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Effects of nitrogen availability on inorganic carbon concentrating mechanisms in marine phytoplankton Corkum, Miranda Elyse 2005

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EFFECTS OF NITROGEN AVAILABILITY ON INORGANIC CARBON CONCENTRATING MECHANISMS IN MARINE PHYTOPLANKTON by MIRANDA ELYSE CORKUM B.Sc, Mount Allison University, 2002 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Botany) THE UNIVERSITY OF BRITISH COLUMBIA APRIL 2005 © Miranda Elyse Corkum, 2005 ABSTRACT Carbon and nitrogen metabolism are tightly coupled through the enzyme RubisCO, but the effects of nitrogen limitation on cellular carbon acquisition are not well understood. This thesis reports measurements of RubisCO and phosphoenolpyruvate carboxylase (PEPC - an enzyme involved in the C 4 photosynthetic pathway) activity in Thalassiosira weissflogii grown under steady-state nitrogen limitation as well as data on the utilization of dissolved inorganic carbon species by this microalgae. These biochemical and physiological parameters were also examined across a natural nitrogen gradient in a field study of phytoplankton assemblages in the Queen Charlotte Sound coastal region. In nitrogen-limited T.weissflogii cultures, RubisCO activity decreased with decreasing growth rate (r=0.821; p<0.001), while PEPC activity remained unchanged over the range of growth rates tested (1.4 to 0.3d"1). / H C 0 3 " values, representing the fraction of dissolved inorganic carbon taken up in the form of HCO3", obtained from 1 4 C uptake experiments decreased from -0.85 to 0.70 as cells became more severely nitrogen-limited (0.682; p<0.001). Carbon uptake kinetics indicated that the decrease in / H C O 3 " was attributed to a decrease in direct HCO3" transport, as extracellular carbonic anhydrase activity appeared unaffected by changes in growth rate. A down-regulation of the activity of the carbon concentrating mechanism (CCM) may serve to control elemental ratios within the cell and maintain metabolic balance under nitrogen-limited conditions. At the seven coastal field stations sampled, there was no relationship between RubisCO activity and nitrate concentration, but a negative trend was observed between PEPC activity and nitrate concentration. The diatom-dominated phytoplankton assemblages studied all utilized HC0 3 "as an exogenous inorganic carbon source and HCO3" uptake occurred mainly via a direct transport mechanism. There was no apparent relationship between /HCO3" values and nitrate concentration at these stations. i i TABLE OF CONTENTS Abstract ii Table of Contents iii List of Tables v List of Figures vi List of Abbreviations vii Acknowledgements viii CHAPTER 1 General Introduction 1 1.1 RubisCO 1 1.2 Carbon Concentrating Mechanisms 2 1.3 Carbonic Anhydrase 3 1.4 CO2 permeability of algal membranes 4 1.5 C 4 photosynthesis 4 1.6 Review of Carbon Concentrating Mechanisms across taxa 5 1.7 Limiting nutrients and dissolved inorganic carbon acquisition 6 1.8 Atmospheric C 0 2 rise - ecological implications 7 1.9 Study organism 8 1.10 Field Studies 8 1.11 Thesis Objectives 10 CHAPTER 2 Effects of nitrogen limitation on inorganic carbon concentrating mechanisms in Thalassiosira weissflogii 11 2.1 Introduction 11 Limiting resources - implications for DIC acquisition 12 Carbon / nitrogen interactions 13 Study Goals and Hypotheses 13 2.2 Materials and Methods 14 Culture Conditions 14 Physiological Assays 16 2.3 Results 20 Growth Rate and Biochemical Parameters 20 RuBP-carboxylase / PEP-carboxylase activity 23 HCO3" vs. C 0 2 Uptake 25 2.4 Discussion 27 Biochemical parameters 27 Regulation of light-saturated photosynthesis - RubisCO 27 CCMs and nitrogen-use efficiency. 28 2.4.4 Summary 30 iii CHAPTER 3 Inorganic carbon acquisition in Queen Charlotte Sound phytoplankton assemblages 31 3.1 Introduction 31 Challenges associated with field studies 31 Importance of field studies 32 Study Goals and Hypotheses 33 3.2 Material and Methods 33 Field site 33 In-situ sampling and isotope disequilibrium experiments 36 3.3 Results 37 Biochemical Parameters 37 RuBP-carboxylase / PEP-carboxylase activity 38 HCOY vs. C 0 2 Uptake 40 3.4 Discussion , 42 RubisCO 42 C 4 photosynthesis 43 HC0 3 "VS C 0 2 uptake 44 Effect of nitrogen on CCM function 45 Summary 46 Final Conclusions 47 Future Research 48 References 49 Appendix A Aquil Recipe 58 Appendix B Permeabilization Buffer 59 LIST OF TABLES Table 2.1: Comparison of biochemical parameters between different nitrogen-limited chemostat culture growth rates and chemostat VS batch cultured cells 20 Table 3.1: Chemical and biological properties of sampling stations 37 Table 3.2: Data analysis of enzyme assays and C/N ratios 38 Table 3.3: Data analysis of isotope disequilibrium experiments and C/N ratios..40 LIST OF FIGURES Figure 2.1: Effect of N-limited growth rate on C/N ratios in T. weissflogii. Results presented for two separate experimental chemostat trials (circles = trial 1; triangles = trial 2) and for independent batch culture experiments (square). 22 Figure 2.2: Effect of N-limited growth rate on a) RubisCO (r=0.821; p<0.001) and b) PEPC activities in T. weissflogii. Results presented for two separate experimental trials (closed circles = trial 1; closed triangles = trial 2) and for independent batch-culture experiments (trial 1 = open circles; trial 2 = open triangles) 24 Figure 2.3: Effect of N-limited growth rate on a) / H C 0 3 " (r=0.682; p<0.001) and b) a (~CA activity) in T. weissflogii. Results presented for two separate ..experimental trials (circles = trial 1; triangles = trial 2) and for independent batch-culture experiments (open squares) 26 Figure 3.1: Map showing location of sampling stations in the Queen Charlotte Sound 35 Figure 3.2: Relationship between PEPC activity and nitrate concentration (r=0.712; p>0.05) 39 Figure 3.3: Time course of 1 4 C assimilation during isotope disequilibrium experiments with phytoplankton assemblages at four of the stations sampled. The dotted line indicates the best fit of the equation assuming only use of CO2 and no contribution of HCO3" 41 VI LIST OF ABBREVIATIONS a external carbonic anhydrase activity CA carbonic anhydrase CCM carbon concentrating mechanism DIC dissolved inorganic carbon / H C O 3 - fraction of DIC taken up in the form of HC0 3 " GF/F glass fiber filter (nominal pore size 0.7/ym) GF/A glass fiber filter (nominal pore size 1.6/ym) OAA oxaloacetic acid PEP phosphoenolpyruvate PEPC phosphoenolpyruvate carboxylase PEPCK phosphoenolpyruvate carboxykinase PAR photosynthetically active radiation (400 to 900nm) POC particulate organic carbon PON particulate organic nitrogen RubisCO ribulose-1,5-bisphosphate carboxylase / oxygenase vii ACKNOWLEDGEMENTS I would like to thank all members of the Tortell / Maldonado lab group for their advice and support, especially the box-office Tortellinis (Julie Granger, Cheryl Martin and Nina Nemcek) Celine Gueguen, Adrian Marchetti and Shannon Harris. I would also like to acknowledge the assistance of EOS technicians Chris Payne and Maureen Soon, as well as the EOS and Botany departmental secretaries (Carol Leven, Veronica Oxtoby, Lebby Balakshin and Judy Heyes). My first oceanographic field cruise was made both successful and memorable thanks to the scientists and crew of the CCGS John P. Tully. I am extremely grateful for the advice offered by my committee members Tony Glass, Peter Jolliffe and Max Taylor. Finally, I would like to thank my supervisor, Dr. Philippe Tortell, for his guidance and support throughout my time at UBC. Chapter 1 General Introduction Though it is now firmly established that marine phytoplankton play a critical role in the global carbon cycle and climate regulation, very few studies have examined the effects of carbon dioxide (CO2) concentrations on oceanic primary production. Research has traditionally focused on other macronutrients such as nitrogen, phosphorous and silica that are typically found in low concentrations in ocean surface waters. In contrast, dissolved inorganic carbon (DIC) is not traditionally considered to be a limiting resource in seawater because of high (~2000uM; Morel et al. 2002) surface concentrations. However, at pH 8.2 in seawater more than 90% of the total DIC pool in the ocean is present in the form of bicarbonate (HC03"), with less than 1% as free C 0 2 (Morel & Herring 1993). Laboratory studies have provided evidence that the growth rate of large marine phytoplankton (particularly diatoms) can potentially be limited by the low CO2 concentrations in seawater (Riebesell et al. 1993). 