Journal of Experimental Botany, Vol. 54, No. 390, pp. 2121-2131,
September 1, 2003
© 2003 Oxford University Press
Determining the role of N remobilization for growth of apple (Malus domestica Borkh.) trees by measuring xylem-sap N flux
Received 3 December 2002; Accepted 3 June 2003
1 Chonbuk National University, Department of Horticulture, Chonju 561-756, Korea 8
2 Agriculture and Agri-Food Canada, Pacific Agri-Food Research Centre, Summerland, BC V0H 1Z0, Canada
3 Macaulay Institute, Cragiebuckler, Aberdeen AB15 8QH, UK
* To whom correspondence should be addressed. Fax: +1 250 494 0755. E-mail: neilsend{at}agr.gc.ca
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Abstract |
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The contribution of N remobilization to the seasonal growth of field-grown Malus domestica (apple) trees was measured using two different techniques. ‘Fuji’ trees grafted on M.9 apple rootstocks were planted in the field and fertilized and irrigated for two growing seasons. During the second year, the trees received 15N-labelled fertilizer and destructive harvests were taken during the spring and summer to determine the pattern of N remobilization and uptake. At the same time, patterns of N translocation in the xylem were measured by sampling saps at each harvest and analysing them for their constituent amino acids and amides. Total water flux through the trunk xylem was also measured throughout the sampling period using the heat balance technique. The flux of amino compounds in the xylem was then calculated to see if this approach could quantify remobilization. Most of the N for leaf growth was provided by remobilization, which lasted for some 40 d following bud-burst. The labelled N was not taken up until 14 d after remobilization had started. The predominant amino compounds recovered in the xylem were Asn, Asp, Arg, and Gln, whose concentration peaked during remobilization, except for Arg whose concentration was highest at bud-break and declined thereafter. The amount of N translocated in the xylem as Asn, Asp and Gln correlated well with the amount of N remobilized (as measured by the recovery of unlabelled N in the new above-ground growth). The data suggest that Arg is translocated predominantly as a consequence of root uptake and they are discussed in relation to measuring N remobilization in field-grown trees.
Key words: Amides, amino acids, field-grown trees, root N uptake, 15N label.
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Introduction |
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Remobilization of N by deciduous trees is a process whereby N stored during winter (in perennial tissues) is translocated in the xylem during the spring and used for leaf and inflorescence growth (Millard, 1996). Remobil ization can occur either before (Millard and Proe, 1991; Rufat and DeJong, 2001) or concurrently with (Millard et al., 1998) root uptake of N from the soil. In Malus domestica (apple) trees, N is remobilized predominantly before there is rapid root uptake from the soil (Millard and Neilsen, 1989; Dong et al., 2001; Neilsen et al., 2001). The remobilized N can account for more than 80% of the N used for the growth of spur leaves of apple by the time of full bloom (Neilsen et al., 1997), with subsequent leaf and shoot growth being more dependent on root uptake (Tromp and Ovaa, 1976; Weinbaum et al., 1984; Neilsen et al., 1997). A number of factors influence the relative contributions of remobilized- and root-supplied N to annual growth, including soil fertility and the previous year’s N supply (Millard, 1996) and timing of any fertilizer applications (Weinbaum et al., 1984).
Assessing the extent that remobilization provides N for the growth of trees in the field is difficult. Previous studies have relied upon either the use of 15N-enriched (Millard, 1996) or depleted (Weinbaum et al., 1984; Weinbaum and van Kessel, 1998) fertilizers, coupled with the destructive harvests of whole trees. An alternative, potentially non-destructive method to assess N remobilization by trees could be to quantify fluxes of N compounds translocated in the xylem. Several studies have shown a peak of N concentration in xylem sap following bud-burst and during leaf-growth, which has been attributed to remobilization (Ferguson et al., 1983; Schneider et al., 1994; Millard et al., 1998). By using 15N to label storage pools, Millard et al. (1998) showed that remobilization in young Betula pendula trees grown in pots led to a 10-fold increase in the concentration of N in the xylem sap, mainly due to elevated levels of Citrulline (Cit) and Glutamine (Gln). In M. domestica trees, the compounds translocated in the xylem as a consequence of remobilization have been shown to be Asparagine (Asn), Aspartic Acid (Asp), and Gln (Malaguti et al., 2001). Knowledge of which compounds are translocated during remobilization might allow the flux of N to be calculated by measuring sap flow and the concentration of the relevant compounds (Malaguti et al., 2001). Such an approach has already been used successfully to quantify fluxes of minerals in the xylem of Picea abies trees (Drambrine et al., 1995) and fluxes of amino acids in the xylem of Juglans nigraxregia (Frak et al., 2002) and Prunus avium (Grassi et al., 2002).
