Seasonal Variations in Nitrate Reductase Activity and Internal N Pools in Intertidal Brown Algae Are Correlated with Ambient Nitrate Concentrations
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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 inter- tidal 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).