Measurement of xylem sap amino acid concentrations in conjunction ...

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, 1689–1699

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 1 Dipartimento di Colture Arboree, Università di Bologna, Via Fanin 46, 40127 Bologna, Italy, 2Macaulay Institute, Craigiebuckler, Aberdeen AB15 8QH, UK and 3Dipartimento AGROSELVITER, Università 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 profiles and whole tree transpiration rates were taken, to determine whether specific amino acids are translocated as a consequence of N remobilization and if remobilization can be quantified by calculating the flux 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 field-grown trees by calculating the flux 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: [email protected] © 2002 Blackwell Publishing Ltd

1996). This source of N is often the first, 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 difficult. 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, field-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 flux of N to be calculated. Such an approach has already been used to quantify mineral fluxes via xylem sap flow 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 profiles and whole tree transpiration rates, in order to determine if: (1) there are specific amino acids translocated by P. avium as a consequence of N remobilization, and (2) remobilization can be quantified by calculating the flux 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.

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 fine 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 0·6 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 0·8 g P, 2·7 g K, 0·9 g Ca, 1·4 g S, 0·3 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 1–2 d. For each individual plant the day the first 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 0·15 g of N as 15NH415NO3 enriched with 15N to 7·75 atom percent excess. Ten destructive harvests were taken between 28 March and 20 June 2000, with 7–14 d intervals

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 flow with heat balance stem gauges (Smith & Allen 1996).

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 five different organs (leaves, axes, stem, coarse and fine 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 V2·0 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 flow 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, 1689–1699

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 0·2 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 0·2 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 (GC–MS).

Analysis of saps by GC–MS 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 0·18 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 GC–MS in the single ion recording mode. The instrumentation used was a Trace 2000 gas chromatograph, fitted 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 ¥ 0·25 mm id ¥ 0·25 mm phase thickness (Phenomenex, Macclesfield, 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 7·5 ∞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 0·18 APE for Asp, 0·21 APE for Glu, 0·37 APE for Asn and 0·15 APE for Gln. The APE

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 profiles 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 fitted 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 significance 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 6·1.3; 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 flux of amino acid and amide N in the xylem, as described by Gomez & Gomez (1984).

RESULTS Tree growth and N remobilization The contrasting N treatments applied during the first year of the experiment resulted in trees with a differing N status. At the end of the first growing season the total leaf N concentration and the number of buds set per plant were significantly 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 final area 56 d after bud burst for both treatments. Both leaf and twig mass continued to increase after leaf area expansion finished, so total new biomass production reached 95% of final 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 significant at P < 0·05 for leaf area (0·89 ± 0·02 m2 for HN and 0·81 ± 0·02 m2 for LN) and for total new biomass production (71·0 ± 1·4 g for HN and 63·0 ± 1·5 g for LN).


1692 G. Grassi et al. Table 1. Parameters measured in Prunus avium trees at the end of the first year of experiment (1999) ± SE

Leaf N before leaf senescence (% DW) Stem height (cm) Stem diameter at the base of the tree (cm) Number of buds

LN

HN

Level of significance

2·1 ± 0·1

2·6 ± 0·1

**

151 ± 4 1·5 ± 0·02

155 ± 3 1·5 ± 0·03

NS NS

31 ± 1

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 significance: NS, not significant, *P £ 0·05, **P £ 0·01.

1 .2

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 finished (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 significant 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 finished, these values changed to 5, 72 and 13%, respectively (Table 2).

0 .6

0 .3 LN HN 0 0

20

40 60 80 Days from bud burst

100

80

(b)

-1

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 finished and were used in subsequent calculations. After these dates, the difference between treatments in the amount of N remobilized was significant at P < 0·001 (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).

