Blackwell Science, LtdOxford, UKPCEPlant, Cell and Environment0016-8025Blackwell Publishing Ltd 2002 25 Original Article Xylem sap amino

acid translocation and N remobilizationG. Grassi et al.

Plant, Cell and Environment (2002) 25, 16891699

Measurement of xylem sap amino acid concentrations in conjunction with whole tree transpiration estimates spring N remobilization by cherry (Prunus avium L.) trees
G. GRASSI1, P. MILLARD2, R. WENDLER2, G. MINOTTA3 & M. TAGLIAVINI1

Dipartimento di Colture Arboree, Universit di Bologna, Via Fanin 46, 40127 Bologna, Italy, 2Macaulay Institute, Craigiebuckler, Aberdeen AB15 8QH, UK and 3Dipartimento AGROSELVITER, Universit di Torino, Italy

ABSTRACT
Prunus avium trees were grown in sand culture for one vegetative season with contrasting N supplies, in order to precondition their N storage capacities. During the spring of the second year a constant amount of 15N was supplied to all the trees, and the recovery of unlabelled N in the new biomass production was used as a direct measure of N remobilization. Destructive harvests were taken during spring to determine the pattern of N remobilization and uptake. Measurements of both xylem sap amino acid proles and whole tree transpiration rates were taken, to determine whether specic amino acids are translocated as a consequence of N remobilization and if remobilization can be quantied by calculating the ux of these amino acids in the xylem. Whereas remobilization started immediately after bud burst, N derived from uptake by root appeared in the leaves only 3 weeks later. The tree internal N status affected both the amount of N remobilization and its dynamics. The concentration of xylem sap amino acids peaked shortly after bud burst, concurrently with the period of fastest remobilization. Few amino acids and amides (Gln, Asn and Asp) were responsible for most of N translocated through the xylem; however, their relative concentration varied over spring, demonstrating that the transport of remobilized N occurred mainly with Gln whereas transport of N taken up from roots occurred mainly with Asn. Coupling measurements of amino acid N in the xylem sap with transpiration values was well correlated with the recovery of unlabelled N in the new biomass production. These results are discussed in relation to the possibility of measuring the spring remobilization of N in eld-grown trees by calculating the ux of N translocation in the xylem. Key-words: asparagine; cherry; glutamine; N remobilization; 15N; transpiration; xylem sap.

INTRODUCTION
Remobilization of stored nitrogen is used by many trees to augment the supply of nutrients from the soil (Millard
Correspondence: Giacomo Grassi. Fax: +39 0512096401; e-mail: grassi@agrsci.unibo.it 2002 Blackwell Publishing Ltd

1996). This source of N is often the rst, and sometimes the only N-source used for growth in the spring and can provide the majority of nitrogen used for growth each year (e.g. Millard & Proe 1991; Neilsen et al. 1997; Weinbaum & van Kessel 1998; Dyckmans & Flessa 2001). However, measurement of the nitrogen storage capacity of a tree is difcult. Studies have often used either 15N enriched (e.g. Millard 1996) or depleted tracers (e.g. Weinbaum et al. 1984; Weinbaum & van Kessel 1998), involving making destructive budgets of tree N. Such studies are either restricted to the use of sand culture for growing small trees, or can be imprecise if N budgets are constructed for large, eld-grown trees. An alternative method to quantify the N storage capacity of a tree might be to measure remobilization of N, because the amount of N remobilized depends upon the amount in store and is unaffected by the current N supply (Millard 1996). Furthermore, storage pools of N can disappear completely by the summer (Coleman et al. 1993; Sauter & van Cleve 1994). Several studies have shown a peak in the concentration of N in the xylem sap during bud burst and leaf growth which was attributed to N remobilization (Ferguson, Eiseman & Leonard 1983; Glavac & Jockheim 1993; Schneider et al. 1994). Using 15N to label storage pools, Millard et al. (1998) showed that remobilization by Betula pendula trees led to a more than 10-fold increase in the concentration of citrulline and glutamine in the xylem sap. The subsequent decrease in concentration of N in the xylem sap was not solely due to dilution, caused as a consequence of higher transpiration rates, but to the end of remobilization (Millard et al. 1998). A similar pattern of amino acid translocation has been measured in the xylem of Malus domestica trees during remobilization (Malaguti et al. 2001). Coupling measurements of the concentration of the amino acids translocated during remobilization with either sap velocity or whole tree transpiration might allow the ux of N to be calculated. Such an approach has already been used to quantify mineral uxes via xylem sap ow in Picea abies trees (Dambrine et al. 1995). This approach could potentially give a non-destructive method to measure N remobilization, and so indirectly the storage capacity of the tree, which does not depend upon the use of tracers. To determine the feasibility of such an approach we grew 1689

1690 G. Grassi et al. Prunus avium trees in sand culture with contrasting N supplies, in order to precondition their N storage capacities. During the spring of the second year we supplied a constant amount of 15N to all the trees and used the recovery of unlabelled N in the new biomass production as a measure of N remobilization. In addition, we measured both xylem sap amino acid proles and whole tree transpiration rates, in order to determine if: (1) there are specic amino acids translocated by P. avium as a consequence of N remobilization, and (2) remobilization can be quantied by calculating the ux of these amino acids in the xylem. The use of trees pre-conditioned to have contrasting amounts of N available for remobilization was to test how robust such a technique could be. between each. One tree from each treatment within each block (four replicates per treatment) was randomly assigned to each harvest. At harvest the trees were destructively sampled, and xylem sap collected as described below. The trees designated for the next harvest then had their transpiration rate measured (as described below) until they in turn were sampled.

