Seasonal Variations in Nitrate Reductase Activity and Internal N Pools in Intertidal Brown Algae Are Correlated with Ambient Nitrate Concentrations

Home , Fucus serratus, Laminaria digitata

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

Seasonal variations in nitrate reductase activity and internal N pools in intertidal brown algae are correlated with 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 and 2Department of Biological Sciences, University of Wisconsin–Milwaukee, 3209 Maryland Avenue, Milwaukee, WI 53211, USA

ABSTRACT Seaweeds in temperate habitats such as Strangford Lough experience large seasonal changes in temperature, Nitrogen metabolism was examined in the intertidal sea- irradiance and nutrient concentration that impose con- weeds Fucus vesiculosus, Fucus serratus, Fucus spiralis straints on their physiology. Furthermore, variations in tidal and Laminaria digitata in a temperate Irish sea lough. emersions also affect nutrient availability and irradiance on - storage, total N content and nitrate reductase Internal NO3 a scale of hours to weeks and often result in a disjunction activity (NRA) were most affected by ambient NO -, with 3 between the optimal light, nutrient availability and tem- highest values in winter, when ambient NO - was maximum, 3 perature for growth. Brown algal species growing at dif- and declined with NO - during summer. In all species, NRA 3 ferent heights in the intertidal zone experience distinct was six times higher in winter than in summer, and was irradiance and emersion regimes that may influence the markedly higher in Fucus species (e.g. 256 Ϯ 33 nmol regulation of nutrient acquisition and assimilation (e.g. NO - min-1 g-1 in F. vesiculosus versus 55 Ϯ 17 nmol NO - 3 3 Thomas, Turpin & Harrison 1987; Phillips & Hurd 2004). min-1 g-1 in L. digitata). Temperature and light were less Identifying responses of macroalgae to daily and seasonal important factors for N metabolism, but influenced in situ fluctuations in irradiance, temperature and nitrogen avail- photosynthesis and respiration rates. NO - assimilating 3 ability is thus critical in understanding the regulation of capacity (calculated from NRA) exceeded N demand (cal- nitrogen metabolism and the role of these productive culated from net photosynthesis rates and C : N ratios) by a macroalgae in near-shore nutrient cycling. factor of 0.7–50.0, yet seaweeds stored significant NO - (up 3 Simple measurements of nutrient uptake are difficult to to 40–86 mol g-1). C : N ratio also increased with height in m make in intertidal species, and can be biased by many the intertidal zone (lowest in L. digitata and highest in F. factors. Alternatively, enzyme activities offer more integra- spiralis), indicating that tidal emersion also significantly tive measures, less biased by instantaneous conditions. constrained N metabolism. These results suggest that, in Nitrate reductase [NR, enzyme class (EC) 1.6.6.1] is often contrast to the tight relationship between N and C metabo- considered the rate-limiting enzyme in inorganic N assimi- lism in many microalgae, N and C metabolism could be lation by algae and is thus a key enzyme in N metabolism. uncoupled in marine macroalgae, which might be an impor- Nitrate reductase activity (NRA) is strongly correlated with tant adaptation to the intertidal environment. N incorporation rates in macroalgae (Davison, Andrews & Stewart 1984). NRA in cultured algae is known to be stimu- Key-words: C : N ratio; macroalgae; nitrate assimilation; - lated by NO3 (Gao, Smith & Alberte 1995; Lartigue & photosynthesis; seasonality. Sherman 2005), and is regulated by light with rapid suppres- sion of NRA in darkness in most algae studied (e.g. Davison INTRODUCTION & Stewart 1984; Gao et al. 1995; Lopes, Oliveira & Colepi- colo 1997; Vergara, Berges & Falkowski 1998; Lartigue & Temperate brown macroalgae form highly productive com- Sherman 2002). NRA is also known to be responsive to munities, accounting for the majority of primary production changes in temperature with narrow temperature range for in many coastal regions and dominating near-shore nutrient optimum NRA (Gao, Smith & Alberte 2000; Berges, Varela cycling (Duggins, Simenstad & Estes 1989). For example, in & Harrison 2002). Therefore, in this study, we monitored Strangford Lough, Northern Ireland, macroalgae account NRA as an indicator of changes in nitrate metabolism in for 98% of algal biomass and 95% of productivity (Birkett, response to these key environmental variables. Dring & Savidge, unpublished results). The vast majority of Changes in NRA over a seasonal cycle have been exam- the macroalgal biomass in the Lough is fucoid algae (Fucus ined in very few macroalgae. To evaluate the factors influ- and Ascophyllum species) and kelps (Laminaria species). encing seasonal changes in N assimilation by macroalgae, it Correspondence: Erica B. Young. Fax: 1 414 229 3926; e-mail: is important to also examine external nutrient availability, [email protected] irradiance and temperature, as well as internal N storage, all

