Seasonal variations in nitrate reductase activity andinternal N pools in intertidal brown algae are correlatedwith ambient nitrate concentrations

ERICA B. YOUNG1,2, MATTHEW J. DRING2, GRAHAM SAVIDGE2, DARYL A. BIRKETT2 & JOHN A. BERGES1,2

1School of Biological Sciences, Queens University of Belfast, Lisburn Road, Belfast, Northern Ireland BT9 7BL, UK and2Department of Biological Sciences, University of Wisconsin–Milwaukee, 3209 Maryland Avenue, Milwaukee, WI 53211, USA

ABSTRACT

Nitrogen metabolism was examined in the intertidal sea-weeds Fucus vesiculosus, Fucus serratus, Fucus spiralisand Laminaria digitata in a temperate Irish sea lough.Internal NO3

- storage, total N content and nitrate reductaseactivity (NRA) were most affected by ambient NO3

-, withhighest values in winter, when ambient NO3

- was maximum,and declined with NO3

- during summer. In all species, NRAwas six times higher in winter than in summer, and wasmarkedly higher in Fucus species (e.g. 256 � 33 nmolNO3

- min-1 g-1 in F. vesiculosus versus 55 � 17 nmol NO3-

min-1 g-1 in L. digitata). Temperature and light were lessimportant factors for N metabolism, but influenced in situphotosynthesis and respiration rates. NO3

- assimilatingcapacity (calculated from NRA) exceeded N demand (cal-culated from net photosynthesis rates and C : N ratios) by afactor of 0.7–50.0, yet seaweeds stored significant NO3

- (upto 40–86 mmol g-1). C : N ratio also increased with height inthe intertidal zone (lowest in L. digitata and highest in F.spiralis), indicating that tidal emersion also significantlyconstrained N metabolism. These results suggest that, incontrast to the tight relationship between N and C metabo-lism in many microalgae, N and C metabolism could beuncoupled in marine macroalgae, which might be an impor-tant adaptation to the intertidal environment.

Key-words: C : N ratio; macroalgae; nitrate assimilation;photosynthesis; seasonality.

INTRODUCTION

Temperate brown macroalgae form highly productive com-munities, accounting for the majority of primary productionin many coastal regions and dominating near-shore nutrientcycling (Duggins, Simenstad & Estes 1989). For example, inStrangford Lough, Northern Ireland, macroalgae accountfor 98% of algal biomass and 95% of productivity (Birkett,Dring & Savidge, unpublished results). The vast majority ofthe macroalgal biomass in the Lough is fucoid algae (Fucusand Ascophyllum species) and kelps (Laminaria species).

Seaweeds in temperate habitats such as StrangfordLough experience large seasonal changes in temperature,irradiance and nutrient concentration that impose con-straints on their physiology. Furthermore, variations in tidalemersions also affect nutrient availability and irradiance ona scale of hours to weeks and often result in a disjunctionbetween the optimal light, nutrient availability and tem-perature for growth. Brown algal species growing at dif-ferent heights in the intertidal zone experience distinctirradiance and emersion regimes that may influence theregulation of nutrient acquisition and assimilation (e.g.Thomas, Turpin & Harrison 1987; Phillips & Hurd 2004).Identifying responses of macroalgae to daily and seasonalfluctuations in irradiance, temperature and nitrogen avail-ability is thus critical in understanding the regulation ofnitrogen metabolism and the role of these productivemacroalgae in near-shore nutrient cycling.

Simple measurements of nutrient uptake are difficult tomake in intertidal species, and can be biased by manyfactors. Alternatively, enzyme activities offer more integra-tive measures, less biased by instantaneous conditions.Nitrate reductase [NR, enzyme class (EC) 1.6.6.1] is oftenconsidered the rate-limiting enzyme in inorganic N assimi-lation by algae and is thus a key enzyme in N metabolism.Nitrate reductase activity (NRA) is strongly correlated withN incorporation rates in macroalgae (Davison, Andrews &Stewart 1984). NRA in cultured algae is known to be stimu-lated by NO3

- (Gao, Smith & Alberte 1995; Lartigue &Sherman 2005), and is regulated by light with rapid suppres-sion of NRA in darkness in most algae studied (e.g. Davison& Stewart 1984; Gao et al. 1995; Lopes, Oliveira & Colepi-colo 1997; Vergara, Berges & Falkowski 1998; Lartigue &Sherman 2002). NRA is also known to be responsive tochanges in temperature with narrow temperature range foroptimum NRA (Gao, Smith & Alberte 2000; Berges, Varela& Harrison 2002). Therefore, in this study, we monitoredNRA as an indicator of changes in nitrate metabolism inresponse to these key environmental variables.

Changes in NRA over a seasonal cycle have been exam-ined in very few macroalgae. To evaluate the factors influ-encing seasonal changes in N assimilation by macroalgae, itis important to also examine external nutrient availability,irradiance and temperature, as well as internal N storage, all

Correspondence: Erica B. Young. Fax: 1 414 229 3926; e-mail:ebyoung@uwm.edu

Plant, Cell and Environment (2007) 30, 764–774 doi: 10.1111/j.1365-3040.2007.01666.x

© 2007 The AuthorsJournal compilation © 2007 Blackwell Publishing Ltd764

factors that may play a role in the regulation of NRA. Ifexternal availability of nitrate is the most important factorregulating uptake and assimilation, then NRA and internalnitrate storage will be closely related to seasonal changesin nitrate concentration. N metabolism in algae is closelylinked to photosynthetic C metabolism (e.g. Vergara et al.1998), and if irradiance is the most important factor influ-encing N metabolism, then NR and internal N storage willbe highest in summer when photosynthesis is not con-strained by irradiance. Temperature is also an importantseasonal environmental variable influencing metabolismand may influence nitrate uptake, storage and NRA.

The aims of the present study were to evaluate the impor-tance of environmental variables influencing N metabolismin intertidal brown algae by making concurrent measure-ments of NRA, total thallus N content, inorganic N storageand photosynthesis, and by comparing these with seasonalnitrate and light availability and temperature in a stronglyseasonal intertidal habitat. Effects of position in the inter-tidal zone on N metabolism were examined by comparingLaminaria and Fucus species. Laminaria digitata grows inthe lower intertidal–subtidal zone, and hence is immersedlonger and experiences greater light attenuation with waterdepth, compared with the intertidal Fucus species, Fucusvesiculosus and Fucus serratus, which grow in the mid-intertidal zone, and Fucus spiralis, which is found evenhigher in the intertidal zone and is emersed for long periodsduring each tidal cycle. In these four brown algal species, weobserved strong seasonal patterns in NRA and internal Nstorage that closely correlate with seasonal changes innitrate availability. In addition, the relationship between Cfixation and N assimilation capacity changes several-foldbetween summer and winter.

MATERIALS AND METHODS

Sampling

Whole thalli of Fucus serratus L., Fucus vesiculosus (L.)Lamour, Fucus spiralis L. and Laminaria digitata (Huds.)Lamour were collected from the intertidal region of‘The Narrows’, Strangford Lough at Portaferry (54°23’N,5°34’W), over the period November 2000–February 2002.Fucus serratus and F. vesiculosus were collected from theshore at low tide, and L. digitata was sampled near themiddle of the day at low tide from the shore or from a boat,as maximum activity was shown to occur during the middleof the day in L. digitata (Davison et al. 1984). Laminariadigitata were sampled by cutting ~30 mm diameter discs outof the thalli, avoiding the meristematic region and theoldest tissue (see Davison & Stewart 1984). Thallus tipswere cut from Fucus thalli by removing 30–40-mm-longterminal tips (trials showed activity was highest in tips).Within a few minutes of all sampling times, tissue sampleswere thoroughly blotted dry, frozen and stored in liquid N2

for later analysis of NRA.Within a few minutes of samplingon the shore, the tissue was thoroughly blotted dry andfrozen and stored in liquid N2 for later analysis.