Daily transpiration measurements

At the beginning of each measurement period, drip emitters were removed and each selected pot was carefully enclosed in a plastic bag. During the period a known amount of nutrient solution was given manually three times a week, and at the same time the plastic bags were opened for a few minutes to avoid the exposure of roots to anoxia. When the trees were harvested subsequently, there was no evidence of any root anoxia. The average daily loss of water by transpiration during each measurement period (expressed as g tree-1 d-1) was calculated from the difference between the sum of the weight of the pot at the beginning of the sampling period plus the weight of the nutrient solution that had been provided, and the weight of the pot at harvest. Additionally, diurnal variations in transpiration were assessed in another set of eight cherry trees (four trees per treatment), grown in the same experimental conditions, by continuously measuring sap ow with heat balance stem gauges (Smith & Allen 1996).

MATERIALS AND METHODS Experimental design

One-year-old Prunus avium L. seedlings (80 in total, provenance northern Italy) were lifted from a nursery while dormant and placed in cold storage, until planted in 14 dm3 pots containing ne sand in May 1999. The trees had previously received a moderate N supply. The pots were arranged outside and placed in four randomized blocks under a shade cloth (which had 50% transmittance). Nitrogen at natural abundance was applied as NH4NO3 once a week, in order to provide each plant with a total of 06 g from May to June, and with either 1 (low nitrogen treatment, LN) or 8 g of N (high nitrogen treatment, HN) from July to October In addition, an automatic, drip emitter irrigation system (four emitters per plant) distributed 1 dm3 of nutrient solution per pot, three times a week. This provided each plant during the whole growing season with 08 g P, 27 g K, 09 g Ca, 14 g S, 03 g Mg, 6 mg B, 3 mg Mn, 3 mg Zn, 1 mg Cu and 45 mg Fe EDDHA (ethylenediamine-di(o-hydroxy-phenylacetic) acid). When required, additional water was given manually. In October 1999 (just before leaf senescence) leaf samples were collected from six randomly chosen plants and their nitrogen concentration measured by a Kjeldahl assay. In December 1999 the stem height, stem diameter (at the base of the tree) and number of buds per plant were measured in all trees. In January 2000 the pots were carefully washed with deionized water to remove any residual nitrogen present in the sand and then transferred to a greenhouse and arranged in four replicate blocks according to their height. Each block contained 10 plants from each treatment. From late February 2000 and throughout the spring the number of open buds on each tree was assessed every 12 d. For each individual plant the day the rst open bud was noted was designated as the date of bud burst. From bud swelling onwards, each plant was re-supplied with a nutrient solution identical to that used in 1999, except that both treatments received three times a week 015 g of N as 15NH415NO3 enriched with 15N to 775 atom percent excess. Ten destructive harvests were taken between 28 March and 20 June 2000, with 714 d intervals

Tree harvesting and analysis

At each harvest the stems of the eight selected plants were cut for collection of xylem sap (see below). The trees were then removed from their pots and the sand was carefully washed from the root system. Trees were separated into ve different organs (leaves, axes, stem, coarse and ne roots), which were dried, weighed and milled to pass through a 5mm screen prior to 15N analysis. A subsample of leaves per each plant was measured for leaf area [with a Hewlett Packard Scan-Jet 6300c scanner (Hewlett Packard, Palo Alto, CA, USA) and Jandel Sigma Scan Pro V20 software (SPSS Inc., Chicago, IL, USA)], and subsequently ovendried to constant weight to obtain leaf mass per area (LMA, g m-2). The total leaf area of each tree was calculated by dividing its leaf dry weight by its LMA. An elemental analyser coupled with a ow mass spectrometer (Finningan deltaplus, Hemel Hempstead, UK) was used for determination of 15N and total N in leaves and axes. The 15 N enrichment was used to calculate the uptake of labelled N taken up during 2000 (Millard& Neilsen 1989). Recovery of unlabelled N in the leaves and axes gave a direct measure of N that had been taken up before 2000 and remobilized for new biomass production during the spring of 2000.

Sap collection
Xylem sap collection was carried out just before harvesting the trees, between 1130 and 1430 h. A portion of the stem,

2002 Blackwell Publishing Ltd, Plant, Cell and Environment, 25, 16891699

Xylem sap amino acid translocation and N remobilization 1691 between 20 and 50 cm above the collar, was collected from the trees. A few centimetres of bark were removed from the higher cut end of the stem to avoid phloem contamination. The stem portion was immediately placed in a Scholander pressure chamber so that the section of wood with bark removed protruded. The pressure in the chamber was slowly increased to a maximum of 02 MPa, and xylem sap exuded was collected with micro-capillary tubes. Initial tests (as described by Malaguti et al. 2001) indicated that pressures up to 02 MPa did not cause any contamination by cellular components. Sap samples were stored at -70 C until analysis of the concentration and 15N enrichment of their individual amino acids by gas chromatography linked to mass spectrometry (GCMS). measured for an individual amino acid had to be greater than the precision value for the corresponding standard before the sample was considered to be enriched.

Data analysis
For the four replicates per treatment, data for leaf area, total new biomass production (leaves and axes), recovery of unlabelled and labelled N, xylem sap amino acid proles and whole tree transpiration were related to the stage of development by using the number of days from bud burst as a measure of time. Data for leaf area, total new biomass production, recovery of unlabelled and labelled N in the new biomass production and transpiration were tted against time using sigmoid functions of Table Curve 2d software (SPSS Inc., Chicago. IL. USA). These functions were chosen because they provided a good and biologically meaningful description of the phenomenon. Data for leaf area, total new biomass production, recovery of unlabelled and labelled N in the new biomass production collected after the 95% of the maximum value was reached (calculated using the functions described above) were subjected to analysis of variance to determine the signicance of the difference between the treatments. Data of amino acid N in xylem sap during and after the period of N remobilization were processed by analysis of variance for the effects of N treatments, time and their interaction using the SPSS for Windows (version 61.3; SPSS Inc.) statistical package. The effect of tree N status on the comparison of the two methods for measuring N remobilization was determined by a comparison of the slope of linear regressions between the amount of labelled N recovered in leaves and the ux of amino acid and amide N in the xylem, as described by Gomez & Gomez (1984).

