www.elsevier.com/locate/jembe

Journal of Experimental Marine Biology and Ecology

303 (2004) 97–114

Effects of nitrogen addition on nitrogen metabolism

and carbon reserves in the temperate seagrass

Posidonia oceanica

Olga Inversa,*, George P. Kraemerb, Marta Pereza, Javier Romeroa

aDepartament d’Ecologia, Facultat de Biolgia, Universitat de Barcelona, Avda. Diagonal 645,

E-08028 Barcelona, SpainbPurchase College, Division of Natural Sciences, State University of New York, Purchase, NY 10577, USA

Received 1 May 2003; received in revised form 6 August 2003; accepted 7 November 2003

Abstract

The effect of repeated N additions on a dense, shallow meadow of Posidonia oceanica (L.) Delile

in the NWMediterranean was studied over a year. N was added biweekly both to the sediment and to

the water column as ammonium and nitrate. The most obvious result of these additions was an

overall increase in N content (% DW) in all tissues of fertilized plants; this increase was maximum in

rhizomes, with values of 5% N reached, which confirmed the storage capacities of these organs.

Fertilization affected the different N fractions in distinct ways. The free amino acid (FAA)

concentration increased the most, particularly in rhizomes and roots, suggesting the function of these

compounds for N storage and, probably, translocation. The non-soluble N fraction also increased

greatly. The total soluble protein (TSP) and the inorganic N forms concentrations were less sensitive

to fertilization, and only increased moderately in a few cases. N assimilation, assessed through in

vivo glutamine synthetase (GS) activity, was maximum in leaves after the peak of growth, which

coincided with the lowest N values in both control and fertilized plants. Thus assimilation was

probably greatest at the period of highest N deficiency. Growth rates did not respond to N

enrichment. Another clear effect of N addition was to decrease carbon reserves. In effect, the

concentration of total non-structural carbohydrate (TNC) greatly decreased in rhizomes of fertilized

plants, coinciding with the increase in FAA. We conclude that increased nitrogen availability can

affect plant survival through the decrease in their carbon reserves, crucial for P. oceanica

overwintering. This interaction between N and C metabolism helps to explain changes in benthic

vegetation after steadily increasing coastal water eutrophication.

D 2004 Elsevier B.V. All rights reserved.

Keywords: Amino acid; Protein; Growth; Glutamine synthetase; Mediterranean; Eutrophication

0022-0981/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.jembe.2003.11.005

* Corresponding author. Tel.: +34-93-40215-06; fax: +34-93-411-14-38.

E-mail address: invers@porthos.bio.ub.es (O. Invers).

O. Invers et al. / J. Exp. Mar. Biol. Ecol. 303 (2004) 97–11498

1. Introduction

Eutrophication of coastal marine waters is occurring worldwide and has generally

detrimental effects on shallow water ecosystems (Nixon, 1995). Eutrophication is

characterized by high seawater nutrient concentrations and low water transparency, both

of which have a negative impact on benthic macrophytes, and especially on seagrasses

(Schramm and Nienhuis, 1996; Short et al., 1995). Thus, it is not surprising that seagrass

meadows have been in steady decline in recent decades (Short and Wyllie-Echevarria,

1996).

Increased nutrient availability affects the ecosystem of the community of submerged

aquatic vegetation in several ways. Excess nutrients promote the proliferation of fast-

growing species, including phytoplankton, epiphytes and opportunistic macroalgae (Sand-

Jensen and Borum, 1991; Duarte, 1995), species that compete with seagrasses for light and

can cause mortality by shading (Silverstein et al., 1986; Short et al., 1995; Hauxwell et al.,

2001). Moreover, increased nutrient availability stimulates seagrass consumption by

herbivores, probably through an increase in the nutritional quality of its food sources

(McGlathery, 1995; Ruiz et al., 2001), which can lead to significant reductions in

photosynthetic biomass and consequently to seagrass mortality.

In addition to causing alterations in the ecosystem, high N availability also has an

effect on individual plants. Moderate increases in nutrients stimulate seagrass growth

and production (Short, 1987; Short et al., 1990; Perez et al., 1991; Kenworthy and

Fonseca, 1992; Alcoverro et al., 1997; Udy and Dennison, 1997; Udy et al., 1999),

although this response shows high inter- and intra-specific variability. However, large

increases in N availability are deleterious to the plants (Burkholder et al., 1992; Short et

al., 1995). Apparently, seagrass is unable to down-regulate N uptake, probably because

of a lack of inhibitory feedback mechanisms (Rabe, 1990; Touchette and Burkholder,

2000). Therefore, under conditions of high N availability, especially when ammonium is

the most abundant form, plant growth and survival can be curtailed by direct ammonium

toxicity (van Katwijk et al., 1997) and by the internal demand of energy and C skeletons

needed for rapid ammonium assimilation (Burkholder et al., 1992). This demand is met

by resources diverted from growth and metabolism maintenance (Marschner, 1995).

Nevertheless, the physiological mechanisms involved in the effects of nutrient increases

on plant status are complex and not yet fully understood (Touchette and Burkholder,

2000).

To research further into the effects of eutrophication on individual plants, and to assess

the physiological response of seagrasses to increased nutrient levels, we conducted an

experimental study with the Mediterranean endemic Posidonia oceanica, a slow-growing

seagrass of major ecological importance. This seagrass shows a clear annual growth

pattern, with a maximum in spring and a minimum in late-summer (Alcoverro et al.,

1995). This pattern is based mainly on the interplay between physical factors (irradiance

and temperature) and an efficient management of reserves through nutrient and C storage

and remobilization following periods of resource excess and shortage, respectively

(Alcoverro et al., 2000, 2001; Invers et al., 2002). Since P. oceanica meadows are affected

by anthropogenic nutrient inputs, which make water quality worse (Marba et al., 1996;

Delgado et al., 1997, 1999; Ruiz et al., 2001), clarification of the physiology of plant–

O. Invers et al. / J. Exp. Mar. Biol. Ecol. 303 (2004) 97–114 99

nutrient interactions is not only relevant in research terms, as indicated above. It can also

assist management and conservation.

We studied the effect of experimental N additions over a year on the main descriptors of

N metabolism, as well as on the C reserves in the different plant organs.

We aimed to:

(i) identify the N pools, as inorganic N, free amino acids (FAA) and total soluble protein

(TSP) at the different organs (i.e. leaves, roots, rhizomes), and in which of those

organs the excess N accumulates;

(ii) determine whether the general annual patterns of growth and nutrient storage are

modified by N addition, and

(iii) evaluate the effects of N addition on carbohydrate storage by testing the hypothesis

that excess leads to a decrease in carbohydrate pools.

2. Material and methods

2.1. Study site and field work

The study was conducted in a dense (ca. 600 shoots m� 2) and shallow (5 m deep) stand

of P. oceanica situated close to the Medes Islands (Girona, NE Spain). Six randomly

selected plots, 50� 50 cm, ca. 20 m apart were permanently marked: three were enriched

with N every 15 days, while three remained as controls. N was added to the sediment using

slow-release commercial fertilizers which contained nitrate and ammonium (molar ratio

1:1; manufactured by AMOSA, Spain). Water surrounding shoots was N-enriched by

placing plaster balls made of an ammonium-nitrate solution and covered by perforated

wax inside the canopy. These balls were prepared to decay completely in about 15 days.

Nutrient loading amounted to 60–100 g N m� 2 month� 1 in the sediment and 10 g N m� 2

month� 1 in the water.

The experiment started in April 1996 and ended in March 1997. Every 2 months, 10

shoots from each plot were punched to measure shoot growth, following a modified

Zieman method (Zieman, 1974; Romero, 1989). One month after marking, these plants

were collected and transported to the laboratory in aerated plastic containers, where they

were kept overnight in aerated seawater collected at the same sampling site and at the

same temperature as in the field. Plants were processed (growth, N analysis, enzymatic

activity, etc.) the following day. This resulted in a total of six tissue samples and growth

measurements (May, July, September and November 1996 and January and March

1997).

2.2. Plant processing

Shoot growth was determined by measuring the distance between the ligula and the

position of the holes of each leaf (see details in Alcoverro et al., 1995) and expressed in

cm2 shoot� 1 day� 1. Shoots were then used to measure inorganic N, FAA and TSP

concentration, in vivo glutamine synthetase (GS) activity, and total N and total non-

structural carbohydrate (TNC) content. For each analysis, several shoots from each plot

O. Invers et al. / J. Exp. Mar. Biol. Ecol. 303 (2004) 97–114100

were pooled, resulting in three independent estimates (one per plot) for each variable.

Before the analysis, epiphytes were removed carefully with a razor blade and the shoots

were then sorted 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), rhizomes (the

uppermost 1 cm), and roots (avoiding the most lignified). Leaf age was determined

following Alcoverro et al. (2000).

Shoots selected for in vivo GS activity measurements were kept in fresh seawater for

immediate assay, while tissues for total N content and TNC determinations were dried at

70 jC until constant weight. The rest of the plant material was frozen in liquid N until

processing.

2.3. Analytical procedures

GS activity was evaluated following the method developed by Kraemer and Mazzella

(1996), based on the in vivo nitrate reductase assay described by Gao et al. (1992), and

Fig. 1. Total N content (%DW) over 1 year in several organs of Posidonia oceanica subjected to nitrogen

enrichment (F) and control (C) treatments: YL, young leaves (0–50 days); IL, intermediate leaves (50–100

days); OL, old leaves (100–150 days); Ri, rhizome; R, root.

O. Invers et al. / J. Exp. Mar. Biol. Ecol. 303 (2004) 97–114 101

following the in vitro GS assay used by Pregnall et al. (1987). To show seasonal shifts

in GS activity (combination of enzyme quantity and activation), pieces of fresh tissue

were incubated for one hour at 20 jC in 14 ml of incubation medium for all sampling

events. The incubation medium was prepared with seawater with additions of hydrox-

ylamine and glutamine as substrates to reach final concentrations of 150 and 50 mM,

respectively. To facilitate the influx of substrates and the efflux of the product, 1-

propanol (3% v/v) was also added to permeabilize tissues. The product (g-glutamyl

hydroxamate) was quantified spectrophotometrically at 540 nm. In vivo GS activity was

expressed as Amol g-glutamyl hydroxamate g� 1DW h� 1, which is equivalent to Amol N

g� 1DW h� 1.

Inorganic N (ammonium, nitrite and nitrate) and TSP were extracted 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). The concentrations of inorganic N

and TSP were measured in the supernatant, previously diluted for the ammonium

and nitrate and 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 established by Ionic Chromatography,

following EPA 9056 procedures, with a KONIK KNK 500-A liquid chromatograph

Table 1

Summary of ANOVA of total nitrogen content, and ammonium and free amino acids (FAA) concentrations in

tissues of control and fertilized shoots of P. oceanica from May 1996 to March 1997

Variable Factor df MS p

Total nitrogen (% dw) Treatment 1 16.04 < 0.001

Time 5 3.47 < 0.001

Tissue 4 31.44 < 0.001

Tr�T 5 0.865 < 0.001

Tr�Ti 4 2.764 < 0.001

T�Ti 20 0.178 < 0.05

Tr�T�Ti 20 0.105 0.307

Error 236 0.621

Ammonium (Amol g dw� 1) Treatment 1 0.343 < 0.01

Time 5 0.692 < 0.001

Tissue 4 1.495 < 0.001

Tr�T 5 0.051 0.179

Tr�Ti 4 0.096 < 0.01

T�Ti 20 0.228 < 0.001

Tr�T�Ti 20 0.062 < 0.01

Error 236 0.148

Free amino acids (Amol g dw� 1) Treatment 1 13884 < 0.01

Time 5 2971 < 0.001

Tissue 4 14520 < 0.001

Tr�T 5 493 < 0.01

Tr�Ti 4 1106 < 0.001

T�Ti 20 235 < 0.001

Tr�T�Ti 20 234 < 0.001

Error 236 959

O. Invers et al. / J. Exp. Mar. Biol. Ecol. 303 (2004) 97–114102

and a WATERS IC-PAK ANIONS column. TSP was determined using the

bicinchoninic acid assay described by Smith et al. (1985) and Brown et al. (1989).

For calculations of N content in soluble proteins, we assumed that TSP averaged 6.25%

N by mass.

For 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 filtered in a microfuge

using low-binding regenerated cellulose Millipore ultra-free filters to exclude peptides

with molecular weights higher than 10,000 D. The FAA content was measured using

an amino acid autoanalyzer via ionic exchange chromatography, following the method

of Spackman et al. (1958). For simplicity, we considered here only the total FAA

concentration. Total N content (% DW) was determined using a Carlo-Erba CNH

elemental autoanalyzer. TNC was measured according to the method described in

Alcoverro et al. (1999), based on Yemm and Williams (1954). Sugars (sucrose and

hexoses) were solubilized from dry and ground tissues by four sequential extractions in

95% (v/v) ethanol at 80 jC for 15 min. The ethanol extracts were evaporated under a

stream of room temperature air and the residues dissolved in deionized water for

Fig. 2. Nitrate + nitrite (triangle symbols) and ammonium (round symbols) concentration (Amol g� 1 DW) over 1

year in several organs of P. oceanica subjected to nitrogen enrichment (F) and control (C) treatments: YL, young

leaves (0–50 days); IL, intermediate leaves (50–100 days); OL, old leaves (100–150 days); Ri, rhizome; R, root.

O. Invers et al. / J. Exp. Mar. Biol. Ecol. 303 (2004) 97–114 103

analysis. Starch was extracted from the ethanol-insoluble residue by keeping it for 24

h in 1 N NaOH. Sugar and starch contents of extracts were determined spectropho-

tometrically using a resorcinol and anthrone assay, respectively, with sucrose as

standard.

2.4. Statistics

The statistical significance of variability found between sampling events (factor ‘time’),

between plant parts (factor ‘tissue’), and between treated and untreated plots (factor

‘treatment’) of the distinct dependent variables (GS activity, inorganic N, FAA and TSP

concentration, total N and TNC content) was tested using three-way, repeated ANOVA,

with two within-subject factors (‘time’ and ‘tissue’) and one between-subject factor

(‘treatment’). Statistical significance of changes in growth was tested by two-way repeated

ANOVA, ‘time’ being the within-subject factor and ‘treatment’ the between-subject factor.

When necessary, significance of differences between experimental conditions was

assessed by the Tukey post-hoc test.

Fig. 3. Free amino acid (FAA) concentration (Amol g� 1 DW) over 1 year in several organs of P. oceanica

subjected to nitrogen enrichment (F) and control (C) treatments: YL, young leaves (0–50 days); IL, intermediate

leaves (50–100 days); OL, old leaves (100–150 days); Ri, rhizome; R, root.

O. Invers et al. / J. Exp. Mar. Biol. Ecol. 303 (2004) 97–114104

3. Results

3.1. Total N content

N addition increased total N content significantly in all the parts of P. oceanica

studied (Fig. 1; Table 1, p < 0.001). The precise effect depended on the tissue and

the time of the year (Table 1, significant interactions, p < 0.01 and p < 0.001,

respectively). In leaves, the increase (yearly averaged) ranged from ca. 13% to

26% (old leaves and young leaves, respectively) above the values of control

plants. Rhizomes showed the highest N content and also the greatest response to

N addition, 55% above control plants (annual average). Roots had the lowest N

content and only responded to treatment from November onwards (eight months

after start of fertilization), when a considerable increase in N content (ca. 43%)

was observed (significant time� treatment interaction, Table 1; p < 0.001). The N

content of leaves and rhizomes showed a similar annual trend under both control

Fig. 4. Total soluble protein (TSP) concentration (Amol g� 1 DW) over 1 year in several organs of P. oceanica

subjected to nitrogen enrichment (F) and control (C) treatments: YL, young leaves (0–50 days); IL, intermediate

leaves (50–100 days); OL, old leaves (100–150 days); Ri, rhizome; R, root.

O. Invers et al. / J. Exp. Mar. Biol. Ecol. 303 (2004) 97–114 105

and fertilized conditions, with a decrease from spring to summer and an increase

in winter.

3.2. Soluble N compounds

All inorganic N forms remained in low concentrations in both control and treated

plants, accounting for a small part of the soluble nitrogen measured, from less than

1% to 5% dw. Ammonium was the main form of inorganic nitrogen, accounting for

50–90% of this pool. It increased significantly in plants from fertilized plots,

throughout the year in roots and only in spring–summer in rhizomes and in young

and intermediate leaves (Fig. 2, Table 1). Nitrate and nitrite accounted for only a small

fraction of total inorganic N compounds, and their concentrations did not change due

to N addition.

FAA concentration in tissues of fertilized plants increased significantly (Fig.

3; Table 1, p < 0.001). Leaf response differed depending on the age class:

young leaves showed a moderate but constant increase (1.6-fold) throughout the

Table 2

Summary of ANOVA of total soluble protein concentration in all tissues of control and fertilized shoots of P.

oceanica from May 1996 to March 1997, and in vivo GS activity in leaves and roots (May 1996 to March 1997)

and of in vivo GS activity in all tissues (from July 1996 to March 1997)

Variable Factor df MS p

Total soluble protein

(mg g dw� 1)

Treatment 1 11407 < 0.01

Time 5 12525 < 0.001

Tissue 4 20751 < 0.001

Tr�T 5 4998 < 0.001

Tr�Ti 4 4012 < 0.001

T�Ti 20 1570 < 0.001

Tr�T�Ti 20 1007 < 0.01

Error 236 2415

In vivo GS activity:

leaves and roots (Amol N g� 1dw h� 1)

Treatment 1 2.0 0.863

Time 5 1027.5 < 0.001

Tissue 3 4946.26 < 0.001

Tr�T 5 218.68 < 0.05

Tr�Ti 3 16.46 0.249

T�Ti 15 130.80 < 0.001

Tr�T�Ti 15 83.19 < 0.01

Error 188 238.7

In vivo GS activity:

all tissues (Amol N g� 1dw h� 1)

Treatment 1 0.7 0.898

Time 4 290.6 < 0.01

Tissue 4 5437.50 < 0.001

Tr�T 4 214.66 < 0.05

Tr�Ti 4 7.27 0.766

T�Ti 16 73.30 < 0.01

Tr�T�Ti 16 83.92 < 0.001

Error 196 244.3

O. Invers et al. / J. Exp. Mar. Biol. Ecol. 303 (2004) 97–114106

year, intermediate leaves only showed this increase in late summer and autumn

(4.7- and 3.2-fold, respectively), while old leaves were only moderately enriched

in winter (2.2-fold in January). FAA concentration in rhizomes was also

moderately higher in fertilized than in control plants (1.6-fold, annual average),

whereas the response in roots was the sharpest and strongly seasonal (ca. 11-fold in

winter).

Fertilization also raised significantly the concentration of TSP in leaves of P. oceanica

from the fertilized plots (Fig. 4; Table 2, p < 0.01), mainly in winter and spring. TSP in

rhizomes and roots did not show any response to treatment.

3.3. In vivo GS activity

N addition, although not having an overall effect on in vivo GS activity (Table 2; Fig.

5), did cause some significant differences between control and fertilized plants. In leaves,

which were the plant parts with the highest GS activity, fertilization seemed to smoothe out

the time course of GS activity (Table 2, significant interaction time� treatment, p < 0.001

in ANOVA restricted to leaf data): the gap between minimum and maximum seasonal

Fig. 5. In vivo GS activity (Amol N g dw� 1 h� 1) over 1 year in several organs of P. oceanica subjected to

nitrogen enrichment (F) and control (C) treatments: YL, young leaves (0–50 days); IL, intermediate leaves (50–

100 days); OL, old leaves (100–150 days); Ri, rhizome; R, root.

O. Invers et al. / J. Exp. Mar. Biol. Ecol. 303 (2004) 97–114 107

values was lower in fertilized than in control plants. N addition also modified the seasonal

pattern of GS activity in roots, inducing maximum values in May, while maximum values

in control plants were found in September. There was also a seasonal increase in this

activity in fertilized plants in spring (in March for rhizomes and in May for young leaves

and roots).

3.4. TNC

N addition significantly reduced the TNC content of P. oceanica tissues (Table 3,

p < 0.01), which was especially sharp in rhizomes, the organs with the highest TNC

content in unmanipulated conditions (Fig. 6). This decrease occurred throughout the

year, though not uniformly (significant interaction treatment� time of two-way

ANOVA restricted to rhizome data, p < 0.005). In spring–summer (from March to

July) the TNC content of rhizomes of fertilized plants was 22% lower than in

controls, while from autumn to winter this decrease was almost 50%. The time

course of TNC content of leaves and roots (of control and fertilized plants) showed

maximum values in summer and low values in autumn–winter, as did the rhizomes

of fertilized plants. However, the rhizomes of controls showed a distinct seasonal

trend, with high values in summer that were maintained up to the end of the annual

cycle.

3.5. Shoot growth

Leaf growth rates were lower in fertilized than in control plants ( p < 0.01, Table 3)

during most of the study period (Fig. 7). However, when these rates were expressed

as specific growth rates (day� 1), differences disappeared because of the smaller size

of fertilized shoots from the very beginning of the experiment (two-way ANOVA,

p < 0.01). Therefore, the differences in growth rates cannot be attributed to fertiliza-

Table 3

Summary of ANOVA of total non-soluble carbohydrates (TNC) content measured in all tissues and leaf growth

measured in control and fertilized shoots of P. oceanica

Variable Factor df MS p

TNC (% dw) Treatment 1 138.5 < 0.01

Time 5 58.6 < 0.001

Tissue 4 619.9 < 0.001

Tr�T 5 6.0 0.075

Tr�Ti 4 68.5 < 0.001

T�Ti 20 20.3 < 0.001

Tr�T�Ti 20 8.0 < 0.001

Error 236 25.94

Growth (cm2 day� 1) Treatment 1 0.902 < 0.01

Time 5 3.026 < 0.001

Tr�T 5 0.016 0.930

Error 495 0.203

Fig. 6. Total non-structural carbohydrate (sucrose + starch) concentration (% DW) over 1 year in several organs of

P. oceanica subjected to nitrogen enrichment (F) and control (C) treatments: YL, young leaves (0–50 days); IL,

intermediate leaves (50–100 days); OL, old leaves (100–150 days); Ri, rhizome; R, root.

O. Invers et al. / J. Exp. Mar. Biol. Ecol. 303 (2004) 97–114108

tion. Treated and untreated plants showed the same clear seasonal pattern of shoot

growth, with maximum values in July and minimum in autumn (from September to

January).

Fig. 7. Leaf growth rates (cm2 day� 1 shoot� 1) from May 1996 to March 1997 in P. oceanica subjected to

nitrogen enrichment (F) and control (C) treatments.

O. Invers et al. / J. Exp. Mar. Biol. Ecol. 303 (2004) 97–114 109

4. Discussion

The increase observed here in total N content has been repeatedly reported in this and

another species as the main response of seagrass to nitrogen additions, whether experi-

mental or not (Duarte, 1990; Perez et al., 1991; Erftemeijer et al., 1994; Udy and

Dennison, 1997; Udy et al., 1999). However, this increase differentially altered the distinct

N pools: TSP and inorganic N compounds were little affected, while the excess of N

incorporated by fertilized plants appeared mostly as FAA. In addition, there was a fraction

of the total N increase in rhizomes unexplained by soluble N compounds. This should

correspond to non-soluble compounds, similar to what occurs in some terrestrial plants

(Nordin and Nasholm, 1997). The response in FAA and non-soluble N pools was more

evident at the period of maximum N content (January, for control plants). At this time of

the year, both categories of N compounds showed their maximum increase with

fertilization (Table 4): FAA increased by from 1.4 to 10-fold (depending on tissue) and

non-soluble N by 2.5-fold (only in rhizomes), suggesting a possible storage role for these

compounds.

In contrast, it seems clear that P. oceanica does not store N in inorganic form. Nitrate

concentration in tissues is very low throughout the year and was not affected by

fertilization, which is consistent with the generally limited capacity of seagrasses to store

nitrate, even under high availability (Touchette and Burkholder, 2000). This limited

capacity contrasts with that of many terrestrial plants and macroalgae to store N as

nitrate (Bidwell, 1974; Vergara, 1993; Lapointe and Duke, 1984). Ammonium concen-

tration was higher and did react to fertilization, although its increase was only a minor

part of total N increase (see Table 4). Ammonium is toxic to seagrasses (van Katwijk et

al., 1997; Touchette and Burkholder, 2000). Given the lack of inhibitory feedback on its

uptake, it is reasonable to assume that the increase of intracellular ammonium caused by

fertilization will be closely followed by an increase in its assimilation into organic

forms. This explains the very small response of ammonium pools to fertilization, which

was effectively reflected in higher FAA concentrations. The first step in assimilation is

the combination of ammonium with glutamate to produce glutamine, which was the

most abundant FAA in roots of fertilized plants. The assimilation process, which is

Table 4

Increase in soluble and non-soluble N (% dw) caused by N addition in young leaves, rhizomes and roots of P.

oceanica. Soluble N was calculated as the sum of N in the form of Free amino acids (FAA), Total soluble protein

(TSP) and inorganic N. Non-soluble N was calculated as the difference between the total N content measured and

the soluble N. The % increase in soluble and non-soluble fractions was calculated as the difference between the

values of control and N-treated plants. The percentage contribution of FAA, TSP and inorganic N to the increase

in soluble N is also shown

Tissue Increase in soluble Contribution to soluble N increase (%) Increase in non-soluble

N (% dw)FAA TSP Inorganic N

N (% dw)

Young leaf 0.98 51.8 47.8 0.3 –

Rhizome 0.41 99.2 – 0.8 1.6

Root 1.3 95.0 4.2 0.7 –

O. Invers et al. / J. Exp. Mar. Biol. Ecol. 303 (2004) 97–114110

energy-dependent, is catalyzed by GS and uses ATP derived from photosynthesis

(Kraemer et al., 1997). Some studies showed that GS activity correlates positively with

N availability (Pregnall et al., 1987; Kraemer et al., 1997), but others reported that GS

enzyme activity does not follow external N availability conditions, and is mostly

controlled by internal requirements (Finneman and Schjoerring, 1998; Thompson and

Valiela, 1999). In our experiment, a significant overall increase in the GS activity of

fertilized plants was not detected, although we found some increase in spring, the period

of maximum leaf elongation, and, consequently, the period of maximum N demand. This

weak relationship between GS activity and N conditions suggests a similar potential N

assimilation in plants, independently of their N environment, as indicated by our

determinations performed with saturating concentrations of substrates. Under natural

conditions, however, GS activities should show differences responding to different

substrate concentrations, given the differences in N content between control and

fertilized plants.

N does not seem to be the growth-limiting factor in the meadow studied, as indicated by

the absence of response of leaf elongation to fertilization. This lack of N limitation may be

due to efficient seasonal nutrient use by the plant, which has an annual cycle characterized

by one period (autumn and winter) in which N supply exceeds the demand for growth,

with the surplus being stored in plant tissues, and one period (spring–summer) in which

the demand for growth exceeds the external supply and the reserves are mobilized

(Alcoverro et al., 2000; Invers et al., 2002). Under conditions of high nitrogen availability,

such as those in the treated plots, internal nutrient imbalances can be induced (or

aggravated), leading to, for instance, C or P limitation (Burkholder et al., 1992; Udy

and Dennison, 1997). In fact, the main effect of fertilization was to enhance the build-up of

N reserves, as shown by the maximum differences in total N content between treated and

untreated plants in autumn and winter. These differences were especially high in rhizomes,

confirming their role as main sites for N storage. The maximum N content found in our

study was 5% DW (rhizomes of fertilized plants in winter), the maximum ever reported for

this species as far as we are aware (see Delgado et al., 1997; Ruiz et al., 2001). It is

probably the upper limit for N storage.

The increase in N availability interfered with carbon metabolism, with a decrease in

TNC content in rhizomes. This decrease in carbon reserves was greatest from autumn to

early spring, coinciding with the maximum N increase caused by fertilization. N

assimilation requires energy and C skeletons; these resources are diverted from carbohy-

drate metabolism, thus explaining the decrease in carbohydrate content when N assim-

ilation is stimulated (see, for Zostera marina, Burkholder et al., 1992; van Katwijk et al.,

1997; and for terrestrial plants, Marschner, 1995). In our study, the diversion of C to

support increased synthesis of FAA as inorganic N is taken up (see above) may largely

explain the decrease observed in TNC in shoots. In fact, there was significant correlation

between the increase in C bound to FAA and the decrease in TNC, indicating the

reduction of rhizome C reserves to support the formation of N stores in all plant tissues

(Fig. 8).

Carbohydrate reserves in temperate seagrasses are crucial for survival during episodes

of low light availability, especially during winter, in which the C balance can be negative

(Pirc, 1985; Burke et al., 1996; Alcoverro et al., 2001). Specifically, the use of internal C

Fig. 8. Linear regression of the decrease in C as TNC (whole shoot) on the increase in C as FAA (rhizomes)

during fertilization (r2 = 0.82; p< 0.05).

O. Invers et al. / J. Exp. Mar. Biol. Ecol. 303 (2004) 97–114 111

reserves allows P. oceanica to maintain a high leaf growth rate while nutrients are

abundant, and develop a dense foliar canopy well before maximum summer irradiance

(Alcoverro et al., 1995). A reduction in these reserves leads to a decrease in the winter

production of leaf tissue required for the acquisition of C and N, probably implying a

deterioration of the plant status and decreased survival (see an example in Alcoverro et al.,

2001). We found no mortality in our fertilized plots, but this was probably due to both a

very healthy plant status and to the relatively short duration and timing of our experiment.

The decrease in C reserves caused by high N availability reported here can interact with

other processes having the same effect. Leaf overgrowth by epiphytes implying shading

(Silverstein et al., 1986; Short et al., 1995; Hauxwell et al., 2001), and photosynthetic

biomass reduction due to herbivory (McGlathery, 1995; Ruiz et al., 2001) are both

reported consequences of nutrient excess. Moreover, both result in a significant reduction

in photosynthetic yield, especially in summer, with subsequent decrease in carbohydrate

storage. The resultant interaction among these processes can compromise seagrass health,

especially of those species such as P. oceanica for which C storage is crucial for winter

survival.

Acknowledgements

We thank Marta Manzanera, Pere Renom, Miquel Angel Rodriguez, Xavier de Pedro

and Fiona Tomas for their help in field and laboratory work. We also thank Dr. Miguel

Angel Mateo for his suggestions. FAA analyses were performed with the help of Pilar

Fernandez and Isidre Casals of the ‘Serveis Cientıfico-Tecnics de la Universitat de

Barcelona’. This research was supported by a grant from the ‘Ministerio de Educacion y

Ciencia’ (PN92) of the Spanish Government, a grant from CICYT (no. MAR98-0356),

and a grant from the ‘Universitat de Barcelona’. AMOSA kindly manufactured on our

request the slow-release fertilizers used. [SS]

O. Invers et al. / J. Exp. Mar. Biol. Ecol. 303 (2004) 97–114112

References

Alcoverro, T., Duarte, C.M., Romero, J., 1995. Annual growth dynamics of Posidonia oceanica: contribution of

large-scale versus local factors to seasonality. Mar. Ecol. Prog. Ser. 120, 203–210.

Alcoverro, T., Romero, J., Duarte, C., Lopez, N.I., 1997. Spatial and temporal variations in nutrient limitation of

seagrass Posidonia oceanica growth in the NW Mediterranean. Mar. Ecol. Prog. Ser. 146, 155–161.

Alcoverro, T., Zimmerman, R.C., Kohrs, D.G., Alberte, R.S., 1999. Resource allocation and sucrose mobilization

in light-limited eelgrass Zostera marina. Mar. Ecol. Prog. Ser. 187, 121–131.

Alcoverro, T., Manzanera, M., Romero, J., 2000. Nutrient mass balance of the seagrass Posidonia oceanica (L.)

Delile: the importance of nutrient retranslocation. Mar. Ecol. Prog. Ser. 194, 13–21.

Alcoverro, T., Manzanera, M., Romero, J., 2001. Annual metabolic carbon balance of the seagrass Posidonia

oceanica (L.) Delile: the importance of carbohydrate reserves. Mar. Ecol. Prog. Ser. 211, 105–116.

Bidwell, R.G.S., 1974. Nitrogen metabolism. In: Giles, N.H., Torrey, J.G. (Eds.), Plant Physiology. Macmillan,

New York, pp. 173–200.

Brown, R.E., Jarvis, K.L., Hyland, K.J., 1989. Protein measurement using Bicinchoninic acid: elimination of

interfering substances. Anal. Biochem. 180, 136–139.

Burke, M.K., Dennison, W.C., Moore, K.A., 1996. Non-structural carbohydrate reserves of eelgrass Zostera

marina. Mar. Ecol. Prog. Ser. 137, 195–201.

Burkholder, J.M., Mason, K.M., Glasgow Jr., H.B. 1992. Water-column nitrate enrichment promotes decline

of eelgrass Zostera marina: evidence from seasonal mesocosm experiments. Mar. Ecol. Prog. Ser. 81,

163–178.

Delgado, O., Grau, A., Pou, S., Riera, F., Massuti, C., Zabala, M., Ballesteros, E., 1997. Seagrass

regression caused by fish cultures in Fornells Bay (Menorca, Western Mediterranean). Oceanol. Acta

20, 557–563.

Delgado, O., Ruiz, J.M., Perez, M., Romero, J., Ballesteros, E., 1999. Effects of fish farming on seagrass

(Posidonia oceanica) in a Mediterranean bay: seagrass decline after organic loading cessation. Oceanol. Acta

22, 109–117.

Duarte, C.M., 1990. Seagrass nutrient content. Mar. Ecol. Prog. Ser. 67, 201–207.

Duarte, C.M., 1995. Submerged aquatic vegetation in relation to different nutrient regimes. Ophelia 41,

87–112.

Erftemeijer, P.L.A., Stapel, J., Smekens, M.J.E., Drossaert, W.M.E., 1994. The limited effect of in situ phospho-

rus and nitrogen addition to seagrass beds on carbonate and terrigenous sediments in South Sulawesi,

Indonesia. J. Exp. Mar. Biol. Ecol. 182, 123–140.

Finneman, J., Schjoerring, J.K., 1998. Ammonium and soluble amide-bound nitrogen in leaves of Brassica

napus as related to glutamine synthetase activity and external N supply. Plant Physiol. Biochem. 36,

339–346.

Gao, Y., Smith, G.J., Alberte, R.S., 1992. Light regulation of nitrate reductase in Ulva fenestrata (Chlorophy-

ceae): I. Influence of light regimes on nitrate reductase activity. Mar. Biol. 112, 691–696.

Grasshoff, K., Ehrhardt, M., Kremling, K., 1983. Methods of Seawater Analysis Verlag Chemie, Weinheim.

Hauxwell, J., Cebrian, J., Furlong, C., Valiela, I., 2001. Macroalgal canopies contribute to eelgrass (Zostera

marina) decline in temperate estuarine ecosystems. Ecol. Washington, D.C. 82 (4), 1007–1022.

Invers, O., Perez, M., Romero, J., 2002. Seasonal nitrogen speciation in temperate seagrass Posidonia oceanica

(L.) Delile. J. Exp. Mar. Biol. Ecol. 273, 219–240.

Kenworthy, W.J., Fonseca, M.S., 1992. The use of fertilizer to enhance growth of trasplanted seagrasses Zostera

marina L. and Halodule wrightii Aschers. J. Exp. Mar. Biol. Ecol. 163, 141–161.

Kraemer, G.P., Mazzella, L., 1996. Nitrogen assimilation and growth dynamics of the mediterranean seagrasses

Posidonia oceanica, Cymodocea nodosa, and Zostera noltii. In: Kuo, J., Phillips, R.C., Walker, D.I., Kirk-

man, H. (Eds.), Seagrass Biology: Proceedings of an International Workshop. Faculty of Science, UWA,

Rottnest Island, Western Australia, pp. 181–190.

Kraemer, G.P., Mazzella, L., Alberte, R.S., 1997. Nitrogen assimilation and partitioning in the Mediterranean

seagrass Posidonia oceanica. P.S.Z.N.I. Mar. Ecol. 18, 175–788.

Lapointe, B.E., Duke, C.S., 1984. Biochemical strategies for growth of Gracilaria tikvahiae (Rhodophyta) in

relation to light intensity and nitrogen availability. J. Phycol. 20, 488–495.

O. Invers et al. / J. Exp. Mar. Biol. Ecol. 303 (2004) 97–114 113

Marba, N., Duarte, C.M., Cebrian, J., Gallegos, M.E., Olsen, B., Sand-Jensen, K., 1996. Growth and population

dynamics of Posidonia oceanica on the Spanish Mediterranean coast: elucidating seagrass decline. Mar. Ecol.

Prog. Ser. 137, 203–213.

Marschner, H., 1995. Mineral Nutrition of Higher Plants Academic Press, London.

McGlathery, K.J., 1995. Nutrient and grazing influences on a tropical seagrass community. Mar. Ecol. Prog. Ser.

122, 239–252.

Nixon, S.W., 1995. Coastal marine eutrophication: a definition, social causes, and future concerns. Ophelia 41,

199–219.

Nordin, A., Nasholm, T., 1997. Nitrogen storage forms in nine boreal understorey plant species. Oecologia 110,

487–492.

Perez, M., Romero, J., Duarte, C., Sand-Jensen, K., 1991. Phosphorus limitation of Cymodocea nodosa growth.

Mar. Biol. 109, 129–133.

Pirc, H., 1985. Growth dynamics in Posidonia oceanica (L.) Delile. I. Seasonal changes of soluble carbohydrates,

starch, free amino acids, nitrogen and organic anions in different parts of the plant. P.S.Z.N.I. Mar. Ecol. 6,

141–165.

Pregnall, A.M., Smith, R.D., Alberte, R.S., 1987. Glutamine synthetase activity and free amino acid pools of

eelgrass (Zostera marina L.) roots anoxia. J. Exp. Mar. Biol. Ecol. 106, 211–228.

Rabe, E., 1990. Stress physiology: the functional significance of the accumulation of N-containing compounds. J.

Hortic. Sci. 65, 231–243.

Romero, J., 1989. Primary production of Posidonia oceanica beds in the Medes Islands (Girona, NE Spain). In:

Boudouresque, C.F., Meinesz, A., Fresi, E., Gravez, V. (Eds.), International Workshop on Posidonia Beds,

vol. 2. GIS Posidonie, Marseille, pp. 85–91.

Ruiz, J.M., Perez, M., Romero, J., 2001. Effects of fish farm loadings on seagrass (Posidonia oceanica) dis-

tribution, growth and photosynthesis. Mar. Pollut. Bull. 42, 749–760.

Sand-Jensen, K., Borum, J., 1991. Interactions among phytoplankton, periphyton, and macrophytes in temperate

freshwaters and estuaries. Aquat. Bot. 41, 137–175.

Schramm, W., Nienhuis, P.H., 1996. Marine Benthic Vegetation. Recent Changes and the Effects of Eutrophi-

cation. Springer, Berlin.

Short, F.T., 1987. Effects of sediment nutrients on seagrasses: literature review and mesocosm experiment. Aquat.

Bot. 27, 41–57.

Short, F.T., Wyllie-Echevarria, S., 1996. Natural and human-induced disturbance of seagrasses. Environ. Con-

serv. 23, 17–27.

Short, F.T., Dennison, W.C., Capone, D.G., 1990. Phosphorus-limited growth of the tropical seagrass Syringo-

dium filiforme in carbonate sediments. Mar. Ecol. Prog. Ser. 62, 169–174.

Short, F.T., Burdick, D.M., Kaldy III, J.E., 1995. Mesocosm experiments quantify the effects of eutrophication on

eelgrass, Zostera marina. Limnol. Oceanogr. 40, 740–749.

Silverstein, K., Chiffings, A.W., McComb, A.J., 1986. The loss of seagrass in Cockburn Sound, Western Australia.

III. The effect of epiphytes on productivity of Posidonia australis Hook F. Aquat. Bot. 24, 355–371.

Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano, M.D., Fujimoto, E.K.,

Goeke, N.M., Olson, B.J., Klenk, D.C., 1985. Measurement of protein using bicinchoninic acid. Anal.

Biochem. 150, 76–85.

Spackman, D.H., Stein, W.H., Moore, S., 1958. Automatic recording apparatus for use in the chromatography of

amino acids. Anal. Chem. 30, 1190–1206.

Thompson, S.M., Valiela, I., 1999. Effect of nitrogen loading on enzyme activity of macroalgae in estuaries in

Waquoit Bay. Bot. Mar. 42, 519–529.

Touchette, B.W., Burkholder, J.M., 2000. Review of nitrogen and phosphorus metabolism in seagrasses. J. Exp.

Mar. Biol. Ecol. 250, 133–167.

Udy, J.M., Dennison, W.C., 1997. Growth and physiological responses of three seagrass species to elevated

sediment nutrients in Moreton Bay, Australia. J. Exp. Mar. Biol. Ecol. 217, 253–277.

Udy, J.W., Dennison, W.C., Lee Long, W.J., McKenzie, L.J., 1999. Responses of seagrass to nutrients in the

Great Barrier Reef, Australia. Mar. Ecol. Prog. Ser. 185, 257–271.

van Katwijk, M.M., Vergeer, L.H.T., Schmitz, G.H.W., Roelofs, J.G.M., 1997. Ammonium toxicity in eelgrass

Zostera marina. Mar. Ecol. Prog. Ser. 157, 159–173.

O. Invers et al. / J. Exp. Mar. Biol. Ecol. 303 (2004) 97–114114

Vergara, J.J., 1993. Modelado del metabolismo de carbono y nitrogeno en rodofıceas. PhD, Malaga.

Yemm, E.W., Williams, A.J., 1954. The estimation of carbohydrates in plant extracts by anthrone. Biochem. J.

57, 508–514.

Zieman, J.C., 1974. Methods for the study of the growth and production of turtle grass, Thalassia testudinum

Konig. Aquaculture 4, 139–143.