Seasonal variations in nitrate reductase activity and internal N pools in intertidal brown algae are correlated with ambient nitrate concentrations

Authors

  • ERICA B. YOUNG,

    Corresponding author
    1. School of Biological Sciences, Queens University of Belfast, Lisburn Road, Belfast, Northern Ireland BT9 7BL, UK and
    2. Department of Biological Sciences, University of Wisconsin–Milwaukee, 3209 Maryland Avenue, Milwaukee, WI 53211, USA
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  • MATTHEW J. DRING,

    1. Department of Biological Sciences, University of Wisconsin–Milwaukee, 3209 Maryland Avenue, Milwaukee, WI 53211, USA
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  • GRAHAM SAVIDGE,

    1. Department of Biological Sciences, University of Wisconsin–Milwaukee, 3209 Maryland Avenue, Milwaukee, WI 53211, USA
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  • DARYL A. BIRKETT,

    1. Department of Biological Sciences, University of Wisconsin–Milwaukee, 3209 Maryland Avenue, Milwaukee, WI 53211, USA
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  • JOHN A. BERGES

    1. School of Biological Sciences, Queens University of Belfast, Lisburn Road, Belfast, Northern Ireland BT9 7BL, UK and
    2. Department of Biological Sciences, University of Wisconsin–Milwaukee, 3209 Maryland Avenue, Milwaukee, WI 53211, USA
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Erica B. Young. Fax: 1 414 229 3926; e-mail: ebyoung@uwm.edu

ABSTRACT

Nitrogen metabolism was examined in the intertidal seaweeds Fucus vesiculosus, Fucus serratus, Fucus spiralis and Laminaria digitata in a temperate Irish sea lough. Internal NO3- storage, total N content and nitrate reductase activity (NRA) were most affected by ambient NO3-, with highest values in winter, when ambient NO3- was maximum, and declined with NO3- during summer. In all species, NRA was six times higher in winter than in summer, and was markedly higher in Fucus species (e.g. 256 ± 33 nmol NO3- min−1 g−1 in F. vesiculosus versus 55 ± 17 nmol NO3- min−1 g−1 in L. digitata). Temperature and light were less important factors for N metabolism, but influenced in situ photosynthesis and respiration rates. NO3- assimilating capacity (calculated from NRA) exceeded N demand (calculated from net photosynthesis rates and C : N ratios) by a factor of 0.7–50.0, yet seaweeds stored significant NO3- (up to 40–86 µmol g−1). C : N ratio also increased with height in the intertidal zone (lowest in L. digitata and highest in F. spiralis), indicating that tidal emersion also significantly constrained N metabolism. These results suggest that, in contrast to the tight relationship between N and C metabolism in many microalgae, N and C metabolism could be uncoupled in marine macroalgae, which might be an important adaptation to the intertidal environment.

INTRODUCTION

Temperate brown macroalgae form highly productive communities, accounting for the majority of primary production in many coastal regions and dominating near-shore nutrient cycling (Duggins, Simenstad & Estes 1989). For example, in Strangford Lough, Northern Ireland, macroalgae account for 98% of algal biomass and 95% of productivity (Birkett, Dring & Savidge, unpublished results). The vast majority of the macroalgal biomass in the Lough is fucoid algae (Fucus and Ascophyllum species) and kelps (Laminaria species).

Seaweeds in temperate habitats such as Strangford Lough experience large seasonal changes in temperature, irradiance and nutrient concentration that impose constraints on their physiology. Furthermore, variations in tidal emersions also affect nutrient availability and irradiance on a scale of hours to weeks and often result in a disjunction between the optimal light, nutrient availability and temperature for growth. Brown algal species growing at different heights in the intertidal zone experience distinct irradiance and emersion regimes that may influence the regulation of nutrient acquisition and assimilation (e.g. Thomas, Turpin & Harrison 1987; Phillips & Hurd 2004). Identifying responses of macroalgae to daily and seasonal fluctuations in irradiance, temperature and nitrogen availability is thus critical in understanding the regulation of nitrogen metabolism and the role of these productive macroalgae in near-shore nutrient cycling.

Simple measurements of nutrient uptake are difficult to make in intertidal species, and can be biased by many factors. Alternatively, enzyme activities offer more integrative measures, less biased by instantaneous conditions. Nitrate reductase [NR, enzyme class (EC) 1.6.6.1] is often considered the rate-limiting enzyme in inorganic N assimilation by algae and is thus a key enzyme in N metabolism. Nitrate reductase activity (NRA) is strongly correlated with N incorporation rates in macroalgae (Davison, Andrews & Stewart 1984). NRA in cultured algae is known to be stimulated by NO3- (Gao, Smith & Alberte 1995; Lartigue & Sherman 2005), and is regulated by light with rapid suppression of NRA in darkness in most algae studied (e.g. Davison & Stewart 1984; Gao et al. 1995; Lopes, Oliveira & Colepicolo 1997; Vergara, Berges & Falkowski 1998; Lartigue & Sherman 2002). NRA is also known to be responsive to changes in temperature with narrow temperature range for optimum NRA (Gao, Smith & Alberte 2000; Berges, Varela & Harrison 2002). Therefore, in this study, we monitored NRA as an indicator of changes in nitrate metabolism in response to these key environmental variables.

Changes in NRA over a seasonal cycle have been examined in very few macroalgae. To evaluate the factors influencing seasonal changes in N assimilation by macroalgae, it is important to also examine external nutrient availability, irradiance and temperature, as well as internal N storage, allfactors that may play a role in the regulation of NRA. If external availability of nitrate is the most important factor regulating uptake and assimilation, then NRA and internal nitrate storage will be closely related to seasonal changes in nitrate concentration. N metabolism in algae is closely linked to photosynthetic C metabolism (e.g. Vergara et al. 1998), and if irradiance is the most important factor influencing N metabolism, then NR and internal N storage will be highest in summer when photosynthesis is not constrained by irradiance. Temperature is also an important seasonal environmental variable influencing metabolism and may influence nitrate uptake, storage and NRA.

The aims of the present study were to evaluate the importance of environmental variables influencing N metabolism in intertidal brown algae by making concurrent measurements of NRA, total thallus N content, inorganic N storage and photosynthesis, and by comparing these with seasonal nitrate and light availability and temperature in a strongly 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 the lower intertidal–subtidal zone, and hence is immersed longer and experiences greater light attenuation with water depth, compared with the intertidal Fucus species, Fucus vesiculosus and Fucus serratus, which grow in the mid-intertidal zone, and Fucus spiralis, which is found even higher in the intertidal zone and is emersed for long periods during each tidal cycle. In these four brown algal species, we observed strong seasonal patterns in NRA and internal N storage that closely correlate with seasonal changes in nitrate availability. In addition, the relationship between C fixation and N assimilation capacity changes several-fold between 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 the shore at low tide, and L. digitata was sampled near the middle of the day at low tide from the shore or from a boat, as maximum activity was shown to occur during the middle of the day in L. digitata (Davison et al. 1984). Laminaria digitata were sampled by cutting ∼30 mm diameter discs out of the thalli, avoiding the meristematic region and the oldest tissue (see Davison & Stewart 1984). Thallus tips were cut from Fucus thalli by removing 30–40-mm-long terminal tips (trials showed activity was highest in tips). Within a few minutes of all sampling times, tissue samples were thoroughly blotted dry, frozen and stored in liquid N2 for later analysis of NRA. Within a few minutes of sampling on the shore, the tissue was thoroughly blotted dry and frozen and stored in liquid N2 for later analysis.

Light, temperature and nitrate data

Surface-incident photosynthetically active radiation (PAR) was measured using a 2π light sensor (Li-Cor, Lincoln, NE, USA) during 2001 and 2002 (Marine Laboratory Database, Queen's University of Belfast 2002). Temperature was measured in surface (∼1 m depth) seawater at The Narrows site [Agri-Food and Biosciences Institute (AFBI) Database 2006]. Nitrate concentration in the Lough was measured in surface samples collected from The Narrows site and was analysed using standard methods (Parsons, Maita & Lalli 1984). Samples for long-term nutrient concentration data were collected during years 1974–1976, 1986–1987 and 1990–1991. Samples were also collected during the study period (2001–2002) to verify the earlier published seasonal NO3- concentration data. Triplicate samples from The Narrows were collected and analysed from single sampling periods over the years 1994–1995 (Service et al. 1996) and 2004–2005 (AFBI Database 2006).

NRA

NRA was estimated using an in vitro assay method described by Young et al. (2005). Frozen thallus samples were ground to a powder in liquid nitrogen and extracted in 200 mmol L−1 potassium phosphate buffer pH 7.9 containing 5 mmol L−1 Na2 ethylenediaminetetraacetic acid (EDTA), 0.3% (w/v) insoluble polyvinyl pyrollidone, 2 mmol L−1dl-dithiothreitol, 3% (w/v) bovine serum albumin (Fraction V) and 1% (v/v) Triton X-100 (all Sigma, St Louis, MO, USA). The assay mixture contained 200 mmol L−1 sodium phosphate buffer pH 7.9 with 200 µmol L−1 NADH (β form, Sigma), 20 µmol L−1 flavin adenine dinucleotide (Sigma), 20% volume as algal extract and 10 mmol L−1 KNO3. The assay was incubated at 12 °C and the reaction terminated by the addition of 1 M zinc acetate. NO2- concentration was measured spectrophotometrically in centrifuged supernatants (Parsons et al. 1984), and activity estimated by linear regression of increasing NO2- concentration over time.

Internal nitrogen pools and tissue N and C content

Several methods used to extract internal inorganic nutrient pools from algae were tested: boiling thallus discs in water for 20 min (Hurd, Harrison & Druel 1996), boiling water added to ground algal tissue, vortexed and incubated for 10 min (after Thoresen, Dortch & Ahmed 1982), boiling thallus pieces in water for 10 min followed by overnight extraction at 4 °C (Naldi & Wheeler 1999) and room temperature ethanol extraction of ground tissue overnight (McGlathery, Pedersen & Borum 1996). The highest internal inorganic N concentrations were obtained by adding 20 mL room temperature Milli-Q water (Millipore, Watford, UK) to samples of ∼50 mg frozen ground thallus tissue in boiling tubes that were vortexed and then placed in a boiling water bath for 45 min, cooled to room temperature, filtered through Whatman GF/A filters (Brent, Middlesex, UK) and stored on ice. Thus, this method was used for all subsequent analyses. The concentrations of NO2-, NO3- and NH4+ were measured in the filtrate within 2 h, without freezing the samples. NO3- was analysed by Cd-column reduction followed by spectrophotometric measurement 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 thallus samples were ground in liquid N2 and dried in a 60 °C oven with desiccant and analysed in a Carlo Erba 1500 NC elemental analyser (CE Elantech, Lakewood, NJ, USA) using acetanilide as a standard and expressing contents as a proportion of dry mass.

Photosynthesis, respiration and N and C assimilation capacity

Photosynthesis and dark respiration rates were also measured during winter (November–March) and summer (June–September) months in F. vesiculosus, F. serratus and L. digitata using large (30 cm diameter × 1 m long) clear plastic chambers suspended 2 m below the Lough surface, containing the whole thalli of a single species. The water in the chambers was continuously circulated by a water pump. A YSI 6000 probe (YSI Inc., Yellow Springs, OH, USA) measuring dissolved oxygen, salinity and temperature was inserted into the chambers and recorded values for these variables at 15 s intervals over 2–3 days. The values were subsequently downloaded, and net oxygen exchange rates over successive 15 min periods were calculated from the oxygen concentrations. Mass-specific net photosynthetic rates for F. vesiculosus, F. serratus and L. digitata were calculated for complete 24 h periods, and the O2 evolution rates were converted to C fixation rates using a photosynthetic 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 groups of monthly values relating to the summer (June–September) and winter (November–March) periods.

Data analysis

Relationships between parameters were examined using Pearson product–moment correlation and analysis of variance (ANOVA) (SigmaPlot version 9.04 and SigmaStat version 3.1; Systat Software Inc., Chicago, IL, USA). Correlation coefficients were compared using Fisher transformations (Zar 1999).

RESULTS

In the three species of Fucus examined and in L. digitata, there were marked differences in NRA at different times in the year (one-way ANOVA, P < 0.001 for each species; Fig. 1a). NRA was highest during the winter and early spring months, peaking in March for F. serratus and F. vesiculosus and in April for L. digitata. Fucus spiralis was not sampled every month, but showed a similar trend to the 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 highest NRA observed in Fucus species (256 ± 33 nmol NO3- 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 different axes scales). The frozen and fresh mass differed less than 1% for all thalli (data not shown) so scaling to fresh or frozen mass will be very similar. The lowest activities in all species were observed in August. In L. digitata, there was no 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.

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 The Narrows, Strangford Lough. Points are means ± SD, n ≥ 4. Note different scale on right-hand side for activity in Laminaria digitata; activity in all Fucus species is plotted on the left-hand axis. For external NO3- concentrations (black triangles), points are means ± 95% confidence limits for 17–33 replicate samples from years 1974–1976, 1986–1987 and 1990–1991. Additional data sets are plotted for 1994–1995 (unfilled triangles), 2001–2002 (unfilled circles) and 2004–2005 (grey triangles), and symbols are means of at least two replicates, ±SD (AFBI 2006). The temperature plot includes a point every 3 h for the year 2000 (AFBI 2006). FW, fresh weight.

The seasonal variation in NRAs was negatively correlated with seawater temperature, which was lowest in February–March (Fig. 1b). Mean water temperature in Strangford Lough is in the range 8 °C (January–February) to 16 °C (July–September) (Fig. 1b; 7–17 °C reported by Stengel & Dring 1997). Seasonal variation in NRA was positively correlated with average water column NO3- concentrations (measured over the period 1974–2005), which peaked during the winter months and were lowest in July (Fig. 1b). NO3- concentrations measured on additional water samples collected during the study period (1999–2002) were 2–8 µmol L−1, therefore, within the ranges shown in Fig. 1b. NO2- concentrations were <0.5 µmol L−1 at all 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 µmol L−1 with no significant changes through the year (P > 0.2, data not shown), while monthly mean PO43− concentrations ranged from 0.48 ± 0.17 µmol L−1 in July to 1.2 ± 0.71 µmol L−1 in December, also with no significant seasonal variation (P > 0.15, data not shown).

The thallus C content was highest in F. spiralis, with over 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. digitata was significantly lower in February than during July–August (P < 0.004), and was always lower throughout 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 months, decreased over the spring with minima during summer, 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 the lowest C : N ratio during winter and F. spiralis the highest in summer (Fig. 2c). In L. digitata, F. vesiculosus and F. serratus, NRA was negatively correlated with thallus C : N ratio and positively correlated with %N (Fig. 3). When compared using Pearson correlation coefficients, the correlation between NR and %N was not significantly stronger than the correlation between NR and C : N (P > 0.37 for all species; r values shown in Figs 3a,b).

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 of four species of brown algae. Points are means ± SD, n ≥ 4. The species symbols are the same for (a), (b) and (c). DW, dry weight.

Figure 3.

Seasonal nitrate reductase activity (NRA) plotted against (a) internal C : N ratio and (b) N content [as % dry weight (DW)] for Fucus vesiculosus, Fucus serratus and Laminaria digitata. Species symbols are the same for (a) and (b). Points are means ± SD, n ≥ 4. Pearson correlation coefficients and P-values are included.

Internal pools of NO3- and NH4+ in the thallus tissue for the 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. digitata stored more NO3- than F. serratus and F. spiralis. The internal NO3- concentration was most variable in L. digitata which stored over 80 µmol NO3- g−1 thallus fresh mass in March, but <2 µmol NO3- g−1 during July–September (Fig. 4a). Variations in internal NO3- concentration in Fucus species were less marked, except for F. vesiculosus, which had 83 µmol NO3- g−1 in February, but less than 20 µmol NO3- g−1 for the rest of the year (Fig. 4b). Fucus serratus showed elevated internal NO3- concentration during January–March (maximum 42 µmol NO3- g−1), but less than 10 µmol NO3- g−1 from April to October (Fig. 4c). Fucus spiralis stored the lowest concentration of NO3- and NH4+, but showed a similar trend with higher internal NO3- concentrations in winter than in summer (Fig. 4d). Internal NO3- storage far exceeded internal NH4+ concentrations (Fig. 4a–d; note different scales for NO3- and NH4+). The highest internal NH4+ concentrations were observed in the thalli during the summer and the lowest during the winter, which was opposite the trend for internal NO3- concentrations. When the total inorganic N content (NO3- + NH4+) from Fig. 4 was compared to the total N content from Fig. 2b, inorganic N accounted for a maximum of 3.2% of the total thallus N in L. digitata in March, 3.8% in F. vesiculosus in February and 2.3% in F. serratus in January.

Figure 4.

Seasonal variation in internal thallus concentration of NO3- (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.

NO3- concentration in the thalli increased with increasing external NO3- concentration. In L. digitata and F. vesiculosus, the internal NO3- concentration was nearly 10 times the external concentration during the peak storage period (winter, Fig. 5a). Internal NO3- concentration increased exponentially as ambient NO3- concentration increased above 7.5 µmol L−1 (January–March, Fig. 1b). In L. digitata, in April and May, there was an increase in NO3- pools despite low external NO3- (2–4 µmol L−1) in those months (Fig. 5a). In F. vesiculosus, 67% of the seasonal variation in NRA could be explained by external NO3- concentration (regression analysis, r = 0.817, P < 0.002; Fig. 5b), but there was no significant correlation between NRA and internal NO3- storage (r = 0.63, P > 0.05; Fig. 5c). In F. serratus, NRA was correlated with both external NO3- (r = 0.82, P < 0.002; Fig. 5b) and internal 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- concentration (r = 0.68, P < 0.03; Fig. 5c). There was no statistical difference in the correlations between NR and internal versus external NO3- concentration for F. serratus and L. digitata (P > 0.43 for both species – Pearson correlation coefficient comparison).

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 brown algae, and the relationship between thallus NRA and internal NO3- concentration (c) over the seasonal cycle. Species symbols are the same for (a), (b) and (c). Correlation coefficients and P-values are included for all the data for Fucus vesiculosus, Fucus serratus and Laminaria digitata. Curves in (a) are exponential models fitted by least squares regression above 4.5 µM for L. digitata[grey line, r2 = 0.94; NO3-int = 0.549 × exp(0.5454 × NO3-ext)], above 1.5 µM for F. serratus[dash-dot line, r2 = 0.85; NO3-int = 1.386 × exp(0.342 × NO3-ext)] and above 7.5 µM 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.

All the macroalgae showed higher net photosynthesis rates in summer than in winter (Table 1). Dark respiration accounted for between 18 and 40% of net daytime photosynthesis rates, with higher respiration rates relative to photosynthesis in summer than in winter. When NRA was used as an estimate of maximum N assimilation rate, and compared to C fixation rates calculated from maximum net photosynthetic oxygen evolution rates for each species during the winter and summer periods (Table 1), the estimated N assimilation capacity was 0.7–50.0 times the estimated C fixation rate for each species. The ratios of estimated C fixation to N assimilation capacity were higher in summer than in winter as the low winter photosynthesis rates coincided with high NRA and thus higher NO3- assimilation capacity (Table 1). This was more pronounced for the Fucus species with at least a 16-fold summer-to-winter difference in C : N assimilation capacity ratio, but this difference was only fourfold in L. digitata. The lowest ratio 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).

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
SpeciesDark respiration
µmol O2 g−1 h−1)
Net photosynthesis
µmol O2 g−1 h−1)
Daily net C fix capacitya
µmol C g−1 d−1)
N assimilation capacityb
µmol N g−1 d−1)
C : N assimilation ratio
(mol mol−1)
C : N in tissuec
(mol mol−1)
SummerWinterSummerWinterSummerWinterSummerWinterSummerWinterSummerWinter
  • 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.

  • b

    N 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).

  • c

    Tissue C : N data from Fig. 2.

  • d

    Photosynthesis rates for F. spiralis were not measured and were based on those for F. vesiculosus.

F. serratus4.661.1612.53.9912215.43747950.3250.019419.413.0
F. vesiculosus3.530.9699.655.1794.126.732613150.2890.020324.417.3
L. digitata3.971.419.587.2290.036.665671.370.54522.39.67
Fucus spiralisd3.530.9699.655.1794.126.72368630.3990.030938.618.0

DISCUSSION

Seasonal nitrate availability

The NRA in four species of intertidal brown algae appeared to be most strongly regulated in response to external NO3- availability; both NO3- and NRA showed marked seasonal variations with winter to early spring maxima. In a study of NRA in Scottish L. digitata by Davison et al. (1984), a similar seasonal pattern was observed, although maximum NRAs were observed later, in April–June, which coincided with an April peak in water NO3- concentrations, later than observed in Strangford Lough (Fig. 1). Summer declines of external NO3- in Strangford Lough can be attributed to increased macroalgal and phytoplankton growth, commonly reported in other temperate habitats (e.g. Wheeler & Srivastava 1984; Pedersen & Borum 1996). NRA in phytoplankton declines rapidly when external NO3- is exhausted (Berges, Cochlan & Harrison 1995). In Strangford Lough, however, there was always some external NO3- available, so that the summer decline in NRA was not due to lack of NO3-. The relative constancy of dissolved PO43− and NH4+, in contrast to NO3-, suggests that N is the most limiting macronutrient, and that NO3- is the dominant inorganic N source for phytoplankton and 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- contributed only a small proportion (<3.9%) of total thallus N content. In these brown algae, significant N could be stored as 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 internal NO3- as external NO3- concentration increased above 7.5 µmol L−1 (Fig. 5a) suggests that the uptake capacity of these macroalgae may not be saturated by ambient NO3- concentrations in the Lough. Previously reported NO3- uptake kinetics for intertidal brown algae (Korb & Gerard 2000; Phillips & Hurd 2004) suggests that, even in winter, NO3- concentrations were below the 20–60 µmol L−1 required to saturate uptake. The relationship between internal NO3- concentration and NRA was unclear, but there may be some saturation of NRA in relation to internal NO3- concentration which varied with species (Fig. 5c). This also suggests that assimilation of internal thallus NO3- is not determined solely by NRA and, conversely, that NRA is not strongly 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 previously for Laminaria saccharina (Wheeler & Weidner 1983). 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 & Johnston 1998; Naldi & Wheeler 1999), although L. digitata in Strangford Lough reached lower %N and higher C : N ratio in summer than L. saccharina in the English Channel (Gevaert et al. 2001). Both thallus total N and NO3- were depleted during the spring when vegetative growth was likely to have accelerated (Chapman & Craigie 1977; Lüning, Wagner & Buchholz 2000; Lehvo, Back & Kiirikki 2001), effectively diluting thallus N reserves (see McGlathery 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 the observed decline in thallus N content. Laminaria digitata produces reproductive sori during the late autumn and winter (Lüning et al. 2000). Winter reproductive growth in Fucus and Laminaria species is an additional sink for N during a period when growth is limited by other factors, and gamete release from Laminaria and Fucus represents a loss of N and C from the thallus, and together with N demands of vegetative growth, may contribute to a spring decline in thallus N. Growth may become limited by N supply during late summer (Chapman & Craigie 1977).

Irradiance

Winter imposes light limitation, and in situ photosynthesis rates varied most strongly with seasonal irradiance. However, although external NO3- concentrations were highest in winter, photosynthesis rates were lower, resulting in a seasonal disjunction between periods of maximum N availability and photosynthesis (Table 1). Such disjunctions have been noted before, particularly at high latitude (Henley & Dunton 1997; Stengel & Dring 1997). C and N assimilation in algae are understood to be closely coupled (Gao et al. 1995; Vergara et al. 1998), but in these brown macroalgae, N uptake, internal NO3- storage and NRA were inversely correlated with seasonal photosynthesis. In winter, when photosynthesis is light limited (Table 1; Stengel & Dring 1997), assimilation of inorganic N into amino acids may be limited by energy and fixed C supply, rather than by NRA. Higher NO3- availability in winter promotes maximum inorganic N uptake and assimilation, for which elevated NRA rates are beneficial. However, as metabolic N demand and photosynthesis rates are low, there is significant winter storage of unassimilated NO3- by the brown algal species examined, particularly L. digitata. In arctic L. solidungula, NO3- uptake kinetics also suggests a ‘storage specialist’ strategy to take up NO3- during winter when it is available and to store significant internal NO3-, even at a time of low growth (Henley & Dunton 1995; Korb & Gerard 2000). In temperate Macrocystis integrifolia, the maximum internal NO3- concentrations were 50–90 µmol NO3- g−1 FW in winter (Wheeler & Srivastava 1984; Hurd et al. 1996) and up to 150 µmol g−1 fresh mass in Laminaria longicruris (Chapman & Craigie 1977). In more extreme arctic conditions, Laminaria species showed spring NO3- storage ∼35 µmol g−1 DW (using a wet-to-dry mass conversion factor for arctic L. saccharina and L. solidungula of 0.125; Henley, personal communication). These values are comparable to the ∼40 µmol NO3- g−1 FW observed in April, and the maximum of 85 µmol NO3- g−1 FW in March in Irish L. digitata (Fig. 4).

Despite the fact that NO3- assimilation requires energy (and is thus closely coupled with photosynthesis and enhanced by light), significant NO3- uptake by algae can occur during the dark, particularly when inorganic N is limiting (Cochlan, Harrison & Denman 1991; Korb & Gerard 2000). Low winter irradiance in Ireland is sufficient to support NO3- uptake, but still limits photosynthesis and growth (Table 1; Stengel & Dring 1997). Although NRA in macroalgae 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 Northern Ireland experience a fivefold change in maximum incident surface irradiance (PAR) from winter to summer (400–2000 µmol photons m−2 s−1), there is no evidence of suppression of NRA by low winter irradiance (Fig. 1). Because of longer immersion, L. digitata will experience lower daily photon doses than the higher intertidal algae, and NRA in the intertidal–subtidal L. digitata was lower than in Fucus species. In a companion study, we found that daily NRA changes in Fucus species were insensitive to irradiance, and that in L. digitata, NRA was suppressed in darkness, but day–night differences were most pronounced in winter, when daytime irradiance was lowest (Young, Dring & Berges unpublished results). The lack of an autumn increase 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 growth and reproductive activity in that species (Gómez & Lüning 2001). From previous studies, it is known that seasonal growth dynamics are different for Laminaria and Fucus species. Growth has been shown to be highest during the late 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 & Dring 1997; Brenchley et al. 1998; Lehvo et al. 2001).

Temperature

The minimum water temperature (∼7 °C) coincided with peak NRAs observed in March (Fig. 1), and low water temperature in winter could influence NRA. The activity of enzymes, including NR, is temperature sensitive, with optimum temperatures for NRA in diverse algae measured in the range of 10–20 °C (Gao et al. 2000; Berges et al. 2002). In this study, we measured NRA at a constant 12 °C throughout the year, a temperature that is close to the average experienced by the algae when submerged, and unlikely to cause inactivation of the enzyme. The in vitro assay at this single temperature means that the NRAs reported in this study are probably underestimates of activity when water is warmer, and overestimates when water is cooler (Young et al. 2005). Low temperatures may require a greater quantity of the enzyme to achieve the same catalytic activity because the enzyme is working below its temperature optimum. Therefore, elevated winter NR enzyme activity may be a component of cold acclimation. Higher activities of NR and other enzymes have been observed at lower temperatures in L. saccharina (Davison & Davison 1987) and in F. vesiculosus, where a similar seasonal pattern of elevated enzyme activities in winter was observed (Collén & Davison 2001). Therefore, low-temperature stimulation of activity may apply to several enzymes, not just to NR. However, based on a Q10 of 2 (discussed by Berges et al. 2002) and a range between winter and summer temperature of ∼10 °C (Fig. 1), one would predict a doubling of NRA to compensate for lower winter temperature. However, the seasonal range of NRA observed is much greater than 2 (∼6 times in both L. digitata and Fucus species). Although all NRA was assayed at 12 °C, in situ NRA in seaweeds will be influenced by temperature, possibly affecting the comparison between NO3- assimilation rate (from NRA in Fig. 1) and photosynthesis rates, presented in Table 1. However, when the seasonal water temperature range (7–17 °C, Fig. 1b) was taken into account, assuming a Q10 of 2, the patterns of summer–winter comparison of C : N assimilation capacity in the four species did not change significantly (data not shown). This supports the idea that seasonal variation in NRA is at most only partially due to temperature acclimation. Respiration rate is likely to be more temperature sensitive than photosynthesis, so the lower respiration relative to photosynthesis rates observed in winter (Table 1) are probably related to lower temperatures.

Position in intertidal zone

Fucus species showed slightly different patterns of N storage and NRA over the seasonal cycle to L. digitata, which is likely to be related to habitat differences. Fucus species growing higher in the intertidal zone showed higher NRA than L. digitata, which could be an adaptation to more prolonged tidal emersion (Murthy, Rao & Reddy 1986). The longer immersion time in the intertidal–subtidal margin allows Laminaria to take up NO3- from the water for longer each day and may explain the higher internal NO3- concentration in L. digitata during winter. Fucus species, growing higher in the intertidal zone, are emersed and thus isolated from the source of NO3- in the seawater, for longer each day. This induces a temporal limitation for NO3- uptake, for which higher uptake rates may compensate (Thomas et al. 1987; Phillips & Hurd 2004). Laminaria digitata may also store more NO3- than do the Fucus species because the NRA is lower, so that NO3- can be assimilated less rapidly after entering the cell, before it is stored (possibly in the vacuole). Despite longer exposure to water column NO3-, intertidal–subtidal L. digitata thalli experience lower irradiance at greater water depth, particularly in winter when irradiance is limiting for growth of brown macroalgae (Stengel & Dring 1997). Winter C : N ratios in L. digitata were lower than in Fucus (Fig. 2c), and might have been a consequence of higher N storage when higher ambient concentration of N was available, and/or low winter C content; lower C : N ratios in L. digitata could also be a consequence of C depletion during light-limited growth. In contrast, F. spiralis, which is found highest in the intertidal zone, showed the lowest N storage and highest summer %C and C : N ratio (Fig. 2c). In a previous survey of intertidal algae, Thomas et al. (1987) also showed a lower %N and higher C : N ratio in thalli with increasing height in the intertidal zone. This trend may relate to the combination of increased irradiance but more restricted access to dissolved inorganic N with height in the intertidal zone.

CONCLUSIONS AND IMPLICATIONS

N uptake, N storage and NRA in temperate intertidal brown algae are likely to be most strongly regulated in response to external NO3- availability, although temperature acclimation may contribute to the seasonal variation in NRA, and light may influence N metabolism more indirectly via C metabolism. When NRA was used as an estimate of N assimilation capacity (see Davison et al. 1984), Fucus species apparently had the capacity to assimilate between 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 measured C : N content ratios in the tissues of between 10 and 40 (Table 1). This suggests an uncoupling of N and C metabolism in the intertidal macroalgae that contrasts with the tight relationship between C and N metabolism reported for some microalgae (e.g. Gao et al. 1995; Vergara et al. 1998). Relative excess of N assimilation capacity may suggest that photosynthesis is significantly limited by resource availability. However, NRAs measured in vitro may overestimate in situ N assimilation capacity in algae exposed to low winter temperatures. Alternatively, these brown algae may require the higher NRA to reduce more NO3- than is actually required for growth and development. NO3- that is taken up by algae can have four distinct fates:

  • 1NO3- is reduced and assimilated into amino acids, which are incorporated into thallus growth (predominant fate during summer, periods of low external NO3- availability but potentially limited by supply of energy and C skeletons in winter).
  • 2NO3- is taken up but stored, possibly in the vacuoles, until thallus N reserves are depleted in spring–summer (an important fate in winter).
  • 3NO3- is taken up, reduced and excreted as NO2- and/or NH4+. In diatoms, reduced inorganic N excretion can represent >50% of NO3- uptake (Collos 1998; Lomas, Rumbley & Glibert 2000). However, in two macroalgal species, negligible NH4+ release was measured following NO3- uptake (Naldi & Wheeler 2002). Release of NO2- can be difficult to assess experimentally because of rapid oxidation of NO2- to NO3- in oxic surface waters.
  • 4NO3- is taken up and assimilated into organic molecules that are excreted and/or ‘leaked’ from healthy or senescent thalli (Naldi & Wheeler 2002; Fong, Fong & Fong 2004; Tyler & McGlathery 2006).

NO3- uptake and release as NO2-, NH4+ or organic N will require greater NRA than may be needed to support N incorporation, and may account for the excess NRAs relative 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 significant for near-shore nutrient cycling (Duggins et al. 1989). It is unknown how seaweeds control the fate of NO3- that is taken up; further exploration of this would contribute both to an understanding of seaweed physiology, but would also clarify the importance and role of these algae in near-shore biogeochemical cycling.

ACKNOWLEDGMENTS

E.B.Y. was supported by a postdoctoral Natural Environment Research Council (NERC) UK grant (GR3/12454), and measurements of macroalgal production in Strangford Lough were supported by NERC UK (GR3/9072). E.B.Y. is grateful to the staff and students at Portaferry Marine Laboratory for field support, particularly D. Rogers and M. Curran for help with boat collections. C and N analysis was carried out by B. Stewart at AFBI, Northern Ireland, with sample preparation assistance from D. Franklin. P. Boyd and L. Gilpin assisted in collection of the long-term nitrate concentration data. A. Mellor (AFBI, Northern Ireland) cheerfully facilitated access to Strangford Lough nutrient data, and temperature data was courtesy of R. Gowan and B. Stewart (AFBI).

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