Analysis of saps by GCMS

Particulate material was removed from xylem sap samples by centrifugation in an MSE Micro-Centaur centrifuge (Thermo ARL UK, Crawley, West Sussex, UK) for 5 min at 5800 g. Xylem sap samples (20 mg) were diluted with 1 cm3 of deionized water. An aliquot of the dilute xylem sap and nor-valine (25 mm3 containing 018 mg nor-valine cm-3) as internal standard were added to a clear glass vial and freeze dried. The free amino acids were converted into their tert-butyldimethylsilyl (t-BDMS) derivatives as described by Millard et al. (1998). The analyses of the derivatives were carried out using GCMS in the single ion recording mode. The instrumentation used was a Trace 2000 gas chromatograph, tted with an AS 2000 autosampler and interfaced to a Finnigan Trace quadrupole mass spectrometer (Thermoquest, Hemel Hempstead, UK). Separation of the tBDMS derivatives was effected using a fused silica Zebron ZB-5 capillary column, 30 m 025 mm id 025 mm phase thickness (Phenomenex, Maccleseld, UK). The temperature programme used was 60 C for 1 min, increased to 225 C at 10 C min-1, held for 1 min, increased to 325 C at 75 C min-1 and then held for 5 min. The injector temperature was held at 240 C and the interface line temperature maintained at 250 C. The mass spectrometer was operated under electron impact ionization mode with an ionization energy of 70 eV and a source temperature of 200 C. The range of ions monitored were the same as those described by Millard et al. (1998). The enrichment of 15N in individual amino acids was calculated from the ratio of the ion monitored at natural abundance and enriched amino acids (Campbell 1974). Amino acid concentrations were calculated using response factors obtained from the analysis of solutions containing known weights of amino acids. Quality control was assured by analysing standard solutions of amino acids. The precision of the atom percent excess (APE) measurements were calculated for each amino acid by measuring, within each analytical run, the variation expressed as mean 2 SD (n = 4) of the ratio of the appropriate ions when analysed at natural abundance. The overall mean precision of the isotope ratio analyses were 018 APE for Asp, 021 APE for Glu, 037 APE for Asn and 015 APE for Gln. The APE

RESULTS Tree growth and N remobilization

The contrasting N treatments applied during the rst year of the experiment resulted in trees with a differing N status. At the end of the rst growing season the total leaf N concentration and the number of buds set per plant were signicantly higher in HN than in LN plants, whereas no differences among treatments were observed in stem height and diameter (Table 1). At the beginning of the second vegetative season leaf area developed earlier in HN than in LN plants; subsequently, leaf area remained slightly higher in HN than in LN plants for all the experimental period (Fig. 1a), reaching 95% of nal area 56 d after bud burst for both treatments. Both leaf and twig mass continued to increase after leaf area expansion nished, so total new biomass production reached 95% of nal mass 74 and 70 d after bud burst for HN and LN plants, respectively (Fig. 1b). After the 95% threshold, the difference between treatments was signicant at P < 005 for leaf area (089 002 m2 for HN and 081 002 m2 for LN) and for total new biomass production (710 14 g for HN and 630 15 g for LN).

2002 Blackwell Publishing Ltd, Plant, Cell and Environment, 25, 16891699

1692 G. Grassi et al.

Table 1. Parameters measured in Prunus avium trees at the end
of the rst year of experiment (1999) SE Level of signicance ** NS NS *

LN Leaf N before leaf senescence (% DW) Stem height (cm) Stem diameter at the base of the tree (cm) Number of buds 21 01 151 4 15 002 31 1

HN 26 01 155 3 15 003 34 1

The concentrations of the three predominant amino acids and amides in the xylem sap were followed during time, and their 15N labelling was used to determine the amount of each translocated as a consequence of N remobilization. Gln and to a lesser extent Asn and Asp were translocated as a consequence of N remobilization, with tree N status having no effect on the form of N translocated during remobilization (Fig. 3a & b). In LN plants some Gln and Asn were also translocated as a consequence of direct

High-nitrogen (HN) and low-nitrogen (LN) treatments refer to the amount of nitrogen received in 1999. Number of replicates: six for leaf N, and 40 for stem height, stem diameter and bud number. Levels of signicance: NS, not signicant, *P 005, **P 001.

1 .2

(a)
0 .9

Total new biomass production (g tree )

Remobilization of N started straight after bud burst in both treatments, reaching 95% of maximum value 62 and 40 d after bud burst for HN and LN plants, respectively (Fig. 2a). These dates were designated as the time when remobilization nished and were used in subsequent calculations. After these dates, the difference between treatments in the amount of N remobilized was signicant at P < 0001 (489 26 mg tree-1 for HN and 237 16 mg tree-1 for LN). At the end of the experiment, the fraction of total N in the new biomass production that came from remobilization was 26% for HN and 14% for LN plants. Recovery of N taken up by roots occurred after remobilization of N had started, reaching the 5% of maximum value at 22 and 20 d after bud burst for HN and LN plants, respectively (Fig. 2b). In the new biomass a slightly higher content of N deriving from root uptake was found in LN in comparison with HN plants; however, after the 95% of maximum value was reached no statistical differences between treatments were recorded (1388 37 mg tree-1 for HN and 1469 62 mg tree-1 for LN).

Leaf area (m tree )

-1

0 .6

0 .3 LN HN 0 0 20 40 60 80 Days from bud burst 100

80

(b)
60

-1

40

Pattern of N translocation in the xylem

The main amino acids and amides recovered in the xylem sap were Gln, Asn and Asp, accounting for 96% of N recovered during the period of N remobilization and 91% of N recovered after remobilization had nished (Table 2). No nitrate was detectable in the xylem saps during the remobilization period. In this period, the total concentration of N in the sap of HN plants was slightly lower in comparison with LN plants, whereas transpiration rates were slightly higher; however, no statistically signicant differences were found between treatments in either N concentration or transpiration. The proportion of the total N in the sap recovered in each amino acid or amide did vary through time. During remobilization Gln, Asn and Asp accounted for about 45, 31 and 20% of the total N recovered in the saps of both treatments. After remobilization had nished, these values changed to 5, 72 and 13%, respectively (Table 2).