1.1 RubisCO: Potential carbon limitation of phytoplankton growth has been attributed to the catalytic inefficiency of Ribulose-1,5-bisphosphate carboxylase. / oxygenase (RubisCO), the central carbon fixing and rate-limiting enzyme in photosynthesis. This enzyme is only able to fix inorganic carbon in the form of CO2 (Cooper er al. 1969), and it has a very slow maximum specific turnover rate and a poor affinity for CO2 attributed, in part, to competition between C 0 2 and 0 2 at the active site 1 (Hartman et al. 1994). The oxygenase function of RubisCO catalyzes the breakdown of organic carbon in the presence of oxygen, utilizing energy without producing useful biosynthetic compounds. The low concentrations of aqueous C 0 2 found in surface seawater (-10 //M) are well below the half-saturation constant of algal RubisCO (Km range -20-200 / /M; Badger et al. 1998). Thus, from a purely biochemical perspective, marine photosynthesis is inherently under saturated with respect to C 0 2 . The catalytic inefficiency of RubisCO observed under modern atmospheric conditions of high 0 2 , low C 0 2 stems from the fact that this highly conserved protein evolved -3 billion years ago in a high CO2, anaerobic atmosphere. Recently it has been proposed that variations in the kinetic properties of RubisCO between major algal and cyanobacterial taxa of different geological ages may be attributed to the evolution of this enzyme toward higher CO2 affinity in response to changes in atmospheric C 0 2 / 0 2 ratios over geological time (Badger et al. 1998;Tortell 2000). 1.2 Carbon Concentrating Mechanisms: Despite the potential for carbon limitation of marine phytoplankton, these organisms are able to maintain rapid growth rates even at low oceanic C 0 2 concentrations. In order to compensate for the catalytic inefficiency of RubisCO in low CO2 environments, phytoplankton and cyanobacteria have evolved various strategies to increase carbon-fixation efficiency. These strategies are collectively known as carbon concentrating mechanisms (CCMs) (see Raven 1997; Badger 2 et al. 1998; Kaplan & Reinhold 1999) since they act to increase the concentration of C 0 2 at the active site of RubisCO, thereby increasing the rate of photosynthetic carbon fixation and suppressing photorespiratory oxygenase activity (Canvin 1990). The CCM is regulated by C 0 2 concentration, and can be induced under conditions of low C 0 2 and repressed under conditions of high C 0 2 (Badger & Price 1992). Not only is there a great deal of discrepancy among phytoplankton in the kinetic properties of RubisCO, there are also differences in the relative efficiencies of their CCMs. As the C 0 2 affinity of RubisCO decreases, phytoplankton appear to rely more heavily on CCMs in an effort to accumulate C 0 2 at the active site of RubisCO (Tortell 2000), 1.3 Carbonic Anhydrase: Despite significant differences among phytoplankton in the nature of the CCM, one common feature of many species is their ability to utilize the abundant HCO3" pool in seawater. Unlike C 0 2 ) the diffusion of the charged HCO3" molecule into the cell is restricted by membrane lipids and by the inside-negative electric potential difference across the plasmalemma (Raven1997). However, species may actively transport HCO3" (and/or C0 2 ) directly into the cell or employ the enzyme carbonic anhydrase (CA), a zinc metalloenzyme that may also contain cobalt or cadmium in the active site (Coleman 1998; Lane & Morel 2000a, 2000b), to catalyze the conversion of HC0 3 " to C 0 2 , with C 0 2 entering the cell (Siiltemeyer 1998). (Although the H C 0 3 " o C 0 2 interconversion occurs spontaneously, it happens very slowly relative to other photosynthetic reactions.) 3 1.4 C 0 2 permeability of algal membranes: The implications of algal membrane CO2 permeability for CCM operation and for the diffusive flux of C0 2 f rom the bulk medium to RubisCO are not fully understood (Raven & Beardall 2003). While a membrane with a high C 0 2 permeability may seem advantageous in allowing C0 2 t o diffuse easily into the cell, the issue of C 0 2 efflux must also be considered. It has been suggested that the energetic costs of CCM operation may be minimized if the membranes between RubisCO and the medium have a low C 0 2 permeability and therefore decreased rates of C 0 2 leakage from cells relative to C fixation (Raven & Johnston 1991). 1.5 C 4 photosynthesis: Among those phytoplankton capable of using HCO3", evidence for a functional unicellular C 4 photosynthetic pathway has recently emerged from studies of a marine diatom (Thalassiosira weissflogii) (Reinfelder et al. 2000; 2004; Morel et al. 2002). It has been proposed that HC0 3 " transported into the cell combines with phosphoenolpyruvate (PEP) in the cytoplasm, in a reaction catalyzed by PEP carboxylase (PEPC), to form the C4 compound oxaloacetic acid (OAA). The OAA is reduced to malate, and malate is then transported into the chloroplast and decarboxylated (via PEP carboxykinase) to regenerate pyruvate and release C 0 2 that can then be fixed by RubisCO. Thus, inorganic carbon is concentrated as a C 4 organic acid that is less likely to diffuse back out of the cell than C 0 2 . Labeling studies have demonstrated that the C 4 pathway is 4 important in carbon accumulation and photosynthetic carbon fixation in diatoms at low levels of C 0 2 (Reinfelder et al. 2004). 1.6 Review of Carbon Concentrating Mechanisms across taxa: To date, laboratory studies of carbon acquisition in phytoplankton have focused mainly on a few model species of cyanobacteria and green algae (see review by Kaplan & Reinhold 1999). Comparatively, much less is known about other ecologically significant groups of marine phytoplankton, such as diatoms. Evidence for direct active uptake of both C 0 2 and HCO3" exists for marine and freshwater cyanobacteria. These organisms may use at least four modes of active DIC uptake: two HCO3"transporters and two C 0 2 uptake systems, although some cyanobaceria ie: Prochlorococcus marinus appear to lack C 0 2 uptake systems entirely (reviewed by Espie et al. 1991; Miller et al. 1991; Badger & Price 2003). While no external CA activity has been detected in cyanobacteria, evidence suggests that the HCO3" transporter itself may contain a transmembrane CA-like subunit (Price & Badger 1989). Both direct and indirect mechanisms of HCO3'uptake have been documented in green algae (reviewed in Sultemeyer et al. 1993; Raven 1997; Amoroso et al. 1998; Heurtas et al. 2000a; Young er al. 2001; Pollock & Colman 2001), in several species of Prymnesiophytes (Nimer et al. 1997; Elzenga et al. 2000; Bhatti et al. 2002; Heurtas et al. 2003) and in a few species of dinoflagellates (Nimer et al. 1997; but see Dason et al. 2004). 5 While diatoms contribute disproportionately to global carbon and silica biogeochemical cycles (Eppley & Peterson 1979; Sarmiento & LeQuere 1996), carbon acquisition strategies in these microalgae are relatively unknown. Evidence from laboratory studies suggests that HCOy utilization is also predominant in this group, although there appears to be considerable variability in carbon acquisition strategies between species and even for one single species, depending on external conditions. Several studies have presented indirect evidence for HC0 3 " transport in diatoms (Burns & Beardall 1987;Colman & Rotatore 1988, 1995; Dixon & Merrett 1988). More direct evidence exists from measurements of CA-catalyzed HCO3" dehydration at the cell surface coupled to a C 0 2 transport system (Rotatore & Colman 1992; Rotatore et al. 1995; Nimer et al. 1997; Sultemeyer et al. 1998; Rost et al. 2003) and from carbon uptake studies (Korb et al. 1997; Elzenga et al. 2000; Burkhardt et al. 2001; Morel et al. 2002). 