum occurred midway through the dark period.
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Irrespective of timing within the light/dark cycle, defoliation resulted in a near exponential decline in NO3– uptake (Fig. 4), comparable in magnitude with that observed for xylem total N concentrations (Fig. 1), and in the disappearance of any diurnal pattern in uptake. Net uptake ceased altogether within 20 h of defoliation in both treatments.
Within 2 h of introducing 15NO3– (4·49 atom % 15N excess) to the flowing nutrient solutions, the Gln-N in xylem exudate of the undefoliated ‘control’ plants attained a similar 15N enrichment (Fig. 5), and remained more or less constant thereafter, with a mean (± s.d.) of 4·75 (± 0·50) atom % excess. This suggests that within 2 h the xylem Gln-N was derived entirely from NO3– absorbed after switching to the 15N-labelled supply. The very low concentrations of amino acids other than Gln in ‘control’ undefoliated plants, and also of Gln in defoliated plants, meant that the 15N enrichment of these species could not be determined accurately.
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Relationship between nitrate uptake and the flux of nitrate in xylem
Rates of water loss from the plants varied little throughout the dark period, with means (± s.d.) of 87 (± 19), 22 (± 13) and 25 (± 17) mg H2O h–1 per plant, respectively, for treatments A, B and C. However, their usefulness in estimating solute fluxes in the xylem is questionable because of the problems associated with multiplication of xylem solute concentrations by measurements of water flux when, as in the present study, xylem fluid is collected using destructive techniques (Schurr, 1998). The choice between taking the water flux measured immediately before xylem collection, or that measured after cutting the plant, as the multiplier in these calculations depends in part on the volume of xylem collected relative to the volume of xylem vessels in the plant following decapitation. Under the present experimental conditions, xylem contents guttated from the plant and water flux during xylem exudate collection was therefore primarily due to root pressure. Water loss prior to exudate collection from defoliated plants in the dark (treatment B) would also have been primarily due to root pressure. Furthermore, the rate of water loss prior to exudate collection from defoliated plants during the light period (25 ± 17 mg H2O h–1 per plant) was similar to that from defoliated plants in the dark (22 ± 13 mg H2O h–1 per plant), implying negligible transpiration in the light from the remaining leaf area after defoliation. It was concluded that the water flux through defoliated plants would be similar prior to and during xylem sampling. Consequently, the mean rate of water loss measured over the 14 h following defoliation was multiplied by concentrations of NO3– given by the curves fitted to data in Fig. 1 to estimate net fluxes of NO3– translocated from the roots of defoliated plants during this period. These values were plotted against net uptake rates of NO3– by defoliated plants given by the curves fitted to the data in Fig. 4. This approach suggested that the flux of NO3– in the xylem was linearly dependent on the rate of NO3– uptake by defoliated plants, irrespective of the timing of defoliation (Fig. 6). Similar assumptions regarding water flux did not hold for the ‘control’ (undefoliated) plants, and NO3– xylem fluxes could not be estimated accurately in this case.
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DISCUSSION |
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Defoliation-induced changes in the xylem Asn : Gln ratio
Previous work on the effects of defoliation on xylem amino acid composition in L. perenne has examined changes occurring after 24 h and later (Bigot et al., 1991). In common with Bigot et al. (1991), we observed a large increase in the Asn : Gln ratio following defoliation. The present study focused on responses evident within 24 h of defoliation. The results show that significant changes in both the composition and flux of N in xylem exudate occur within 4 h of a single severe defoliation. However, the concentration of all amino acids in the xylem exudate declined after defoliation, and the increase in Asn : Gln is accounted for by a disproportionately high reduction in Gln levels. A decline in xylem amino acid concentrations and changes in amino acid composition following defoliation have also been reported for nodulated and non-nodulated Medicago sativa (Kim et al., 1993). The changes observed in M. sativa are similar to those observed here for L. perenne in so far as the most prevalent amino acid prior to defoliation showed the proportionately greatest decline (Asn in M. sativa, Gln in L. perenne).
The xylem exudates collected both in this study and in that of Bigot et al. (1991) were from the cut ends of L. perenne plants defoliated to a height of 4 cm. Exudates would therefore have passed through the meristematic region of the leaves prior to collection. Consequently, the compounds measured correspond to those loaded into the xylem and which are subsequently not taken up by the leaf meristem. Although the term ‘xylem fluid’ has been used to describe the fluid collected, the possibility that some back-flow in the phloem contributed to the collected fluid cannot be ignored. Leaf elongation zones are recognized as sinks for both C and N (Gastal and Nelson, 1994; De Visser et al., 1997; Schnyder and De Visser, 1999). The extent of the growth zone and cell expansion and cell production have all been shown to be reduced following defoliation of L. perenne (Schäufele and Schnyder, 2000). This suggests that absorption of compounds by the leaf meristem may be reduced following defoliation and that the measured xylem compounds would more closely represent those loaded into xylem vessels. However, a small but significant increase in leaf elongation rate of L. perenne in the first 24 h following defoliation has also been observed (Morvan-Bertrand et al., 2001), suggesting leaf meristem absorption from the xylem may in fact be greater in defoliated compared with undefoliated plants. Indeed, Morvan-Bertrand et al. (2001) showed that the net deposition rate of nitrate in the elongating leaf bases of L. perenne increased following defoliation. The increased Asn : Gln ratio observed in the xylem of defoliated plants may therefore equally be due to changes in resorption from the xylem by the leaf meristem as to a change in the source of nitrogen (current uptake or mobilization of stores) being loaded into the xylem. Unfortunately, in the current study, the low concentrations of amino acids in the xylem of defoliated plants precluded accurate discrimination of their nitrogen content into pre- and post-defoliation sources.
Any contribution by mobilization of N stores to the increased Asn : Gln ratio in the xylem samples of defoliated plants may well peak after the time scale of the current experiment. Activities of endopeptidase, aminopeptidase and carboxypeptidase enzymes in the roots of L. perenne peak 4 d after defoliation (Ourry et al., 1989), and the maximum impact of N mobilization on xylem amino acid composition might be expected to coincide with this or follow shortly afterwards. Increased endopeptidase activity, concomitant with an increased Asn content, has been reported in maize plants that were carbon-limited as a result of prolonged darkness (Brouquisse et al., 1998). These authors considered that the increase in Asn accounted for most of the N released by protein breakdown. An increased Asn : Gln ratio resulting from a greater increase in Asn compared with Gln has been observed over a 6 d period in leaf sections of Lolium temulentum during senesence in the dark (Thomas, 1978).
Results for the undefoliated ‘control’ plants, showing the rapid incorporation of 15NO3 from the root bathing solution into xylem Gln, suggest that the most prevalent xylem amino acid completely turns over within 4 h. Rapid turnover would be advantageous if the shoot–root cycling of amino acids is, as has been suggested, acting as a signal for the regulation of N uptake (Cooper and Clarkson, 1989; Muller et al., 1995).
Relationship between NO3– uptake and xylem N following defoliation
The severe decline in NO3– uptake observed over 12 h following defoliation is in agreement with a number of comparable studies of grass species receiving adequate N supplies (Clement et al., 1978b; Bakken et al., 1998). This response is consistent over a wide range of photoperiods, and for defoliation at different times within the diurnal cycle. However, when NO3– is supplied immediately following defoliation to N-deficient plants, uptake rates appear to increase over several hours and then stabilize at high levels (Macduff et al., 1989). The near exponential decline in NO3– uptake following defoliation has been attributed to a decrease or termination in the supply of photosynthate to the roots, and hence the progressive exhaustion of available carbohydrate pools (Clement et al., 1978b; Richards, 1993). From the standpoint of carbohydrate translocation from the shoot to the roots there are obvious analogies between the impact of defoliation and darkness. The supply of photosynthate to the roots has been causally linked to the diurnal pattern of nitrate uptake in Glycine max (Delhon et al., 1996), the dark-induced decline in uptake being associated with decreased phloem transport of carbohydrate. Delhon et al. (1996) rejected alternative explanations for the darkness-induced decline in uptake which invoked negative feedback mechanisms effected by (1) dark accumulation of nitrate and/amino acids in the roots, or (2) arising from decreased nitrate reduction in the leaves. Whether these mechanisms can be disregarded entirely in the case of nitrate uptake following defoliation remains an open question.
As with the xylem amino acids, the nitrate contents of the sampled xylem exudates will represent that which has been loaded into the xylem and which was not taken up by the leaf meristem. Following defoliation, only a modest proportion of the flux of nitrate from root uptake appears in the flux of nitrate in the xylem above the leaf meristem. This may be explained, in part, by increased deposition rates of nitrate in the elongating leaf bases of L. perenne following defoliation (Morvan-Bertrand et al., 2001). Extrapolation of the linear relationship between the flux of nitrate from root uptake and the flux of nitrate in the xylem above the leaf meristem gives a translocation rate of approx. 2 µg NO3–-N h–1 per plant at zero root NO3– uptake. Root cytosolic NO3– activity is maintained at constant levels, even under conditions where uptake rate varies (Miller and Smith, 1996; van der Leij et al., 1998). The xylem N flux at zero net uptake may therefore represent the translocation rate of NO3– derived from the vacuolar compartment of the root, as opposed to that directly from uptake. Given that an unknown quantity of nitrate may have been resorbed from the xylem by the leaf meristem, the value of the flux from the vacuole to the xylem of 2 µg NO3–-N h–1 per plant represents a minimum value. It has been shown that vacuolar NO3– in barley roots is capable of supplying the N demands of the shoot and maintaining root cytosolic NO3– concentrations for 24 h following total withdrawal of external NO3– (van der Leij et al., 1998). However, the same study showed that xylem NO3– activity declined gradually over this period, implying that the flux of NO3– from the vacuolar compartment into the xylem also declines quite rapidly following the cessation of NO3– uptake. Therefore, the value of 2 µg NO3–-N h–1 per plant as the minimum nitrate flux from the vacuole to the xylem should be regarded only as a first estimate.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSLITERATURE CITED |
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Results lend support to our first hypothesis, namely that defoliation-induced changes in the xylem amino acid composition of Lolium perenne plants do occur within the first 24 h, irrespective of the phasing of defoliation with respect to light/dark cycles. There was no support for the second hypothesis: the concentration of all amino acids in the xylem exudate declined after defoliation, and the increase in Asn : Gln was accounted for by a disproportionately high reduction in Gln levels.
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ACKNOWLEDGEMENTS |
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We thank Mr A. Hepburn and Mrs M. M. Procee of the Macaulay Institute for GC-MS analysis and Mrs S. M. McIntyre for nitrate analysis. We also thank Dr R. De Visser for comments on an earlier version of this manuscript. We thank the Scottish Executive Environment and Rural Affairs Department (SEERAD) and the Biotechnology and Biological Sciences Research Council (BBSRC) of the United Kingdom for funding this work.
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LITERATURE CITED |
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