© 2007 The Authors 764 Journal compilation © 2007 Blackwell Publishing Ltd Seasonal N metabolism in brown algae 765 factors that may play a role in the regulation of NRA. If Light, temperature and nitrate data external availability of nitrate is the most important factor Surface-incident photosynthetically active radiation (PAR) regulating uptake and assimilation, then NRA and internal was measured using a 2p light sensor (Li-Cor, Lincoln, NE, nitrate storage will be closely related to seasonal changes USA) during 2001 and 2002 (Marine Laboratory Database, in nitrate concentration. N metabolism in algae is closely Queen’s University of Belfast 2002).Temperature was mea- linked to photosynthetic C metabolism (e.g. Vergara et al. sured in surface (~1 m depth) seawater at The Narrows site 1998), and if irradiance is the most important factor influ- [Agri-Food and Biosciences Institute (AFBI) Database encing N metabolism, then NR and internal N storage will 2006]. Nitrate concentration in the Lough was measured in be highest in summer when photosynthesis is not con- surface samples collected from The Narrows site and was strained by irradiance. Temperature is also an important analysed using standard methods (Parsons, Maita & Lalli seasonal environmental variable influencing metabolism 1984). Samples for long-term nutrient concentration data and may influence nitrate uptake, storage and NRA. were collected during years 1974–1976, 1986–1987 and The aims of the present study were to evaluate the impor- 1990–1991. Samples were also collected during the study tance of environmental variables influencing N metabolism period (2001–2002) to verify the earlier published seasonal in intertidal brown algae by making concurrent measure- NO - concentration data. Triplicate samples from The ments of NRA, total thallus N content, inorganic N storage 3 Narrows were collected and analysed from single sampling and photosynthesis, and by comparing these with seasonal periods over the years 1994–1995 (Service et al. 1996) and nitrate and light availability and temperature in a strongly 2004–2005 (AFBI Database 2006). seasonal intertidal habitat. Effects of position in the intertidal zone on N metabolism were examined by comparing Laminaria and Fucus species. Laminaria digitata grows in NRA the lower intertidal–subtidal zone, and hence is immersed longer and experiences greater light attenuation with water NRA was estimated using an in vitro assay method depth, compared with the intertidal Fucus species, Fucus described by Young et al. (2005). Frozen thallus samples vesiculosus and Fucus serratus, which grow in the mid- were ground to a powder in liquid nitrogen and extracted -1 intertidal zone, and Fucus spiralis, which is found even in 200 mmol L potassium phosphate buffer pH 7.9 con- -1 higher in the intertidal zone and is emersed for long periods taining 5 mmol L Na2 ethylenediaminetetraacetic acid during each tidal cycle. In these four brown algal species, we (EDTA), 0.3% (w/v) insoluble polyvinyl pyrollidone, -1 observed strong seasonal patterns in NRA and internal N 2 mmol L dl-dithiothreitol, 3% (w/v) bovine serum storage that closely correlate with seasonal changes in albumin (Fraction V) and 1% (v/v) Triton X-100 (all Sigma, nitrate availability. In addition, the relationship between C St Louis, MO, USA). The assay mixture contained -1 fixation and N assimilation capacity changes several-fold 200 mmol L sodium phosphate buffer pH 7.9 with -1 -1 between summer and winter. 200 mmol L NADH (b form, Sigma), 20 mmol L flavin adenine dinucleotide (Sigma), 20% volume as algal extract -1 and 10 mmol L KNO3. The assay was incubated at 12 °C MATERIALS AND METHODS and the reaction terminated by the addition of 1 M zinc - acetate. NO2 concentration was measured spectrophoto- Sampling metrically in centrifuged supernatants (Parsons et al. 1984), Whole thalli of Fucus serratus L., Fucus vesiculosus (L.) and activity estimated by linear regression of increasing - Lamour, Fucus spiralis L. and Laminaria digitata (Huds.) NO2 concentration over time. Lamour were collected from the intertidal region of ‘The Narrows’, Strangford Lough at Portaferry (54°23’N, Internal nitrogen pools and tissue 5°34’W), over the period November 2000–February 2002. N and C content Fucus serratus and F. vesiculosus were collected from the shore at low tide, and L. digitata was sampled near the Several methods used to extract internal inorganic nutrient middle of the day at low tide from the shore or from a boat, pools from algae were tested: boiling thallus discs in water as maximum activity was shown to occur during the middle for 20 min (Hurd, Harrison & Druel 1996), boiling water of the day in L. digitata (Davison et al. 1984). Laminaria added to ground algal tissue, vortexed and incubated for digitata were sampled by cutting ~30 mm diameter discs out 10 min (after Thoresen, Dortch & Ahmed 1982), boiling of the thalli, avoiding the meristematic region and the thallus pieces in water for 10 min followed by overnight oldest tissue (see Davison & Stewart 1984). Thallus tips extraction at 4 °C (Naldi & Wheeler 1999) and room tem- were cut from Fucus thalli by removing 30–40-mm-long perature ethanol extraction of ground tissue overnight terminal tips (trials showed activity was highest in tips). (McGlathery, Pedersen & Borum 1996). The highest Within a few minutes of all sampling times, tissue samples internal inorganic N concentrations were obtained by were thoroughly blotted dry, frozen and stored in liquid N2 adding 20 mL room temperature Milli-Q water (Millipore, for later analysis of NRA.Within a few minutes of sampling Watford, UK) to samples of ~50 mg frozen ground thallus on the shore, the tissue was thoroughly blotted dry and tissue in boiling tubes that were vortexed and then placed frozen and stored in liquid N2 for later analysis. in a boiling water bath for 45 min, cooled to room © 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd, Plant, Cell and Environment, 30, 764–774 766 E. B. Young et al.

temperature, ﬁltered through Whatman GF/A ﬁlters 350 80 (Brent, Middlesex, UK) and stored on ice.Thus, this method (a) L. digitata 300 Fucus serratus 70 was used for all subsequent analyses. The concentrations of Fucus vesiculosus

- - + 60 FW) NO2 ,NO3 and NH4 were measured in the ﬁltrate within FW) 250 Fucus spiralis –1 - –1 g