This experiment was carried out in order to see if such an approach would work for quantifying N remobilization in M. domestica and, thus, to characterize the suite of amino compounds associated with remobilization so as to obviate the need for 15N labelling in future experiments. Trees were grown in an orchard and provided with contrasting N supplies over 2 years to precondition their N storage capacity. In the second year of the experiment, all the fertilizer N was labelled with 15N in order to quantify root uptake of N during spring growth. Growing leaves and shoots were sampled to measure N remobilization and xylem sap flux and N composition were measured in order to: (1) determine the impact of the tree N supply on remobilization and the translocation of N in the xylem; (2) calculate the flux of remobilized N; and (3) compare the calculated flux of remobilized N with the amount measured by leaf sampling.
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Materials and methods |
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Plant material and treatments
One hundred and eight nursery apple trees (M. domestica Borkh. cv. Fuji), grafted on M.9 clonal rootstocks in the spring of 1998, with stem diameters of 9–15 mm measured 30 cm above the bud union, were lifted from the nursery when dormant, and planted in an Osoyoos loamy sand soil (Wittneben, 1986) on 25 May 1999, at the Pacific Agri-Food Research Centre, Summerland, Canada (49° 33' 39" N, 119° 38' 39" W). The trees were irrigated daily with two 4 dm3 h–1 pressure-compensating emitters per tree, spaced in the tree row at 25 cm either side of the trunk. Irrigation was applied to meet daily requirements, based on the demand as estimated by the previous day’s evaporation from an atmometer (ET Gauge Co., Loveland, CO, USA) linked through a data logger to irrigation controls. Nitrogen was injected into the irrigation water through injectors (model 283, Mazzei Injector Corp., Bakersfield, CA, USA) to give a mean concentration of N at the emitter of either 30 (low-N) or 150 (high-N) mg dm–3 as Ca(NO3)2. The trees were laid out in six replicate blocks and allocated to the N treatment and harvest (a total of nine) randomly within each block. Applications of N started on 25 May and continued until 20 July, and irrigation until 25 September. Irrigation and N applications were resumed on 27 March 2000 (at bud-burst) and continued until their respective harvest dates, with the trees receiving the same treatments as the previous year, except that all the N was supplied as Ca(15NO3)2 (enriched to 2.25 atom% 15N).
Tree harvests
Nine successive destructive harvests were made in 2000 (Table 1). On each occasion all above-ground growth was divided into the current year’s growth (spur leaves, shoot and shoot leaves, and reproductive tissues) and previous year’s growth (stem and branches). All samples were oven dried, weighed, and milled, prior to 15N analysis. A Tracer MAT continuous flow mass spectrometer (Finningan MAT, Hemel Hempstead, UK) was used for the determination of 15N and total N. The 15N enrichment was used to calculate the amount of labelled N taken up from the fertilizer applied in 2000, as described by Millard and Neilsen (1989).
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Sap-flow measurement
Eight sap-flow gauges of 19 mm diameter (Dynamax Inc., Houston, TX, USA), which operate on the theory of heat balance, were used to measure rates of whole-plant water flux (Baker and van Bavel, 1987). Four of the six replicate trees selected for the next harvest for each treatment were fitted with a gauge and monitored continuously, until they, in turn, were harvested. Measurements started prior to the first harvest, but no sap movement was detected until after the second harvest on 11 April. The gauges were installed in mid-afternoon, to take advantage of any diurnal shrinkage, which would allow for a tighter fit, at about 30 cm above the graft union. To insulate the gauges, a section of the trunk from 10 cm above to 10 cm below the gauges was wrapped with two layers of aluminium foil, followed by three layers of aluminized bubble-wrap and finally another three layers of aluminium foil. Each end of the insulation was sealed with electrical tape. To prevent sunlight from affecting the sensitive energy balance reading, the exposed trunk (a 20 cm section above the gauge and all the section below the gauge to the ground level) was covered with three layers of aluminium foil.
