Uncoupling nitrogen requirements for spring growth. from root uptake in a young evergreen shrub (Rhododendron ferrugineum)

Transcription

1 Research Uncoupling nitrogen requirements for spring growth Blackwell Science, Ltd from root uptake in a young evergreen shrub (Rhododendron ferrugineum) T. Lamaze 1, F. Pasche 1,2 and A. Pornon 2 1 Centre d Etudes Spatiales de la Biosphère, CNES-CNRS-IRD-UMR 5639, Université Paul Sabatier, F Toulouse cedex 4, France; 2 Laboratoire Evolution et Diversité Biologique, CNRS-FRE 2629, Université Paul Sabatier, F Toulouse cedex 4, France Abstract Author for correspondence: A. Pornon Fax: Pornon@cict.fr Received: 28 February 2003 Accepted: 6 May 2003 doi: /j x Internal cycling of nitrogen (N) was investigated in a subalpine field population of the evergreen shrub Rhododendron ferrugineum during spring growth. The foliar nitrogen of 5-yr-old-plants was directly labeled with 15 N and subsequently traced to all plant compartments. In addition, 15 N-ammonium uptake was estimated in glasshouse experiments. Before shoot growth, redistribution of 15 N occurred in the plant without net N transfer. During spring development, the decreases in both leaf 15 N and total N were almost identical in terms of percentage, and most of the 15 N withdrawn from the leaf compartments was recovered in the growing shoots. Net changes in the N contents of the various leaf and woody compartments indicate that internal remobilization (especially from 1-yr-old leaves) could have met most of the N needs of new shoot growth. Simultaneously, the rate of mineral N uptake was very low. Thus, leaves in young plants provide N for new shoots (by contrast with old individuals) and allow, with woody tissues, almost complete uncoupling of N requirement for spring growth from root uptake. Key words: Internal cycling, remobilization, leaf 15 N labeling, root uptake, Rhododendron ferrugineum, spring growth, evergreen. New Phytologist (2003) 159: Introduction Nutrient for new growth can be derived from root uptake or internal recycling. Nutrient remobilization from stored reserves can contribute a large proportion of the annual supply required for reproductive and vegetative growth (Nambiar & Fife, 1991; Sanchez et al., 1991). The tissues used to store nutrients, the periods of remobilization, the storage capacity and the ways used to replenish reserves through root uptake or foliar resorption are diverse (Karlsson, 1994; Munson et al., 1995; Proe et al., 2000). Using nutrient remobilization, a plant can temporarily uncouple growth and nutrient uptake and release vegetative and reproductive growth from competition for exogenous nutrients (Millard & Proe, 1991). For nitrogen (N), which is the element that most often limits plant growth in many terrestrial ecosystems, it has been demonstrated for a range of tree species that early season growth is primarily supported by internal remobilization followed by late-season root uptake (Munoz et al., 1993; Tagliavini et al., 1997; Malik & Timmer, 1998). The potential contribution of internally redistributed N to the seasonal requirements of growth was estimated to be about 83% in Rhododendron lapponicum nonreproductive branches (Karlsson, 1994). At the extreme, one can imagine that a plant could be able to achieve total shoot growth at the expense of N storage and with minimum synchronous root nitrogen uptake. This has been described in the graminoid, Eriophorun vaginatum (Jonasson & Chapin, 1985). Evergreenness and/or high nutrient resorption efficiency can explain the success of certain species in poor nutrient environments (Aerts, 1990). Differences between species in (1) the amplitude and the period of nitrogen remobilization, (2) the nature of the organs and tissues involved in storage and (3) the ability to more or less uncouple growth and uptake could New Phytologist (2003) 159:

2 638 Research play major roles in regulating interspecific interactions and vegetation functioning processes. These differences could represent strategies variable in time and complementary in the use of endogenous reserves vs exogenous resources and thus favor species coexistence and diversity in plant communities. Such temporal complementary strategies could be of prime importance, for example in cold environments in which soil resources are temporally very variable but with low fertility in spring when many plants have to grow and reproduce during a short period (Golberg & Novoplansky, 1997). The fact that direct assessment of N storage and remobilization from the different compartments is scarce (Millard, 1996) could be due to the lack of suitable tools to