20 LN HN 0 0 20 40 60 80 Days from bud burst 100

Figure 1. Pattern of (a) leaf area and (b) total new biomass production (leaves + axes) during 2000 in Prunus avium trees, in relation to days from bud burst. High-nitrogen (HN) and lownitrogen (LN) treatments refer to the amount of nitrogen received in 1999. Data points in (a) are tted with sigmoid curves with the following equations: y = -004 + 088/(1 + exp((x - 306)/101)) for LN plants (r2 = 094, P < 0001); y = 0018 + 090/(1 + exp((x 304)/966)) for HN plants (r2 = 093, P < 0001). Data points in (b) are tted with sigmoid curves with the following equations: y = -298 + 692/(1 + exp((x - 425)/129)) for LN plants (r2 = 097, P < 0001); y = -253 + 758/(1 + exp((x - 427)/129)) for HN plants (r2 = 098, P < 0001).

2002 Blackwell Publishing Ltd, Plant, Cell and Environment, 25, 16891699

Xylem sap amino acid translocation and N remobilization 1693

Table 2. Comparison of the recovery of N in amino acids in the xylem sap of Prunus avium trees harvested during the period of N
remobilization and after remobilization had nished in 2000. High-nitrogen (HN) and low-nitrogen (LN) treatments refer to the amount of nitrogen received in 1999. N treatment HN Time (days from bud burst) Amino acid Alanine Asparagine Aspartic GABA Glutamic Glutamine Glycine Isoleucine Leucine Phenylalanine Proline Serine Threonine Valine Total Transpiration 10 2 09 01 488 44 295 71 24 05 73 16 673 127 07 02 58 07 25 03 94 16 134 31 56 14 168 22 110 16 1532 202 150 51 63 2 11 01 850 29 163 39 04 01 19 06 57 21 05 01 03 01 01 00 01 00 16 06 12 04 22 07 06 01 117 37 315 13 LN 12 1 07 02 666 115 428 93 12 02 58 12 986 194 05 01 58 00 30 03 73 06 81 13 65 14 205 06 105 05 2150 363 84 15 59 2 15 02 685 17 119 25 05 01 17 03 47 16 06 01 02 01 02 00 01 00 11 02 13 03 17 05 05 01 945 23 300 33 Level of signicance N time N time

NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS

* *** *** *** *** *** NS *** *** *** *** *** *** *** *** ***

NS NS NS * NS NS NS NS NS NS NS NS NS NS NS NS

Values are given as g amino acid N g xylem sap1 and are mean SE of four replicates. Mean daily transpiration (g tree1 d1) SE of the selected plants is also shown. Levels of signicance: NS, not signicant, *P 005, ***P 0001.

N uptake by roots, while remobilization was in progress (Fig. 3d).

Calculating the ux of remobilized N

Having determined the temporal pattern of amino acid concentrations in the xylem sap, whole tree transpiration for each sampling period was measured, so that the total amount of N translocated through the xylem could be calculated. The mean daily transpiration water loss calculated for each sampling period (Fig. 4a) followed the increase in leaf area as the canopy grew (Fig. 1a). Because of the earlier development of their leaf area, HN plants showed a higher transpiration than LN plants during the period of intense remobilization (in the rst 34 weeks). However, after 95% of maximum transpiration was reached no statistical differences between treatments were observed (307 17 g tree-1 d-1 for HN and 294 8 g tree-1 d-1 for LN). Measurement of the diurnal variation in sap ux showed that the great majority of total ux occurred between 0700 and 1900 h (Fig. 4b). For each sampling period, data on the concentration of remobilized N in the xylem sap (Fig. 3a & b) were multiplied by the total transpiration measured on the same plant, to calculate N remobilization for that period. Only the N present in the three main amino acid and amides (Gln, Asn and Asp), which accounted for 96% of total N recovered in xylem sap during the period of N remobilization (Table 2), was considered in this analysis. For each treatment, the mean value of N remobilization estimated for the

individual plants in a sampling period was added to the mean value of N remobilization of the previous period, in order to calculate the cumulative N remobilized. This procedure was then repeated until the end of the period of N remobilization for each treatment (62 and 40 d from bud burst for HN and LN trees, respectively). Figure 5 shows the amount of N remobilized by HN and LN trees during the remobilization period, as measured by the recovery of unlabelled N in their new biomass production (Fig. 2a), compared with the amount calculated by the ux of amino acid in their xylem. The comparison showed a good agreement between the two methods, and the slopes of the linear regressions for the two N treatments (084 for the LN trees and 109 for the HN trees) were not signicantly different. During the calculation of N ux through the xylem we assumed that the concentration of N in the xylem sap which was taken at the end of the sampling period was representative of the mean concentration of the whole period. This assumption involves two possible errors. First, marked diurnal variations in xylem sap N concentration, possibly due to variation in transpiration rates, would mean that at the time of sap collection the concentration of xylem N was signicantly different from other periods of the day. A separate study on P. avium trees indicated that in spring xylem sap N concentration during the night was signicantly higher (60% more, P < 001) than during the day. However, if we consider that in the trees used in our experiment transpiration during the night period (19000700 h) was negligible in comparison with

2002 Blackwell Publishing Ltd, Plant, Cell and Environment, 25, 16891699

1694 G. Grassi et al. separate set of 20 trees, 30 ( 2) days after bud burst; sap samples were collected from 3 or 4 replicate trees per treatment in three different periods: 07001100, 11001500 and 15001900 h. The concentration of N in Gln, Asn and Asp showed a relatively small, and statistically insignicant, decline during the central part of the day (Table 3); furthermore, because of the higher transpiration rates, the total ux of N through the xylem was higher during the middle of the day, the period when saps were collected from the main experiment. Therefore, we concluded that any errors associated with a diurnal variation in sap composition were negligible. The second possible error could be due to marked variations in amino acid concentrations during the sampling period. This occurred during the rst 3 weeks following bud burst, when a peak in concentration was evident (Fig. 3). This may have led to an overestimation of remobilization of N if the saps were collected during the ascending part of the peak and to an underestimation if they were collected as the peak declined. However, these two errors would partly compensate for each other, and so the overall error was considered probably to be negligible.