Light, temperature and nitrate data

Surface-incident photosynthetically active radiation (PAR)was measured using a 2p light sensor (Li-Cor, Lincoln, NE,USA) during 2001 and 2002 (Marine Laboratory Database,Queen’s University of Belfast 2002).Temperature was mea-sured in surface (~1 m depth) seawater at The Narrows site[Agri-Food and Biosciences Institute (AFBI) Database2006]. Nitrate concentration in the Lough was measured insurface samples collected from The Narrows site and wasanalysed using standard methods (Parsons, Maita & Lalli1984). Samples for long-term nutrient concentration datawere collected during years 1974–1976, 1986–1987 and1990–1991. Samples were also collected during the studyperiod (2001–2002) to verify the earlier published seasonalNO3

- concentration data. Triplicate samples from TheNarrows were collected and analysed from single samplingperiods over the years 1994–1995 (Service et al. 1996) and2004–2005 (AFBI Database 2006).

NRA

NRA was estimated using an in vitro assay methoddescribed by Young et al. (2005). Frozen thallus sampleswere ground to a powder in liquid nitrogen and extractedin 200 mmol L-1 potassium phosphate buffer pH 7.9 con-taining 5 mmol L-1 Na2 ethylenediaminetetraacetic acid(EDTA), 0.3% (w/v) insoluble polyvinyl pyrollidone,2 mmol L-1 dl-dithiothreitol, 3% (w/v) bovine serumalbumin (Fraction V) and 1% (v/v) Triton X-100 (all Sigma,St Louis, MO, USA). The assay mixture contained200 mmol L-1 sodium phosphate buffer pH 7.9 with200 mmol L-1 NADH (b form, Sigma), 20 mmol L-1 flavinadenine dinucleotide (Sigma), 20% volume as algal extractand 10 mmol L-1 KNO3. The assay was incubated at 12 °Cand the reaction terminated by the addition of 1 M zincacetate. NO2

- concentration was measured spectrophoto-metrically in centrifuged supernatants (Parsons et al. 1984),and activity estimated by linear regression of increasingNO2

- concentration over time.

Internal nitrogen pools and tissueN and C content

Several methods used to extract internal inorganic nutrientpools from algae were tested: boiling thallus discs in waterfor 20 min (Hurd, Harrison & Druel 1996), boiling wateradded to ground algal tissue, vortexed and incubated for10 min (after Thoresen, Dortch & Ahmed 1982), boilingthallus pieces in water for 10 min followed by overnightextraction at 4 °C (Naldi & Wheeler 1999) and room tem-perature ethanol extraction of ground tissue overnight(McGlathery, Pedersen & Borum 1996). The highestinternal inorganic N concentrations were obtained byadding 20 mL room temperature Milli-Q water (Millipore,Watford, UK) to samples of ~50 mg frozen ground thallustissue in boiling tubes that were vortexed and then placedin a boiling water bath for 45 min, cooled to room

Seasonal N metabolism in brown algae 765

© 2007 The AuthorsJournal compilation © 2007 Blackwell Publishing Ltd, Plant, Cell and Environment, 30, 764–774

temperature, filtered through Whatman GF/A filters(Brent, Middlesex, UK) and stored on ice.Thus, this methodwas used for all subsequent analyses. The concentrations ofNO2

-, NO3- and NH4

+ were measured in the filtrate within2 h, without freezing the samples. NO3

- was analysed byCd-column reduction followed by spectrophotometric mea-surement of NO2

-, and NH4+ was estimated by the phenol-

hypochlorite method, both according to Parsons et al.(1984). To determine tissue C and N content, frozen thallussamples were ground in liquid N2 and dried in a 60 °C ovenwith desiccant and analysed in a Carlo Erba 1500 NCelemental analyser (CE Elantech, Lakewood, NJ, USA)using acetanilide as a standard and expressing contents as aproportion of dry mass.

Photosynthesis, respiration and N and Cassimilation capacity

Photosynthesis and dark respiration rates were also mea-sured during winter (November–March) and summer(June–September) months in F. vesiculosus, F. serratus andL. digitata using large (30 cm diameter ¥ 1 m long) clearplastic chambers suspended 2 m below the Lough surface,containing the whole thalli of a single species. The water inthe chambers was continuously circulated by a water pump.A YSI 6000 probe (YSI Inc., Yellow Springs, OH, USA)measuring dissolved oxygen, salinity and temperature wasinserted into the chambers and recorded values for thesevariables at 15 s intervals over 2–3 days. The values weresubsequently downloaded, and net oxygen exchange ratesover successive 15 min periods were calculated from theoxygen concentrations. Mass-specific net photosyntheticrates for F. vesiculosus, F. serratus and L. digitata were cal-culated for complete 24 h periods, and the O2 evolutionrates were converted to C fixation rates using a photosyn-thetic quotient of 1.2. C fixation rates were compared to theN assimilation capacity estimated from NRA for eachspecies. For comparison, the values were averaged overgroups of monthly values relating to the summer (June–September) and winter (November–March) periods.

Data analysis

Relationships between parameters were examined usingPearson product–moment correlation and analysis of vari-ance (ANOVA) (SigmaPlot version 9.04 and SigmaStatversion 3.1; Systat Software Inc., Chicago, IL, USA). Corre-lation coefficients were compared using Fisher transforma-tions (Zar 1999).

RESULTS

In the three species of Fucus examined and in L. digitata,there were marked differences in NRA at different times inthe year (one-way ANOVA, P < 0.001 for each species;Fig. 1a). NRA was highest during the winter and earlyspring months, peaking in March for F. serratus and F.

vesiculosus and in April for L. digitata. Fucus spiralis wasnot sampled every month, but showed a similar trend to theother species with highest NRAs in January and April. Thelowest NRAs were observed from late summer into autumnwith the minimum values in August for all species. Thehighest NRA observed in Fucus species (256 � 33 nmolNO3

- min-1 g-1 frozen mass) was five times higher than in L.digitata (55 � 17 nmol NO3

- min-1 g-1) (Fig. 1a; note differ-ent axes scales). The frozen and fresh mass differed lessthan 1% for all thalli (data not shown) so scaling to fresh orfrozen mass will be very similar. The lowest activities in allspecies were observed in August. In L. digitata, there was noevidence of an autumn increase in NRA as there was in allthree species of Fucus, and the NRA of L. digitata did notrise significantly until February.

The seasonal variation in NRAs was negatively corre-lated with seawater temperature, which was lowest inFebruary–March (Fig. 1b). Mean water temperature inStrangford Lough is in the range 8 °C (January–February)to 16 °C (July–September) (Fig. 1b; 7–17 °C reported byStengel & Dring 1997). Seasonal variation in NRA was

J F M A M J J A S O N D N

O3

– (m

mol L

–1)

0

2

4

6

8

10

12

Sea s

urf

ace t

em

pera

ture

(oC

)

8

10

12

14

16

18

20

NO3

–1974–1991

NO3

–2004–2005

NO3

–1994–1995

NO3

–2001–2002

Temperature

(b)

NR

A L

. dig

itata

(nm

ol N

O3

– m

in–1 g

–1 F

W)

0

10

20

30

40

50

60

70

80

NR

A F

ucus

sp

ecie

s

(nm

ol N

O3

– m

in–1 g

–1 F

W)

0

50

100

150

200

250

300

350L. digitataFucus serratusFucus vesiculosusFucus spiralis

(a)

Figure 1. Seasonal variation in nitrate reductase activity(NRA) in four brown algal species (a) and external NO3

-

availability and surface (~1 m) sea temperature (b) at TheNarrows, Strangford Lough. Points are means � SD, n � 4. Notedifferent scale on right-hand side for activity in Laminariadigitata; activity in all Fucus species is plotted on the left-handaxis. For external NO3

- concentrations (black triangles), pointsare means � 95% confidence limits for 17–33 replicate samplesfrom years 1974–1976, 1986–1987 and 1990–1991. Additional datasets are plotted for 1994–1995 (unfilled triangles), 2001–2002(unfilled circles) and 2004–2005 (grey triangles), and symbols aremeans of at least two replicates, �SD (AFBI 2006). Thetemperature plot includes a point every 3 h for the year 2000(AFBI 2006). FW, fresh weight.

766 E. B. Young et al.

© 2007 The AuthorsJournal compilation © 2007 Blackwell Publishing Ltd, Plant, Cell and Environment, 30, 764–774

positively correlated with average water column NO3- con-

centrations (measured over the period 1974–2005), whichpeaked during the winter months and were lowest in July(Fig. 1b). NO3

- concentrations measured on additionalwater samples collected during the study period (1999–2002) were 2–8 mmol L-1, therefore, within the rangesshown in Fig. 1b. NO2

- concentrations were <0.5 mmol L-1 atall times. NH4

+ and PO43- concentrations (from the 1974–

1991 data set) varied less than NO3- over the seasonal cycle.

Monthly mean NH4+ concentrations ranged from 0.9 � 0.4

to 3.1 � 2.1 mmol L-1 with no significant changes throughthe year (P > 0.2, data not shown), while monthly meanPO4

3- concentrations ranged from 0.48 � 0.17 mmol L-1 inJuly to 1.2 � 0.71 mmol L-1 in December, also with no sig-nificant seasonal variation (P > 0.15, data not shown).

The thallus C content was highest in F. spiralis, with over40% of dry mass as C in July (Fig. 2a), but C content did notchange significantly through the year in F. vesiculosus and F.serratus (P > 0.05) (Fig. 2a). In contrast, the C content of L.digitata was significantly lower in February than duringJuly–August (P < 0.004), and was always lower throughoutthe year than in the Fucus species. The N content [as % dryweight (DW)] varied with season in all species (P < 0.001,Fig. 2). The N content (% DW) was highest in the wintermonths, decreased over the spring with minima duringsummer, and increased slightly during autumn (Fig. 2b),with a similar pattern to that for NO3

- availability and NRA(Fig. 1). The highest thallus N contents were recorded in L.digitata during January–May, whereas the lowest were in F.spiralis in August. Consequently, L. digitata showed thelowest C : N ratio during winter and F. spiralis the highest insummer (Fig. 2c). In L. digitata, F. vesiculosus and F. serra-tus, NRA was negatively correlated with thallus C : N ratioand positively correlated with %N (Fig. 3).When comparedusing Pearson correlation coefficients, the correlationbetween NR and %N was not significantly stronger thanthe correlation between NR and C : N (P > 0.37 for allspecies; r values shown in Figs 3a,b).

Internal pools of NO3- and NH4

+ in the thallus tissue forthe four species varied over the year (one-way ANOVA,P < 0.001 for each species for both NH4

+ and NO3-; Fig. 4).

Internal NO3- concentration in the thalli showed a similar

trend to external NO3-, with higher values in the winter

and lowest in late summer. Fucus vesiculosus and L. digi-tata stored more NO3

- than F. serratus and F. spiralis. Theinternal NO3

- concentration was most variable in L. digi-tata which stored over 80 mmol NO3

- g-1 thallus fresh massin March, but <2 mmol NO3

- g-1 during July–September(Fig. 4a). Variations in internal NO3

- concentration inFucus species were less marked, except for F. vesiculosus,which had 83 mmol NO3

- g-1 in February, but less than20 mmol NO3

- g-1 for the rest of the year (Fig. 4b). Fucusserratus showed elevated internal NO3

- concentrationduring January–March (maximum 42 mmol NO3

- g-1), butless than 10 mmol NO3

- g-1 from April to October (Fig. 4c).Fucus spiralis stored the lowest concentration of NO3

- andNH4

+, but showed a similar trend with higher internalNO3

- concentrations in winter than in summer (Fig. 4d).

Internal NO3- storage far exceeded internal NH4

+ concen-trations (Fig. 4a–d; note different scales for NO3

- andNH4

+). The highest internal NH4+ concentrations were

observed in the thalli during the summer and the lowestduring the winter, which was opposite the trend for inter-nal NO3

- concentrations. When the total inorganic Ncontent (NO3

- + NH4+) from Fig. 4 was compared to the

total N content from Fig. 2b, inorganic N accounted for amaximum of 3.2% of the total thallus N in L. digitata inMarch, 3.8% in F. vesiculosus in February and 2.3% in F.serratus in January.

NO3- concentration in the thalli increased with increas-

ing external NO3- concentration. In L. digitata and F.

vesiculosus, the internal NO3- concentration was nearly 10

times the external concentration during the peak storage

C c

on

ten

t (%

DW

)

0

10

20

30

40

N c

on

ten

t (%

DW

)

0

1

2

3

4

Fucus spiralis Fucus vesiculosus Fucus serratus Laminaria digitata

J F M A M J J A S O N D

C:N

ratio (

mol m

ol–

1)

0

10

20

30

40

50

60

(a)

(b)

(c)

Figure 2. Seasonal changes in internal C content (a), N content(b), both as % dry mass, and molar C : N ratio (c) of the thalli offour species of brown algae. Points are means � SD, n � 4. Thespecies symbols are the same for (a), (b) and (c). DW, dry weight.

Seasonal N metabolism in brown algae 767

© 2007 The AuthorsJournal compilation © 2007 Blackwell Publishing Ltd, Plant, Cell and Environment, 30, 764–774

period (winter, Fig. 5a). Internal NO3- concentration

increased exponentially as ambient NO3- concentration

increased above 7.5 mmol L-1 (January–March, Fig. 1b). InL. digitata, in April and May, there was an increase inNO3

- pools despite low external NO3- (2–4 mmol L-1)

in those months (Fig. 5a). In F. vesiculosus, 67% of theseasonal variation in NRA could be explained by externalNO3

- concentration (regression analysis, r = 0.817,P < 0.002; Fig. 5b), but there was no significant correlationbetween NRA and internal NO3

- storage (r = 0.63,P > 0.05; Fig. 5c). In F. serratus, NRA was correlated withboth external NO3

- (r = 0.82, P < 0.002; Fig. 5b) and inter-nal NO3

- concentration (r = 0.69, P < 0.02; Fig. 5c). In L.digitata, NRA was also correlated with external NO3

-

(r = 0.64, P < 0.05; Fig. 5b) and internal NO3- concentra-

tion (r = 0.68, P < 0.03; Fig. 5c). There was no statistical dif-ference in the correlations between NR and internalversus external NO3

- concentration for F. serratus and L.digitata (P > 0.43 for both species – Pearson correlationcoefficient comparison).

All the macroalgae showed higher net photosynthesisrates in summer than in winter (Table 1). Dark respirationaccounted for between 18 and 40% of net daytime photo-synthesis rates, with higher respiration rates relative to pho-tosynthesis in summer than in winter. When NRA was usedas an estimate of maximum N assimilation rate, and com-pared to C fixation rates calculated from maximum netphotosynthetic oxygen evolution rates for each speciesduring the winter and summer periods (Table 1), the esti-mated N assimilation capacity was 0.7–50.0 times the esti-mated C fixation rate for each species. The ratios ofestimated C fixation to N assimilation capacity were higherin summer than in winter as the low winter photosynthesisrates coincided with high NRA and thus higher NO3

-

C:N ratio 0 10 20 30 40

NR

A

(nm

ol N

O3

– m

in–1 g

–1)

0

50

100

150

200

250

300

(a)

(b)

F. serratus, r = – 0.81, P < 0.003

F. vesiculosus, r = – 0.70, P < 0.025

L. digitata, r = – 0.76, P < 0.015

N content (% DW)

1 2 3 4

NR

A

(nm

ol N

O3

– m

in–1 g

–1)

0

50

100

150

200

250

300 F. vesiculosus, r = 0.83, P < 0.004

F. serratus, r = 0.824, P < 0.002

L. digitata, r = 0.899, P < 0.0005

Figure 3. Seasonal nitrate reductase activity (NRA) plottedagainst (a) internal C : N ratio and (b) N content [as % dryweight (DW)] for Fucus vesiculosus, Fucus serratus andLaminaria digitata. Species symbols are the same for (a) and (b).Points are means � SD, n � 4. Pearson correlation coefficientsand P-values are included.

Inte

rnal N

O3

– (m

mo

l g

–1

FW

)

0

20

40

60

80

100

Inte

rnal N

H4

+

(mm

ol g

–1 F

W)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Laminaria digitata

0.0

0.5

1.0

1.5

2.0

2.5

0

20

40

60

80

100 Fucus vesiculosus

0

20

40

60

80

100

0.0

0.5

1.0

1.5

2.0

2.5Fucus serratus

J F M A M J J A S O N D

0

20

40

60

80

100

0.0

0.5

1.0

1.5

2.0

2.5Fucus spiralis

(a)

(b)

(c)

(d)

Figure 4. Seasonal variation in internal thallus concentration ofNO3

- (filled circles) and NH4+ (open triangles) for the four

species of brown algae. Note the different scales for NO3- and

NH4+. Points are means � SD, n � 4. FW, fresh weight.

768 E. B. Young et al.

© 2007 The AuthorsJournal compilation © 2007 Blackwell Publishing Ltd, Plant, Cell and Environment, 30, 764–774

Inte

rna

l N

O3

– (m m

ol g

–1 F

W)

0

20

40

60

80

100

External NO3

– (mmol L–1)

0 2 4 6 8 10

NR

A(n

mo

l N

O3–

min

–1 g

–1 F

W)

0

100

200

300

F. vesiculosus, r = 0.608, P > 0.05

F. serratus, r = 0.871, P < 0.0005

L. digitata, r = 0.873, P < 0.0005

F. spiralis

Internal NO3

– (mmol g–1 FW)

0 20 40 60 80 100

NR

A(n

mo

l N

O3

– m

in–

1 g

–1 F

W)

0

100

200

300

(a)

(b)F. serratus: r = 0.823, P < 0.002

L. digitata: r = 0.64, P < 0.05

F. vesiculosus: r = 0.817, P < 0.002

(c)F. vesiculosus r = 0.631, P < 0.05

F. serratus r = 0.691, P < 0.02

L. digitata r = 0.683, P < 0.03

Figure 5. The relationship between external NO3- availability

(from Fig. 1b) and thallus internal NO3- concentrations (a) and

nitrate reductase activity (NRA) (b) in four species of brownalgae, and the relationship between thallus NRA and internalNO3

- concentration (c) over the seasonal cycle. Species symbolsare the same for (a), (b) and (c). Correlation coefficients andP-values are included for all the data for Fucus vesiculosus,Fucus serratus and Laminaria digitata. Curves in (a) areexponential models fitted by least squares regression above4.5 mM for L. digitata [grey line, r2 = 0.94; NO3

-int = 0.549 ¥

exp(0.5454 ¥ NO3-

ext)], above 1.5 mM for F. serratus [dash-dotline, r2 = 0.85; NO3

-int = 1.386 ¥ exp(0.342 ¥ NO3

-ext)] and

above 7.5 mM for F. vesiculosus [dashed line, r2 = 0.96;NO3

-int = 7E-7 ¥ exp(1.979 ¥ NO3

-ext)]. All points are means � SD,

n � 4. FW, fresh weight.

Tab

le1.

Res

pira

tion

and

net

phot

osyn

thes

isra

tes

ofbr

own

alga

efr

omSt

rang

ford

Lou

gh,‘

The

Nar

row

s’re

gion

and

com

pari

son

ofN

assi

mila

tion

capa

city

valu

eses

tim

ated

from

mon

thly

nitr

ate

redu

ctas

eac

tivi

ties

(NR

As)

ofL

amin

aria

digi

tata

,Fuc

usve

sicu

losu

san

dF

ucus

serr

atus

Spec

ies

Dar

kre

spir

atio

nmm

olO

2g-1

h-1)

Net

phot

osyn

thes

ismm

olO

2g-1

h-1)

Dai

lyne

tC

fixca

paci

tya

mmol

Cg-1

d-1)

Nas

sim

ilati

onca

paci

tyb

mmol

Ng-1

d-1)

C:N

assi

mila

tion

rati

o(m

olm

ol-1

)C

:Nin

tiss

uec

(mol

mol

-1)

Sum

mer

Win

ter

Sum

mer

Win

ter

Sum

mer

Win

ter

Sum

mer

Win

ter

Sum

mer

Win

ter

Sum

mer

Win

ter

F.se

rrat

us4.

661.

1612

.53.

9912

215

.437

479

50.

325

0.01

9419

.413

.0F.

vesi

culo

sus

3.53

0.96

99.

655.

1794

.126

.732

613

150.

289

0.02

0324

.417

.3L

.dig

itata

3.97

1.41

9.58

7.22

90.0

36.6

6567

1.37

0.54

522

.39.

67F

ucus

spir

alis

d3.

530.

969

9.65

5.17

94.1

26.7

236

863

0.39

90.

0309

38.6

18.0

Sum

mer

data

are

valu

esav

erag

edov

erJu

ne–S

epte

mbe

r,an

dw

inte

rda

taar

eav

erag

esof

Nov

embe

r–M

arch

valu

es.

a Cal

cula

ted

usin

gda

ytim

eph

otos

ynth

esis

and

dark

resp

irat

ion

valu

es;n

etox

ygen

evol

utio

nes

tim

ates

base

don

15h

dayl

ight

(sum

mer

)an

d9

hda

ylig

ht(w

inte

r).O

2ev

olut

ion

was

conv

erte

dto

Cfix

atio

nus

ing

aph

otos

ynth

etic

quot

ient

(mol

eO

2ev

olve

dpe

rm

ole

Cfix

ed)

of1.

2.b N

assi

mila

tion

capa

city

esti

mat

esfr

omm

ean

nitr

ate

redu

ctas

e(N

R)

valu

esav

erag

edfo

rsu

mm

er(J

une–

Sept

embe

r)an

dw

inte

r(N

ovem

ber–

Mar

ch)

mon

ths,

from

Fig.

1a.F

orF

ucus

spec

ies,

inw

hich

nitr

ate

redu

ctas

eac

tivi

ty(N

RA

)is

nots

uppr

esse

din

the

dark

,NO

3-as

sim

ilati

onis

assu

med

tooc

cur

24h

ada

y,w

hile

inL

.dig

itata

,NR

Ais

supp

ress

edin

dark

ness

,so

NO

3-as

sim

ilati

onw

ason

lyca

lcul

ated

tooc

cur

duri

ngda

ylig

htho

urs

(You

nget

al.u

npub

lishe

dre

sult

s).

c Tis

sue

C:N

data

from

Fig.

2.d P

hoto

synt

hesi

sra

tes

for

F.sp

iral

isw

ere

not

mea

sure

dan

dw

ere

base

don

thos

efo

rF.

vesi

culo

sus.

Seasonal N metabolism in brown algae 769

© 2007 The AuthorsJournal compilation © 2007 Blackwell Publishing Ltd, Plant, Cell and Environment, 30, 764–774

assimilation capacity (Table 1). This was more pronouncedfor the Fucus species with at least a 16-fold summer-to-winter difference in C : N assimilation capacity ratio, butthis difference was only fourfold in L. digitata. The lowestratio of C : N content of thalli in winter was close to 10 in L.digitata, and the highest in summer was >38 in F. spiralis(Table 1).

DISCUSSION

Seasonal nitrate availability

The NRA in four species of intertidal brown algaeappeared to be most strongly regulated in response toexternal NO3

- availability; both NO3- and NRA showed

marked seasonal variations with winter to early springmaxima. In a study of NRA in Scottish L. digitata byDavison et al. (1984), a similar seasonal pattern wasobserved, although maximum NRAs were observed later, inApril–June, which coincided with an April peak in waterNO3

- concentrations, later than observed in StrangfordLough (Fig. 1). Summer declines of external NO3

- inStrangford Lough can be attributed to increased macroalgaland phytoplankton growth, commonly reported in othertemperate habitats (e.g. Wheeler & Srivastava 1984; Peder-sen & Borum 1996). NRA in phytoplankton declinesrapidly when external NO3

- is exhausted (Berges, Cochlan& Harrison 1995). In Strangford Lough, however, there wasalways some external NO3

- available, so that the summerdecline in NRA was not due to lack of NO3

-. The relativeconstancy of dissolved PO4

3- and NH4+, in contrast to NO3

-,suggests that N is the most limiting macronutrient, and thatNO3

- is the dominant inorganic N source for phytoplanktonand macroalgal growth in Strangford Lough.

As external NO3- availability increased during the

winter, the macroalgae stored more NO3- in the thallus

tissue (Fig. 4). However, when adjusting for mass[DW = 0.125 ¥ fresh weight (FW)], internal NO3

- contrib-uted only a small proportion (<3.9%) of total thallus Ncontent. In these brown algae, significant N could be storedas protein, as seen in Laminaria solidungula (Pueschel &Korb 2001), although this was not the case in a green alga(McGlathery, Pedersen & Borum 1996). The rise in internalNO3

- as external NO3- concentration increased above

7.5 mmol L-1 (Fig. 5a) suggests that the uptake capacity ofthese macroalgae may not be saturated by ambient NO3

-

concentrations in the Lough. Previously reported NO3-

uptake kinetics for intertidal brown algae (Korb & Gerard2000; Phillips & Hurd 2004) suggests that, even in winter,NO3

- concentrations were below the 20–60 mmol L-1

required to saturate uptake.The relationship between inter-nal NO3

- concentration and NRA was unclear, but theremay be some saturation of NRA in relation to internalNO3

- concentration which varied with species (Fig. 5c).Thisalso suggests that assimilation of internal thallus NO3

- is notdetermined solely by NRA and, conversely, that NRA is notstrongly regulated by intracellular NO3

- availability. If NO3-

is not immediately assimilated, significant NO3- may be

stored in the vacuole, a major site of NO3- storage in higher

plants (Granstedt & Huffaker 1982), thus removing NO3-

from the site of NRA in the cytosol.Total thallus N content (% DW) was correlated with

external NO3- in all species (Figs 1–2), as reported previ-

ously for Laminaria saccharina (Wheeler & Weidner 1983).The total N content in all thalli were similar to previousreports of macroalgae (Hernández et al. 1993; McGlatheryet al. 1996; Pedersen & Borum 1996; Brenchley, Raven &Johnston 1998; Naldi & Wheeler 1999), although L. digitatain Strangford Lough reached lower %N and higher C : Nratio in summer than L. saccharina in the English Channel(Gevaert et al. 2001). Both thallus total N and NO3

- weredepleted during the spring when vegetative growth waslikely to have accelerated (Chapman & Craigie 1977;Lüning, Wagner & Buchholz 2000; Lehvo, Back & Kiirikki2001), effectively diluting thallus N reserves (see McGlath-ery et al. 1996). Reproduction is another sink for thallus N.In F. vesiculosus, reproductive growth occurs in winter,and gametes are released in May–June (Berger, Malm& Kautsky 2001; Lehvo et al. 2001), coinciding with theobserved decline in thallus N content. Laminaria digitataproduces reproductive sori during the late autumn andwinter (Lüning et al. 2000). Winter reproductive growth inFucus and Laminaria species is an additional sink for Nduring a period when growth is limited by other factors, andgamete release from Laminaria and Fucus represents a lossof N and C from the thallus, and together with N demandsof vegetative growth, may contribute to a spring decline inthallus N. Growth may become limited by N supply duringlate summer (Chapman & Craigie 1977).

Irradiance

Winter imposes light limitation, and in situ photosynthesisrates varied most strongly with seasonal irradiance.However, although external NO3

- concentrations werehighest in winter, photosynthesis rates were lower, resultingin a seasonal disjunction between periods of maximum Navailability and photosynthesis (Table 1). Such disjunctionshave been noted before, particularly at high latitude(Henley & Dunton 1997; Stengel & Dring 1997). C and Nassimilation in algae are understood to be closely coupled(Gao et al. 1995; Vergara et al. 1998), but in these brownmacroalgae, N uptake, internal NO3

- storage and NRAwere inversely correlated with seasonal photosynthesis. Inwinter, when photosynthesis is light limited (Table 1;Stengel & Dring 1997), assimilation of inorganic N intoamino acids may be limited by energy and fixed C supply,rather than by NRA. Higher NO3

- availability in winterpromotes maximum inorganic N uptake and assimilation,for which elevated NRA rates are beneficial. However, asmetabolic N demand and photosynthesis rates are low,there is significant winter storage of unassimilated NO3

- bythe brown algal species examined, particularly L. digitata. Inarctic L. solidungula, NO3

- uptake kinetics also suggests a‘storage specialist’ strategy to take up NO3

- during winterwhen it is available and to store significant internal NO3

-,

770 E. B. Young et al.

© 2007 The AuthorsJournal compilation © 2007 Blackwell Publishing Ltd, Plant, Cell and Environment, 30, 764–774

even at a time of low growth (Henley & Dunton 1995; Korb& Gerard 2000). In temperate Macrocystis integrifolia, themaximum internal NO3

- concentrations were 50–90 mmolNO3

- g-1 FW in winter (Wheeler & Srivastava 1984; Hurdet al. 1996) and up to 150 mmol g-1 fresh mass in Laminarialongicruris (Chapman & Craigie 1977). In more extremearctic conditions, Laminaria species showed spring NO3

-

storage ~35 mmol g-1 DW (using a wet-to-dry mass conver-sion factor for arctic L. saccharina and L. solidungula of0.125; Henley, personal communication). These values arecomparable to the ~40 mmol NO3

- g-1 FW observed in April,and the maximum of 85 mmol NO3

- g-1 FW in March in IrishL. digitata (Fig. 4).

Despite the fact that NO3- assimilation requires energy

(and is thus closely coupled with photosynthesis andenhanced by light), significant NO3

- uptake by algae canoccur during the dark, particularly when inorganic N islimiting (Cochlan, Harrison & Denman 1991; Korb &Gerard 2000). Low winter irradiance in Ireland is sufficientto support NO3

- uptake, but still limits photosynthesis andgrowth (Table 1; Stengel & Dring 1997). Although NRA inmacroalgae can respond rapidly to irradiance (e.g. Davison& Stewart 1984; Gao et al. 1995; Lopes et al. 1997; Lartigue& Sherman 2002), and intertidal macroalgae in NorthernIreland experience a fivefold change in maximum incidentsurface irradiance (PAR) from winter to summer (400–2000 mmol photons m-2 s-1), there is no evidence of suppres-sion of NRA by low winter irradiance (Fig. 1). Because oflonger immersion, L. digitata will experience lower dailyphoton doses than the higher intertidal algae, and NRA inthe intertidal–subtidal L. digitata was lower than in Fucusspecies. In a companion study, we found that daily NRAchanges in Fucus species were insensitive to irradiance, andthat in L. digitata, NRA was suppressed in darkness, butday–night differences were most pronounced in winter,when daytime irradiance was lowest (Young, Dring &Berges unpublished results). The lack of an autumnincrease in NRA and internal NO3

- concentration in L.digitata may be related to low irradiance in the intertidal–subtidal region, or to the dynamics of low autumn growthand reproductive activity in that species (Gómez & Lüning2001). From previous studies, it is known that seasonalgrowth dynamics are different for Laminaria and Fucusspecies. Growth has been shown to be highest during thelate winter–early summer in Laminaria species (Chapman& Craigie 1977; Davison et al. 1984; Henley & Dunton 1997;Sjøtun, Fredriksen & Rueness 1998; Gómez & Lüning 2001)but in spring–summer for fucoid algae (Stengel & Dring1997; Brenchley et al. 1998; Lehvo et al. 2001).

Temperature

The minimum water temperature (~7 °C) coincided withpeak NRAs observed in March (Fig. 1), and low water tem-perature in winter could influence NRA. The activity ofenzymes, including NR, is temperature sensitive, withoptimum temperatures for NRA in diverse algae measuredin the range of 10–20 °C (Gao et al. 2000; Berges et al. 2002).

In this study, we measured NRA at a constant 12 °Cthroughout the year, a temperature that is close to theaverage experienced by the algae when submerged, andunlikely to cause inactivation of the enzyme. The in vitroassay at this single temperature means that the NRAsreported in this study are probably underestimates of activ-ity when water is warmer, and overestimates when water iscooler (Young et al. 2005). Low temperatures may require agreater quantity of the enzyme to achieve the same catalyticactivity because the enzyme is working below its tempera-ture optimum. Therefore, elevated winter NR enzymeactivity may be a component of cold acclimation. Higheractivities of NR and other enzymes have been observed atlower temperatures in L. saccharina (Davison & Davison1987) and in F. vesiculosus, where a similar seasonal patternof elevated enzyme activities in winter was observed(Collén & Davison 2001). Therefore, low-temperaturestimulation of activity may apply to several enzymes, notjust to NR. However, based on a Q10 of 2 (discussed byBerges et al. 2002) and a range between winter and summertemperature of ~10 °C (Fig. 1), one would predict a dou-bling of NRA to compensate for lower winter temperature.However, the seasonal range of NRA observed is muchgreater than 2 (~6 times in both L. digitata and Fucusspecies). Although all NRA was assayed at 12 °C, in situNRA in seaweeds will be influenced by temperature, pos-sibly affecting the comparison between NO3

- assimilationrate (from NRA in Fig. 1) and photosynthesis rates, pre-sented in Table 1. However, when the seasonal water tem-perature range (7–17 °C, Fig. 1b) was taken into account,assuming a Q10 of 2, the patterns of summer–winter com-parison of C : N assimilation capacity in the four species didnot change significantly (data not shown). This supports theidea that seasonal variation in NRA is at most only partiallydue to temperature acclimation. Respiration rate is likelyto be more temperature sensitive than photosynthesis, sothe lower respiration relative to photosynthesis ratesobserved in winter (Table 1) are probably related to lowertemperatures.

Position in intertidal zone

Fucus species showed slightly different patterns of Nstorage and NRA over the seasonal cycle to L. digitata,which is likely to be related to habitat differences. Fucusspecies growing higher in the intertidal zone showed higherNRA than L. digitata, which could be an adaptation to moreprolonged tidal emersion (Murthy, Rao & Reddy 1986).Thelonger immersion time in the intertidal–subtidal marginallows Laminaria to take up NO3

- from the water for longereach day and may explain the higher internal NO3

- concen-tration in L. digitata during winter. Fucus species, growinghigher in the intertidal zone, are emersed and thus isolatedfrom the source of NO3

- in the seawater, for longer eachday. This induces a temporal limitation for NO3

- uptake, forwhich higher uptake rates may compensate (Thomas et al.1987; Phillips & Hurd 2004). Laminaria digitata may alsostore more NO3

- than do the Fucus species because the

Seasonal N metabolism in brown algae 771

© 2007 The AuthorsJournal compilation © 2007 Blackwell Publishing Ltd, Plant, Cell and Environment, 30, 764–774

NRA is lower, so that NO3- can be assimilated less rapidly

after entering the cell, before it is stored (possibly in thevacuole). Despite longer exposure to water column NO3

-,intertidal–subtidal L. digitata thalli experience lower irradi-ance at greater water depth, particularly in winter whenirradiance is limiting for growth of brown macroalgae(Stengel & Dring 1997). Winter C : N ratios in L. digitatawere lower than in Fucus (Fig. 2c), and might have been aconsequence of higher N storage when higher ambientconcentration of N was available, and/or low winter Ccontent; lower C : N ratios in L. digitata could also be aconsequence of C depletion during light-limited growth. Incontrast, F. spiralis, which is found highest in the intertidalzone, showed the lowest N storage and highest summer %Cand C : N ratio (Fig. 2c). In a previous survey of intertidalalgae, Thomas et al. (1987) also showed a lower %N andhigher C : N ratio in thalli with increasing height in theintertidal zone. This trend may relate to the combination ofincreased irradiance but more restricted access to dissolvedinorganic N with height in the intertidal zone.

CONCLUSIONS AND IMPLICATIONS

N uptake, N storage and NRA in temperate intertidalbrown algae are likely to be most strongly regulated inresponse to external NO3

- availability, although tempera-ture acclimation may contribute to the seasonal variation inNRA, and light may influence N metabolism more indi-rectly via C metabolism. When NRA was used as an esti-mate of N assimilation capacity (see Davison et al. 1984),Fucus species apparently had the capacity to assimilatebetween 2.5 and 50.0 times more N than C was fixed, and L.digitata up to 1.8 times more N than C, despite measuredC : N content ratios in the tissues of between 10 and 40(Table 1). This suggests an uncoupling of N and C metabo-lism in the intertidal macroalgae that contrasts with thetight relationship between C and N metabolism reportedfor some microalgae (e.g. Gao et al. 1995; Vergara et al.1998). Relative excess of N assimilation capacity maysuggest that photosynthesis is significantly limited byresource availability. However, NRAs measured in vitromay overestimate in situ N assimilation capacity in algaeexposed to low winter temperatures. Alternatively, thesebrown algae may require the higher NRA to reduce moreNO3

- than is actually required for growth and development.NO3

- that is taken up by algae can have four distinct fates:

1 NO3- is reduced and assimilated into amino acids, which

are incorporated into thallus growth (predominant fateduring summer, periods of low external NO3

- availabilitybut potentially limited by supply of energy and Cskeletons in winter).

2 NO3- is taken up but stored, possibly in the vacuoles, until

thallus N reserves are depleted in spring–summer (animportant fate in winter).

3 NO3- is taken up, reduced and excreted as NO2

- and/orNH4

+. In diatoms, reduced inorganic N excretion canrepresent >50% of NO3

- uptake (Collos 1998; Lomas,

Rumbley & Glibert 2000). However, in two macroalgalspecies, negligible NH4

+ release was measured followingNO3

- uptake (Naldi & Wheeler 2002). Release of NO2-

can be difficult to assess experimentally because of rapidoxidation of NO2

- to NO3- in oxic surface waters.

4 NO3- is taken up and assimilated into organic molecules

that are excreted and/or ‘leaked’ from healthy or senes-cent thalli (Naldi & Wheeler 2002; Fong, Fong & Fong2004; Tyler & McGlathery 2006).

NO3- uptake and release as NO2

-, NH4+ or organic N will

require greater NRA than may be needed to support Nincorporation, and may account for the excess NRAs rela-tive to rates of photosynthetic C fixation. In temperate near-shore ecosystems dominated by brown macroalgal biomass,algal NO3

- uptake and release as organic N could be signifi-cant for near-shore nutrient cycling (Duggins et al. 1989). Itis unknown how seaweeds control the fate of NO3

- that istaken up; further exploration of this would contribute bothto an understanding of seaweed physiology, but would alsoclarify the importance and role of these algae in near-shorebiogeochemical cycling.

ACKNOWLEDGMENTS

E.B.Y. was supported by a postdoctoral Natural Environ-ment Research Council (NERC) UK grant (GR3/12454),and measurements of macroalgal production in StrangfordLough were supported by NERC UK (GR3/9072). E.B.Y. isgrateful to the staff and students at Portaferry MarineLaboratory for field support, particularly D. Rogers and M.Curran for help with boat collections. C and N analysis wascarried out by B. Stewart at AFBI, Northern Ireland, withsample preparation assistance from D. Franklin. P. Boydand L. Gilpin assisted in collection of the long-term nitrateconcentration data. A. Mellor (AFBI, Northern Ireland)cheerfully facilitated access to Strangford Lough nutrientdata, and temperature data was courtesy of R. Gowan andB. Stewart (AFBI).

REFERENCES

AFBI Database (2006) Agri-Food and Biosciences Institute,Northern Ireland – Coastal monitoring programme. http://www.afbini.gov.uk/index/services/specialist-advice/coastal-monitoring.htm

Berger R., Malm T. & Kautsky L. (2001) Two reproductive strate-gies in Baltic Fucus vesiculosus (Phaeophyceae). EuropeanJournal of Phycology 36, 265–273.

Berges J.A., Cochlan W.P. & Harrison P.J. (1995) Laboratory andfield responses of algal nitrate reductase to diel periodicity inirradiance, nitrate exhaustion, and the presence of ammonium.Marine Ecology Progress Series 124, 259–269.

Berges J.A., Varela D.E. & Harrison P.J. (2002) Effects of tempera-ture on growth rate, cell composition and nitrogen metabolism inthe marine diatom Thalassiosira pseudonana (Bacillari-ophyceae). Marine Ecology Progress Series 225, 139–146.

Brenchley J.L., Raven J.A. & Johnston A.M. (1998) Carbon andnitrogen allocation in two intertidal fucoids: Fucus serratus andHimenthalia elongata (Phaeophyta). European Journal ofPhycology 33, 307–313.

772 E. B. Young et al.

© 2007 The AuthorsJournal compilation © 2007 Blackwell Publishing Ltd, Plant, Cell and Environment, 30, 764–774

Chapman A.R.O. & Craigie J.S. (1977) Seasonal growth inLaminaria longicruris: relations with dissolved inorganic nutri-ents and internal reserves of nitrogen. Marine Biology 40, 197–205.

Cochlan W.P., Harrison P.J. & Denman K.L. (1991) Diel periodicityof nitrogen uptake by marine phytoplankton in nitrate-rich envi-ronments. Limnology and Oceanography 36, 1689–1700.

Collén J. & Davison I.R. (2001) Seasonality and thermal acclima-tion of reactive oxygen metabolism in Fucus vesiculosus (Phaeo-phyceae). Journal of Phycology 37, 474–481.

Collos Y. (1998) Nitrate uptake, nitrite release and uptake, and newproduction estimates. Marine Ecology Progress Series 171, 293–301.

Davison I.R. & Stewart W.D.P. (1984) Studies on nitrate re-ductase activity in Laminaria digitata (Huds.) Lamour. II. Therole of nitrate availability in the regulation of enzyme activity.Journal of Experimental Marine Biology and Ecology 79, 65–78.

Davison I.R. & Davison J.O. (1987) The effect of growth tempera-ture on enzyme activities in the brown alga Laminaria saccha-rina. British Phycological Journal 22, 77–87.

Davison I.R., Andrews M. & Stewart W.D.P. (1984) Regulation ofgrowth in Laminaria digitata: use of in vivo nitrate reductaseactivities as an indicator of nitrogen limitation in field popula-tions of Laminaria spp. Marine Biology 84, 207–217.

Duggins D.O., Simenstad C.A. & Estes J.A. (1989) Magnification ofsecondary production by kelp detritus in coastal marine ecosys-tems. Science 245, 170–173.

Fong P., Fong J.J. & Fong C.R. (2004) Growth, nutrient storage, andrelease of dissolved organic nitrogen by Enteromorpha intestina-lis in response to pulses of nitrogen and phosphorus. AquaticBotany 78, 83–95.

Gao Y., Smith G.J. & Alberte R.S. (1995) Induction of nitratereductase activity by light and NO3

- in the marine diatom Skel-etonema costatum. Plant Physiology 108, 71–71.

Gao Y., Smith G.J. & Alberte R.S. (2000) Temperature dependenceof nitrate reductase activity in marine phytoplankton: biochemi-cal analysis and ecological, implications. Journal of Phycology36, 304–313.

Gevaert F., Davoult D., Creach A., Kling R., Janquin M.-A.,Seuront L. & Lemoine Y. (2001) Carbon and nitrogen content ofLaminaria saccharina in the eastern English Channel: biometricsand seasonal variations. Journal of the Marine Biological Asso-ciation of the United Kingdom 81, 727–734.

Granstedt R.C. & Huffaker R.C. (1982) Identification of the leafvacuole as a major nitrate storage pool. Plant Physiology 70,410–413.

Gómez I. & Lüning K. (2001) Constant short-day treatment ofoutdoor-cultivated Laminaria digitata prevents summer drop ingrowth rate. European Journal of Phycology 36, 391–395.

Henley W.J. & Dunton K.H. (1995) A seasonal comparison ofcarbon, nitrogen, and pigment content in Laminaria solidungulaand L. saccharina (Phaeophyta) in the Alaskan Arctic. Journal ofPhycology 31, 325–331.

Henley W.J. & Dunton K.H. (1997) Effects of nitrogen supply andcontinuous darkness on growth and photosynthesis of the arctickelp Laminaria solidungula. Limnology and Oceanography 42,209–216.

Hernández I., Corzo A., Gordillo F.J., Robles M.D., Saez E.,Fernández J.A. & Niell F.X. (1993) Seasonal cycle of the game-tophytic form of Porphyra umbilicalis – nitrogen and carbon.Marine Ecology Progress Series 99, 301–311.

Hurd C.L., Harrison P.J. & Druel L.D. (1996) Effect of seawatervelocity on inorganic nitrogen uptake by morphologicallydistinct forms of Macrocystis integrifolia. Marine Biology 126,205–214.

Korb R.E. & Gerard V.A. (2000) Nitrogen assimilation character-istics of polar seaweeds from differing nutrient environments.Marine Ecology Progress Series 198, 83–92.

Lartigue J. & Sherman T.D. (2002) Field assays for measuringnitrate reductase activity in Entermorpha sp. (Chlorophyceae),Ulva sp. (Chlorophyceae) and Gelidium sp. (Rhodophyceae).Journal of Phycology 38, 971–982.

Lartigue J. & Sherman T.D. (2005) Response of Enteromorpha sp.(Chlorophyceae) to a nitrate pulse: nitrate uptake, inorganicnitrogen storage, and nitrate reductase activity. Marine EcologyProgress Series 292, 147–157.

Lehvo A., Back S. & Kiirikki M. (2001) Growth of Fucus vesiculo-sus L. (Phaeophyta) in the northern Baltic proper: energy andnitrogen storage in seasonal environment. Botanica Marina 44,345–350.

Lomas M.W., Rumbley C.J. & Glibert P.M. (2000) Ammoniumrelease by nitrogen sufficient diatoms in response to rapidincreases in irradiance. Journal of Plankton Research 22, 2351–2366.

Lopes P.F., Oliveira M.C. & Colepicolo P. (1997) Diurnal fluctua-tion of nitrate reductase activity in the marine red algaGracilaria tenuistipitata (Rhodophyta). Journal of Phycology 33,225–231.

Lüning K., Wagner A. & Buchholz C. (2000) Evidence for inhibi-tors of sporangium formation in Laminaria digitata (Phaeo-phyceae) during the season of rapid growth. Journal ofPhycology 36, 1129–1134.

Marine Laboratory Database, Queen’s University of Belfast (2002)PAR data. http://www.qub.ac.uk/bb/mbs (accessed 2004).

McGlathery K.J., Pedersen M.F. & Borum J. (1996) Changes inintracellular nitrogen pools and feedback controls on nitrogenuptake in Chaetomorpha linum (Chlorophyta). Journal ofPhycology 32, 393–401.

Murthy M.S., Rao A.S. & Reddy E.R. (1986) Dynamics of nitratereductase activity in two intertidal algae under desiccation.Botanica Marina 29, 471–474.

Naldi M. & Wheeler P.A. (1999) Changes in nitrogen pools in Ulvafenestrata (Chlorophyta) and Gracilaria pacifica (Rhodophyta)under nitrate and ammonium enrichment. Journal of Phycology35, 70–77.

Naldi M. & Wheeler P.A. (2002) 15N measurements of ammoniumand nitrate uptake by Ulva fenestrata (Chlorophyta) andGracilaria pacifica (Rhodophyta): comparison of net nutrientdisappearance, release of ammonium and nitrate, and 15N accu-mulation in algal tissue. Journal of Phycology 38, 135–144.

Parsons T.R., Maita Y. & Lalli C.M. (1984) A Manual of Chemicaland Biological Methods For Seawater Analysis, p. 173. PergamonPress, Oxford, UK.

Pedersen M.F. & Borum J. (1996) Nutrient control of algal growthin estuarine waters. Nutrient limitation and the importance ofnitrogen requirements and nitrogen storage among phytoplank-ton and species of macroalgae. Marine Ecology Progress Series142, 261–272.

Phillips J.C. & Hurd C.L. (2004) Kinetics of nitrate, ammonium,and urea uptake by four intertidal seaweeds from New Zealand.Journal of Phycology 40, 534–545.

Pueschel C.M. & Korb R.E. (2001) Storage of nitrogen in the formof protein bodies in the kelp Laminaria solidungula. MarineEcology Progress Series 218, 107–114.

Service M., Durrant A.E., Mills J.A., Taylor J.E. & Faughey D.(1996) The trophic status of two northern Irish Sea loughs.Journal of Coastal Conservation 2, 159–168.

Sjøtun K., Fredriksen S. & Rueness J. (1998) Effect of canopy andwave exposure on growth in Laminaria hyperborea (Laminari-aceae: Phaeophyta). European Journal of Phycology 33, 337–343.

Seasonal N metabolism in brown algae 773

© 2007 The AuthorsJournal compilation © 2007 Blackwell Publishing Ltd, Plant, Cell and Environment, 30, 764–774

Stengel D.B. & Dring M.J. (1997) Morphology and in situ growthrate of plants of Ascophyllum nodosum (Phaeophyta) from dif-ferent shore levels and response of plants to vertical transplan-tation. European Journal of Phycology 32, 193–202.

Thomas T.E., Turpin D.H. & Harrison P.J. (1987) Desiccationenhanced nitrogen uptake rates in intertidal seaweeds. MarineBiology 94, 293–298.

Thoresen S.S., Dortch Q. & Ahmed S.I. (1982) Comparison ofmethods for extracting intracellular pools of inorganic nitrogenfrom marine phytoplankton. Journal of Plankton Research 4,695–704.

Tyler A.C. & McGlathery K.J. (2006) Uptake and release of nitro-gen by the macroalgae Gracilaria vermiculophylla (Rhodo-phyta). Journal of Phycology 42, 515–525.

Vergara J.J., Berges J.A. & Falkowski P.G. (1998) Diel periodicity ofnitrate reductase activity and protein levels in the marine diatomThalassiosira weissflogii (Bacillariophyceae). Journal of Phycol-ogy 34, 952–961.

Wheeler W.N. & Weidner M. (1983) Effects of external inorganicnitrogen concentration on metabolism, growth and activities ofkey carbon and nitrogen assimilatory enzymes of Laminariasaccharina (Phaeophyceae) in culture. Journal of Phycology 19,92–96.

Wheeler W.N. & Srivastava L.M. (1984) Seasonal nitrate physiol-ogy of Macrocystis integrifolia Bory. Journal of ExperimentalMarine Biology and Ecology 76, 35–50.

Young E.B., Lavery P.S., van Elven B., Dring M.J. & Berges J.A.(2005) Dissolved inorganic nitrogen profiles and nitrate reduc-tase activity in macroalgal epiphytes within seagrass meadows.Marine Ecology Progress Series 288, 103–114.

Zar J.H. (1999) Biostatistical Analysis, 4th edn. Prentice Hall,Englewood Cliffs, NJ, USA.

Received 23 January 2007; received in revised form 14 March 2007;accepted for publication 16 March 2007

774 E. B. Young et al.

© 2007 The AuthorsJournal compilation © 2007 Blackwell Publishing Ltd, Plant, Cell and Environment, 30, 764–774