investigate internal transfer in field populations. It has been shown that using changes in pool size from sequential samplings along the growth season can lead to underestimation of remobilization (Jonasson, 1989; Proe et al., 2000). Indeed, it does not allow clear differentiation of internal remobilization from root uptake (Proe et al., 2000) which is likely partly responsible for the discrepancies in N remobilization values reported in the literature (Jonasson, 1995; Millard, 1996). Soil enrichment with 15 N has been extensively and successfully used in artificial environments to distinguish uptake from remobilization, and to trace N in the plant and elucidate sink source interactions (Sanchez et al., 1991; Millard & Proe, 1993; Munoz et al., 1993; Proe & Millard, 1994; Thornton et al., 1995; Millard, 1996). Unfortunately, it is practically impossible to obtain a reliable quantitative picture of N movements in the plant soil system in natural field conditions. This is because the 15 N introduced in the soil will be diluted by the natural N, leading to unknown 15 N abundance in the soil compartment, and because the tracer can be immobilized by soil microorganisms and/or leached (Mead & Preston, 1994). In a previous study on a subalpine population of 40-yr-old Rhododendron ferrugineum (an evergreen shrub), we showed that, despite internal N cycling between the different organs in the plant, leaves did not constitute a net source of N supporting shoot growth (Pasche et al., 2002). This was assumed to be mainly provided by endogenous N stored in the large woody tissues. In young R. ferrugineum with smaller stems and roots, leaf compartments make up a larger proportion of total plant biomass. Our working hypothesis is that, in young individuals, unlike old ones, the mature leaves constitute a net source of N for the growing shoot. If this is true, it would appear that in R. ferrugineum the mechanism of N remobilization supporting shoot growth is age-dependent. In the present study, we quantitatively investigated internal cycling of nitrogen in young (about 5 yr old) R. ferrugineum. The N pool of the leaves was directly labeled with 15 N and traced through all plant compartments during spring growth. Simultaneously, 15 N-ammonium uptake was also estimated in a glasshouse experiment. Our main objectives were to quantify N fluxes within the plant and to estimate the contribution of all potential N storage sites (especially leaf compartments) to the current shoot production in young plants with reduced wood stores. Materials and Methods Plants The evergreen shrub R. ferrugineum L. (Ericaceae) reaches a height of cm and is widely distributed in the Alps and Pyrenees at altitudes between 1600 m and 2200 m (Ozenda, 1985). Reproduction and vegetative growth start 2 wk after snow thawing and are synchronous, increasing the probability of competition between the two for nutrients. Site The study was conducted at a subalpine heath site just above the timberline ( Herbe Soulette pass, N, 0 52 E, c m above sea level) located in the central Pyrenees about 150 km south of Toulouse, France. Soils are Typical Haplorthod to Umbric Dystrochrept with a ph (H 2 O) of 4.3 and C : N ratio of 14. Leaf 15 N labeling The experiment was carried out in the field during the 1999 growing season (May July). Before bud break, 80 seed-sired individuals of similar size (roughly 5-yr-old shrubs about 10 cm high and bearing one branch) were randomly selected and tagged. Because plants must be older than yr to develop layering, all individuals selected were isolated genets comprising roots, a single stem, two leaf generations (1-yr-old (L1) and 2- yr-old (L2) leaves) and a vegetative bud. Half of the plants were used for 15 N labeling of L2 leaves and the other half for 15 N labeling of L1 leaves. Labeling was performed as described in Pasche et al. (2002): on 14th May 1999 (time T0 ), all L1 and L2 leaves (depending on the treatment) of an individual were labeled by deposition of 4 µl of 15 NH 4 Cl (50 mm, 15 N abundance of 99 atom%) on the abaxial face of the leaves. Sampling In order to estimate the amount of 15 N actually loaded into the leaves, a first harvest was performed 2 d after T0 (16th May, T0). To study N dynamics, three harvests were made in the shoot development period: before bud break (21 May, T1), at the beginning of the current shoot growth (15 June, T2) and at the end of the growing period (24 July, T3). At least 10 individuals per treatment were collected at each harvest. Some L2 leaves that fell during the experiment were collected and used to estimate the amount of N that was lost in the litter. When recovered, they were treated and analysed like the other leaves. Five additional individuals were used to determine the natural 15 N abundance in the tissues. New Phytologist (2003) 159:

3 Research 639 Plants were carefully lifted in the field with a ball of earth left on the roots and transferred to the laboratory at 0 2 C. There, the roots were cleared of soil, rinsed with tap water and excised from the branch. The branches were separated into four compartments: current year shoots (Sh0), L1 leaves, L2 leaves and stem. The tissues were dried at 70 C for 48 h and ground to a fine powder (< 1 µm). The compartments of each individual were treated separately for analysis of total N and 15 N abundance using a continuous-flow isotope ratio mass spectrometer coupled with an elemental analyser (model ANCA-MS; Europa Scientific, Crewe, UK). Mineral 15 N-Nitrogen uptake by the plant The experiment was carried out in 1999 as described previously by Pasche et al. (2002). Briefly, 20 young R. ferrugineum plants (about 5 yr old) were carefully collected in the field with a ball of earth and were transferred to the laboratory s glasshouse. Environmental conditions were natural light, and day/night cycles of 25 C, 50% relative humidity (r.h.)/20 C, 70% r.h. The root systems of half of the plants were washed free of soil before transplanting into individual pots of sand. The other half was potted with their ball of earth (natural soil). The pots ( m 3 ) were placed in trays containing a complete nutrient solution with a ph of 4.5. The basal culture solution contained the following macronutrients (mol m 3 ): KNO 3, 0.1; (NH 4 ) 2 SO 4, 0.05; MgSO 4, 0.25; CaSO 4, 0.4; CaCl 2, 0.25; KH 2 PO 4, 0.25; KCl, 1. The solution also contained the following micronutrients (mmol m 3 ): MnSO 4 H 2 O, 9; ZnSO 4 H 2 O, 0.75; CuSO 4 (H 2 O) 5, 0.3; H 3 O 3 B, 45; MoO 3, 0.5; ethylenediaminetetraacetic acid (EDTA)Na 2 Fe, 90. The solution was renewed three times a week to prevent nutrient depletion and ph changes. The characteristics of the solution did not change notably between two renewals (not shown). At the beginning of shoot growth (beginning of July), plants were supplied with the same nutrient solution as previously except for N which was only present as NH 4 + (0.2 mol m 3 enriched with 15 N up to 50 atom%) because R. ferrugineum preferentially uses NH 4 + during spring growth (Pasche et al., 2002). At the end of shoot growth (20 d later), plants were sampled and treated as for field experiments. Data analysis The amount of 15 N in excess in each plant compartment was calculated as the product of m, the dry mass of the compartment with c, the total nitrogen concentration (%) and e, the 15 N in excess. Isotopic excess was calculated as the difference between the 15 N abundance in the compartments of labeled plants and natural 15 N abundance in control plants (0.365%). Differences between phenological stages were statistically tested for each parameter and each plant compartment by Fig. 1 Dry mass of current year s shoot (triangles), 1-yr-old (squares) and 2-yr-old (circles) leaf cohorts of Rhododendron ferrugineum during spring growth (Sh0, shoots; L1, 1-yr-old leaves; L2, 2-yr-old leaves). The sampling times were: 14 May (T0), 21 May (T1), 15 June (T2) and 24 July (T3). Values are means ± SD of 20 individuals analysed separately. Values sharing the same letter are not significantly different at P < 0.05 (one-way ANOVA followed by a Tukey HDS multiple-range test). performing one-way ANOVA (Systat, 1997). Percentages of 15 N were arcsine transformed before being analysed. Comparisons of mean values were carried out using the Tukey HDS multiplerange test. Results Organ biomass and nitrogen contents Changes in mean dry mass of mature leaves and growing shoots on single branches during the period of study are shown in Fig. 1. The current-year shoots (Sh0) started growth at the beginning of June (after T1). Before this date, the Sh0 compartment represented only the apical bud but after T1, it differentiated into leaves, stems and new buds. Leaves were fully expanded by late July (T3). The mean dry weight of compartment Sh0 strongly increased between T2 and T3. During shoot growth (T1 T3), the mean dry mass of compartment L2 decreased significantly, mainly as a result of leaf shedding (Fig. 1 and Table 1). The dry mass of compartment L1 significantly decreased during the phase of rapid shoot development (T2 T3) because of the mass reduction of individual leaves, while the number of L1 leaves was not significantly altered (Table 1). Mean dry mass of root and stem compartments did not significantly change through the growth period (490 ± 110 mg and 655 ± 155 mg dry wt, respectively). Shoot development was accompanied by very high N accumulation in compartment Sh0 (from 0.33 mg at T1 to 5.53 mg at T3; Fig. 2). The amount of N in woody stem and roots decreased significantly as shoots developed (from 4.45 mg and 2.79 mg to 3.34 mg and 1.91 mg, respectively). As a result, N originating from net remobilization of woody New Phytologist (2003) 159:

4 640 Research Table 1 Changes in numbers and mass of mature leaves of roughly 5-yr-old Rhododendron ferrugineum plants during spring growth in the field Characteristics 1-yr-old leaf (L1) 2-yr-old leaf (L2) Sampling time Number of leaves per plant (n = 20) Mean dry mass per leaf (mg) Number of leaves per plant (n = 20) Mean dry mass per leaf (mg) T ± 3.6 a 34.8 ± a 5.36 ± 2.68 a ± 9.57 a n = 201 n = 104 T ± 3.59 a ± a 5.20 ± 2.61 a ± 9.85 a n = 190 n = 96 T ± 3.43 a 38.2 ± a 4.87 ± 2.44 a ± a n = 152 n = 100 T ± 3.38 a 25.1 ± b 3.76 ± 1.88 b ± a n = 140 n = 66 Values are means ± SD. Values sharing the same letter are not significantly different at P < 0.05 (one-way ANOVA followed by a Tukey HDS multiple-range test). average in fallen leaves). Thus, taking into account the loss of N due to L2 leaf abscission, the L1 plus L2 leaf compartments were potentially able to supply shoot growth with mg N. 15 N partitioning Fig. 2 Total nitrogen content in the compartments of Rhododendron ferrugineum during spring growth. The sampling times were: 14 May (T0), 21 May (T1), 15 June (T2) and 24 July (T3). Values are means ± SD of 20 individuals analysed separately. Values sharing the same letter are not significantly different at P < 0.05 (one-way ANOVA followed by a Tukey HDS multiple-range test). Triangles, shoots (Sh0); squares, 1-yr-old leaves (L1); circles, 2-yr-old leaves (L2); asterisks (dash-dot line), stem; cross (dashed line), root; solid line at top of figure gives total. tissue reserves can contribute to the N accumulated in Sh0 compartment during growth by up to 2 mg. Between T1 and T3, the N content of compartments L1 and L2 dropped from 5.07 mg and 1.51 mg to 2.71 mg and 0.79 mg, respectively. Since no L1 leaves fell until time T3, the amount of N that left compartment L1 (2.36 mg) was potentially available for shoot growth. Owing to leaf fall, the decrease in the N content of compartment L2 was not entirely due to recycling. The quantity of N contained in L2 leaves that fell between times T1 and T3 was estimated at 0.23 mg (this value was obtained by multiplying the biomass of a single leaf at time T3 with an N concentration of 0.65% on a dry wt basis observed as an Two days after 15 + NH 4 application (time T0), 100% of the amount of 15 N theoretically provided to either the L1 or the L2 leaves was recovered in these compartments. In the following, all the percentages of 15 N are expressed on the basis of the amounts recovered at T0. At the end of the experiment (T3), the recovery of 15 N in the plants was higher than 80% and 70% for L1 and L2 labeling, respectively (Fig. 3), despite a large redistribution of the tracer among the various organs and leaf shedding. After L1 labeling, before spring growth (period T0 T1), some tracer left the L1 compartment (19%) and was recovered in stem (10%), root (4%) and in the Sh0 (3%) compartments. During growth (period T1 T3), compartment L1 lost 35% of its 15 N, which accumulated mainly in growing shoots (28%). The 15 N content of root and stem increased during the T0 T1 period and decreased during the T1 T3 period. Thus, during the experiment (70 d), 55% of the tracer left compartment L1. Around 31% appeared in the Sh0 compartment but only traces were found in compartment L2. Some 15 N accumulated in stems (4%) but only a little accumulated in roots (1%). In the case of L2 labeling, between T0 and T1, a large portion of the 15 N withdrawn from compartment L2 (35%) accumulated in compartment L1 (17%), the remainder being found in stem (6%), root (4%) and Sh0 (2%) compartments. Subsequently, 15 N losses from compartment L2 were from combined translocation and leaf fall. During Sh0 shoot growth (period T1 T3), the increase in tracer in compartment Sh0 (14%) was mainly supplied by compartment L2 although some 15 N could be derived from compartment L1. New Phytologist (2003) 159:

5 Research 641 Indeed, the amount of tracer in compartment L1 (17% at T1) steadily decreased until T3 (9%). Mineral ( 15 N) uptake When 15 N-ammonium was supplied to plants growing on sand or on natural soil in the glasshouse, very low amounts of labeled N were recovered in the various plant compartments at the end of shoot development (Table 2). Total N contents in the plant compartments (including new shoots) were quite similar for plants transplanted into sand or with a ball of earth left on the roots (minimizing handing). Fig. 3 Time-dependent change in the amount of 15 N in excess in the different plant compartments of Rhododendron ferrugineum, during spring growth. One-yr-old leaves (a) or 2-yr-old leaves (b) were labelled before growth on 14 May 1999 by depositing 2 2 µl of a solution containing 15 NH 4 Cl (50 mm). Values are the means ± SD from 10 individuals analysed separately. Values sharing the same letter are not significantly different at P < 0.05 (Data were arcsine transformed before being analysed by one-way ANOVA followed by a Tukey HDS multiple-range test). Triangles, shoots (Sh0); open squares, 1-yr-old leaves (L1); circles, 2-yr-old leaves (L2); closed squares, root; crosses, stem; solid line at top of figure gives total. Discussion We investigated the contribution that remobilization of foliar N made to spring growth in young evergreen shrubs in the field by direct labeling of leaf N with 15 NH 4 +. The innocuousness of the labeling method has previously been demonstrated (Pasche et al., 2002). Both the very high recovery of tracer in the plant after the 70-d outdoor experiment, and the large proportion of 15 N that moved from the labeled leaves to other organs suggested that most of the 15 N-ammonium supplied to the leaves was assimilated into organic forms (since it is the form for translocation). This, and the absence of significant changes in the 15 N abundance in leaves between T1 and T3, support the hypothesis that labeled-n was mainly homogeneously diluted in the leaf N pool so that it follows the same fate as endogenous N (neglecting isotope discrimination). Thus, our method can be used satisfactorily to trace leaf N reallocation in field experiments. Nevertheless, some 15 N was not recovered in the plant system and leaf fall cannot entirely account for this. It is likely that a small fraction of the 15 N-nitrogen provided to the leaves was not assimilated and was progressively leached away (rough calculation gives a little more than 10% for both L1 and L2 labeling). From the distribution of tracer in the different plant compartments at T1, a diagrammatic scheme of N circulation can be deduced (Fig. 4a) (Devienne et al., 1994). It shows that, before spring growth (period T0 T1), no net N transfer occurred between plant compartments although there was intense internal N cycling, as indicated by 15 N redistribution, as previously demonstrated for other plants (Cooper & Clarkson, 1989). The exchanges between compartments consist of equivalent influx and efflux. Efflux of N from compartment L1 was primarily directed and accumulated in stem, root and Sh0 (bud) compartments while N from compartment L2 was able to reach compartment L1 directly. Plants supplied with 15 NH 4 + and grown on sand or maintained on natural soil in a greenhouse displayed very low uptake and transport of 15 N during shoot development (Table 2), in agreement with the results of Pasche et al. (2002). Such a low uptake cannot be attributed to previous New Phytologist (2003) 159:

6 642 Research Exogenous 15 N (µg) Nitrogen content Total N (mg) Sand (n = 8) Natural soil (n = 8) Sand (n = 8) Natural soil (n = 8) Shoots 6.15 ± 0.28 a 1.33 ± 1.46 b 2.46 ± 0.64 a 2.65 ± 0.38 a Leaves 3.18 ± 0.45 a 0.07 ± 0.15 b 4.85 ± 0.20 a 4.27 ± 0.35 a Stems ± 1.2 a 0.18 ± 0.3 b 3.15 ± 0.15 a 2.74 ± 0.35 a Roots 39.4 ± 6.31 a 1.04 ± 0.7 b 3.12 ± 0.46 a 2.66 ± 0.87 a Table 2 Amount of total N and 15 N (mean ± SD) in different plant parts of 5-yr-old Rhododendron ferrugineum growing on sand or in natural soil supplied with a complete nutrient solution containing 200 µm of 15 NH 4 + ( 15 N isotopic excess of 50 atom%) during shoot growth Values of total N and exogenous 15 N between compartments of plants growing on sand or in natural soil sharing the same letter are not significantly different at P < 0.05 (t-test). Fig. 4 Diagrammatic scheme of N circulation before (a, period T0 T1) and during (b, period T1 T3) growth deduced from simultaneous changes in total N and 15 N content of the various plant compartments. Values in (b) were calculated from total N budget and represent the potential N remobilization from the various compartments (expressed in mg per plant). fine-roots loss or damage during the excavation procedure in the case of plants grown on natural soil since their roots were not manipulated before the experiment. It can be assumed that the amount of exogenous 15 N absorbed by the plant was underestimated