2 h, without freezing the samples. NO3 was analysed by g 50 –1 –1 Cd-column reduction followed by spectrophotometric mea- species 200 40 min min

- + – L. digitata 3 surement of NO2 , and NH4 was estimated by the phenol- –

3 150 hypochlorite method, both according to Parsons et al. Fucus 30 100 NRA (1984). To determine tissue C and N content, frozen thallus 20 NRA (nmol NO samples were ground in liquid N2 and dried in a 60 °C oven (nmol NO 50 10 with desiccant and analysed in a Carlo Erba 1500 NC 0 0 elemental analyser (CE Elantech, Lakewood, NJ, USA) – NO3 1974–1991 (b) – using acetanilide as a standard and expressing contents as a 12 NO3 2004–2005 20 C) o – NO3 1994–1995

proportion of dry mass. ) 10 – 18

–1 NO3 2001–2002 8 Temperature 16 mol L

Photosynthesis, respiration and N and C m 6 14 ( – assimilation capacity 3 4 12

Photosynthesis and dark respiration rates were also mea- NO 2 10

sured during winter (November–March) and summer ( Sea surface temperature 0 8 (June–September) months in F. vesiculosus, F. serratus and L. digitata using large (30 cm diameter ¥ 1 m long) clear JFMAMJJASOND plastic chambers suspended 2 m below the Lough surface, Figure 1. Seasonal variation in nitrate reductase activity containing the whole thalli of a single species. The water in - (NRA) in four brown algal species (a) and external NO3 the chambers was continuously circulated by a water pump. availability and surface (~1 m) sea temperature (b) at The A YSI 6000 probe (YSI Inc., Yellow Springs, OH, USA) Narrows, Strangford Lough. Points are means Ϯ SD, n Ն 4. Note measuring dissolved oxygen, salinity and temperature was different scale on right-hand side for activity in Laminaria inserted into the chambers and recorded values for these digitata; activity in all Fucus species is plotted on the left-hand - variables at 15 s intervals over 2–3 days. The values were axis. For external NO3 concentrations (black triangles), points Ϯ subsequently downloaded, and net oxygen exchange rates are means 95% confidence limits for 17–33 replicate samples from years 1974–1976, 1986–1987 and 1990–1991. Additional data over successive 15 min periods were calculated from the sets are plotted for 1994–1995 (unfilled triangles), 2001–2002 oxygen concentrations. Mass-specific net photosynthetic (unfilled circles) and 2004–2005 (grey triangles), and symbols are rates for F. vesiculosus, F. serratus and L. digitata were cal- means of at least two replicates, ϮSD (AFBI 2006). The culated for complete 24 h periods, and the O2 evolution temperature plot includes a point every 3 h for the year 2000 rates were converted to C fixation rates using a photosyn- (AFBI 2006). FW, fresh weight. thetic quotient of 1.2. C fixation rates were compared to the N assimilation capacity estimated from NRA for each species. For comparison, the values were averaged over vesiculosus and in April for L. digitata. Fucus spiralis was groups of monthly values relating to the summer (June– not sampled every month, but showed a similar trend to the September) and winter (November–March) periods. other species with highest NRAs in January and April. The lowest NRAs were observed from late summer into autumn with the minimum values in August for all species. The Data analysis highest NRA observed in Fucus species (256 Ϯ 33 nmol - -1 -1 NO3 min g frozen mass) was five times higher than in L. Relationships between parameters were examined using - -1 -1 digitata (55 Ϯ 17 nmol NO3 min g ) (Fig. 1a; note differ- Pearson product–moment correlation and analysis of vari- ent axes scales). The frozen and fresh mass differed less ance (ANOVA) (SigmaPlot version 9.04 and SigmaStat than 1% for all thalli (data not shown) so scaling to fresh or version 3.1; Systat Software Inc., Chicago, IL, USA). Corre- frozen mass will be very similar. The lowest activities in all lation coefficients were compared using Fisher transforma- species were observed in August. In L. digitata, there was no tions (Zar 1999). evidence of an autumn increase in NRA as there was in all three species of Fucus, and the NRA of L. digitata did not rise significantly until February. RESULTS The seasonal variation in NRAs was negatively corre- In the three species of Fucus examined and in L. digitata, lated with seawater temperature, which was lowest in there were marked differences in NRA at different times in February–March (Fig. 1b). Mean water temperature in the year (one-way ANOVA, P < 0.001 for each species; Strangford Lough is in the range 8 °C (January–February) Fig. 1a). NRA was highest during the winter and early to 16 °C (July–September) (Fig. 1b; 7–17 °C reported by spring months, peaking in March for F. serratus and F. Stengel & Dring 1997). Seasonal variation in NRA was © 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd, Plant, Cell and Environment, 30, 764–774 Seasonal N metabolism in brown algae 767

- positively correlated with average water column NO3 con- (a) centrations (measured over the period 1974–2005), which 40 peaked during the winter months and were lowest in July - (Fig. 1b). NO3 concentrations measured on additional water samples collected during the study period (1999– 30 2002) were 2–8 mmol L-1, therefore, within the ranges - -1 shown in Fig. 1b. NO2 concentrations were <0.5 mmol L at + 3- 20 all times. NH4 and PO4 concentrations (from the 1974– Fucus spiralis - 1991 data set) varied less than NO3 over the seasonal cycle. Fucus vesiculosus + Ϯ C content (% DW) Monthly mean NH4 concentrations ranged from 0.9 0.4 10 Fucus serratus -1 to 3.1 Ϯ 2.1 mmol L with no significant changes through Laminaria digitata the year (P > 0.2, data not shown), while monthly mean 3- -1 0 PO4 concentrations ranged from 0.48 Ϯ 0.17 mmol L in (b) July to 1.2 Ϯ 0.71 mmol L-1 in December, also with no sig- 4 nificant seasonal variation (P > 0.15, data not shown). The thallus C content was highest in F. spiralis, with over 3 40% of dry mass as C in July (Fig. 2a), but C content did not change significantly through the year in F.vesiculosus and F. serratus (P > 0.05) (Fig. 2a). In contrast, the C content of L. 2 digitata was significantly lower in February than during < July–August (P 0.004), and was always lower throughout N content (% DW) 1 the year than in the Fucus species. The N content [as % dry weight (DW)] varied with season in all species (P < 0.001, Fig. 2). The N content (% DW) was highest in the winter 0 (c) months, decreased over the spring with minima during 60 summer, and increased slightly during autumn (Fig. 2b), )

- –1 50 with a similar pattern to that for NO3 availability and NRA (Fig. 1). The highest thallus N contents were recorded in L. 40 digitata during January–May, whereas the lowest were in F. spiralis in August. Consequently, L. digitata showed the 30 lowest C : N ratio during winter and F.spiralis the highest in summer (Fig. 2c). In L. digitata, F. vesiculosus and F. serra- 20

tus, NRA was negatively correlated with thallus C : N ratio C:N ratio (mol mol and positively correlated with %N (Fig. 3).When compared 10 using Pearson correlation coefﬁcients, the correlation 0 between NR and %N was not signiﬁcantly stronger than the correlation between NR and C : N (P > 0.37 for all JFMAMJJASOND species; r values shown in Figs 3a,b). - + Figure 2. Seasonal changes in internal C content (a), N content Internal pools of NO3 and NH4 in the thallus tissue for (b), both as % dry mass, and molar C : N ratio (c) of the thalli of the four species varied over the year (one-way ANOVA, four species of brown algae. Points are means Ϯ SD, n Ն 4. The + - P < 0.001 for each species for both NH4 and NO3 ; Fig. 4). species symbols are the same for (a), (b) and (c). DW, dry weight. - 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- Internal NO3 storage far exceeded internal NH4 concen- - - tata stored more NO3 than F. serratus and F. spiralis.The trations (Fig. 4a–d; note different scales for NO3 and - + + internal NO3 concentration was most variable in L. digi- NH4 ). The highest internal NH4 concentrations were - -1 tata which stored over 80 mmol NO3 g thallus fresh mass observed in the thalli during the summer and the lowest - -1 in March, but <2 mmol NO3 g during July–September during the winter, which was opposite the trend for inter- - - (Fig. 4a). Variations in internal NO3 concentration in nal NO3 concentrations. When the total inorganic N - + Fucus species were less marked, except for F. vesiculosus, content (NO3 + NH4 ) from Fig. 4 was compared to the - -1 which had 83 mmol NO3 g in February, but less than total N content from Fig. 2b, inorganic N accounted for a - -1 20 mmol NO3 g for the rest of the year (Fig. 4b). Fucus maximum of 3.2% of the total thallus N in L. digitata in - serratus showed elevated internal NO3 concentration March, 3.8% in F. vesiculosus in February and 2.3% in F. - -1 during January–March (maximum 42 mmol NO3 g ), but serratus in January. - -1 - less than 10 mmol NO3 g from April to October (Fig. 4c). NO3 concentration in the thalli increased with increas- - - Fucus spiralis stored the lowest concentration of NO3 and ing external NO3 concentration. In L. digitata and F. + - NH4 , but showed a similar trend with higher internal vesiculosus, the internal NO3 concentration was nearly 10 - NO3 concentrations in winter than in summer (Fig. 4d). times the external concentration during the peak storage © 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd, Plant, Cell and Environment, 30, 764–774 768 E. B. Young et al.

(a) All the macroalgae showed higher net photosynthesis 300 rates in summer than in winter (Table 1). Dark respiration F. serratus, r = – 0.81, P < 0.003 F. vesiculosus, r = – 0.70, P < 0.025 accounted for between 18 and 40% of net daytime photo- 250 L. digitata, r = – 0.76, P < 0.015

) synthesis rates, with higher respiration rates relative to pho- –1 tosynthesis in summer than in winter. When NRA was used g

–1 200 as an estimate of maximum N assimilation rate, and com-

min pared to C fixation rates calculated from maximum net – 150 3 photosynthetic oxygen evolution rates for each species NRA 100 during the winter and summer periods (Table 1), the estimated N assimilation capacity was 0.7–50.0 times the esti- (nmol NO 50 mated C fixation rate for each species. The ratios of estimated C fixation to N assimilation capacity were higher 0 in summer than in winter as the low winter photosynthesis 0 10203040 - rates coincided with high NRA and thus higher NO3 C:N ratio (b)

300 F. vesiculosus, r = 0.83, P < 0.004 3.0 F. serratus, r = 0.824, P < 0.002 100 (a) Laminaria digitata L. digitata, r = 0.899, P < 0.0005 2.5

) 250

–1 80

g 2.0

–1 200 60 1.5 min

– 150 3

NRA 40 1.0 100 20 0.5

(nmol NO (nmol 50 0 0.0 0 100 (b) Fucus vesiculosus 2.5 1234 80 2.0 N content (% DW) FW) 60 1.5 FW) –1 –1 Figure 3. Seasonal nitrate reductase activity (NRA) plotted 40 against (a) internal C : N ratio and (b) N content [as % dry 1.0

weight (DW)] for Fucus vesiculosus, Fucus serratus and mol g 20 mol g 0.5 m Laminaria digitata. Species symbols are the same for (a) and (b). m

( 0 ( – Points are means Ϯ SD, n Ն 4. Pearson correlation coefﬁcients +

3 0.0 4 and P-values are included. 100 (c) Fucus serratus 2.5 80 - 2.0 period (winter, Fig. 5a). Internal NO3 concentration - 60 increased exponentially as ambient NO3 concentration 1.5 Internal NO -1 Internal NH increased above 7.5 mmol L (January–March, Fig. 1b). In 40 L. digitata, in April and May, there was an increase in 1.0 - - -1 20 NO3 pools despite low external NO3 (2–4 mmol L ) 0.5 in those months (Fig. 5a). In F. vesiculosus, 67% of the 0 seasonal variation in NRA could be explained by external 0.0 100 - (d) NO3 concentration (regression analysis, r = 0.817, Fucus spiralis 2.5 P < 0.002; Fig. 5b), but there was no significant correlation 80 - 2.0 between NRA and internal NO3 storage (r = 0.63, 60 P > 0.05; Fig. 5c). In F. serratus, NRA was correlated with 1.5 - < both external NO3 (r = 0.82, P 0.002; Fig. 5b) and inter- 40 - 1.0 nal NO3 concentration (r = 0.69, P < 0.02; Fig. 5c). In L. - 20 digitata, NRA was also correlated with external NO3 0.5 - (r = 0.64, P < 0.05; Fig. 5b) and internal NO3 concentra- 0 tion (r = 0.68, P < 0.03; Fig. 5c). There was no statistical dif- 0.0 ference in the correlations between NR and internal JFMAMJJASOND versus external NO - concentration for F. serratus and L. 3 Figure 4. Seasonal variation in internal thallus concentration of digitata (P > 0.43 for both species – Pearson correlation - + NO3 (filled circles) and NH4 (open triangles) for the four - coefficient comparison). species of brown algae. Note the different scales for NO3 and + NH4 . Points are means Ϯ SD, n Ն 4. FW, fresh weight. © 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd, Plant, Cell and Environment, 30, 764–774 exp(0.5454 fo i.1)adtalsitra NO internal thallus and 1b) Fig. (from NO bv 7.5 above line, la,adterltosi ewe hlu R n internal and NRA brown thallus NO of between species relationship four the in and algae, (b) (NRA) activity reductase nitrate n r h aefr() b n c.Creaincefiinsand coefficients Correlation (c). and (b) P (a), for same the are xoeta oesfitdb es qae ersinabove regression squares 4.5 least by fitted models exponential serratus Fucus ora oplto 07BakelPbihn Ltd, Publishing Blackwell 2007 © compilation Journal Authors The 2007 © 5. Figure vle r nlddfraltedt for data the all for included are -values Ն NRA NRA m 3 3

.F,fehweight. fresh FW, 4. – –1 –1 - -

r – –1 Mfor – –1 –1

int m

2 (nmol NO min g FW) ocnrto c vrtesaoa yl.Seissymbols Species cycle. seasonal the over (c) concentration 3 (nmol NO3 min g FW) Internal NO3 ( mol g FW) = = 100 200 300 100 200 300 100 .5 NO 0.85; 7E 20 40 60 80 m 0 0 0 .digitata L. ¥ Mfor h eainhpbtenetra NO external between relationship The - 0246810 7 NO 204060801000 F. vesiculosus: F. vesiculosus: L. digitata: F. serratus: ¥ and exp(1.979 3 - 3 .vesiculosus F. - ext int aiai digitata Laminaria ] bv 1.5 above )], F. spiralis digitata, L. F. serratus, F. vesiculosus, = ge line, [grey Internal NO 1.386

r F. serratus F. serratus

F. vesiculosus L. digitata

r =

¥ = External NO

0.64, 0.64,

r NO 0.823,

¥ =

0.817, exp(0.342 3 dse line, [dashed - P r r m ext r 2

< 0.05 <

= = P Mfor = r

] l onsaemeans are points All )].

0.873, < 0.002 < 3 r = 0.871, 0.871, r .4 NO 0.94;

– =

( P uvsi a are (a) in Curves .

= 0.683,

3 r 0.691,

< 0.002 < - 0.608, 0.608,

m = 3 ocnrtos()and (a) concentrations ¥ .serratus F. mol g

uu vesiculosus Fucus – 0.631, 0.631, ( P NO P

P m

< < r P < 0.03 < 3

mol L 2 -

P 0.0005 3 0.0005 int < 0.02 - = –1

ext P > FW) 0.96; = < 0.05

]and )] 0.05 0.05 3 0.549 - [dash-dot –1 availability ) ln,Cl n Environment, and Cell Plant, ¥ , Ϯ (c) (b) (a) SD,

Table 1. Respiration and net photosynthesis rates of brown algae from Strangford Lough, ‘The Narrows’ region and comparison of N assimilation capacity values estimated from monthly nitrate reductase activities (NRAs) of Laminaria digitata, Fucus vesiculosus and Fucus serratus 30, a b c Dark respiration Net photosynthesis Daily net C ﬁx capacity N assimilation capacity C : N assimilation ratio C : N in tissue algae brown in metabolism N Seasonal 764–774 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 mmol O2 g h ) mmol O2 g h ) mmol C g d ) mmol N g d ) (mol mol ) (mol mol )

Species Summer Winter Summer Winter Summer Winter Summer Winter Summer Winter Summer Winter

F. serratus 4.66 1.16 12.5 3.99 122 15.4 374 795 0.325 0.0194 19.4 13.0 F. vesiculosus 3.53 0.969 9.65 5.17 94.1 26.7 326 1315 0.289 0.0203 24.4 17.3 L. digitata 3.97 1.41 9.58 7.22 90.0 36.6 65 67 1.37 0.545 22.3 9.67 Fucus spiralisd 3.53 0.969 9.65 5.17 94.1 26.7 236 863 0.399 0.0309 38.6 18.0

Summer data are values averaged over June–September, and winter data are averages of November–March values. a Calculated using daytime photosynthesis and dark respiration values; net oxygen evolution estimates based on 15 h daylight (summer) and 9 h daylight (winter). O2 evolution was converted

to C ﬁxation using a photosynthetic quotient (mole O2 evolved per mole C ﬁxed) of 1.2. bN assimilation capacity estimates from mean nitrate reductase (NR) values averaged for summer (June–September) and winter (November–March) months, from Fig. 1a. For Fucus species, - - in which nitrate reductase activity (NRA) is not suppressed in the dark, NO3 assimilation is assumed to occur 24 h a day, while in L. digitata, NRA is suppressed in darkness, so NO3 assimilation was only calculated to occur during daylight hours (Young et al. unpublished results). cTissue C : N data from Fig. 2.

dPhotosynthesis rates for F. spiralis were not measured and were based on those for F. vesiculosus. 769 770 E. B. Young et al.

- assimilation capacity (Table 1). This was more pronounced stored in the vacuole, a major site of NO3 storage in higher - for the Fucus species with at least a 16-fold summer-to- plants (Granstedt & Huffaker 1982), thus removing NO3 winter difference in C : N assimilation capacity ratio, but from the site of NRA in the cytosol. this difference was only fourfold in L. digitata. The lowest Total thallus N content (% DW) was correlated with - ratio of C : N content of thalli in winter was close to 10 in L. external NO3 in all species (Figs 1–2), as reported previ- digitata, and the highest in summer was >38 in F. spiralis ously for Laminaria saccharina (Wheeler & Weidner 1983). (Table 1). The total N content in all thalli were similar to previous reports of macroalgae (Hernández et al. 1993; McGlathery et al. 1996; Pedersen & Borum 1996; Brenchley, Raven & DISCUSSION Johnston 1998; Naldi & Wheeler 1999), although L. digitata Seasonal nitrate availability in Strangford Lough reached lower %N and higher C : N ratio in summer than L. saccharina in the English Channel - The NRA in four species of intertidal brown algae (Gevaert et al. 2001). Both thallus total N and NO3 were appeared to be most strongly regulated in response to depleted during the spring when vegetative growth was - - external NO3 availability; both NO3 and NRA showed likely to have accelerated (Chapman & Craigie 1977; marked seasonal variations with winter to early spring Lüning, Wagner & Buchholz 2000; Lehvo, Back & Kiirikki maxima. In a study of NRA in Scottish L. digitata by 2001), effectively diluting thallus N reserves (see McGlath- Davison et al. (1984), a similar seasonal pattern was ery et al. 1996). Reproduction is another sink for thallus N. observed, although maximum NRAs were observed later, in In F. vesiculosus, reproductive growth occurs in winter, April–June, which coincided with an April peak in water and gametes are released in May–June (Berger, Malm - NO3 concentrations, later than observed in Strangford & Kautsky 2001; Lehvo et al. 2001), coinciding with the - Lough (Fig. 1). Summer declines of external NO3 in observed decline in thallus N content. Laminaria digitata Strangford Lough can be attributed to increased macroalgal produces reproductive sori during the late autumn and and phytoplankton growth, commonly reported in other winter (Lüning et al. 2000). Winter reproductive growth in temperate habitats (e.g. Wheeler & Srivastava 1984; Peder- Fucus and Laminaria species is an additional sink for N sen & Borum 1996). NRA in phytoplankton declines during a period when growth is limited by other factors, and - rapidly when external NO3 is exhausted (Berges, Cochlan gamete release from Laminaria and Fucus represents a loss & Harrison 1995). In Strangford Lough, however, there was of N and C from the thallus, and together with N demands - always some external NO3 available, so that the summer of vegetative growth, may contribute to a spring decline in - decline in NRA was not due to lack of NO3 . The relative thallus N. Growth may become limited by N supply during 3- + - constancy of dissolved PO4 and NH4 , in contrast to NO3 , late summer (Chapman & Craigie 1977). suggests that N is the most limiting macronutrient, and that - NO3 is the dominant inorganic N source for phytoplankton Irradiance and macroalgal growth in Strangford Lough. - As external NO3 availability increased during the Winter imposes light limitation, and in situ photosynthesis - winter, the macroalgae stored more NO3 in the thallus rates varied most strongly with seasonal irradiance. - tissue (Fig. 4). However, when adjusting for mass However, although external NO3 concentrations were - [DW = 0.125 ¥ fresh weight (FW)], internal NO3 contrib- highest in winter, photosynthesis rates were lower, resulting uted only a small proportion (<3.9%) of total thallus N in a seasonal disjunction between periods of maximum N content. In these brown algae, significant N could be stored availability and photosynthesis (Table 1). Such disjunctions as protein, as seen in Laminaria solidungula (Pueschel & have been noted before, particularly at high latitude Korb 2001), although this was not the case in a green alga (Henley & Dunton 1997; Stengel & Dring 1997). C and N (McGlathery, Pedersen & Borum 1996). The rise in internal assimilation in algae are understood to be closely coupled - - NO3 as external NO3 concentration increased above (Gao et al. 1995; Vergara et al. 1998), but in these brown -1 - 7.5 mmol L (Fig. 5a) suggests that the uptake capacity of macroalgae, N uptake, internal NO3 storage and NRA - these macroalgae may not be saturated by ambient NO3 were inversely correlated with seasonal photosynthesis. In - concentrations in the Lough. Previously reported NO3 winter, when photosynthesis is light limited (Table 1; uptake kinetics for intertidal brown algae (Korb & Gerard Stengel & Dring 1997), assimilation of inorganic N into 2000; Phillips & Hurd 2004) suggests that, even in winter, amino acids may be limited by energy and fixed C supply, - -1 - NO3 concentrations were below the 20–60 mmol L rather than by NRA. Higher NO3 availability in winter required to saturate uptake.The relationship between inter- promotes maximum inorganic N uptake and assimilation, - nal NO3 concentration and NRA was unclear, but there for which elevated NRA rates are beneficial. However, as may be some saturation of NRA in relation to internal metabolic N demand and photosynthesis rates are low, - - NO3 concentration which varied with species (Fig. 5c).This there is significant winter storage of unassimilated NO3 by - also suggests that assimilation of internal thallus NO3 is not the brown algal species examined, particularly L. digitata.In - determined solely by NRA and, conversely,that NRA is not arctic L. solidungula,NO3 uptake kinetics also suggests a - - - strongly regulated by intracellular NO3 availability. If NO3 ‘storage specialist’ strategy to take up NO3 during winter - - is not immediately assimilated, significant NO3 may be when it is available and to store significant internal NO3 , © 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd, Plant, Cell and Environment, 30, 764–774 Seasonal N metabolism in brown algae 771 even at a time of low growth (Henley & Dunton 1995; Korb In this study, we measured NRA at a constant 12 °C & Gerard 2000). In temperate Macrocystis integrifolia, the throughout the year, a temperature that is close to the - maximum internal NO3 concentrations were 50–90 mmol average experienced by the algae when submerged, and - -1 NO3 g FW in winter (Wheeler & Srivastava 1984; Hurd unlikely to cause inactivation of the enzyme. The in vitro et al. 1996) and up to 150 mmol g-1 fresh mass in Laminaria assay at this single temperature means that the NRAs longicruris (Chapman & Craigie 1977). In more extreme reported in this study are probably underestimates of activ- - arctic conditions, Laminaria species showed spring NO3 ity when water is warmer, and overestimates when water is storage ~35 mmol g-1 DW (using a wet-to-dry mass conver- cooler (Young et al. 2005). Low temperatures may require a sion factor for arctic L. saccharina and L. solidungula of greater quantity of the enzyme to achieve the same catalytic 0.125; Henley, personal communication). These values are activity because the enzyme is working below its tempera- - -1 comparable to the ~40 mmol NO3 g FW observed in April, ture optimum. Therefore, elevated winter NR enzyme - -1 and the maximum of 85 mmol NO3 g FW in March in Irish activity may be a component of cold acclimation. Higher L. digitata (Fig. 4). activities of NR and other enzymes have been observed at - Despite the fact that NO3 assimilation requires energy lower temperatures in L. saccharina (Davison & Davison (and is thus closely coupled with photosynthesis and 1987) and in F. vesiculosus, where a similar seasonal pattern - enhanced by light), significant NO3 uptake by algae can of elevated enzyme activities in winter was observed occur during the dark, particularly when inorganic N is (Collén & Davison 2001). Therefore, low-temperature limiting (Cochlan, Harrison & Denman 1991; Korb & stimulation of activity may apply to several enzymes, not

Gerard 2000). Low winter irradiance in Ireland is sufficient just to NR. However, based on a Q10 of 2 (discussed by - to support NO3 uptake, but still limits photosynthesis and Berges et al. 2002) and a range between winter and summer growth (Table 1; Stengel & Dring 1997). Although NRA in temperature of ~10 °C (Fig. 1), one would predict a dou- macroalgae can respond rapidly to irradiance (e.g. Davison bling of NRA to compensate for lower winter temperature. & Stewart 1984; Gao et al. 1995; Lopes et al. 1997; Lartigue However, the seasonal range of NRA observed is much & Sherman 2002), and intertidal macroalgae in Northern greater than 2 (~6 times in both L. digitata and Fucus Ireland experience a fivefold change in maximum incident species). Although all NRA was assayed at 12 °C, in situ surface irradiance (PAR) from winter to summer (400– NRA in seaweeds will be influenced by temperature, pos- -2 -1 - 2000 mmol photons m s ), there is no evidence of suppres- sibly affecting the comparison between NO3 assimilation sion of NRA by low winter irradiance (Fig. 1). Because of rate (from NRA in Fig. 1) and photosynthesis rates, pre- longer immersion, L. digitata will experience lower daily sented in Table 1. However, when the seasonal water tem- photon doses than the higher intertidal algae, and NRA in perature range (7–17 °C, Fig. 1b) was taken into account, the intertidal–subtidal L. digitata was lower than in Fucus assuming a Q10 of 2, the patterns of summer–winter com- species. In a companion study, we found that daily NRA parison of C : N assimilation capacity in the four species did changes in Fucus species were insensitive to irradiance, and not change significantly (data not shown).This supports the that in L. digitata, NRA was suppressed in darkness, but idea that seasonal variation in NRA is at most only partially day–night differences were most pronounced in winter, due to temperature acclimation. Respiration rate is likely when daytime irradiance was lowest (Young, Dring & to be more temperature sensitive than photosynthesis, so Berges unpublished results). The lack of an autumn the lower respiration relative to photosynthesis rates - increase in NRA and internal NO3 concentration in L. observed in winter (Table 1) are probably related to lower digitata may be related to low irradiance in the intertidal– temperatures. subtidal region, or to the dynamics of low autumn growth and reproductive activity in that species (Gómez & Lüning Position in intertidal zone 2001). From previous studies, it is known that seasonal growth dynamics are different for Laminaria and Fucus Fucus species showed slightly different patterns of N species. Growth has been shown to be highest during the storage and NRA over the seasonal cycle to L. digitata, late winter–early summer in Laminaria species (Chapman which is likely to be related to habitat differences. Fucus & Craigie 1977; Davison et al. 1984; Henley & Dunton 1997; species growing higher in the intertidal zone showed higher Sjøtun, Fredriksen & Rueness 1998; Gómez & Lüning 2001) NRA than L. digitata, which could be an adaptation to more but in spring–summer for fucoid algae (Stengel & Dring prolonged tidal emersion (Murthy,Rao & Reddy 1986).The 1997; Brenchley et al. 1998; Lehvo et al. 2001). longer immersion time in the intertidal–subtidal margin - allows Laminaria to take up NO3 from the water for longer - Temperature each day and may explain the higher internal NO3 concentration in L. digitata during winter. Fucus species, growing The minimum water temperature (~7 °C) coincided with higher in the intertidal zone, are emersed and thus isolated - peak NRAs observed in March (Fig. 1), and low water tem- from the source of NO3 in the seawater, for longer each - perature in winter could influence NRA. The activity of day.This induces a temporal limitation for NO3 uptake, for enzymes, including NR, is temperature sensitive, with which higher uptake rates may compensate (Thomas et al. optimum temperatures for NRA in diverse algae measured 1987; Phillips & Hurd 2004). Laminaria digitata may also - in the range of 10–20 °C (Gao et al. 2000; Berges et al. 2002). store more NO3 than do the Fucus species because the © 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd, Plant, Cell and Environment, 30, 764–774 772 E. B. Young et al.

- NRA is lower, so that NO3 can be assimilated less rapidly Rumbley & Glibert 2000). However, in two macroalgal + after entering the cell, before it is stored (possibly in the species, negligible NH4 release was measured following - - - vacuole). Despite longer exposure to water column NO3 , NO3 uptake (Naldi & Wheeler 2002). Release of NO2 intertidal–subtidal L. digitata thalli experience lower irradi- can be difficult to assess experimentally because of rapid - - ance at greater water depth, particularly in winter when oxidation of NO2 to NO3 in oxic surface waters. - irradiance is limiting for growth of brown macroalgae 4 NO3 is taken up and assimilated into organic molecules (Stengel & Dring 1997). Winter C : N ratios in L. digitata that are excreted and/or ‘leaked’ from healthy or senes- were lower than in Fucus (Fig. 2c), and might have been a cent thalli (Naldi & Wheeler 2002; Fong, Fong & Fong consequence of higher N storage when higher ambient 2004; Tyler & McGlathery 2006). concentration of N was available, and/or low winter C NO - uptake and release as NO -,NH+ or organic N will content; lower C : N ratios in L. digitata could also be a 3 2 4 require greater NRA than may be needed to support N consequence of C depletion during light-limited growth. In incorporation, and may account for the excess NRAs rela- contrast, F. spiralis, which is found highest in the intertidal tive to rates of photosynthetic C fixation. In temperate near- zone, showed the lowest N storage and highest summer %C shore ecosystems dominated by brown macroalgal biomass, and C : N ratio (Fig. 2c). In a previous survey of intertidal algal NO - uptake and release as organic N could be signifi- algae, Thomas et al. (1987) also showed a lower %N and 3 cant for near-shore nutrient cycling (Duggins et al. 1989). It higher C : N ratio in thalli with increasing height in the is unknown how seaweeds control the fate of NO - that is intertidal zone. This trend may relate to the combination of 3 taken up; further exploration of this would contribute both increased irradiance but more restricted access to dissolved to an understanding of seaweed physiology, but would also inorganic N with height in the intertidal zone. clarify the importance and role of these algae in near-shore biogeochemical cycling. CONCLUSIONS AND IMPLICATIONS ACKNOWLEDGMENTS N uptake, N storage and NRA in temperate intertidal brown algae are likely to be most strongly regulated in E.B.Y. was supported by a postdoctoral Natural Environ- - response to external NO3 availability, although tempera- ment Research Council (NERC) UK grant (GR3/12454), ture acclimation may contribute to the seasonal variation in and measurements of macroalgal production in Strangford NRA, and light may influence N metabolism more indi- Lough were supported by NERC UK (GR3/9072). E.B.Y.is rectly via C metabolism. When NRA was used as an esti- grateful to the staff and students at Portaferry Marine mate of N assimilation capacity (see Davison et al. 1984), Laboratory for field support, particularly D. Rogers and M. Fucus species apparently had the capacity to assimilate Curran for help with boat collections. C and N analysis was between 2.5 and 50.0 times more N than C was fixed, and L. carried out by B. 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