1.7 Limiting nutrients and dissolved inorganic carbon acquisition: Phytoplankton must expend considerable resources to acquire inorganic carbon. For example, energy is required for the active transport of carbon into cells, thus low light may impair the ability of phytoplankton to actively concentrate carbon. Cells that produce carbonic anhydrase may have increased trace metal requirements such as zinc, cobalt and cadmium (Coleman 1998; Lane & Morel 2000a, 2000b) because these metals are cofactors in this enzyme. Indeed, laboratory studies have shown an interaction between zinc and C 0 2 limitation 6 mediated through carbonic anhydrase (Morel et al. 1994). Since RubisCO represents a large nitrogen sink for the cell, accounting for up to 30% of cellular protein (Ellis 1979), the availability of nitrogen may affect the efficiency of cellular carbon fixation. In summary, the ability of each species to obtain and use DIC may depend on the availability of other potentially limiting resources such as light, trace metals and nitrogen (Raven & Johnston 1991; Beardall etal. 1998). 1.8 Atmospheric C0 2 rise - ecological implications: If resource availability does affect inorganic carbon acquisition, the physiological mechanism of carbon acquisition should influence the growth response of natural phytoplankton assemblages to changing levels of C 0 2 . It is expected that by the end of the next century, we will observe a doubling of surface ocean C 0 2 levels (from current partial pressure values of ~360ppm up to ~750ppm; Houghton et al. 1995). The implications of this predicted increase in C 0 2 concentration for the functioning and productivity of marine ecosystems are unknown. However, preliminary studies have shown that shifts in phytoplankton community composition may occur with changes in C 0 2 concentration (Tortell et al. 2002). Field incubation experiments in an Equatorial Pacific phytoplankton assemblage revealed that the relative abundance of diatoms decreased significantly under low [C02] relative to high [C02] while the abundance of Phaeocystis, a colonial prymnesiophyte, increased at low [C0 2] (Tortell et al. 2002). While this evidence suggests that certain phytoplankton taxa may be better adapted for survival in low C 0 2 conditions than others, the nature of this 7 competitive edge remains unclear. Factors that influence phytoplankton species distribution and succession are important because the relative contributions of different phytoplankton species or groups (ie: silicifying vs calcifying phytoplankton) to the carbon pump are highly variable (Rost et al. 2003). 1.9 Study organism: Thalassiosira weissflogii is a particularly attractive model diatom as it is easy to grow in the lab, specifically in stressful chemostat culture conditions (Li & Goldman 1981). Previous studies of DIC uptake strategies in this species provide evidence of highly efficient uptake systems for both C 0 2 and HC0 3 " (Nimer et al. 1997; Lane & Morel 2000a; Burkhardt et al. 2001; Morel et al. 2002). More controversial evidence of a C 4 photosynthetic pathway (Reinfelder et al. 2000; 2004; Morel et al. 2002) has been presented for this diatom. However, other than studies of the impact of zinc on carbon uptake in T. weissflogii (Morel et al. 1994), there is overall little information on the impacts of other resources on microalgal CCMs. 1.10 Field Studies: In addition to physiological studies of carbon uptake strategies employed by model species in the laboratory, field studies are also important. However, field research is often expensive, time consuming and associated with many logistical challenges. Additionally, it can be difficult to interpret experimental results since natural phytoplankton assemblages are composed of many different 8 species that experience considerable variation in growth conditions (ie: light levels, C 0 2 and nutrient concentrations). As a result of these complexities, there have been relatively few field studies investigating the pathways of inorganic carbon assimilation and fixation in natural plankton communities (Berman-Frank etal. 1994, 1996, 1998; Tortell et a/. 1997, 2002; Tortell & Morel 2002; Cassaref al. 2004). Although regular fluctuations in nutrient and C 0 2 concentrations do occur in natural coastal systems, the specific impacts of resource limitation on carbon uptake mechanisms are not well understood. Despite the difficulties associated with field work, understanding the mechanisms of carbon uptake occurring in the field is important as it is these natural mixes of phytoplankton that are controlling primary productivity in the oceans. 9 1.11 Thesis Objectives: Specifically, my research goals were to: 1) Investigate the effect of nitrogen limitation on activities of RubsiCO and PEPC in Thalassiosira weissflogii. 2) Investigate the effect of nitrogen limitation on the species of dissolved inorganic carbon taken up by Thalassiosira weissflogii (ie: HC03" vs. C0 2 contribution). 3) Observe activities of RubisCO and PEPC across a nitrogen gradient in the Queen Charlotte Sound coastal region. 4) Investigate the source of inorganic carbon utilized by phytoplankton assemblages in the Queen Charlotte Sound coastal region. 10 Chapter 2 Effects of nitrogen limitation on inorganic carbon concentrating mechanisms in Thalassiosira weissflogii 2.1 Introduction Laboratory studies have provided evidence that the growth rate of large marine phytoplankton, particularly diatoms, can be limited by CO2 diffusion (Riebesell et al. 1993). This finding has been attributed to the catalytic inefficiency of Ribulose-1,5-bisphosphate carboxylase / oxygenase (RubisCO), which is only able to fix inorganic carbon in the form of CO2 (Cooper et al. 1969). Despite the potential for carbon limitation of marine phytoplankton growth, these organisms are able to maintain rapid growth rates under low oceanic CO2 concentrations with the use of carbon concentrating mechanisms (CCMs) (Canvin 1990; Raven 1997; Badger et a/. 1998; Kaplan & Reinhold 1999). Phytoplankton may actively transport HC0 3 " (and/or CO2) from the external medium directly into the cell or employ the enzyme carbonic anhydrase (CA) to catalyze the conversion of HCO3" to C 0 2 , with CO2 entering the cell (Sultemeyer 1998). Additionally, species may use HC0 3 " in a C 4 photosynthetic pathway that functions as an intracellular component of the CCM (Reinfelder et al. 2000; 2004; Morel et al. 2002). 11 2.1.1 Limiting resources - implications for DIC acquisition: Phytoplankton must expend considerable resources in order to operate CCMs. For example, energy is required for the active transport of inorganic carbon into cells (Raven & Lucas 1985), thus low levels of PAR (photosynthetically active radiation: 400 to 700nM) may impair the ability of phytoplankton to actively concentrate carbon. The long-term effects of acclimation to growth-limiting irradience on the function of the CCM have been investigated in a cyanobacterium (Beardall 1991) and in a few species of macroalgae (Kubler & Raven 1994, 1995). More recently, Young and Beardall (2004) demonstrated that CCM investment in the microalgae Dunaliella tertiolecta was limited by sub-saturating irradiance during growth. Even under high photon flux densities, photosynthetic energy harvesting can be compromised by macronutrient and iron deficiency (Martin er al. 1989; Greene etal. 1992; Sunda & Huntsman 1997; Geideref al. 1998; Young & Beardall 2003). Cells that produce carbonic anhydrase may have increased trace metal requirements such as zinc, cobalt and cadmium (Coleman 1998; Lane & Morel 2000a, 2000b) because these metals are cofactors in this enzyme. Indeed, laboratory studies have shown an interaction between zinc and CO2 limitation mediated through carbonic anhydrase (Morel etal. 1994). Also, since RubisCO represents a large nitrogen sink for the cell, the availability of nitrogen may affect the efficiency of cellular carbon fixation. Understanding how the availability of other potentially limiting resources affects the ability of each species to obtain 12 and use DIC is necessary in order to appreciate the ecological relevance of CCMs in natural phytoplankton populations. 2.1.2 Carbon / nitrogen interactions: Since carbon fixation is expensive in terms of nitrogen due to the high nitrogen requirements associated with RubisCO, there is the potential for carbon-nitrogen interactions. It is firmly established that nitrogen deficiency causes a decrease in photosynthetic capacity through effects on both the enzymatic component of the photosynthetic apparatus and light-harvesting pigment protein complexes (Eppley & Renger 1974; see review by Turpin 1991; Geider et al. 1998; Keller et al. 1999). The negative impact of nitrogen limitation on the cellular content and activity of RubisCO is also well documented (Lapointe & Duke 1984; Falkowski etal. 1989; Beardall etal. 1991; Geider etal. 1998). Although it has been suggested that CCM activity may improve nitrogen-use efficiency by increasing the CO2 concentration at the active site of RubisCO (Raven & Johnston 1991), the effects of nitrogen limitation on regulation of CCM function are not well understood (Beardall etal. 1982; 1991; Beardall & Giordiano 2002; Spalding et al. 2002; Young & Beardall 2004). 