800

(a)
Unlabelled N (mg tree )
-1 15

600

400

200 LN HN 0 0 20 40 60 80 Days from bud burst 100

2000

(b)
Labelled N (mg tree )
-1

1600 1200 800 400 0 0 20 40 60 80 100 Days from bud burst

DISCUSSION Tree growth, N status and N remobilization

The N treatments applied during the rst year of the experiment did not have any effect upon tree growth. This was probably due to the N treatments being applied from July to October At this time most of the N taken up by the roots is partitioned to the perennial organs, thereby increasing their N storage capacity, as demonstrated in Prunus persica (Tagliavini, Millard & Quartieri 1998) and Pyrus communis (Quartieri, Millard & Tagliavini 2002 ). There was, however, an effect of high N supply on leaf phenology, which was exerted through both a delay in autumn leaf abscission and earlier bud burst (data not shown) and an earlier leaf growth. Tree internal N status affected both the amount of N remobilization, which at the end of the experiment was more than double in HN than in LN trees, and its dynamics, with HN trees relying on N remobilization for a much longer period than LN plants.

15

LN HN

Figure 2. Recovery of (a) unlabelled N and (b) labelled 15N in

the new biomass production (leaves + axes), in relation to days from bud burst at harvest of each tree in 2000. High-nitrogen (HN) and low-nitrogen (LN) treatments refer to the amount of nitrogen received in 1999. Data points in (a) are tted with sigmoid curves with the following equations: y = -682 + 314/(1 + exp((x - 123)/ 942)) for LN plants (r2 = 071, P < 0001); y = -859 + 1378/ (1 + exp((x + 736)/1874)) for HN plants (r2 = 086, P < 0001). Data points in (b) are tted with sigmoid curves with the following equations: y = -638 + 1619/(1 + exp((x - 494)/125)) for LN plants (r2 = 097, P < 0001); y = -352 + 1553/(1 + exp( (x - 536)/ 12)) for HN plants (r2 = 097, P < 0001).

Amino acid translocation during remobilization

Three types of tree species can be identied from the dominant N compounds in their xylem saps: allantonin, amide and citrulline translocators (Sauter 1981). We found that in P. avium few amino acids and amides were responsible for N translocation in the xylem. Nitrate has been reported to be a signicant component of xylem saps of several tree species (Stadler, Gebauer & Schulze 1993; Glavac & Jochheim 1993 ; Prima Putra & Botton 1998 ; Toselli et al. 1999), although in each case higher concentrations of amino acids were recovered than of nitrate. We found no evidence for nitrate translocation in the xylem of P. avium during the

that occurring during the light period (07001900 h) (typically less than 2% of total daily transpiration, see Fig. 4b), we concluded that the resulting ux of N during the night was also negligible. We thus concentrated our attention to the light period, in order to check if concomitant variations in xylem sap N concentration and transpiration between 0700 and 1900 h could have produced errors in our calculations. The pattern of N in Gln, Asn and Asp during the light period was checked, by collecting xylem saps from a

2002 Blackwell Publishing Ltd, Plant, Cell and Environment, 25, 16891699

Xylem sap amino acid translocation and N remobilization 1695

1200 (a) HN plants 900 Aspartic Asparagine Glutamine

1200

(c) HN plants
900

600

600

Aspartic Asparagine Glutamine

300

300

Unlabelled N (m gN g sap)

0 0 20 40 60 80 Days from bud burst 100

Labelled 15N (mgN g-1 sap)

0 0 20 40 60 80 Days from bud burst (d) LN plants 900 Aspartic Asparagine Glutamine 100

-1

1200 (b) LN plants 900 Aspartic Asparagine Glutamine

1200

600 300

600

300

0 0 20 40 60 80 100 Days from bud burst

0 0 20 40 60 80 Days from bud burst 100

Figure 3. Concentration of (a and b) unlabelled N and (c and d) labelled 15N in aspartic acid, asparagine and glutamine in the xylem sap
of Prunus avium trees in relation to days from bud burst. High-nitrogen (HN) and low-nitrogen (LN) treatments refer to the amount of nitrogen received in 1999.

remobilization period. In this period, Gln accounted for nearly half of the total amino acid N in the xylem sap followed by Asn and Asp, whereas only negligible concentrations of other amino acids were found. Therefore, P. avium was predominantly an amide translocator, as has been reported for a range of other tree species. For example, Gln has been reported to be the predominant form of organic N in xylem saps of Prunus spp. (Andersen et al. 1995 ), Populus and Salix spp. (Sauter 1981; Sauter & van Cleve 1994; Schneider et al. 1994), Eucalyptus spp. (Adams, Attiwell & Wang-Liang 1995), Actinidia deliciosa (Clark & Smith 1991), Vitis vinifera (Peuke 2000), Pinus spp. (Barnes 1963; Plassard, Bonafos & Touraine 2000) and Picea abies (Weber et al. 1998). Asparagine was the predominant amino acid in Prunus dulcis (Yousse, Brown & Weinbaum 2000), Malus domestica (Tromp & Ovaa 1985; Malaguti et al. 2001) and Sorbus aucuparia (Prima Putra & Botton 1998). Increases in the Gln concentration in xylem saps of Populus canadensis in the spring have been correlated with the turnover of ray parenchyma storage protein vesicles (Sauter & van Cleve 1994). These authors suggested that as Gln was a minor component of the storage protein but the

predominant form of N in the sap during remobilization, protein turnover must result in Gln synthesis before release into the xylem. Tree N status had no statistically signicant effect upon the amino acid concentration or composition of the xylem saps. In contrast, Yousse et al. (2000) found that fertilization of P. dulcis increased both tree N status and xylem sap amino acid concentrations. However, in our study HN plants had an earlier leaf area development and consequently higher initial transpiration rates compared to LN trees, so the results may be at least in part the consequence of a different dilution of the saps between treatments. The peak in concentration of amino acids found in xylem sap shortly after bud burst was predominantly due to remobilization of N. The subsequent decrease in xylem sap amino acid and amide N concentration was caused only in part by dilution, as a consequence of higher transpiration as leaves grew. Whereas the sap N concentration showed a 12-fold decrease between day 10 and 20 from bud burst, transpiration increased by only six times; subsequently, N concentration remained constant despite a large increase in transpiration. Furthermore, qualitative differences in sap

2002 Blackwell Publishing Ltd, Plant, Cell and Environment, 25, 16891699

1696 G. Grassi et al. (Malaguti et al. 2001), suggesting that these changes are species-specic.

500

(a)
Transpiration (g tree d )
-1

400 300 200 100 0 0 20 40 60 80 Days from bud burst 100 LN HN

Measurement of remobilization by sap ux

The calculation of the ux of spring N remobilization was well correlated with the recovery of unlabelled N in the new biomass production, for both HN and for LN trees (Fig. 5). The slopes and the intercepts of the regressions indicate that the method employed in the calculations is suitable to predict remobilization of N. We have therefore demonstrated that the remobilization of N during spring can be quantied by calculating the ux of amino acids in the xylem. After the remobilization of N has nished, however, the ux of amino acid N through xylem overestimated the recovery of N in the new biomass production (data not shown). There are several possible problems with developing this technique for use with larger trees. For example, it has been suggested that the phloem might play a role in translocating remobilized N. This has been demonstrated in those species that during the winter store N in foliage (e.g. evergreen conifers, Nambiar & Fife 1991; Schneider et al. 1996) or in twigs adjacent to buds (e.g. Sambucus nigra, NsimbaLumaki & Peumans 1986), whereas we found that P. avium stores the majority of N in the roots (data not shown). Another potential problem with scaling from the small trees we used in the present study to larger, eld-grown trees could be the sampling of the xylem sap. Several studies have reported variations in the composition of xylem sap when sampled from different positions within the tree

-1

40 HN LN 30

(b)

Sap flux (g h )

-1

20

10

0 1 2 4 5 7 9 10 12 13 15 17 18 20 22 Hour
Figure 4. (a) Daily mean transpiration rates by Prunus avium
trees in relation to days from bud burst. Data points are tted with sigmoid curves with the following equations: y = -096 + 2948/ (1 + exp((x - 263)/490)) for LN plants (r2 = 091, P < 0001) and y = -127 + 3176/(1 + exp((x - 235)/703)) for HN plants (r2 = 092, P < 0001). (b) Daily courses of sap ux measured 30 2 d from bud burst (averages of four trees per treatment).

Calculated N remobilization (mg tree-1)

800

600

400

200 LN HN 0 0 200 400 600

-1

composition during and after remobilization were evident. The proportion of the total sap N recovered in Gln showed a 10-fold decrease following remobilization, whereas the proportion of total N found in Asn doubled. This demonstrated that the changes in xylem sap composition during spring reect a shift in N sources from remobilization of internal reserves to uptake by root and therefore that remobilization of N occurred through specic amino acids, partially different from those used for transport of N taken up from roots. Similar qualitative changes in xylem sap composition during spring were also observed in Betula pendula (Millard et al. 1998), but not in Malus domestica

800

Measured N remobilization (mg tree )

Figure 5. Comparison of the amount of N remobilized by HN and LN Prunus avium trees during the remobilization period (until 62 and 40 d after bud burst for HN and LN plants, respectively), as measured by the recovery of unlabelled N in their new biomass production, with the amount calculated by the ux of amino acid in their xylem sap. The thin line indicates the 1 : 1 relationship. The regression equations are: y = 084x + 238 for LN plants (r2 = 090, P < 0001) and y = 109x + 188 for HN plants (r2 = 081, P < 0001).

2002 Blackwell Publishing Ltd, Plant, Cell and Environment, 25, 16891699

Xylem sap amino acid translocation and N remobilization 1697

Table 3. Diurnal pattern of amino acid N in the xylem sap, transpiration and ux of N in Prunus avium trees on 26th April 2000 (30 2 d
from bud burst). High-nitrogen (HN) and low-nitrogen (LN) treatments refer to the amount of nitrogen received in 1999. Concentration of N in Asp, Asn and Glu (mg N g sap-1) Period of the day (h) 07001100 (n = 3) 11001500 (n = 4) 15001900 (n = 3) Total Level of signicance N time N time NS NS NS HN 2961 38 2028 83 2334 80 LN 3191 45 2198 71 3345 47

Transpiration (g) HN 396 27 1078 15 447 98 1921 12 LN 350 28 1003 16 353 34 1706 12 NS *** NS

Flux of N in the xylem (mg N tree-1) HN 115 08 190 57 87 19 392 26 LN 110 13 187 54 115 05 412 23 NS NS NS

Values are mean SE. Levels of signicance: NS, not signicant; ***P 0001.

(Glavac et al. 1989; Smith & Shortle 2001), as well as spatial variations in sap ux density (Loustau, Domec & Bosc 1998; Lu, Muller & Chacko 2000). If a signicant exchange of N compounds occurs between the xylem sap and surrounding tissues as the sap ascends the tree, then measuring sap velocity and composition at only one point might not reect the ux of remobilized N reaching the buds and new biomass production. The processes of N remobilization and uptake in spring showed markedly different dynamics. During the rst 3 weeks after bud burst, when about 60% of N remobilization had already occurred in both treatments, practically no root N uptake had occurred. The temporal separation of these processes varies between species. For example, N uptake and remobilization are concurrent in Betula pendula (Millard et al. 1998), whereas in Acer pseudoplatanus (Millard and Proe 1991), Pyrus communis (Tagliavini, Quartieri & Millard 1997) and Prunus persica (Rufat & DeJong 2001) the majority of N recovered in the new biomass production during the rst 2530 d following bud burst is supplied by remobilization. In contrast, there is almost no N uptake by Sorbus aucuparia before remobilization is complete (Millard et al. 2001). The temporal separation of uptake by root from remobilization would make estimating the ux of remobilized N in the xylem easier, and avoid the need to use isotopes. In the current study, translocation of some Gln, Asn and Asp as a consequence of uptake by root was measured during the period of remobilization by LN trees. This demonstrated that when trees had a low N status and were provided with abundant N in the spring, they were capable of translocating N from uptake by root in the same form as that from remobilization. For species exhibiting concurrent uptake and remobilization of N, this could cause a considerable error in estimating the ux of remobilized N in the xylem sap, unless isotopic tracers were used. Millard et al. (1998) raised the possibility of measuring the spring remobilization of N in eld-grown trees by calculating the ux of N translocation in the xylem during

spring, without the use of tracers. Data from this study suggest that, given that the amount of transpiration can be measured precisely, such an approach could be feasible for eld-grown P. avium trees. However, such an approach would probably not work for species exhibiting concurrent N uptake and remobilization in the spring. Furthermore, it would be necessary to know which amino acids were translocated as a consequence of remobilization. If these were the same as those resulting from uptake by root and assimilation of N (e.g. Malus domestica, Malaguti et al. 2001) then such an approach would probably not be feasible.

ACKNOWLEDGMENTS
Dip. Colture Arborce Univ. Bologna, publication no. 1768. P.M. thanks Manaaki Whenua Landcare Research, New Zealand for funding a Senior Research Fellowship which enabled him to nd the time to contribute to the writing of the paper, and David Whitehead for all his support and encouragement while in New Zealand. The authors also like to thank P. Gioacchini and R. Croce for technical help and A. Peressotti (University of Udine) for providing the heat balance stem gauges. The research was funded in part by Italian Ministry of University and Research (MURST, projects ex 40% and ex 60%) and by SEERAD through their grant-in-aid to the Macaulay Institute.

REFERENCES
Adams M.A., Attiwell P.M. & Wang-Liang M. (1995) Effects of phosphorus supply on growth and nitrogen fractions in xylem sap and foliage of Eucalyptus regnans (F. Muell.), E. nitens (Maiden) and E. globulus (Labill.) seedlings: implications for herbivory. Trees 9, 324331. Andersen P.C., Brodbeck B.V. & Mizel R.F. III (1995) Diurnal variations in tension, osmolarity and the composition of nitrogen and carbon assimilated in xylem uid of Prunus persica, Vitis

2002 Blackwell Publishing Ltd, Plant, Cell and Environment, 25, 16891699

1698 G. Grassi et al.

hybrid and Pyrus communis. Journal of the American Society of Horticultural Science 120, 600606. Barnes R.L. (1963) Organic nitrogen compounds in tree xylem sap. Forest Science 9, 98102. Campbell I.M. (1974) Review: Incorporation and dilution values their calculation in mass spectrally assayed stable isotope labelling experiments. Bioorganic Chemistry 3, 386397). Clark C.J. & Smith G.S. (1991) Seasonal variation of nitrogenous compounds in components of the kiwifruit vine. Annals of Botany 68, 441450. Coleman G.D., Englert J.M., Chen T.H.H. & Fuchigami L.H. (1993) Physiological and environmental requirements for poplar (Populus deltoides) bark storage protein degradation. Plant Physiology 102, 5359. Dambrine E., Martin F., Carisey N., Granier A., Hallgren J.E. & Bishop K. (1995) Xylem sap composition: a tool for investigating mineral uptake and cycling in adult spruce. Plant and Soil 169, 233241. Dyckmans J. & Flessa H. (2001) Inuence of tree internal N status on uptake and translocation of C and N in beech: a dual 13C and 15N labelling approach. Tree Physiology 21, 395401. Ferguson A.R., Eiseman J.A. & Leonard J.A. (1983) Xylem sap from Actinidia chinensis: seasonal changes in composition. Annals of Botany 51, 823833. Glavac V. & Jochheim H. (1993) A contribution to understanding the internal nitrogen budget of beech (Fagus sylvatica L.). Trees 7, 237241. Glavac V., Koenies H., Jochheim H. & Ebben U. (1989) Mineralcomposition of the xylem sap from beech (Fagus sylvatica L.) and variations of its ion concentration along the axial gradient. Angewandte Botanik 63, 471486. Gomez K.A. & Gomez A.A. (1984) Statistical Procedures for Agricultural Research. Wiley Interscience, Singapore. Loustau D., Domec J.C. & Bosc A. (1998) Interpreting the variations in xylem sap ux density within the trunk of Maritime pine (Pinus pinaster Ait.): application of a model for calculating water ows at tree and stand levels. Annales des Sciences Forestieres 55, 2946. Lu P., Muller W.J. & Chacko E.K. (2000) spatial variations in the xylem sap ux density in the trunk of orchard-grown, mature mango trees under changing soil water conditions. Tree Physiology 20, 683692. Malaguti D., Millard P., Wendler R., Hepburn A. & Tagliavini M. (2001) Translocation of amino acids in the xylem of apple (Malus domestica Borkh.) trees in spring as a consequence of both N remobilization and uptake by root. Journal of Experimental Botany 52, 16651671. Millard P. (1996) Ecophysiology of the internal cycling of nitrogen for tree growth. Journal of Plant Nutrition and Soil Science 159, 110. Millard P. & Neilsen G.H. (1989) The inuence of nitrogen supply on the uptake and remobilization of stored N for the seasonal growth of apple trees. Annals of Botany 63, 301309. Millard P. & Proe M.F. (1991) Leaf demography and the seasonal internal cycling of nitrogen in sycamore (Acer pseudoplatanus L.) seedlings in relation to nitrogen supply. New Phytologist 117, 587596. Millard P., Hester A., Wendler R. & Baillie G. (2001) Interspecic defoliation responses of trees depend on sites of winter nitrogen storage. Functional Ecology 15, 535543. Millard P., Wendler R., Hepburn A. & Smith A. (1998) Variations in the amino acid composition of xylem sap of Betula pendula Roth. trees due to remobilization of stored N in the spring. Plant, Cell and Environment 21, 715722. Nambiar E.K.S. & Fife D.N. (1991) Nutrient retranslocation in temperate conifers. Tree Physiology 9, 185207. Neilsen D., Millard P., Neilsen G.H. & Hogue E.J. (1997) Sources of N used for leaf growth in a high density apple (Malus domestica) orchard irrigated with ammonium nitrate solution. Tree Physiology 17, 733739. Nsimba-Lubaki M. & Peumans W.J. (1986) Seasonal uctuations of lectins in the barks of elderberry (Sambucus nigra) and black locust (Robinia pseudoacacia). Plant Physiology 80, 747 751. Peuke A.D. (2000) The chemical composition of xylem sap in Vitis vinifera L. cv. Riesling during vegetative growth on three different Franconian vineyard soils and as inuenced by nitrogen fertilizer. American Journal of Enology and Viticulture 51, 329 339. Plassard C., Bonafos B. & Touraine B. (2000) Differential effects of mineral and organic N sources, and of ectomycorrhizal infection by Hebeloma cylindrosporanum, on growth and N utilization in Pinus pinaster. Plant, Cell and Environment 23, 1195 1205. Prima Putra D. & Botton B. (1998) Organic and inorganic compounds of xylem exudates from ve woody plants at the stage of bud breaking. Journal of Plant Physiology 153, 670676. Quartieri M., Millard P. & Tagliavini M. (2002) Storage and remobilization of nitrogen by pear (Pyrus communis) trees as affected by timing of N supply. European Journal of Agronomy 17, 105110. Rufat J. & deJong T.M. (2001) Estimating seasonal nitrogen dynamics in peach trees in response to nitrogen availability. Tree Physiology 21, 11331140. Sauter J.J. (1981) Season variation in amino acids and amides in the xylem sap of Salix. Zeitschnift fr Panzenphysiologie 98, 377391. Sauter J.J. & van Cleve B. (1994) Storage, mobilization and interrelations of starch, sugars, protein and fat in the ray storage tissue of poplar trees. Trees 8, 297304. Schneider S., Gessler A., Weber P., von Sengbusch D., Hanemann U. & Rennenberg H. (1996) Soluble N compounds in trees exposed to high loads of N: a comparison of spruce (Pices abies) and beech (Fagus sylvatica) grown under eld conditions. New Phytologist 134, 103114. Schneider A., Kreuzwieser J., Schupp R., Sauter J. J. & Rennenberg H. (1994) Thiol and amino acid composition of the xylem sap of poplar trees (Populus canadensis robusta). Canadian Journal of Botany 72, 347351. Smith D.M. & Allen S.J. (1996) Measurements of sap ow in plant stems. Journal of Experimental Botany 47, 18331844. Smith K.T. & Shortle W.C. (2001) Conservation of element concentration in xylem sap of red spruce. Trees 15, 148153. Stadler J., Gebauer G. & Schulze E.D. (1993) The inuence of ammonium on nitrate uptake and assimilation in 2-year-old ash and oak trees a tracer study with N-15. Isotopenpraxis 29, 85 92. Tagliavini M., Millard P. & Quartieri M. (1998) Storage of foliarabsorbed nitrogen and remobilization for spring growth in young nectarine (Prunus persica var. nectarina) trees. Tree Physiology 18, 203207. Tagliavini M., Quartieri M. & Millard P. (1997) Uptake by root of nitrogen and remobilization of stored nitrogen for spring leaf growth and fruiting in pear (Pyrus communis L.) trees. Plant and Soil 195, 137142. Toselli M., Flore J.A., Marangoni B. & Masia A. (1999) Effects of root zone temperature on nitrogen accumulation by non bearing apple trees. Journal of Horticultural Science and Biotechnology. 74, 118124. Tromp J. & Ovaa J.C. (1985) Response of young apple trees to time of nitrogen fertilization with respect to the nitrogen, potassium, and calcium levels in xylem sap, new biomass production,

2002 Blackwell Publishing Ltd, Plant, Cell and Environment, 25, 16891699

Xylem sap amino acid translocation and N remobilization 1699

and the tree as a whole. Journal of Plant Physiology 119, 301 309. Weber P., Stoerner H., Gessler A., Schneider S., von Sengbusch D., Hanemann U., Rennenberg H. & von Sengbusch D. (1998) Metabolic responses of Norway spruce (Picea abies) trees to long-term forest management practices and acute (NH4)2SO4 fertilization: transport of soluble non-protein nitrogen compounds in xylem and phloem. New Phytologist 140, 461475. Weinbaum S. & van Kessel C. (1998) Quantitative estimates of uptake and internal cycling of N-14 labelled fertilizer in mature walnut trees. Tree Physiology 18, 795801. Weinbaum S.A., Klein I., Broadbent F.E., Micke W.C. & Muroaka T.T. (1984) Effects of time of nitrogen application and soil texture on the availabilty of isotopically labelled fertilizer nitrogen to reproductive and vegetative growth of mature almond trees. Journal of the American Society of Horticultural Science 109, 339343. Yousse F., Brown P.H. & Weinbaum S.A. (2000) Relationship between tree nitrogen status, xylem and phloem sap amino acid concentrations, and apparent soil nitrogen uptake by almond trees (Prunus dulcis). Journal of Horticultural Science and Biotechnology 75, 6268. Received 17 May 2002; accepted for publication 5 July 2002

2002 Blackwell Publishing Ltd, Plant, Cell and Environment, 25, 16891699