because some fine roots were lost at the end of the experiment when they were removed from the substrate for tissue analysis. However, the amount of N taken up by the fine roots but not transported to the other parts of the plants was likely to be small. Thus, it appears that mineral N is weakly absorbed during spring growth in young R. ferrugineum. It has been reported that, like some other species (Högberg, 1990; Chalot & Brun, 1998; Näsholm et al., 1998) ericaceous species associated with ericoid mycorrhizal fungi can take up organic N compounds (Read, 1996). However in young R. ferrugineum, total N contents in the various compartments (including new shoots) were identical for plants grown on sand or on natural soil containing organic N (Table 2). This demonstrates that during spring growth in this species, absorption of both organic and mineral N is low so that it must be assumed that most of the N necessary for current shoot development is derived from endogenous N remobilization. The net contribution of L1 and L2 leaf reserves to the N required for spring shoot growth was potentially 56% (2.9 mg of the total 5.2 mg required for full shoot growth; Fig. 4b), which is in the range of values for the percentage of resorption obtained for evergreen and deciduous species (Karlsson, 1994; Eckstein et al., 1998). The L1 and L2 compartments contributed roughly 80% and 20% of this remobilized N, respectively. This agrees with findings of Nambiar & Fife (1991) that N remobilization from nonsenescing leaves can be important in evergreen species. It also supports the hypothesis of Pornon et al. (1998) that half of the N requirement for shoot growth could depend upon leaf remobilization in R. ferrugineum growing in the Alps. During Sh0 shoot development (between T1 and T3), the percentage decreases in 15 N and total N contents of compartments L1 and L2 were proportionally almost identical (c. 45% and 46% for N and 15 N, respectively, in compartment L1, and 47% for both N and 15 N in compartment L2). Thus, there was no change in L1 and L2 15 N abundance during N leaf remobilization. The simplest hypothesis to explain these similar turnover times for 15 N and endogenous N in leaves is that exchanges between the leaf compartments and their surrounding environments were mainly unidirectional effluxes (including N lost to the litter) (Devienne et al., 1994). During growth, shoots act as a strong sink for N (Fig. 4b). The amount of new shoot N derived from remobilization of leaf N can also be quantified from the amounts of 15 N that appeared in the new shoots. About 31% and 17% of the initial L1 and L2 N contents, respectively, were transported to the new shoots during spring growth. This gives a total amount of 1.85 mg N, which is less than that hypothesized from the use of net changes in leaf N content (2.9 mg). This New Phytologist (2003) 159:

7 Research 643 is surprising since the latter method often leads to underestimation of the contribution of remobilization. Such a discrepancy may be linked to the disappearance of some 15 N initially contained in the leaves. Indeed, if some 15 N is not assimilated after deposition and leached away, the 15 N excess in the labeled leaf would be overestimated and the proportion of new shoot N originating from mature leaf underestimated. Whatever the exact evaluation of leaf N translocation, it is clear that mature leaves do provide a large part of the N required for spring shoot growth in R. ferrugineum. By contrast, Jonasson (1989) found that old leaves of R. lapponicum did not directly contribute to the nutrition of expanding new leaves. In addition, in our previous study on adult R. ferrugineum shrubs (Pasche et al., 2002), we found that mature (L1 and L2) leaves did not constitute a net source of N during the period of shoot growth. Two reasons could explain this strong discrepancy. (1) By contrast with our previous study, in the present one some L2 leaves fell during shoot growth so that it is possible that the new leaves were supplied by N resorbed from these senescing L2 leaves. This suggests that the processes of nutrient remobilization from L2 leaves depends on a close synchronization between leaf senescence and shoot growth. (2) Our previous study used old plants and suggested that the major N source for the growing shoots of adult shrubs was endogenous N stored in the woody tissues (stem and root). Woody tissues make up a higher proportion of total biomass in older than in younger plants. Indeed, the low storage capacity of woody tissues in the young plant was compensated by a greater contribution of the leaf compartment for N remobilization (especially L1 leaves). These results indicate that the evergreen shrub could use alternative methods for storing nutrients depending on leaf phenology or developmental stage. One of the most frequently cited differences between deciduous and evergreen species is that the main storage site for N during autumn and winter is the foliage in evergreen species (Nambiar & Fife, 1991; Helmisaari, 1995) and the perennial woody tissues for deciduous species (Millard, 1996). Our studies show that net remobilization of N from stems and roots can reach 2 mg, contributing potentially more than 38% to the net source of N for the growing shoots (Fig. 4b). This observation suggests that N storage in woody tissues can be important in young evergreen plants with relatively small woody compartments. Exogenous N uptake was low during spring growth and, interestingly, net endogenous N reallocation calculated using net changes in N content in leaf and woody tissues accounted for about 94% of the amount used for spring shoot growth. Thus, our work suggests (after Jonasson & Chapin (1985) for a grass) that an evergreen (woody species) can achieve its entire spring growth almost exclusively from its internal reserves. Later in summer and autumn, uptake of N by roots may be important for replenishing the stores, as demonstrated for other species (Millard, 1996). Net reallocation means that the N content of the various internal store compartments decreased since exogenous N did not replenish the N remobilized from old tissues during the period of study. High values of N remobilization from old tissues used to support growth of new tissues in trees have already been reported (Millard, 1996). For example, it has been reported that internal cycling provided up to 93% and 83% of N used for growth in Prunus persica (Munoz et al., 1993) and Pinus sylvestris (Proe et al., 2000), respectively. However, the value found for P. persica concerned only the start of flower and shoot development. Later in the growing season, the contribution of N reallocation strongly decreased. For a range of tree species, early growth is largely supported by nutrient remobilization (Tagliavini et al., 1997; Malik & Timmer, 1998) but later, when the winter stores are exhausted, the N used for shoot growth comes from root uptake. By contrast, in our study a value of 94% accounted for N remobilization during the whole period of spring growth (until late July). The value of 83% obtained for P. sylvestris included the fraction of N that left old tissues but was simultaneously replaced by exogenous N coming from root uptake (15%). Thus, in the present study, the net contribution of old tissues to new tissues was 68%. In conclusion, a shrub (R. ferrugineum) suited to a stressful environment with low soil N resources at the start of the growing season (Pornon & Doche, 1995) is able to support spring shoot development mainly through remobilization of internal N. This could allow the plant to partly release vegetative growth from competition for the exogenous nutrient. While old plants with large woody tissues are assumed to essentially use stems and roots (and not the leaves) as storage sites, in young plants, with less woody reserves, mature leaves contribute largely to the nutrition of expanding new leaves. This strategy could explain why despite having a very slow growth rate and experiencing strong competition from grasses during the first 5 10 yr of its life, in many sites the shrub makes up a subclimax vegetation occupying wide areas of the subalpine level of European mountains. Acknowledgements We thank Dr A. Gojon and P. Tillard (INRA-Montpellier) for the 15 N analyses and Dr A. Mangin for technical support (CNRS-Moulis). The PhD thesis of F. Pasche was supported by a grant from the Pyrenees National Park. References Aerts R Nutrient use efficiency in evergreen and deciduous species from heathlands. Oecologia 84: Chalot M, Brun A Physiology of organic nitrogen acquisition by ectomycorrhizal fungi and ectomycorrhizas. FEMS Microbiology Review 22: Cooper HD, Clarkson DT Cycling of amino-nitrogen and other nutrients between shoots and roots in cereals. Journal of Experimental Botany 40: New Phytologist (2003) 159:

8 644 Research Devienne F, Mary B, Lamaze T Nitrate transport in intact wheat roots II. Long-term effects of NO 3 concentration in the nutrient solution on unidirectional fluxes and distribution within the tissues. Journal of Experimental Botany 45: Eckstein RL, Karlsson PS, Weih M The significance of leaf resorption of leaf resources for shoot growth in evergreen and deciduous woody plant from subartic environment. Oikos 81: Golberg D, Novoplansky A On the relative importance of competition in improductive environments. Journal of Ecology 85: Helmisaari HS Nutrient cycling in Pinus sylvestris stands in eastern Finland. Plant and Soil : Högberg P N natural abundance as a possible marker of the ectomycorrhizal habit of trees in mixed African woodlands. New Phytologist 115: Jonasson S Implications of leaf longevity, leaf nutrient re-absorption and translocation for resource economy of five evergreen plant species. Oikos 56: Jonasson S Resource allocation in relation to leaf retention time of the wintergreen Rhododendron lapponicum. Ecology 76: Jonasson S, Chapin FS Significance of sequential leaf development for nutrient balance of the cotton sedge, Eriophorun vaginatum L. Oecologia 67: Karlsson PS The significance of internal nutrient cycling in branches for growth and reproduction of Rhododendron lapponicum. Oikos 70: Malik V, Timmer VR Biomass partitioning and nitrogen retranslocation in black spruce seedlings on competitive mixewood sites: a bioassay study. Canadian Journal of Forest Research 28: Mead DJ, Preston CM Distribution and retranslocation of 15 N in lodgepole pine over eight growing seasons. Tree Physiology 14: Millard P Ecophysiology of the internal cycling of nitrogen for the tree growth. Journal of Plant Nutrition and Soil Sciences 159: Millard P, Proe MF Leaf demography and the seasonal internal cycling of nitrogen in sycamore (Acer pseudoplatanus L.) seedlings in relation to nitrogen supply. New Phytologist 117: Millard P, Proe MF Nitrogen uptake, partitioning and internal cycling in Picea sitchensis (Bong.) Carr. As influenced by nitrogen supply. New Phytologist 125: Munoz N, Guerry J, Legaz F, Primo-Millo E Seasonal uptake of 15 N-nitrate and distribution of absorbed nitrogen in peach trees. Plant and Soil 150: Munson AD, Margolis HA, Brand DG Seasonal nutrient dynamics in white pine and white spruce in response to environmental manipulation. Tree Physiology 15: Nambiar SEK, Fife DN Nutrient retranslocation in temperate conifers. Tree Physiology 9: Näsholm T, Nordin A, Giesler R, Högberg M, Högberg P Boreal forest plants take up organic nitrogen. Nature 392: Ozenda P La Végétation de la Chaîne Alpine dans L espace Montagnard Européen. Paris, France: Masson. Pasche F, Pornon A, Lamaze T Do mature leaves provide a net source of nitrogen supporting shoot growth in Rhododendron L.? New Phytologist 154: Pornon A, Doche B Minéralisation et nitrification de l azote dans les différentes stades de colonisation des landes subalpines à Rhododendron ferrugineum L. (Alpes du Nord; France). Comptes Rendus de l Académie des Sciences 318: Pornon A, Doche B, Escavarage N Cycle interne de l azote dans une nanophanérophyte subalpine (Rhododendron ferrugineum) se développant sur sols pauvres. Ecologie 29: Proe MF, Millard P Relationships between nutrient supply nitrogen partitioning and growth in young Sitka spruce (Picea sitchensis). Tree Physiology 14: Proe MF, Midwood AJ, Graig J Use of stable isotopes to quantify nitrogen, potassium and magnesium dynamics in young Scots pine (Pinus sylvestris). New Phytologist 146: Read DJ The structure and function of the ericoid mycorrhizal root. Annal of Botany 77: Sanchez EE, Righetti L, Sugar D, Lombard PB Recycling of nitrogen in field-grown Comice pears. Journal of Horticultural Science 66: Systat Systat user s guide. Evaston, IL, USA: Systat Inc. Tagliavini M, Millard P, Quartieri M Storage of foliar-absorbed nitrogen and remobilisation for spring growth in young nectarine (Prunus persica var. nectarina) trees. Tree Physiology 18: Thornton B, Millard P, Tyler MR Effects of nitrogen supply on the seasonal re-mobilization of nitrogen in Ulex europaeus. New Phytologist 130: About New Phytologist New Phytologist is owned by a non-profit-making charitable trust dedicated to the promotion of plant science. Regular papers, Letters, Research reviews, Rapid reports and Methods papers are encouraged. Complete information is available at All the following are free essential colour costs, 25 offprints as well as a PDF (i.e. an electronic version) for each article, online summaries and ToC alerts (go to the website and click on 'Journal online') You can take out a personal subscription to the journal for a fraction of the institutional price. Rates start at 86 in Europe/$145 in the USA & Canada for the online edition (go to the website and click on 'Subscribe') If you have any questions, do get in touch with Central Office (newphytol@lancaster.ac.uk; tel ) or, for a local contact in North America, the USA Office (newphytol@ornl.gov; tel ) New Phytologist (2003) 159: