Seasonal nitrogen speciation in temperate seagrass Posidonia oceanica (L.) Delile

Journal of Experimental Marine Biology and Ecology 273 (2002) 219 – 240 www.elsevier.com/locate/jembe

Seasonal nitrogen speciation in temperate seagrass Posidonia oceanica (L.) Delile Olga Invers *, Marta Pe´rez 1, Javier Romero Faculty of Biology, Department of Ecology, University of Barcelona, Av. Diagonal, 645, 08028, Barcelona, Spain Received 3 December 2001; received in revised form 28 January 2002; accepted 5 May 2002

Abstract To better understand some basic aspects of the nitrogen economy in Posidonia oceanica and, specifically, the seasonality of the processes of storage, translocation and assimilation, we examined nitrogen speciation into soluble compounds, both inorganic (nitrates, nitrites and ammonium) and organic (free amino acids, FAA, and total soluble protein, TSP), and the nitrogen assimilation potential (through the glutamine synthetase activity measurement) in the leaves, rhizomes and roots of P. oceanica over a 1-year cycle. Only a limited amount of inorganic nitrogen was found, accounting for less than 3.3% of the total nitrogen content, and it was mostly in the form of ammonium. Nitrate and nitrite concentrations were very low, always below 7.2 Amol g 1 dw in annual average. Among the organic soluble fractions, FAAs were the most abundant, accounting for up to 50% of N pools. Rhizomes were the organs in which FAA concentrations reached their maximum value. The leaves showed higher nitrogen assimilation potential than the roots and this assimilation potential was highest during and after the period of maximum leaf growth, probably corresponding to the assimilation of both new and recycled nitrogen. Our results suggest that 5% of the total nitrogen assimilation occurs in roots and 79% in leaves on an annual average. In addition, rhizomes contributed to the total shoot nitrogen assimilation by 32 – 54% between autumn and spring. Rhizomes appear as key organs in the nitrogen economy of the plant, not only as a major site for nitrogen assimilation but also as an organ for nitrogen storage. This storage, mostly in the form of FAA, occurs during periods of high availability and low demand (winter). This stored nitrogen can supply up to 33% of plant demands during the moment of maximum leaf growth (i.e. late spring). D 2002 Elsevier Science B.V. All rights reserved. Keywords: Amino acid; Growth; GS activity; Inorganic nitrogen; Mediterranean; Protein; Storage

*

Corresponding author. Tel.: +34-93-402-15-06; fax: +34-93-411-14-38. E-mail address: [email protected] (O. Invers). 1 Tel.: +34-93-402-15-06; fax: +34-93-411-14-38. 0022-0981/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 0 9 8 1 ( 0 2 ) 0 0 1 6 7 - 3

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1. Introduction The elucidation of nutrient – plant interactions is basic to a better understanding of ecosystem function, especially in those environments in which nutrients are known to limit primary production, as in most marine habitats (Valiela, 1995). This view is largely supported by an increasing amount of evidence about the stimulating effect of nutrient additions on plant growth (e.g. Short, 1987; Howarth, 1988) and also of functional modifications following eutrophication, e.g. changes in epiphyte loading, changes in herbivore activity (see Williams and Ruckelshaus, 1993; Short et al., 1995; Valentine and Heck, 2001; Ruiz et al., 2001). However, the effects of nutrient availability on plant physiology are, in many cases, far more complex than simple dose –response kinetics. Marine plants, from phytoplankton cells to large seaweeds and vascular plants, have evolved in an environment where nutrient supply is generally limited, at least temporally, and have developed a number of adaptive mechanisms to compensate for this limitation. A wellknown example is the capacity of a number of marine plants to uptake nutrients, when available, well above immediate plant requirements for growth (luxury consumption) and to store them until they are consumed when the nutrient supply does not meet growth needs (Bird et al., 1982; Jones et al., 1996 and citations therein). The variety of mechanisms to accommodate nutrient scarcity increases with plant complexity. Hence, marine vascular plants, which present remarkable histological, anatomical and morphological differentiation (e.g. den Hartog, 1970; Phillips and McRoy, 1980), are those with the highest potential to develop complex responses to nutrient shortage and are thus interesting models for the assessment of plant – nutrient interactions. Particularly, seagrasses can achieve high production rates in oligotrophic waters (Hemminga, 1998) and this can be attributed to certain specific features. The existence of perennial organs, such as rhizomes, can extend the storage/consumption interplay over long periods of time, resulting in a greater independence from the external medium than in other marine plant groups (Pedersen and Borum, 1993; Alcoverro et al., 1997; Sand-Jensen and Borum, 1991). Finally, the possibility of nutrient uptake through roots allows seagrasses to exploit pore-water nutrients which are not available to most marine producers (except for a few species of algae; Iizumi and Hattori, 1982; Thursby and Harlin, 1982; Short and McRoy, 1984; Hemminga et al., 1994; Pedersen et al., 1997; Hemminga, 1998). Moreover, seagrass rhizosphere has been demonstrated to be hosting an active nitrogen fixation in tropical areas (Patriquin, 1972; Capone and Penhale, 1979; Capone and Budin, 1982), thus increasing nitrogen availability to the plant. Nutrient conservation through partial resorption of leaf nutrient stocks before leaf loss is also considered as an important mechanism that helps to decrease external nutrient demand, reducing the plant’s dependence on external nutrient availability (Pedersen and Borum, 1993). In a recent paper, Hemminga et al. (1999) indicate that such a mechanism is only of moderate importance, accounting on an average, about 20% of the annual nitrogen demands. However, this figure increases to 40% of the annual nitrogen demands of the Mediterranean species Posidonia oceanica (Lepoint et al., 2000; Alcoverro et al., 2000). The leaves of this species present the longest lifespan known so far in seagrasses (up to more than 300 days: Romero, 1989b), suggesting that leaf longevity could represent an additional advantage in nutrient conservation (Hemminga et al., 1999; Alcoverro et al., 2000; Escudero et al., 1992).

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Like other seagrasses inhabiting temperate waters, P. oceanica is subjected to a seasonally changing nutrient availability, with relatively high water column nutrient concentrations in fall – winter (up to 3.5 AM nitrate and 0.85 AM phosphate) and very low concentrations in spring – summer (0.8 AM nitrate and < 0.01 AM phosphate, Ballesteros, 1989; Vidondo and Duarte, 1995). In late fall – winter, growth rates are low and nutrient uptake exceeds the demand causing an increase in nutrient content in plant tissues. In contrast, from March to July, growth is very active and, due to the reduced nutrient availability in the water column, nutrient demand must be met, at least in part, by internal pools (Pedersen and Borum, 1993; Pe´rez and Romero, 1994; Pedersen et al., 1997; Alcoverro et al., 1997, 2000; Stapel and Hemminga, 1997). In this period, therefore, nutrient concentration in plant tissues drops, clearly leading to an eventual limitation of plant growth. Despite the importance in the nutrient economy of the plant in the above mentioned process, the physiological bases are still poorly understood. A large part of the available knowledge has been obtained using total nutrient concentrations and plant mass balances (e.g. Pedersen and Borum, 1993; Erftemeijer and Middelburg, 1995; Stapel and Hemminga, 1997; Alcoverro et al., 1997, 2000; Hemminga et al., 1999) while only a few attempts at a closer look at the dynamics of metabolic compounds have been reported so far (e.g. Pirc and Wollenweber, 1988; Udy and Dennison, 1997; Udy et al., 1999; Kraemer et al., 1997; Kraemer and Mazzella, 1999); yet nitrogen metabolism includes a wide variety of processes (uptake, translocation, storage) and reactions (reduction, assimilation) in which a large number of chemical species of N participate, from single inorganics to macromolecules. We use P. oceanica here as an example of a long living seagrass species inhabiting oligotrophic waters and we attempt to elucidate some of the mechanisms allowing this species to achieve high growth rates in the relatively low nutrient waters of the Mediterranean sea (Margalef, 1985). To do that, we examine, during a 1-year cycle, some basic aspects of nitrogen metabolism like assimilation and speciation into different nitrogen soluble compounds (inorganic nitrogen, free amino acids and proteins) and how these compounds are distributed among the different plant organs (leaves, rhizomes and roots). Our final aim is to better understand some basic aspects of the nitrogen economy in P. oceanica and specifically: (i) the relative importance of the different nitrogen soluble compounds for nitrogen storage and translocation between different plant parts; (ii) the role of the different plant parts in the processes of uptake, assimilation and storage of nitrogen; and (iii) the extent to which nitrogen uptake and assimilation are coupled.

2. Material and methods The indicators related with nitrogen metabolism chosen were: total nitrogen content in tissues, nitrogen speciation into different soluble compounds (inorganic nitrogen, free amino acids and proteins) and nitrogen assimilation (expressed as in vivo glutamine synthetase activity). These variables were followed during a 1-year cycle (from March 1996 to March 1997) in a dense, shallow P. oceanica stand (600 shoots m 2 at a depth of 5 m) close to the Medes Islands (Girona, NE Spain; see for more details, Alcoverro et al.,

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1995). The parts of the plant considered were: leaves of different ages, rhizomes and roots. Also, some key plant features (leaf growth and epiphyte load) were assessed. 2.1. Sample design and plant processing Three plots, 50  50 cm, were chosen at random within the seagrass bed and permanently marked in March 1996. From this date until March 1997, the plots were visited once every 2 months (i.e. seven sampling events). To estimate growth, 1 month before collection 10 shoots of each plot were individually identified and punched, following a modified Zieman method (Zieman, 1974; Romero, 1989a). At collection time, marked shoots were harvested by scuba diving and transported to the laboratory in aerated plastic containers. Plants were kept overnight in an aerated seawater collected at the same sampling site and maintained at field temperature. Sample processing was performed within 24 h. Growth (leaf production, cm2 day 1) was determined in 10 shoots per plot by measuring the distance between the ligula and the position of the holes at the moment of collection (see details in Alcoverro et al., 1995). After growth had been measured, these shoots were used for inorganic nitrogen, total soluble protein (TSP), free amino acid concentration (FAA) GS in vivo activity, and total nitrogen content determinations and for epiphyte biomass evaluation. This resulted in three independent estimates (one per plot) for each variable at each sampling event. Epiphytes were carefully removed with a razor blade and then every shoot was divided into the following parts: young leaf tissue (0– 50 days old), intermediate leaf tissue (50 – 100 days old), old leaf tissue (100 –150 days old) following Alcoverro et al. (2000), rhizomes (the uppermost 1 cm) and roots (avoiding the most lignified ones). Shoots selected for in vivo GS activity measures were kept in fresh seawater until performing the assay; plant tissues for total nitrogen content analyses were dried at 70 jC to constant weight and the rest of the plant material was kept frozen in liquid nitrogen until processing. 2.2. Analytical procedures As glutamine synthetase is the enzyme catalyzing the first step (i.e. the incorporation of ammonium into glutamate producing glutamine) of nitrogen assimilation in plants, its activity has already been used as an indicator of nitrogen assimilation in seagrasses (Kraemer et al., 1997; Kraemer and Mazzella, 1999). We determined the in vivo GS activity, applying the assay developed by Kraemer and Mazzella (1996), based on previous works by Gao et al. (1992) and Pregnall et al. (1987). So, pieces of fresh tissue were incubated at 20 jC for all sampling events to show seasonal shifts of GS activity (combination of enzyme quantity and activation). Incubation lasted for 1 h in 14 ml of incubation medium. The incubation medium was prepared with seawater to which hydroxylamine and glutamine were added as substrates to reach final concentrations of 150 and 50 mM, respectively. 1-Propanol (3% v/v) was also added to permeabilize tissues, which allow the influx of substrates and the efflux of the product, the g-glutamyl hydroxamate, which was quantified spectrophotometricaly at 540 nm. In vivo GS activity

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was expressed as Amol g-glutamyl hydroxamate gdw 1 h 1 which is equivalent to Amol N gdw 1 h 1 (see Kraemer et al., 1997). Inorganic nitrogen (ammonium, nitrate and nitrite) and total soluble proteins (TSP) were extracted from tissues by grinding frozen tissues in 20-ml deionized water. The extract was then centrifuged for 15 min at 11,600 rpm (Lapointe and Duke, 1984); protein, nitrate, nitrite and ammonium concentrations were determined in the supernatant previously diluted for ammonium, and nitrate + nitrite (1:2 and 1:5, respectively). Ammonium concentration was measured using a Technicon II Autoanalyzer (Koroleff method, Grasshoff et al., 1983). Nitrate and nitrite concentrations were measured by Ionic Chromatography following EPA 9056 reglamentation with a KONIK KNK 500-A liquid chromatograph and a WATERS IC-PAK ANIONS column. Total soluble protein was determined using bicinchoninic acid, assay based on Smith et al. (1985) and Brown et al. (1989). For free amino acid (FAA) determination, frozen tissues were ground in 20 ml of 0.05 N HCl and then centrifuged for 5 min at 10,000 rpm. The supernatant was then filtered in a microfuge using low-binding, regenerated cellulose Millipore ultrafree filters to exclude peptides with molecular weights higher than 10,000 Da. Free amino acid

Table 1 Summary of ANOVA results Variable

Factor

df

MS

p

Total N (% dw)

Time Tissue TT error Time Tissue TT error Time Tissue TT error Time Tissue TT error Time Tissue TT error Time Tissue TT error

6 4 24 68 6 4 24 68 6 4 24 68 6 4 24 68 6 4 24 68 6 4 24 68

2.899 13.92 0.268 0.362 709 1372 150 66 434 850 146 38 129 112 11 14 624 6200 158 156 4122 7058 724 635

< 0.001 < 0.001 < 0.05

Inorganic nitrogen (Amol g

Ammonium (Amol g

dw)

1

dw)

1

Nitrate + nitrite (Amol g

Total free AA (mg g

1

dw)

1

dw)

Total soluble protein (mg g

1

dw)

< 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.05 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.01

The dependent variables were: total nitrogen content, inorganic nitrogen (sum of ammonium, nitrate and nitrite), ammonium, nitrate + nitrite, free amino acids and total soluble protein concentrations. Independent variables (‘‘factor’’) were, in all cases, the different plant parts (three leaf age classes, rhizomes and roots: ‘‘tissue’’) and sampling event (‘‘time’’).

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concentration in tissues was measured using an amino acid autoanalyzer with ionic exchange chromatography, following the method of Spackman et al. (1958). For the sake of simplicity, we considered here only the total FAA concentration. Total nitrogen content (% dw) in plant tissues was determined using a Carlo-Erba CNH elemental autoanalyzer. 2.3. Estimates and statistics Nitrogen incorporation by seagrass leaves (mg N gdw 1 day 1), for any interval between successive sampling events, was calculated as the product of the total

Fig. 1. Nitrogen content (% dw) in P. oceanica tissues. (a) Leaves: YL, young leaves; IL, intermediate leaves; OL, old leaves. (b) Below-ground parts: Ri, rhizomes; R, roots. Vertical bars correspond to standard error of the mean (n = 3).

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nitrogen content measured in young leaves and growth (leaf elongation) corrected for the corresponding leaf specific weight (dry weight per unit area, Alcoverro et al., 1995). Nitrogen assimilation potential (Ag N h 1 shoot 1) was estimated by multiplying the specific GS activities measured for each plant part by its respective weight at the moment of sampling (Kraemer et al., 1997; Alcoverro et al., 1995). To extrapolate these values to a daily basis (mg N day 1 shoot 1), we assumed that nitrogen assimilation only took place during the hours in which irradiance reaching the plant was above Isat (saturating irradiance for photosynthesis), due to energetic constraints of N assimilation (Dennison and Alberte, 1985; Pregnall et al., 1987, in Kraemer et al., 1997). Isat values were those reported by Alcoverro et al. (1998) for the same site and

Fig. 2. Inorganic nitrogen (nitrate + nitrite + ammonium) concentration in P. oceanica tissues. (a) Leaves: YL, young leaves; IL, intermediate leaves; OL, old leaves. (b) Below-ground parts: Ri, rhizomes; R, roots. Vertical bars correspond to standard error of the mean (n = 3).

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the number of daily hours of irradiance above Isat were computed as in Alcoverro et al. (2001). NAP estimates, using GS activity measured at constant temperature (20 jC for all the sampling events), did not reflect in situ nitrogen assimilation but was used to assess seasonal changes in GS activity independently of the temperature and to evaluate the role of each plant part in the nitrogen assimilation of a shoot. The statistical significance of variability found among sampling events (factor ‘time’) and that of differences between plant parts (factor ‘tissue’) in GS activity, inorganic nitrogen, FAA, TSP and total nitrogen content on tissues were tested using a two-way repeated measures ANOVA, with two within subjects factors (‘time’ and ‘tissue’). Changes of growth were tested by a one-way repeated measures ANOVA, with ‘time’ as the within subject factor. Statistical relationships between some variables were explored using linear

Fig. 3. Ammonium concentration in P. oceanica tissues. (a) Leaves: YL, young leaves; IL, intermediate leaves; OL, old leaves. (b) Below-ground parts: Ri, rhizomes; R, roots. Vertical bars correspond to standard error of the mean (n = 3).

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regressions. Whenever necessary, significance of differences between given experimental conditions was assessed using the Tukey posthoc test.

3. Results 3.1. Total nitrogen content and nitrogen speciation Total nitrogen content significantly differed among tissues with maximum values in rhizomes and minimum in roots (Table 1, p < 0.001; Fig. 1). In leaves, values were intermediate and varied with leaf age, being higher in young and intermediate leaves and

Fig. 4. Nitrate + nitrite concentration in P. oceanica tissues. (a) Leaves: YL, young leaves; IL, intermediate leaves; OL, old leaves. (b) Below-ground parts: Ri, rhizomes; R, roots. Vertical bars correspond to standard error of the mean (n = 3).

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lower in the old ones. Nitrogen content changed seasonally in leaves and rhizomes with maximum values in winter and minimum values in summer while it was constant throughout the year in roots. Inorganic nitrogen concentration (sum of ammonium, nitrate and nitrite) in tissues was low in general, the roots being the plant part with the lowest values and the young leaves with the highest values (Fig. 2; Table 1, p < 0.001; Tukey test, p < 0.01). This variable changed seasonally in leaves with seasonality depending on leaf age (Table 1). Intermediate and young leaves showed a distinct maximum in May and minimum values in September while maximum values for old leaves were detected in March, although this maximum did not appear the following year. Inorganic nitrogen concentration in rhizomes and roots were relatively constant along the year.

Fig. 5. Free amino acid (FAA) concentration in P. oceanica tissues. (a) Leaves: YL, young leaves; IL, intermediate leaves; OL, old leaves. (b) Below-ground parts: Ri, rhizomes; R, roots. Vertical bars correspond to standard error of the mean (n = 3).

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Ammonium was, except in a few cases, the most abundant form of inorganic nitrogen in tissues specially in underground organs where it generally accounted for more than 80% of the total inorganic nitrogen with a well-marked seasonality (Fig. 3). Maximum values of nitrate + nitrite concentration were detected in leaves from samples taken in spring 1996, although this maximum in nitrates was not detected in the same month of the following year (Fig. 4).The highest free amino acid concentration (FAA), in annual average, was found in rhizomes and the lowest in roots (Table 1; Tukey test, p < 0.001); FAA concentration in leaves followed leaf age, with the lowest values in the oldest leaves (Fig. 5). Seasonal differences were significant with maximum values of FAA generally found in winter, although time course in this variable showed significant differences among plant parts (e.g. almost constant values in roots during all the years and a relative second maximum in rhizomes in September).

Fig. 6. Total soluble protein (TSP) concentration in P. oceanica tissues. (a) Leaves: YL, young leaves; IL, intermediate leaves; OL, old leaves. (b) Below-ground parts: Ri, rhizomes; R, roots. Vertical bars correspond to standard error of the mean (n = 3).

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The different plant parts showed significant differences in total soluble protein (TSP) concentration (Fig. 6, Table 1, p < 0.001), with the ranking: leaves > rhizomes>roots, on an annual average. TSP concentration decreased from March to July in all plant parts and increased from July onwards, although in a quite irregular manner (significant interaction, p < 0.001). The soluble compounds (sum of inorganic nitrogen, FAA and TSP) accounted for about half of the total nitrogen content of the plant. Inorganic nitrogen represented only a small fraction of the total nitrogen, from 0.6% of total N (in rhizomes) to 3.2% (in young leaves). Thus, FAA and TSP were the main forms of soluble nitrogen. In leaves, TSP was the most abundant form in summer and autumn specially in old leaves while in the roots, the relative contribution of TSP to nitrogen pools peaked in spring and winter. In the case of

Fig. 7. Total nitrogen content (symbols) and nitrogen content in form of soluble compounds (bars) in different parts of P. oceanica. (a) Leaves: YL (white), young leaves; IL (grey), intermediate leaves; OL (black), old leaves. (b) Below-ground parts: Ri (white), rhizomes; R (grey), roots.

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Fig. 8. Growth as leaf elongation (open symbols) and N incorporation into new leaf tissue (black symbols) from May 1996 to March 1997. Vertical bars correspond to standard error of the mean.

the rhizomes, FAA was the most abundant fraction of soluble nitrogen throughout the year (Fig. 7). 3.2. Growth, nitrogen incorporation and potential assimilation Growth rates of P. oceanica leaves were higher in late spring –summer than in the rest of the year (Fig. 8, Table 2, p < 0.001), reaching its maximum in July. Nitrogen

Table 2 Summary of ANOVA results Variable

Factor

df

MS

p

In vivo GS activity (without rhizome)

Time Tissue TT error Time Tissue TT error Time error

6 3 18 54 4 4 16 48 5 110

885.0 2579.1 111.64 73.7 474.3 2848.6 96.80 84.3 1.661 0.064

< 0.001 < 0.001 < 0.001

In vivo GS activity (July – March 97 with rhizome)

Growth (cm2 day

1

) (May 96 – March 97)

< 0.01 < 0.001 < 0.01 < 0.001

The dependent variables were GS activity and leaf growth. Due to the missing data of GS for rhizomes in the first two sampling events, data have been analysed in two ways: all the sampling events (only data for the three leaf age classes and for roots) and only the last five sampling events (data for the three leaf age classes, for roots and for rhizomes). Independent variables (‘‘factor’’) were, for GS, the different plant parts (‘‘tissue’’) and the sampling event (‘‘time’’), while for leaf growth it was only the factor ‘‘time’’.

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incorporation into new leaf tissue, expressed as mg N shoot 1 day 1, followed the same seasonal trend with relatively low values from September to January (Fig. 8). The highest values of in vivo GS activity were found in leaves with the annual means being sevenfold higher than those found in rhizomes and 21-fold higher than those found in roots (Fig. 9; Table 2, p < 0.001). In vivo GS activity of leaves decreased with age and changed seasonally with a seasonal pattern coherent among age classes showing low values in spring followed by a maximum in late summer. Roots showed a trend similar to that of leaves, although with much lower values; maximum activity in September was followed by a sharp decrease in November. In contrast, GS activity in rhizomes was maximum in January (Table 2, significant interaction time  tissue, p < 0.01).

Fig. 9. In vivo GS activity measured in P. oceanica tissues. (a) Leaves: YL, young leaves; IL, intermediate leaves; OL, old leaves; (b) below-ground parts: Ri, rhizomes; R, roots. Vertical bars correspond to standard error of the mean (n = 3).

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Fig. 10. Nitrogen assimilation potential (NAP) estimated for P. oceanica, including the contribution of leaves, leaves + roots and whole shoot (leaves + rhizome + roots).

Nitrogen assimilation potential (NAP), considering leaves, rhizomes and roots, was maximum throughout the summer and decreased in winter (Fig. 10). Leaves contributed the most to the NAP, accounting for nearly 87% (September) to nearly 60% (January) of the whole shoot NAP; among leaves, the oldest age class had the lowest contribution. Roots showed low values of NAP throughout the entire period with a slight increase in September. Data for rhizome GS activity in March and May 1996 were not available; therefore, no NAP estimates for either rhizomes or the whole shoot were possible for these months. Rhizomes had NAP values midway between those found in leaves and roots with the highest values in January and March 1997, resulting in a substantial contribution of this organ to whole shoot NAP from November to March (about 30%). On an annual basis, leaves accounted for 79%, rhizomes for 16% (average from July to March) and roots for 5% of the whole shoot NAP of P. oceanica.

4. Discussion Results presented here about seasonality in nitrogen content and incorporation of P. oceanica indicate that, for nitrogen dynamics, there are at least two periods. The first one is in late fall – winter, with relatively high nitrogen availability in the water and in the sediment (Alcoverro et al., 1995) and low leaf growth rates; during this period, nitrogen content in leaves increases, indicating that uptake exceeds demand for growth. The second period is in spring– summer, with an active leaf growth and a low nitrogen availability; during this period nutrient demand exceeds uptake and part of this demand is met by internal pools (reserves), with a subsequent decrease in tissue nitrogen content. These findings agree well with previous views on nitrogen dynamics (e.g. Pedersen and Borum,

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1993; Pedersen et al., 1997; Alcoverro et al., 1997, 2000; Stapel and Hemminga, 1997). Moreover, our results shed some additional light on this basic scheme. Resorption of nitrogen from old leaves takes place before they are shed and it is used in newly growing meristems. Averaged across different seagrass species, this source seems to account for about 20% of total N requirements (Stapel and Hemminga, 1997), with a much higher percentage (40%) in P. oceanica (Alcoverro et al., 2000). However, storage in rhizomes which can represent a second internal source has been mentioned but rarely evaluated (Lepoint et al., 2000; Alcoverro et al., 2000). Here we found a difference between the maximum nitrogen content in rhizomes at the beginning of the growth period (i.e. March) and the minimum at the end (July) of 2.2% N (relative to dry weight). For the usual rhizome biomass found at our study site (Alcoverro et al., 2001) and considering only the upper, most active 1 cm of rhizomes, this represents 4.34 mg N shoot 1. This amount can meet 33% of the shoot nitrogen requirement during the growth period (from May to July) and is equivalent to 19% of the total annual N requirements for leaf growth, a value which is higher than that previously reported for P. oceanica (Alcoverro et al., 2000). The seasonal shift of consumption/storage periods could be interpreted as a strategy to obtain independence from the environmental fluctuations in nitrogen availability, as occurs in some terrestrial grasses and marsh plants (Lipson et al., 1996; Farahbakhshazad and Morrison, 1997; Weber and Braendle, 1994). Despite significant seasonal behaviour, the values reached during the periods with high nitrogen content and low nitrogen content show a great interannual variability. For example, the nitrogen content in rhizomes measured in March 1996 is higher than that measured in March 1997. Similarly, Alcoverro et al. (2000) also found clear differences between the nitrogen content in winter of 2 consecutive years. We suggest that nitrogen storage in rhizomes probably reflects interannual changes in nitrogen availability and demand, and also interannual changes in the energy (from light) available for nitrogen assimilation. This suggestion is supported by the low values of nitrogen content found in rhizomes by Alcoverro et al. (2000) in 1992 compared with those of this work which coincides with low values of light availability and low values of net carbon balance of P. oceanica in 1992 (Renom et al., 2000) that indicate conditions of low energy for nitrogen assimilation. The major role of rhizomes in the nitrogen metabolism in P. oceanica seems to be confirmed by the fact that they show a higher nitrogen content than the leaves, the opposite of what has been reported in other seagrasses (Zostera marina, Pedersen and Borum, 1993; Cymodocea nodosa, Pirc and Wollenweber, 1988; Pe´rez et al., 1994; Kraemer and Mazzella, 1999; Z. noltii, Pirc and Wollenweber, 1988; Kraemer and Mazzella, 1999, and Amphibolis antarctica, Pedersen et al., 1997). Another peculiarity of the nitrogen metabolism in this species is the limited nitrogen assimilation capacity of roots. The role of roots in P. oceanica (viz. are they metabolically active or do they act as mere anchoring system?) continues to be a controversial issue. While in other species nutrient uptake (and assimilation) through roots has been well documented (e.g. Z. marina, Iizumi and Hattori, 1982; Short and McRoy, 1984; Pregnall et al., 1987; Hemminga et al., 1994; Z. noltii, Kraemer and Mazzella, 1999; A. antarctica, Pedersen et al., 1997), this is not yet the case for P. oceanica. The low and constant values of total nitrogen content and FAA concentration suggest a limited contribution to the

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overall nitrogen metabolism. However, roots are undoubtedly metabolically active in terms of nitrogen (see phosphorus, Invers et al., 1995), since they show some capacity for nitrogen assimilation; the same conclusion was reached by Kraemer and Mazzella (1996) using an in vitro assay. However, this capacity seems limited and is much lower not only than those of leaves and rhizomes, but also lower than those found in roots of other seagrass species (e.g. Z. marina, Pregnall et al., 1987). In contrast, assimilation in rhizomes was relatively high, mainly from autumn to spring, accounting for as much as 32 –54% of total NAP during that period. This fact is relatively surprising since the morphology of those massive organs (low surface to volume ratio) and the dense coating of the axis with scales (remaining from shed leaf bases, see Manzanera et al., 1998) suggest that rhizome uptake of nitrogen should be very low. We suggest that the nitrogen assimilated into the rhizomes emanates from the roots because it is unlikely that ammonium could come from the leaves due to the high nitrogen assimilation potential found there. Since ammonium is the most abundant form of inorganic nitrogen in sediments (Zimmerman et al., 1987; Hemminga et al., 1994; Hemminga, 1998), our assumption is consistent with the seasonal variation of GS activity found in rhizomes which reaches its maximum level coinciding with the period of high ammonium concentration in sediment pore water (i.e. winter; Ballesteros, 1989; Vidondo and Duarte, 1995; Pedersen and Borum, 1993). The heavy burden imposed on the carbon balance of P. oceanica by the respiratory demand of the high biomass of rhizomes has been pointed out in previous works (Hemminga, 1998; Alcoverro et al., 2001). The uses of rhizomes (mainly storage but also assimilation) in the nitrogen economy of the plant could, at least in part, be the benefit counteracting such a heavy load. Among the different forms of nitrogen within the plant, inorganic compounds are the least abundant; in effect, less than 1% of total N was found as inorganic nitrogen. This is not surprising for ammonium (see below) but contrasts with the well-known accumulation of NO3 in vacuoles reported in terrestrial plants and in some macroalgae (Bidwell, 1974; Millard, 1988; Lapointe and Duke, 1984 and citations therein; Vergara, 1993). The reasons for this are unknown but it may be attributed either to a fast response in nitrate reductase activity at increased nitrate availability (Touchette and Burkholder, 2000) or to low nitrate uptake due to the prevalence of ammonium forms in the pore water or close to the sediment (Zimmerman et al., 1987; Hemminga et al., 1994; Hemminga, 1998). Ammonium concentration in tissues is also very low, as is to be expected given its toxicity (Bidwell, 1974). Ammonium assimilation probably follows uptake quite closely; this is coherent with the fact that FAA are the compounds that increase the most during the period of maximum uptake (winter and spring, Alcoverro et al., 2000). FAA, besides being the first step in nitrogen assimilation, is the largest N pool at the moment of maximum N reserves, indicating that amino acids are the preferential storage forms. However, soluble proteins also account for an important part of nitrogen storage (13% and 25% in leaves and rhizomes, respectively) as well as nonsoluble compounds (28% and 45% in leaves and rhizomes, respectively). Those nonsoluble compounds were estimated as the difference between total nitrogen content (after subtracting 1% of total nitrogen content considered as structural nitrogen, see Duarte, 1990) and total nitrogen in the form of soluble compounds. Translocation probably also takes place in the form of FAA, as suggested by the low

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concentration of FAA in old leaves at the moment in which N resorption is occurring (summer – early autumn, Alcoverro et al., 2000). The role of amino acids as storage and transport compounds is well known in terrestrial plants (Bidwell, 1974; Lea, 1994), and amines (asparagine and glutamine) are the compounds chiefly involved in these processes; according to our results, asparagine was the most abundant amino acid in all P. oceanica parts considered and accounted for most of the seasonal FAA variation (see also Pirc and Wollenweber, 1988). A major part of the nitrogen assimilation takes place in leaves which accounts for 60– 87% of total NAP, depending on the season, confirming the role of leaves as important sites for nitrogen acquisition as already reported for this and other species (Zimmerman et al., 1987; Pedersen and Borum, 1992; Stapel et al., 1996; Kraemer et al., 1997; Kraemer and Mazzella, 1999, among others). Nitrogen assimilation potential in leaves is maximum in September, well before nitrogen availability in the water column reaches its seasonal maximum. In fact, September is the period in which nitrogen retranslocation is at its maximum (Alcoverro et al., 2000). Therefore, the seasonal pattern of NAP (at constant temperature, i.e. potential GS activity) seems to be more closely controlled by internal than by external nitrogen availability in that period of the year and should reflect the nitrogen recycling within the plant from protein breakdown (Bidwell, 1974; Lea, 1994). Due to the long life span of the leaves of this species (up to 300 days, Romero, 1989b), changes in nitrogen metabolism due to leaf ageing are apparent. Young and intermediate leaves show pronounced elongation, which implies a high nitrogen demand; total nitrogen content and concentrations of soluble nitrogen forms are high and nitrogen assimilation is actively taking place (Kraemer and Mazzella, 1996; Kraemer et al., 1997). In contrast, old leaves do not elongate at all and present low nitrogen content and concentrations of soluble nitrogen forms as well as low nitrogen assimilation. This decrease in nitrogen compounds can be caused by leaching and also by an important translocation to young leaves (Lepoint et al., 2000). So, leaves shift from being nitrogen sinks to sources as they age, contributing to nitrogen conservation within the plant (Borum et al., 1989; Pedersen and Borum, 1993; Stapel and Hemminga, 1997; Hemminga et al., 1999; Alcoverro et al., 2000). In summary, P. oceanica is able to maintain a very high production in the generally oligotrophic conditions of the Mediterranean sea. The success of this species in nutrient-poor waters seems to rely more on an effective nitrogen conservation strategy than on a high uptake of nitrogen by roots, which seems to be a more common strategy in other seagrasses (Hemminga, 1998). There are basically two features that allow this conservation strategy: a nutrient resorption from old leaves (Alcoverro et al., 2000; Lepoint et al., 2000) due to the long life span of leaves, and a high capacity for nitrogen storage in rhizomes, enhanced by a high rhizome biomass (Romero et al., 1994). While the importance of leaves in nutrient conservation seems relatively constant from one year to another, the magnitude of nitrogen storage in rhizomes exhibits interannual fluctuations. These fluctuations probably depend on the surplus of the nitrogen acquired and assimilated relative to nitrogen required for growth, which in turn depends on both nitrogen availability and energetic restrictions of the assimilatory process.

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Acknowledgements We thank Marta Manzanera, Pere Renom, Miquel Angel Rodriguez, Xavier de Pedro and Fiona Toma`s for their help in field and laboratory work. We also thank Dr. George Kraemer for teaching the techniques of GS activity and for other suggestions, and to Dr. Miguel Angel Mateo for his suggestions. FAA analyses were performed with the help of Pilar Ferna´ndez and Isidre Casals of the ‘Serveis Cientı´fico-Te`cnics de la Universitat de Barcelona’. This research was supported by a grant from the Ministerio de Educacio´n y Ciencia (PN92) of the Spanish Government, a grant from CICYT (no. MAR98-0356) and a grant from the University of Barcelona. [SS]

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