2.1.3 Study Goals and Hypotheses: The main goal of this research was to investigate the effects of nitrogen limitation on CCM function in the diatom Thalassiosira weissflogii grown under ambient CO2 concentrations. Measurements of RubisCO and PEPC activities 13 were made using cells cultured under varying degrees of nitrogen limitation. In addition, 1 4 C uptake experiments were used to quantify the CO2 vs. HC0 3 " contribution to photosynthesis. My main hypothesis was that CCM function would be upregulated under increasing levels of nitrogen limitation. Specifically, I expected to observe a decrease in RubisCO activity, but an increase in PEPC and extracellular carbonic anhydrase activity and direct HCO3" transport with decreasing nitrogen-limited growth rate. 2.2 Materials and Methods 2.2.1 Culture Conditions: The diatom Thalassiosira weissflogii [(Grunow) G. Fryxell et Hasle 1977] obtained from the Canadian Center for the Culture of Microorganisms (Isolate ACTIN, CCMP1336 from Gardiner's Island, NY) was cultured in Aquil / synthetic ocean water medium with f/2 concentrations of trace elements and vitamins (Price et al. 1988/89: Appendix A) at a temperature of -21 ±1°C. The cultures were illuminated continuously with ~200//mol photons-m"2-s"1. Only milli-Q water (18.2 Q) was used in the preparation of the culture medium and all medium carboys, culture vessels and tubing were acid washed and sterilized using both autoclave and microwave methods. In non-aerated, unstirred nutrient-replete batch cultures, cells were harvested during the exponential phase of growth when cell density was - 4 x 1 0 4 cells-ml"1. Growth rates (JJ, day"1) were calculated as the slope of the natural log of fluorescence (in vivo chlorophyll) values over time (Brand & Guillard 1981). 14 Nitrogen-limited cells were obtained using a continuous chemostat culture method. In a common batch culture, it is impossible to separate the effects of an overall deterioration in cell metabolism occurring in an aging culture from the effects of nutrient limitation. In a continuous chemostat culture, however, the effects of nutrient (ie: nitrogen) limitation on photosynthesis can be studied without the compounding variables inherent in batch culture experiments (Li & Goldman 1981). The chemostat is a flow-through system in which individual cells are suspended in a constant volume at (or near) a steady state of growth established by the continuous flow of fresh (nitrogen-containing) media into the culture vessel, and the continual removal of part of the culture. In this way cells are maintained at exponential growth rates under constant and limiting concentrations of nitrogen. Continuous chemostat cultures were grown in 1-L glass side-arm flasks, stirred with a Teflon coated stir bar and constantly aerated with H2SO4 scrubbed, water-saturated air passed through a 0.2/vm Acrodisc filter capsule. Initial culture medium contained 50//M of NaN0 3 , 10ycvM of NaH 2 P0 4 -H 2 0 and 100//M of Na 2 Si0 3 -9H 2 0; fresh media was delivered continuously to the culture vessel at pre-set dilution rates controlled by multi-channel Manostat peristaltic pumps. Constant volume within the culture vessel was maintained via an overflow arm as new media was introduced. Dilution rates (D) were calculated using daily estimates of the volume of effluent collected (E) and total culture volume (V) where D = EA/. Cultures were monitored daily (using overflow samples) for fluorescence (Fluorometer: Turner 10-AU), cell abundance and cell size (Coulter 15 particle count and size analyzer: Model Z2) to ascertain steady-state conditions. Nitrate concentrations were measured regularly using a Nitric Oxide Analyzer (Antek: Model 7020). Chemostats were sacrificed for experiments after cultures had maintained steady-state growth (determined via stable fluorescence and cell density ± 15%) for a minimum of five generation times. Growth rates in steady-state chemostat cultures were assumed to equal the dilution rates (Li & Goldman 1981). Two independent chemostat trials were conducted: the first trial took place over a six month period while the second trial was completed over the span of two months. In trial one, the number of experimental replicates at each growth rate is inconsistent due to chemostat complications such as inconsistent flow rates caused by air bubbles in tubing and a power outage in the culture incubator. There were two experimental replicates completed for each growth rate in trial two. 2.2.2 Physiological Assays: a) Particulate Organic Carbon and Particulate Organic Nitrogen: A 20ml sample from log phase batch culture or sacrificed chemostat was filtered through a 15mm combusted (450°C for 4.5 h) glass-fiber filter (GF/F nominal pore size 0.7/vm). The filters were dried at 55°C for a minimum of 24 h and analyzed using a Carlo Erba NCS elemental analyzer NA1500. Both filter and container blanks were subtracted from the samples (UC Davis Facility; Maureen Soon, UBC). 16 b) RuBP-carboxylase / PEP-carboxylase activity: 250ml of log-phase batch or chemostat culture was concentrated onto a 25mm glass-fiber filter (GF/A nominal pore size 1 .6/YITI) and immediately resuspended into 4ml of ice-cold extraction buffer (modified from Maclntyre & Geider 1996: Appendix B). Cells were disrupted by homogenization for 3*40sec periods in an ice-bath. Crude cell extracts were clarified using a microcentrifuge at 14 OOOxg for 2><10s, and the supernatants were retained for enzyme assays. Enzyme activities were determined by RubisCO / PEP-dependent 1 4 C fixation (assay modified from Smith et al. 1983; Maclntyre & Geider 1996; Hatch et al. 1978). Seven 200//I aliquots of cell homogenate were sampled into microtubes: 3 each for RuBP and PEP plus 1 blank. Following a pre-incubation period of 15 minutes at 25°C in the dark, the reaction was initiated by the addition of either 20//I of RuBP stock (23mM) or 20/vl of PEP stock (50mM). RuBP and PEP stock were omitted for the blanks. After samples were incubated for 3 minutes on the bench, 5/J\ of 50jL/Ci/ml_ N a H 1 4 C 0 3 solution was added. Reactions were stopped after 30 minutes by the addition of 100//L of 6N HCI, and samples were left shaking in a fume hood overnight to drive off any unfixed 1 4 C . Degassed samples were transferred to 7ml scintillation vials and 5ml scintillation cocktail (Fisher Scintisafe 50%) was added. After mixing, the 1 4 C activity in samples was measured by standard liquid scintillation procedures (Beckman Coulter LS 6500 Scintillation Counter). 1 4 C counts (disintegrations per minute) were normalized to total protein in samples. Protein was measured using a Bio-Rad® Protein Assay. 17 c) HCOj" VS CCb Uptake - Isotope disequilibrium technique: To examine inorganic carbon uptake by nitrogen-limited T.weissflogii cells during steady state photosynthesis, the isotope disequilibrium technique described by Elzenga etal. (2000) was employed with several minor modifications (see Tortell & Morel 2002): 500ml of steady-state nitrogen-limited chemostat culture was concentrated onto a 5yt/m polycarbonate filter and re-suspended into 14ml Bicine seawater buffer (20mM Bicine, pH 8.5 ± 0.05). The cell suspension was stored in a 15°C water bath until used. 2.5ml of concentrated cell suspension (~ 2x10 6 cells-ml"1) was acclimated at 15°C for 4 minutes in a rapidly stirred, thermostated O2 electrode chamber. Cell suspensions were continually mixed with a Teflon-coated magnetic stir bar and illuminated by a Kodak Carousel 4200 slide-projector bulb with a photon flux of ~400jt/E-m'2-s"1. Radiolabeled carbon uptake was initiated by injecting 10/yCi 1 4 C solution into the electrode chamber. Immediately prior to experiments the 1 4 C spike solution was prepared by diluting 10/vCi primary isotope stock (NEN Di/ponf/l/yCi-//!"1; ~50mCi-mmol"1) into 90/yl of pH 7.0 (± 0.05) 50mM HEPES buffer. 