The Flow32TM sap-flow system (Dynamax, Inc.) was used to record data and supply the power to the heaters. It was equipped with a data logger (CR10X, Campbell Scientific Corp., Logan, UT), and a voltage regulator (model AVR3/6, Dynamax, Inc.) with the power down mode adopted to minimize heat build-up during the night when the sap flow was minimal. The power to the heaters was set at 0.26 W for the period between early spring and mid May, and then increased to 0.31 W because the tree canopy was larger. The data logger recorded every min and stored estimates of mean water flux every 15 min. Two deep-cycle batteries connected in parallel were used to supply power to the data logger and a total of eight gauges. Minimum sheath conductance was determined as the mean value between 03.00 h and 05.00 h, when whole-plant sap flow was assumed to be at its slowest. Preliminary gauge calibration was carried out in the greenhouse, using similar apple trees potted in 10 dm3 containers, and indicated a close relationship between the cumulative flow and the observed weight loss due to transpiration.
Xylem sap collection
Xylem sap was collected from six replicate trees from each treatment between 07.00 h and 08.30 h, just before their destructive harvest. To facilitate the collection of sap, the canopy of each of the 12 trees to be sampled was covered with a plastic bag after sunset the previous evening. This step was designed to improve their water status, by stopping any transpiration during the night caused by the especially high evaporation conditions typical of a semi-arid region. Preliminary tests showed that, without covering the trees, it was not possible to collect the required amount (>5 mm3) of sap, even with a high extraction pressure (>1 MPa).
Two branches of 80–110 cm length were selected at a height of 90–110 cm above ground from each tree. Two 2-year-old sections of 20 cm length were collected from the base of each branch and a few cm of bark were removed from the cut end. The exposed wood was washed with double-distilled water to avoid contamination from the phloem. The stem portion was immediately placed in a Scholander pressure chamber (Soil Moisture Equipment Corp., Santa Barbara, CA, USA) so that the section of wood, with bark removed, protruded. The chamber was pressurized to 
0.1 MPa and xylem sap was collected using a micropipette. The two samples (typically 50 mm3 each) from each tree were pooled and then stored at –80 °C until analysis. A previous study with apple trees (Malaguti et al., 2001) showed that no detectable ATP (an indication of cytoplasmic contamination) was found when a pressure of 0.2 MPa was used to collect the sap.
To check for any diurnal variation in the concentration of sap amino acids, additional samples were taken at the ‘tight-cluster’ stage. Twelve trees were sampled, as described above, by selecting four replicates at three separate times on the same day (08.00 h, 12.00 h and 18.00 h).
Analysis of xylem sap
Particulate material was removed from xylem sap samples by centrifugation in an MSE Micro-Centaur centrifuge for 5 min at 5800 g. Xylem sap samples (20 mg) were diluted with 1 cm3 demineralized water. A 40 mm3 aliquot of the dilute xylem sap and nor-valine (50 mm3 containing 0.05 µg) as an internal standard were added to a 1-CWV clear glass crimp-top-tapered vial (Chromacol Ltd, Welwyn Garden City, England) and freeze dried. The derivatization reagent (80 mm3), consisting of N-methyl-(tert-butyldimethylsilyl) trifluoroacetamide containing 1% tert-butyldimethylsilyl chloride (Sigma-Aldrich, Gillingham, England) in acetonitrile (1:4, v:v), was added to the dried material and left at room temperature for 10 min. The solution was then heated at 70 °C for 35 min to convert the free amino acids to their tert-butyldimethylsilyl derivatives (t-BDMS). The analyses of the derivatives were carried out using a Trace 2000 gas chromatograph, fitted with an AS 2000 autosampler, in the single ion recording (SIR) mode. The t-BDMS derivatives were separated using a fused silica Zebron ZB-5 capillary column, 30 m x 0.25 mm internal diameter x 0.25 µm phase thickness (Phenomenex, Macclesfield, England). The column was operated with a temperature program of 60 °C for 1 min, increased to 225 °C at 10 °C min–1, held for 1 min, increased to 325 °C at 7.5 °C min–1 and held for 5 min. The sample was introduced to the column using a splitless technique (splitless for 1 min followed by an 80:1 split), with an injector temperature held at 240 °C.
Arginine was determined as the N-heptafluorobutryl n-butyl ester (MacKenzie and Tenaschuk, 1979). Xylem sap samples (15 mg) containing nor-valine (50 mm3 H2O containing 0.36 µg) as an internal standard were freeze-dried and esterified by heating at 100 °C for 30 min in 120 mm3 reagent (acetyl chloride:n-butanol, 1:10, v:v). After evaporating excess reagent, the residue was acylated for 10 min at 150 °C using 120 mm3 heptafluorobutyric anhydride. After cooling, the excess reagent was evaporated; the residue dissolved in 100 mm3 ethyl acetate and the solution analysed by GC using the same conditions as described above, except that the column temperature program was changed to 60 °C for 1 min, increased to 225 °C at 10 °C min–1, held for 2 min and increased to 300 °C at 25 °C min–1 and held for 1 min. Amino acid concentrations were calculated using response factors derived from the analysis of solutions containing known weights of amino acids. Quality control was assured by analysing standard solutions of amino acids.
Statistical analysis
The effects of sample time and N-fertilizer treatments on tree growth, N remobilization, N uptake and xylem sap amino acid concentrations were determined by analysis of variance using SAS General Linear Model procedures (SAS Institute Inc., Cary NC, 1985). The relationships between sap N flux and tissue N content were determined using SAS- regression procedures (SAS Institute Inc., Cary NC, 1985).
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Results |
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Leaf and shoot growth and N remobilization
During 1999 the low-N trees received 13.5 g N tree–1 at planting and 5.1 g N tree–1 with their irrigation. High-N trees received the same amount at planting and 25.7 g N tree–1 through irrigation. Despite these differences in N supply there was no significant difference in the N status of the trees. The N content of leaves (sampled from mid-terminal, 1999 extension growth) in August (prior to any leaf senescence), was 28±0.9 mg g–1 and 31±0.5 mg g–1 for the low-N and high-N trees, respectively. The following spring, spur leaves grew quickly during the period between tight-cluster (day of the year (DOY) 110) and petal-fall (DOY 140), irrespective of N treatments (Fig. 1a). By petal fall, more than 75% of their final growth was achieved. By contrast, the rapid growth of shoot tissues (shoots and shoot leaves) did not occur until the growth rate of the spur leaves had declined (Fig. 1b). Rapid growth of the reproductive tissues (flower buds, flowers and fruit) was not seen until mid June, following fruit cell expansion (data not shown). The different N treatments had no significant (P >0.05) effect upon the total dry weight of all the tissues produced during 2000 (Fig. 1c).
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Remobilization of N (as measured by recovery of unlabelled N) for the growth of spur leaves started by tight-cluster (DOY 109), reached a maximum of about 2000 mg N tree–1 by petal fall and levelled off thereafter (Fig. 2a). In shoot tissues, however, the amounts of unlabelled N kept increasing throughout the experimental period (Fig. 2b), probably as a consequence of the uptake of mineralized N from the soil. However, any uptake of unlabelled N from the soil probably only occurred after fruit set as indicated by the significant difference (P <0.05) between the two fertilizer treatments after DOY 150. Before then, N supply had no significant effect on the recovery of unlabelled N. It is likely, therefore, that the N supply had no effect upon the amount of N remobilized for the growth of either spur leaves or shoot tissues. The reproductive tissues did not become a substantial sink for unlabelled N until the completion of fruit-cell division (approximately DOY 160) and by the end of the experiment accounted for approximately one-sixth of the total, unlabelled N recovered in the new, above-ground growth (data not shown). By the last sampling time (late July, DOY 206), the low-N trees had accumulated more unlabelled N in new above-ground growth than those receiving high N (Fig. 2c), although the difference was not significant (P >0.05).
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Fertilizer (labelled) N was recovered in spur leaves by the pink growth stage (DOY 123) (Fig. 3a), 14 d after remobilization of N had started (Fig. 2a). N supply had a significant (P <0.01) effect upon the labelled N content of spur leaves. By the final harvest, labelled N had provided 8% of the N in the spur leaves of low-N trees and 27% in the high-N trees. A similar trend was found for the uptake of labelled N into shoot tissues (Fig. 3b), which contributed 13% and 39% of the total N recovered by the end of the experiment in low-N and high-N trees, respectively. The contribution of the labelled fertilizer N to the total N content of shoot leaves was relatively small (<18%) until after petal fall (DOY 150), confirming that N remobilization was probably unaffected by current season uptake. Low root uptake of labelled N from fertilizer in the low-N treatment was partially offset by uptake from the soil N pool (Fig. 2b). The reproductive tissues did not accumulate labelled N until after fruit set. By the end of the experiment they contained 12.4% (low-N) and 17.8% (high-N) of the total recovered in the new, above-ground growth.
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Flux of N translocation in the xylem
Sap flux in the xylem increased from the stage of bud-break until July, with the maximum daily flux being around 4 dm3 tree–1 (Fig. 4). There was no significant effect of N treatment on the daily sap flux.
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The predominant amino acids recovered from the xylem sap were Asn, Asp, Arg and Gln, between them accounting for about 95% of N recovered, regardless of when the sap was sampled (Table 2). As there were very few significant differences in individual amino acid or amide concentrations in response to the low-N and the high-N treatments, the values in Table 2 were pooled across N treatments. There were large quantitative and qualitative differences in the composition of the xylem sap throughout the experiment. The pooled data showed that during remobilization Asn, Asp, Arg, and Gln accounted for 28%, 19%, 28%, and 19% of the total N recovered in the saps, and that after remobilization had finished these values changed to 25%, 36%, 29%, and 5%, respectively (Table 2). There was also a substantial decrease in the total concentration of N in the sap once remobilization had finished.
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The concentrations of Asn, Asp and Gln in the xylem sap changed in a similar pattern over time, with an increase in the maximum between ‘half-an-inch green’ (DOY 102) and ‘tight-cluster stage’ (DOY 109) and a decrease thereafter (Fig. 5). The maximum values of Asn, Asp and Gln were 145, 108 and 110 µg N g–1 sap, respectively. By contrast, Arg did not follow the pattern shown by other amino acids and amides. The concentration of Arg was highest (171 µg N g–1 sap) on the first sampling date (bud break), and then, with several fluctuations, decreased thereafter (Fig. 5). When trees were sampled at different times during the day (at tight-cluster, DOY 109 when sap N concentrations were maximal), no significant diurnal differences in the concentration of dominant amino acids were found. The total concentration of Asn, Asp, Arg, and Gln together was 413±97, 405±85 and 417±6 µg g–1 sap, at 08.00 h, 12.00 h and 18.00 h, respectively.
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N sap flux versus N remobilized
The flux rate for N in each of the four main amino acid and amides in the xylem for each sampling period was calculated. The sap flux rate (g d–1 tree–1) was multiplied by the amino acid or amide-N concentration (µg N g–1 sap). Values of the sap flux rate for each sampling time were obtained from the regression made between daily sap flux and time, pooling the low-N and high-N treatments (Fig. 4). The calculated flux of N in the xylem as Asn, Asp and Gln peaked around full-bloom at DOY 130 (Fig. 6). Thereafter, their flux rate declined. By contrast, the flux rate of Arg peaked at full bloom, decreased until fruit set (DOY 158), but then increased again to give a value at DOY 206 that was three times the flux found at full bloom.
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To identify the amount of N translocated in the xylem as a consequence of remobilization, curves were fitted to the data for the flux of N in each of the amino acids and amides in Fig. 6. The area under the curves for Asp, Asn, Gln and Arg up to DOY 158 were then integrated and summed. These compounds were chosen because they accounted for most of the translocated N and because a previous study (Malaguti et al., 2001) had shown them to be the amino acids and amides likely to be involved in N remobilization in M. domestica. Integration up to DOY 158 was chosen because, by then, the recovery of unlabelled N in the new growth above ground had shown that N remobilization had finished (Fig. 2). Figure 7 shows a comparison of the amount of N remobilized up to DOY 158, as measured by the recovery of unlabelled N in new above-ground growth (Fig. 2), with the amount calculated by the flux of N in Asp, Asn and Gln, with and without Arg, in the xylem for the same period (Fig. 6; Table 3). The slope of the two regression lines indicated that sap amino acid flux including Arg overestimated remobilization (slope=1.54) compared with sap amino acid flux without Arg (slope=1.01).
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Discussion |
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N remobilization and uptake
Young M. domestica trees rely heavily upon remobilization of N for their growth in the spring (Neilsen et al., 1997, 2001). The trees in the current experiment were evidently replete with N at planting, because there was no growth response to the N treatments applied in either year. This may have been a consequence of the fact that, as in many species of tree, remobilization uses N taken up by the root over several previous years (Weinbaum and van Kessel, 1998). The high-N treatment was also possibly supra-optimal for N uptake by trees in irrigated and fertilized orchards (Neilsen and Neilsen, 2002). In a previous experiment, the uptake of N by newly planted M. domestica trees was not limited even when the soil-solution N concentrations were between 10 and 20 mg dm–3 (Neilsen et al., 2001), lower levels than the treatment concentrations in the present study. As a consequence, the treatments applied in the current study had no effect upon N remobilization one year after planting, but did affect N uptake in the second year.
Measurement of remobilization by sap flux
The amount of N remobilization, estimated by coupling the sap-flux measurements and the concentration of N as Asp, Asn and Gln translocated in the xylem, was in agreement with that measured by recovery of the unlabelled N in the new growth. However, for this method to be successful a number of assumptions must be made.
First, it is assumed that there is no diurnal or spatial variation in the concentration of N compounds in the xylem. These data showed no diurnal variation in sap N concentrations. However, other species of tree, such as Pryus communis and Prunus persica (Andersen et al., 1995) do show marked diurnal variations in sap N composition. Several studies have also reported spatial variations in the compositon of xylem sap when sampled from different positions in the tree (Glavac et al., 1989; Smith and Shortle, 2001). In addition there are reports of spatial variations in sap flux density, although these have been under conditions where soil moisture supply may have been less than adequate (Loustau et al., 1998; Lu et al., 2000). If a significant exchange of N compounds occurs between the xylem sap and surrounding tissues, the composition of the sap at one point might not reflect the flux of remobilized N reaching the buds and growing leaves. In the current study, the influence of any such spatial effects was reduced by collecting and pooling sap from two separate branches, which represented a significant proportion of the tree canopy. However, if attempting to use this method, the presence of any diurnal or spatial pattern of amino acid and amide concentrations in the xylem would need to be evaluated, particularly for larger trees.
The second main assumption of the method is that translocation of one or more N compounds are specific to remobilization. Several studies using 15N labelling have demonstrated translocation of specific compounds during remobilization: Cit in B. pendula (Millard et al., 1998); Arg and, to a lesser extent, Gaba, Gln and Cit in Juglans nigraxregia (Frak et al., 2002), and Gln, Asn and Asp in P. avium (Grassi et al., 2002). In the present study, the fertilizer applied with the irrigation was not sufficiently enriched with 15N to permit the determination of the labelling pattern of individual amino acids in the xylem sap. Thus, it is not certain that the translocation of Asp, Asn and Gln up to DOY 158 was due solely to remobilization. This assumption that these three compounds were probably specific to remobilization was based upon the 15N-labelling pattern reported by Malaguti et al. (2001), who applied labelled fertilizer the year before sampling the xylem sap of potted M. domestica trees. However, Malaguti et al. (2001) were not able to use their 15N labelling to determine if translocation of Asp, Asn and Gln were exclusively due to remobilization, without any contribution from root uptake. Furthermore, their study did not quantify the Arg concentrations or enrichments and so it could not assess the contribution of Arg translocation to N remobilization.
In the present study, the flux of N in Asp, Asn and Gln corresponded well with the amount of N remobilization, as measured by the recovery of N at natural abundance in the new growth. Also, the rapid increase in the xylem sap flux of Arg after DOY 158 suggested that nitrate taken up and assimilated by the roots was translocated in this form. However, there was a discrepancy between the total flux of N measured in the xylem and the N recovered in the new above ground growth. By DOY 158 a total flux of 4068 mg N tree–1 was calculated in the xylem from Asp, Asn and Gln. This figure compares well with the recovery of 4134 mg N tree–1 as unlabelled N in the new growth (averaged across both the treatments). The xylem flux of Arg over the same period was calculated as 2148 mg N tree–1. However, the uptake of labelled N was only 789 mg and 328 mg from the high-N and low-N trees, respectively. Therefore, the flux of Arg would have overestimated root uptake. This discrepancy is possibly a consequence of a shoot-to-root translocation of N. Several studies with herbaceous species have demonstrated that a considerable proportion of the N recovered in xylem sap has been recycled (via the phloem) from shoots to roots and then reloaded into the xylem (Simpson et al., 1982; Cooper and Clarkson, 1989; Jeschke and Hartung, 2000). Such internal recycling of N has also been shown to occur in conifer trees (Weber et al., 1998). It has been suggested that because there is an inverse correlation between amino–N concentrations in the phloem and nitrate uptake by roots, such internal recycling of N is involved in the regulation of nitrate uptake by trees (Gessler et al., 1998; Weber et al., 1998; Youssefi et al., 2000). In the present experiment, it is possible that a proportion of the N translocated during remobilization was recycled back to the roots and so measured more than once as a flux in the xylem. As a consequence, these data are not sufficient for it to be certain that Arg translocation was only due to root uptake.
A final potential error in measuring N remobilization by sap flux is the accuracy of using the heat balance method to measure whole-tree transpiration. Previous attempts to measure the sap flux of amino acids and amides have used potted trees, where transpiration could be measured by weighing the trees (Frak et al., 2002; Grassi et al., 2002). There are several potential errors in the heat balance method (Grime and Sinclair, 1999). These include heat storage in the stem due to the changes in stem temperature (Shackel et al., 1992), particularly for trunks with a diameter greater than 3 cm (Weibel and Boersma, 1995), and poor contact between the gauge and the stem (Weibel and Devos, 1994). However, despite these potential problems the heat balance method has been successfully used for sap flow measurements in M. domestica, to give errors of less than 4% of those measured by gravimetic weight loss (Weibel and Boersma, 1995). It was not possible to check the accuracy of the sap flow in these field-grown trees gravimetrically. However, in the current experiment, the trees had trunk diameters less than 3 cm and great care was taken to ensure good contact between the stem and the gauge. Therefore, from the data in the present study it would appear that if there were any systematic errors in estimating sap flux, then they were compensated for by errors in the other measurement techniques used.
Given the assumptions that are necessary for this method to work, the potential errors involved in the measurements, and the possibility of recycling of N from xylem to phloem and back to the xylem, it is perhaps surprising that such good agreement was found between the calculated flux of remobilized N and the unlabelled N recovered in new growth. This is the first time that such a method has been attempted for field-grown trees. N remobilization appears to be a source-driven process (Millard et al., 2001), and so all N allocated to storage in deciduous trees in the autumn is remobilized the following spring, irrespective of the current N supply (for a review see Millard, 1996). Therefore, despite the potential errors involved in the measurements, the calculation of the flux of remobilized N should allow the N storage capacity of a tree to be determined. This could be used to determine either the impact of management or environmental change on the ability of trees to store N. Even if the results are only relative, the method should allow a good comparison between trees from different treatments and offer significant advantages over the traditional methods using isotopes and destructive tree harvests.
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Acknowledgements |
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We thank the Washington Tree Fruit Research Commission, the Agriculture and Agri-Food Canada MII Programme and the Scottish Executive Environment and Rural Affairs Department for funding this study. Our gratitude is also extended to David Gregory, Brian Drought, Andrea Martin, Linda Herbert, Steve Losso, Michael Beulah, and Bill Rabbie for their help with the tree sampling, and Alan Hepburn for the GC analyses. This paper is Summerland contribution No. 2197.
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