0 .9

2

-1

Leaf area (m tree )

(a)

60

40

20 LN HN 0 0

20


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 fitted with sigmoid curves with the following equations: y = -0·04 + 0·88/(1 + exp(–(x - 30·6)/10·1)) for LN plants (r2 = 0·94, P < 0·001); y = 0·018 + 0·90/(1 + exp(–(x 30·4)/9·66)) for HN plants (r2 = 0·93, P < 0·001). Data points in (b) are fitted with sigmoid curves with the following equations: y = -2·98 + 69·2/(1 + exp(–(x - 42·5)/12·9)) for LN plants (r2 = 0·97, P < 0·001); y = -2·53 + 75·8/(1 + exp(–(x - 42·7)/12·9)) for HN plants (r2 = 0·98, P < 0·001).


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 finished 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 0·9 ± 0·1 488 ± 44 295 ± 71 2·4 ± 0·5 7·3 ± 1·6 673 ± 127 0·7 ± 0·2 5·8 ± 0·7 2·5 ± 0·3 9·4 ± 1·6 13·4 ± 3·1 5·6 ± 1·4 16·8 ± 2·2 11·0 ± 1·6 1532 ± 202 15·0 ± 5·1

LN

Level of significance

63 ± 2

12 ± 1

59 ± 2

N

time

N × time

1·1 ± 0·1 85·0 ± 29 16·3 ± 3·9 0·4 ± 0·1 1·9 ± 0·6 5·7 ± 2·1 0·5 ± 0·1 0·3 ± 0·1 0·1 ± 0·0 0·1 ± 0·0 1·6 ± 0·6 1·2 ± 0·4 2·2 ± 0·7 0·6 ± 0·1 117 ± 37 315 ± 13

0·7 ± 0·2 666 ± 115 428 ± 93 1·2 ± 0·2 5·8 ± 1·2 986 ± 194 0·5 ± 0·1 5·8 ± 0·0 3·0 ± 0·3 7·3 ± 0·6 8·1 ± 1·3 6·5 ± 1·4 20·5 ± 0·6 10·5 ± 0·5 2150 ± 363 8·4 ± 1·5

1·5 ± 0·2 68·5 ± 17 11·9 ± 2·5 0·5 ± 0·1 1·7 ± 0·3 4·7 ± 1·6 0·6 ± 0·1 0·2 ± 0·1 0·2 ± 0·0 0·1 ± 0·0 1·1 ± 0·2 1·3 ± 0·3 1·7 ± 0·5 0·5 ± 0·1 94·5 ± 23 300 ± 33

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 µg amino acid N g xylem sap−1 and are mean ± SE of four replicates. Mean daily transpiration (g tree−1 d−1) ± SE of the selected plants is also shown. Levels of significance: NS, not significant, *P ≤ 0·05, ***P ≤ 0·001.

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

Calculating the flux 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 first 3–4 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 flux showed that the great majority of total flux 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 flux 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 (0·84 for the LN trees and 1·09 for the HN trees) were not significantly different. During the calculation of N flux 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 significantly 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 significantly higher (60% more, P < 0·01) than during the day. However, if we consider that in the trees used in our experiment transpiration during the night period (1900–0700 h) was negligible in comparison with


1694 G. Grassi et al.

1600

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: 0700–1100, 1100–1500 and 1500–1900 h. The concentration of N in Gln, Asn and Asp showed a relatively small, and statistically insignificant, decline during the central part of the day (Table 3); furthermore, because of the higher transpiration rates, the total flux 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 first 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.

1200

DISCUSSION

800

15

-1

Unlabelled N (mg tree )

(a) 600

400

200 LN HN 0 0

20


100

2000

Tree growth, N status and N remobilization

15

-1

Labelled N (mg tree )

(b)

800 400

LN HN

0 0

20

40

60

80

100

Days from bud burst 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 fitted with sigmoid curves with the following equations: y = -68·2 + 314/(1 + exp(–(x - 12·3)/ 9·42)) for LN plants (r2 = 0·71, P < 0·001); y = -859 + 1378/ (1 + exp(–(x + 7·36)/18·74)) for HN plants (r2 = 0·86, P < 0·001). Data points in (b) are fitted with sigmoid curves with the following equations: y = -63·8 + 1619/(1 + exp(–(x - 49·4)/12·5)) for LN plants (r2 = 0·97, P < 0·001); y = -35·2 + 1553/(1 + exp(– (x - 53·6)/ 12)) for HN plants (r2 = 0·97, P < 0·001).

that occurring during the light period (0700–1900 h) (typically less than 2% of total daily transpiration, see Fig. 4b), we concluded that the resulting flux 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

The N treatments applied during the first 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.

Amino acid translocation during remobilization Three types of tree species can be identified 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 significant 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


Xylem sap amino acid translocation and N remobilization 1695

1200

1200

(a) HN plants

(c) HN plants

900

900

Aspartic Asparagine Glutamine

600

300

300

0 0

20


100

Labelled 15N (mgN g-1 sap)

-1

Unlabelled N (m gN g sap)

Aspartic Asparagine Glutamine

600

0 0

20


100

1200

1200 (b) LN plants 900 Aspartic Asparagine Glutamine

600

(d) LN plants 900 Aspartic Asparagine Glutamine

600

300

300

0

0

0

20

40

60

80

0

100

Days from bud burst

20


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 (Youssefi, 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 significant effect upon the amino acid concentration or composition of the xylem saps. In contrast, Youssefi 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


1696 G. Grassi et al.

500 400

Measurement of remobilization by sap flux

-1

-1

Transpiration (g tree d )

(a)

300 200 LN HN

100 0 0

20


100

40

(b)

HN LN 30 -1

Sap flux (g h )

(Malaguti et al. 2001), suggesting that these changes are species-specific.

20

10

The calculation of the flux 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 quantified by calculating the flux of amino acids in the xylem. After the remobilization of N has finished, however, the flux 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, field-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 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 fitted with sigmoid curves with the following equations: y = -0·96 + 294·8/ (1 + exp(–(x - 26·3)/4·90)) for LN plants (r2 = 0·91, P < 0·001) and y = -12·7 + 317·6/(1 + exp(–(x - 23·5)/7·03)) for HN plants (r2 = 0·92, P < 0·001). (b) Daily courses of sap flux measured 30 ± 2 d from bud burst (averages of four trees per treatment).

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 reflect a shift in N sources – from remobilization of internal reserves to uptake by root – and therefore that remobilization of N occurred through specific 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

Calculated N remobilization (mg tree-1)

0

800

600

400

200 LN HN 0 0

200

400

600

800 -1

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 flux of amino acid in their xylem sap. The thin line indicates the 1 : 1 relationship. The regression equations are: y = 0·84x + 23·8 for LN plants (r2 = 0·90, P < 0·001) and y = 1·09x + 18·8 for HN plants (r2 = 0·81, P < 0·001).


Xylem sap amino acid translocation and N remobilization 1697 Table 3. Diurnal pattern of amino acid N in the xylem sap, transpiration and flux 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)

Transpiration (g)

Period of the day (h)

HN

LN

HN

LN

HN

LN

0700–1100 (n = 3) 1100–1500 (n = 4) 1500–1900 (n = 3)

296·1 ± 38 202·8 ± 83 233·4 ± 80

319·1 ± 45 219·8 ± 71 334·5 ± 47

39·6 ± 2·7 107·8 ± 15 44·7 ± 9·8

35·0 ± 2·8 100·3 ± 16 35·3 ± 3·4

11·5 ± 0·8 19·0 ± 5·7 8·7 ± 1·9

11·0 ± 1·3 18·7 ± 5·4 11·5 ± 0·5

192·1 ± 12

170·6 ± 12

39·2 ± 2·6

41·2 ± 2·3

Total Level of significance N time N ¥ time

NS NS NS

Flux of N in the xylem (mg N tree-1)

NS *** NS

NS NS NS

Values are mean ± SE. Levels of significance: NS, not significant; ***P £ 0·001.

(Glavac et al. 1989; Smith & Shortle 2001), as well as spatial variations in sap flux density (Loustau, Domec & Bosc 1998; Lu, Muller & Chacko 2000). If a significant 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 reflect the flux 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 first 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 first 25–30 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 flux 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 flux 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 field-grown trees by calculating the flux 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 field-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 find 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.

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