200//I samples were removed from the electrode chamber at intervals between 5 and 180 seconds and rapidly dispensed into scintillation vials containing ml 50% HCI (~6N) in order to track the time course of cellular 1 4 C incorporation into organic matter. The acid solution immediately stopped photosynthesis, converting all inorganic carbon to CO2. Samples were left to degas on a shaker table overnight in a fume hood in order to remove all unfixed carbon. After degassing was complete, 5ml scintillation 18 cocktail (Fisher Scintisafe 50%) was added to each vial, and the 1 4 C activity in samples was measured using liquid scintillation procedures. Blank samples, consisting of 1 4 C added to bicine buffer without cells, were also run in order to correct for small amounts of residual inorganic carbon present in samples. All experiments were run in duplicate. To analyze the results of isotope disequilibrium experiments, a model fit was used to extract HC0 3 " / C 0 2 uptake rates and external carbonic anhydrase activity from the 1 4 C time course data. Equations given by Elzenga er al. (2000) were fit to data by non-linear regression using Sigma Plot: DPWIt = Vt*d -f)/a*(a*t+49*(1 -exp(a*t))) + Vt*f/(a*1.16VW1.16Vt-.24*(1 -exp(-(a*1 A6Yt))) C 0 2 contribution HC0 3 " contribution Where: DPM t = fixed radioactivity at time (t) Vt = total rate of fixation of carbon in acid stable compounds / = fraction of HC0 3 " taken up by the cells 1 - / = fraction of C 0 2 taken up by the cells a = rate constant of equilibration (-carbonic anhydrase activity) 19 2.3 Results 2.3.1 Growth Rate and Biochemical Parameters: Table 2.1: Comparison of biochemical parameters between different nitrogen-limited chemostat culture growth rates and chemostat vs. batch culture cells ± standard error. Parameter Growth-rate dependence (p<0.05) N-limited chemostat cells Batch-cultures P value chemostat vs. batch cells Residual N0 3 " (/vM) No 0.53±0.21 N/A N/A Fluor/Cell Density ratio (x1Cr5) Yes (p<0.001) 8.71±2.55 62.2±13.0 p<0.001 Cell Volume (fl-ceir1) No 82.51±5.58 132.47±1.18 p<0.001 C/N ratio No 7.8110.745 5.87±0.047 p<0.05 Several biochemical parameters provided evidence that the steady-state growth of chemostat-cultured cells was in fact limited by nitrogen supply (Table 2.1). Measurements made using the Nitric Oxide Analyzer confirmed that less than 1/vM residual nitrate remained in all steady-state chemostat cultures, irrespective of growth rate. By comparison, autoanalyzer measurements (performed by Chris Payne, UBC) confirmed that other potentially limiting nutrients (ie: phosphorous and silica) were present in excess quantities ([P] = 2.6/yM ± 0.29; [Si] = 86.2//M ± 3.3). There was no significant change in cell volume or C/N ratio (Fig 2.1) between growth rates. However the steady-state ratio of fluorescence to cell density decreased more than two-fold between the highest (1.4-d"1) and lowest (0.3-d"1) growth rates. Unstirred, non-aerated batch cultures had significantly higher fluorescence/cell density ratios and larger cell volumes than nitrogen-limited 20 chemostat cells. The C/N ratio in batch-cultured cells was significantly lower than that observed in chemostat-grown cells over the range of growth rates tested (Fig 2.1). C/N ratios of chemostat cells were also elevated with respect to the Redfield ratio, a representative of the average elemental composition of marine phytoplankton (Redfield 1934). 21 Figure 2.1: Effect of N-limited growth rate on C/N ratios in T. weissflogii. Results presented for two separate experimental chemostat trials (circles = trial 1; triangles = trial 2) and for independent batch culture experiments (square) ± standard error. 0.2 0.4 0.6 0.8 1.0. 1.2 1.4 1.6 Growth rate (d"1) 22 2.3.2 RuBP-carboxylase / PEP-carboxylase activity: There was a significant correlation between RubisCO activity and growth rate (r=0.821; p<0.001) in the chemostat cultures (Fig 2.2a). Results demonstrate a 5-fold decrease in activity from highest to lowest nitrogen-supply rate (1.4-d"1 to 0.3-d"1), and activities appeared to plateau at growth rates above -1.1 -d"1. Non-aerated, unstirred batch cultured cells (growth rate -1.1 -d"1) had higher RubisCO activities than chemostat-cultured cells at an equivalent growth rate. While RubisCO activity was strongly correlated with growth rate, there appeared to be no such trend in PEPC activity (Fig 2.2b). PEPC activity remained relatively unchanged throughout the range of growth rates tested. Although PEPC activity did not appear to be regulated by nitrogen-supply, activities across all flow rates were maintained at rates comparable to those of RubisCO. 23 Figure 2.2: Effect of N-limited growth rate on a) RubisCO (r=0.821; p<0.001) and b) PEPC activities in T. weissflogii. Results presented for two separate experimental trials (closed circles = trial 1; closed triangles = trial 2) and for independent batch-culture experiments (trial 1 = open circles; trial 2 = open triangles) ± standard error. 3e-11 2e-11 H ^ 2e-11 -\ 2e-11 5e-12 0.4 0.6 0.8 1.0 1.2 Growth Rate (d"1) 1.4 1.6 24 2.3.3 HC0 3 " vs. C 0 2 Uptake: There was a significant correlation between / H C 0 3 " (the fraction of HC0 3 " taken up by the cells) and growth rate (r = 0.682; p<0.001), with / H C 0 3 " values decreasing from -0.85 - 0.65 as growth rate decreased from 1.4 to 0.3-d"1 (Fig 2.3a). Non-stirred, unaerated batch cultures had / H C 0 3 " values -10% greater than chemostat cultured cells at equivalent growth rates (-1.1-d"1). Values of a (analogous to carbonic anhydrase activity) were not correlated with growth rate, and carbonic anhydrase activities at all growth rates were elevated relative to batch cultured cells (Fig 2.3b). 25 Figure 2.3: Effect of N-limited growth rate on a) / H C 0 3 " (r=0.682; p<0.001) and b) a (~CA activity) in T. weissflogii. Results presented for two separate experimental trials (circles = trial 1; triangles = trial 2) and for independent batch-culture experiments (open squares) ± standard error. 0.6 0.10 0.08 0.06 0.04 0.02 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 G r o w t h rate ( d 1 ) 26 2.4 Discussion 2.4.1 Biochemical parameters: When algal growth is limited by nitrogen availability, cells typically exhibit elevated C/N ratios, decreased levels of chlorophyll, nitrogen and protein per cell and decreased cellular rates of photosynthesis and fluorescence yield (Eppley & Renger 1974; see review by Turpin 1991; Geider er al. 1998; Keller et al. 1999). While C/N ratios in my chemostat-cultured cells were elevated relative both to the Redfield ratio and to the batch culture samples, I did not observe the linear increase in C/N with decreasing nitrogen supply as I would have expected. Neither the amount of C-cell"1 nor N-cell"1 was significantly affected by growth rate in my chemostat cultures. While stable values of C-cell"1 with decreasing growth rate are consistent with the literature, the fact that I did not see a decrease in N-cell"1 is puzzling. These C/N ratios should be interpreted with caution as the measured values of total POC and PON on my filters were at or near the threshold detection limit of the instruments. A more rigorous sampling protocol, including the collection of more cell biomass on filters, may help to clarify this result. 2.4.2 Regulation of light-saturated photosynthesis - RubisCO: RubisCO accounts for a major portion of total cell protein, and my results suggest that RubisCO activity in T.weissflogii is strongly regulated by nitrogen supply or, alternatively, by growth rate. This finding is consistent with previous studies that have shown a decrease in both cellular RubisCO content and/or 27 activity under nitrogen-limitation (Beardall et a/. 1982; Lapointe & Duke 84; Osborne & Geider 1986; Falkowski etal. 1989; Plumley & Schmidt 1989; Beardall et al. 1991; Geider et al. 1998). I did not quantify the amount of RubisCO present in my nitrogen-limited chemostat cells, so the assumption that observed changes in RubisCO activity represent changes in the amount of the enzyme present presumes that activation and deactivation of the enzyme is not regulating the rate of photosynthesis at saturating irradiences (Maclntyre et al. 1997; Geider etal. 1998). The relationship between enzyme activity and protein content is complicated by the fact that large pools of RubisCO are catalytically inactive (LaCoste-Royale & Gibbs 1987; McKay & Gibbs 1991; Badger et al 1998), possibly due to the role of this enzyme as a nitrogen store (Ekman et al. 1989; Falkowski et al. 1989; Plumley & Schmidt 1989; Geider et al. 1993; Garcia-Ferris & Moreno 1994). Determination of the quantity of RubisCO present using the Western blot technique in addition to enzyme activity would help to clarify this result for T. weissflogii. 2.4.3 CCMs and nitrogen-use efficiency: Although carbon and nitrogen metabolism are tightly coupled through RubsiCO, the relationship between activity of the CCM and nitrogen-limited growth rate is not well understood. It has been proposed that CCMs may increase cellular nitrogen-use efficiency (rate of C 0 2 fixation per unit of nitrogen in RubisCO) by increasing the concentration of CO2 next to the active site of RubisCO, thereby decreasing the enzyme's wasteful photorespiratory reactions 28 (Raven & Johnston 1991; Beardall etal. 1982, 1991). Spalding etal. (2002) report a transiently decreased expression of both small and large subunits of RubisCO when the CCM is induced under low C 0 2 conditions, and an enhanced affinity for DIC with increasing nitrogen-stress has recently been demonstrated in Dunaliella tertiolecta (Young & Beardall 2004). However, earlier studies on the response of the CCM to nitrogen-limitation have produced mixed results, including an apparent decrease in affinity for C 0 2 in two species of green microalgae (Beardall etal. 1982, 1991; Beardall & Giordano 2002). My results do not provide evidence of an upregulation of CCM activity under increasing nitrogen-stress in 7. weissflogii. While PEPC activity was maintained at high levels (comparable to those of RubisCO) throughout chemostat cultures at all growth rates, there was no apparent regulation of PEPC activity with growth rate. This suggests that the C 4 photosynthetic pathway is not directly influenced by nitrogen supply. In contrast, data obtained from 1 4 C uptake experiments illustrated that while HC0 3 " uptake accounted for a substantial proportion (more than 65%) of the total inorganic carbon taken up even at the lowest growth rates, the HCO3" contribution in fact decreased as cells became more severely nitrogen-limited or, alternatively, as their growth rates decreased. The absence of a corresponding decline in carbonic anhydrase activity (implied from values of a) established that the observed decrease in / H C 0 3 " was attributed to a decrease in direct HCO3" transport. It is possible that this reduction in direct HC0 3 " uptake may be due to the decreased carbon demand of cells that are growing more slowly. 29 2.4.4 Summary: In summary, these results suggest that as nitrogen supply and RubisCO activity decreases, CCM activity in T. weissflogii remains unchanged with respect to the function of the internal C 4 photosynthetic pathway, but is actually down-regulated with respect to direct HCO3" transport from the outside medium. Since carbon-fixation rates associated with slower growth will create a lower demand for net DIC-uptake, it is possible that nutrient acquisition mechanisms, including CCMs, may be co-regulated in order to control elemental ratios and maintain metabolic balance during periods of nutrient stress. Investigating the effects of phosphate limitation on the function of the CCM may help to distinguish the impacts of decreased nitrogen-supply from growth rate effects. 30 Chapter 3 Inorganic carbon acquisition in Queen Charlotte Sound phytoplankton assemblages 3.1 Introduction Despite the established importance of marine phytoplankton in the global carbon cycle, the physiological mechanisms of dissolved inorganic carbon (DIC) acquisition in phytoplankton remain poorly understood. In particular, the extent to which phytoplankton utilize C 0 2 vs. HCO3" as sources of inorganic carbon for photosynthesis, and active vs. diffusion-based mechanisms of DIC uptake is currently debated. Virtually all studies of carbon acquisition in phytoplankton to date have focused on a handful of model organisms grown in monospecific, nutrient-replete laboratory batch cultures (ie: Korb et al. 1997; Siiltemeyer et al. 1998; Elzenga et al. 2000; Burkhardt et al. 2001; Morel et al. 2002; Rost et al. 2003). While these controlled studies help us to understand the complex physiological processes involved in carbon acquisition at a cellular level, the results do not necessarily explain processes occurring in complex natural phytoplankton assemblages. 3.1.1 Challenges associated with field studies: As a result of the complex and logistically challenging nature of field work, the pathways of inorganic carbon assimilation and fixation in natural phytoplankton assemblages remain poorly studied (Berman-Frank et al. 1994, 1996, 1998; Tortell etal. 1997, 2000; Tortell & Morel 2002; Cassaref a/. 2004). 31 Oceanographic field studies are particularly difficult as they are often extremely expensive, time consuming and hampered by unfavorable weather conditions that may interfere with or prohibit sampling altogether. Experimental procedures that are easily carried out in a laboratory are often logistically difficult to reproduce at sea. For example, the low biomass of natural phytoplankton assemblages can make it difficult to obtain enough cells to perform experiments or make measurements. Interpretation of experimental results can be challenging since natural phytoplankton assemblages are composed of many different species whose carbon uptake systems have not yet been studied. Furthermore, these communities experience considerable variation in growth conditions (ie: light levels, CO2 and nutrient concentrations) and the specific impacts of resource limitation on carbon uptake mechanisms are not well understood. 3.1.2 Importance of field studies: Despite their inherent limitations, field experiments are important because it is natural mixes of phytoplankton that are controlling primary productivity in the oceans. Coastal marine waters experience regular fluctuations in nutrient and CO2 concentrations that can influence phytoplankton growth. In the lab I examined the function of the carbon concentrating mechanism over a range of nitrogen concentrations in one species of marine diatom. This complementary field study is a preliminary step to examine carbon uptake in natural coastal phytoplankton assemblages over a nitrogen gradient. 32 3.1.3 Study Goals and Hypotheses: The goals of this field study of phytoplankton assemblages in the Queen Charlotte Sound coastal region were to: 1) Examine trends in activities of enzymes involved in dissolved inorganic carbon assimilation (RubisCO and PEPC) across a nitrogen gradient. 2) Investigate the source of dissolved inorganic carbon utilized by phytoplankton assemblages. My main hypothesis was that CCM function would be upregulated in regions where nitrogen levels were low. Specifically, I expected to observe a decrease in RubisCO activity, but an increase in PEPC and extracellular carbonic anhydrase activity and direct HCO3" transport in those phytoplankton assemblages found in areas with low levels of nitrate as compared to those assemblages growing in areas with high nitrate concentrations. 3.2 Material and Methods 3.2.1 Field site. Field sampling was conducted from August 11-19 of 2004 in coastal waters of the Queen Charlotte Sound aboard the CCGS John P. Tully. Sampling sites were located along two cross-shelf transects extending into offshore waters and along-shore following the shelf break (Fig 3.1). Seasonally intense upwelling events carry nutrient and C 0 2 - r i c h deep waters to the surface in this coastal region, supporting high levels of primary productivity dominated mainly by large 33 (>307vM) solitary and chain-forming diatoms (Lalli & Parsons 1994). A large degree of spatial and temporal variability exists both in nutrient levels and phytoplankton biomass in these waters. At each station, nutrient concentrations were determined with a shipboard autoanalyzer, and total chlorophyll a (Chi a) was determined by fluorometric analysis later in the lab (Lindsay Richier, Varela lab, UVic: method in Parsons etal. 1984). 34 3.2.2 In-situ sampling and isotope disequilibrium experiments: a) Enzyme activity: All samples were collected from the chlorophyll maximum (depth ranging from -0.2 - 22.6m) using 10L Niskin bottles on a rosette. Several 1L phytoplankton samples taken from Niskin bottles were concentrated onto 25mm glass-fiber filters (GF/A: nominal pore size 1.6/ym) and frozen in liquid nitrogen for subsequent laboratory analysis of RubisCO and PEPC expression. Enzyme assays were conducted as described in methods section of Chapter 2 with one minor modifcation: 10^ /1 of 50//Ci-ml"1 NaH 1 4C03 solution was added instead of 5//I in order to compensate for lower biomass on filters. b) Particulate Organic Carbon and Particulate Organic Nitrogen: A 500mL sample was filtered through a 25mm combusted (450°C for 4.5 h) glass-fiber filter (GF/F: nominal pore size 0.7/vm) Whatman® filter and rinsed with 10ml of nitrogen-free synthetic ocean water (SOW). The filters were stored in a glass dessicator for the duration of the cruise then dried at 55°C for 24hrs when back in the lab. C/N ratios were determined using an isotope ratio mass spectrometer (U.C. Davis Facility). c) Isotope diseguilibrium experiments: Phytoplankton were collected by passing water directly from 2*101. Niskin bottles through 3pm in-line polycarbonate filters (filtered only by gravity). Microalgae were re-suspended off filters into 14ml of seawater buffer containing 20mM Bicine adjusted to pH 8.5 (±0.05) and isotope disequilibrium experiments were immediately begun. Experiments were conducted as described in methods section of Chapter 2 with one minor modification: 20//I of 50/yCi-ml"1 NaH 1 4C03 solution was added instead 36 of 10/YI in order to compensate for lower biomass in cell suspension. Leftover cell-suspension was fixed with formaldehyde to a final concentration of 2% for later determination of phytoplankton community taxonomic composition. Phytoplankton were categorized into two main groups based on size: cells <10uM (mainly nanoflagellates and small diatoms) and cells >10uM (mainly larger diatoms and dinoflagellates). 3.3 Results 3.3.1 Biochemical Parameters: Table 3.1: Chemical and biological properties of sampling stations. Station Sampling Nitrate Silicate Phosphate Chi a ID Depth (MM) (uM) (MM) (ug-L/1) (dbar) GB5 19.9 5.7 11.5 0.76 1.96 MB2 22.6 5.5 11.8 0.75 1.20 SP4 8.6 0.2 5.5 0.38 1.50 SS2.5 20.4 3.5 10.4 0.65 0.96 CPE 0.2 17.6 33.3 1.68 5.50 SS6 14.9 4.4 9.2 0.66 1.77 G3 12.6 1.1 12.8 0.46 Over the seven stations sampled, nitrate concentrations were positively correlated with measurements of Chi a (r = 0.934; p<0.01; Table 3.1). With the exception of the highly productive inshore station CPE, nitrate levels ranged from 0.2-5.7uM and Chi a levels were between 0.96-1.96ug-L"1. There were no clear differences between onshore and offshore siteswith respect to phytoplankton biomass or nutrient concentrations. Microscopic examination of phytoplankton samples revealed that diatoms >30uM, including Coscinodiscus spp., 37 Cylindrotheca spp., and Pseudo-nitzschia spp., dominated at all stations sampled except for station SP4, where nanoflagellates and small diatoms <10uM were more abundant. 3.3.2 RuBP-carboxylase / PEP-carboxylase activity: Table 3.2: Data analysis of enzyme assays and C/N ratios ± standard error. Station C/N RubisCO Activity PEPC Activity ID (x10-12) (x10 1 3) (Mol C fixed-ug (Mol C fixed-ug extract protein"1 -hr"1) extract protein"1 -hr~1) GB5 6.08 0.78 ±0.014 0.33 ± 0.022 MB2 5.92 1.36 ±0.030 3.92 ± 0.951 SP4 5.22 1.09 ±0.013 7.01 ±0.933 SS2.5 5.31 0.32 ± 0.032 1.43 ±0.534 CPE 5.84 1.37 ±0.012 0.18 ±0.046 SS6 5.28 2.21 ±0.014 4.90 ± 1.44 G3 5.84 2.19 ±0.033 6.35 ± 1.91 C/N ratios varied little between stations, ranging from -5.2 to -6.1 (Table 3.2). There was significant variation in RubisCO activity between stations (p<0.001), with a seven-fold difference between the highest and lowest measured activities. However, these data do not support any correlation between RubisCO activity and C/N ratio, nitrate concentration or level of Chi a. Measurements of RubisCO activity obtained in the field, ranging from 0.32 - 2.21 Mol C fixed-ug extract protein"1-hr"1, were of the same order of magnitude as those observed in lab-grown monocultures of T. weissflogii at the lowest growth rates (0.3-d'1; see Results Chapter 2). The relationship between PEPC activity and nitrate concentration was on the threshold of statistical significance (p~0.05), with PEPC activity increasing as 38 Figure 3.2: Relationship between PEPC activity and nitrate concentration (r=0.712; p~0.05) ± standard error. , r - 1e-12 .E 8e-13 5 10 Nitrate (uM L"1) 15 20 nitrate concentration decreased (Fig 3.2). Field measurements of PEPC activities were approximately one order of magnitude lower than those of lab-grown T.weissflogii growing at 0.3-d"1 (see Results Chapter 2). 3.3.3 HC0 3 " vs. C 0 2 Uptake: Table 3.3 Data analysis of isotope disequilibrium experiments and C/N ratios. Station ID C/N / H C 0 3 - a GB5 6.08 — — MB2 5.92 — — SP4 5.22 0.9298±0.030 0.0272±0.022 SS2.5 5.31 0.8762±0.024 0.0299±0.007 CPE 5.84 0.8903±0.012 0.0272±0.004 SS6 5.28 0.8885±0.012 0.0332±0.005 G3 5.84 0.8589±0.027 0.0272±0.005 At all five stations where disequilibrium experiments were performed, / H C O 3 " values indicate that more than 86% of the total carbon demand could be accounted for by HCO3" uptake (Table 3.3; Fig 3.3). Low values of a (external CA activity) suggest that at these stations HC03" was taken up mainly via direct active transport. While /HC0 3 " values were not significantly correlated with nitrate concentration or C/N ratios, the highest /HC0 3 " value (obtained at station SP4) was associated with the lowest nitrate concentration and C/N ratio, and the highest PEPC activity. Additionally, this was the only station dominated by small diatoms and nanoflagellates rather than large diatoms. 40 Figure 3.3: Time course of C assimilation during isotope disequilibrium experiments with phytoplankton assemblages at four of the stations sampled (± standard error). The dotted line indicates the best fit of the equation assuming only use of CO2 and no contribution of HCO3". Time (s) 41 3.4 Discussion 3.4.1 RubisCO: Since RubisCO is only able to fix inorganic carbon in the form of C 0 2 (Cooper et al. 1969) this enzyme is undersaturated with respect to the concentrations of C 0 2 found in surface seawater (Badger et al. 1998). RubisCO represents a large nitrogen-sink for the cell (Ellis 1979), and while the effect of nitrogen-limitation on the cellular content and activity of RubisCO in lab-grown phytoplankton cultures is well documented (Lapointe & Duke 1984; Falkowski et al. 1989; Beardall etal. 1991; Geider etal. 1998; also see Results Chapter 2), it is unclear how combined RubisCO activities of complex phytoplankton assemblages vary over nitrogen gradients in the field. Despite the significant correlation between nitrate and Chi a levels, RubisCO activity did not vary predictably over the range of nitrogen concentrations reported in this field study. This is not the result I would have expected based on the findings of my lab experiments. However, my nitrogen-limited chemostat cultures of T. weissflogii had residual nitrate levels less than 1//M at all experimental growth rates tested (see Results Chapter 2), while five of the seven stations sampled had nitrate concentrations >3.5/vM-L"1. Since the half-saturation constant for nitrate uptake in coastal marine phytoplankton ranges from 0.98 to 4.2 JJM (Raymont, J.E.G.) it is possible that the nitrate levels at these field stations may not have been low enough to elicit a change in the cellular content of RubisCO. Alternatively, variations in cellular growth stage (ie: exponential vs. stationary growth phase) may have influenced RubisCO activity. 42 3.4.2 C 4 photosynthesis: Since carbon-fixation is expensive in terms of nitrogen, the availability of nitrogen may affect the efficiency of cellular carbon fixation (Raven & Johnston 1991). Laboratory labeling studies have demonstrated that a unicellular C 4 photosynthetic pathway is important in carbon accumulation and photosynthetic carbon fixation in diatoms at low levels of C 0 2 and Zn (Reinfelder et a/.2000, 2004; Morel et al. 2002). However, the relationship between PEPC activity and nitrate concentration remains uncertain. PEPC activities did not appear to be regulated by nitrogen supply in nitrogen-limited chemostat cultures of T. weissflogii (see Results Chapter 2). The negative trend between PEPC activity and nitrate concentrations observed at the seven stations sampled in this field study suggests that PEPC activity may be up-regulated when nitrogen levels are low. The induction of PEPC activity at low levels of nitrogen may help to save nitrogen in RubisCO by making the enzyme more efficient. These RubisCO and PEPC enzyme data should be interpreted with caution as there was only one sample obtained per station and the activities measured, particularly of PEPC, were low compared to lab-grown T. weissflogii cultures. It is possible that these low activities may be explained by variations in the phytoplankton growth phase. While lab cultures were sampled for enzyme assays during the exponential phase of growth, phytoplankton sampled in the field may have been in stationary phase. More rigorous sampling, including the collection of more phytoplankton biomass on filters over a range of depths (ie. depth profiles), may help to clarify the results of these enzyme assays. 43 3.4.3 HC03"VS C0 2 uptake: All phytoplankton assemblages studied utilized HC0 3 "as an exogenous inorganic carbon source. The observed kinetics of 1 4 C incorporation suggest that carbon uptake occurred mainly via a direct transport mechanism. Laboratory studies on inorganic carbon acquisition in diatoms have shown that HC0 3 " utilization is common in this group (Burns & Beardall 1987; Nimer era/. 1997), although there appears to be considerable variability in carbon acquisition strategies between species and even for one single species, depending on external conditions (Elzenga et al. 2000; Burkhardt et al. 2001; Morel et al. 2002). Additionally, the use of HC0 3 "as a carbon source in natural diatom-dominated phytoplankton assemblages has been demonstrated (Tortell et al. 1997; Tortell et al. 2000; Tortell & Morel 2002; Cassar et al. 2004). However, reports on the contribution of direct active HC0 3 " uptake vs. indirect, CA-mediated HC0 3"use are mixed. Evidence of external CA expression in coastal Pacific phytoplankton assemblages was demonstrated by Tortell et al. (2000). Subsequently, data from isotope disequilibrium experiments performed in the eastern subtropical and Equatorial Pacific region showed that diatom-dominated assemblages expressed external CA and transported C 0 2 derived from the catalyzed dehydration of HC0 3 " (Tortell & Morel 2002), although authors acknowledged that direct HC0 3 " transport may have been present. In contrast, negligible CA activity was detected in diatom-dominated Southern Ocean phytoplankton assemblages (Cassar et al. 2004). These authors report data from 44 isotope disequilibrium experiments confirming that 50% of the DIC was obtained through direct HC0 3 " uptake, the other half being active or passive CO2 uptake. It is interesting to note that in three of the five stations sampled, the model rate-constant for HCO3" / CO2 equilibration was not different from uncatalyzed rates, implying that absolutely no CA activity was present at these stations. In support of this finding, we were unable to detect any CA activity in these samples using membrane inlet mass spectrometry (MIMS) (measurements made by P. Tortell). However, a large amount of phytoplankton biomass is required to make reliable measurements of CA activity via MIMS, so it is possible that there simply was not enough phytoplankton biomass on the filters. 3.4.4 Effect of nitrogen on CCM function: While the function of the CCM has been studied in relation to CO2 concentrations, the relationship between nitrogen availability and active inorganic carbon uptake mechanisms remains unclear. Results from my lab experiments with chemostat-cultured T. weissflogii suggest that while HC0 3 " uptake remains high even under extreme nitrogen-limitation, HC0 3 " transport may in fact be down-regulated with decreasing nitrogen-supply. In this field study, / H C 0 3 " values were not correlated with nitrate concentrations or C/N ratios, although station SP4 had the lowest nitrate concentration and C/N ratio and the highest PEPC activity and / H C O 3 " values of all the stations sampled. Interestingly, this was also the only station dominated by small diatoms and nanoflagellates, suggesting that a more detailed investigation of phytoplankton community 45 structure (ie. mainly small diatoms or mainly nanoflagellates) may be important for the interpretation of these results. It is difficult to draw any conclusions on the relationship between nitrate-supply and HCOy uptake at these stations since, as previously discussed, five of the seven stations sampled had nitrate concentrations that were potentially non-limiting. 3.4.5 Summary: In summary, while RubisCO activity was not regulated by nitrogen supply at the stations sampled in this field study, the negative trend observed between nitrate concentration and PEPC activity suggests that the function of the C 4 photosynthetic pathway may have been enhanced under low levels of nitrogen. Inorganic carbon acquisition occurred mainly via direct HC0 3 " transport at all stations sampled and did not appear to be influenced by nitrogen levels. 46 FINAL CONCLUSIONS 1) RubisCO activity was positively correlated with nitrogen-limited growth rate in T. weissflogii. The function of the C 4 photosynthetic pathway, as determined by PEPC activity, was not influenced by nitrogen-limited growth rate. 2) HCO3" uptake accounted for 65 - 85% of the total dissolved inorganic carbon taken up by T. weissfloggii over the range of growth rates tested. The decrease in / H C O 3 " (the fraction of dissolved inorganic carbon taken up in the form of HCO3") observed with decreasing growth rate was attributed to a decrease in direct HCO3" transport. 3) There was no relationship between RubisCO activity and nitrate concentration at the seven diatom-dominated field stations sampled, but a negative trend was observed between PEPC activity and nitrate concentration. 4) All phytoplankton assemblages studied utilized HC0 3 "as an exogenous inorganic carbon source and HCO3" uptake occurred mainly via a direct active transport mechanism. 47 FUTURE RESEARCH This thesis has provided preliminary physiological data on the effects of nitrogen supply on carbon-uptake systems in T. weissflogii and in coastal phytoplankton assemblages of the Queen Charlotte Sound. Suggestions for future research include: 1) Assessment of the effects of CO2 concentration on carbon acquisition in nitrogen-limited phytoplankton. 2) Investigation of silica (Si)-limitation on carbon-uptake mechanisms of cells grown under high and low CO2. 3) Characterization of N and Si / carbon interactions for different phytoplankton species representing different taxonomic / functional groups (ie: coccolithophores, nanoflagellates). 4) Analysis of the expression of particular genes involved in DIC acquisition under nutrient / carbon limitation (using the recently sequenced genome of T. pseudonana). 5) Assessment of effects of C02on competitive interactions in nutrient-limited phytoplankton. Competition experiments could be conducted in the laboratory (using chemostats) as well as in the field (using incubation experiments) and could help to predict potential long-term ecological shifts that may result from future variations in atmospheric CO2 levels. 48 References Amoroso, G., D. 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In press. 57 Appendix A Aquil Culture Medium (Price etal. 1988/89) Substance Final Concentration (M) Aquil Salts NaCI 4.20 x 10"1 N a 2 S 0 4 2.88 x 10"2 KCI 9.39 x lO" 3 NaHCOa 2.38 x 10-3 KBr 8.40 x 10'4 H 3 B O 3 4.85 x 10-4 NaF 7.14 x10" 5 MgCI 2-6H 20 5.46 x 10"2 CaCI 2-2H 20 1.05 x 10 - 2 SrCI 2-6H 20 6.38 x 10"5 Nutrients NaH 2 P0 4 -H 2 0 1.00 x 10~5 NaN0 3 3.00 x10" 4 Na 2 Si0 3 -9H 2 0 1.00 x ;10"4 Trace metals Na 2EDTA 5.00 x 10"6 FeCI 3-6H 20 4.51 x 10 - 7 ZnS0 4 -7H 2 0 4.00 x10" 9 MnCI 2-4H 20 2.30 x 10 - 8 CoCI 2-6H 20 2.50 x l O ' 9 CuS0 4 -5H 2 0 9.97 x l O - 1 0 Na 2Mo04-2H 20 1.00 x 10 - 7 Na 2 Se0 3 1.00 x 10 - 8 Vitamins B-|2 5.50 x 10' 7g-L' 1 Biotin 5.00 x 10-7g-l_-1 Thiamine HCI 1.00 x 10" 4g-L 1 Appendix B Permeabilization Buffer (Modified from Maclntyre etal. 1996) 50mM Bicine (pH 7.8) [163g-mol"1; 815mg-100mr1] 1mM EDTA [200//I 0.5M EDTA-IOOml"1; 0.5M EDTA = 4.653g-25ml"1] 10mM MgCI2 [1ml 1.0M MgCI2-100ml"1; 1.0M MgCI2= 5.083g-25mf1] 1.5M Glycerol [=glycerin; 92g-mol"1; 1.26gml"1; 11ml-100ml"1] 10 mM NaHC0 3 [84-1 OOmf1] 5mg BSA (bovine serum albumen) * Adjust pH with NaOH (to pH 7.8) 5mM DTT [154g-mof1; 77mg-100mr1] 0.1% Triton-X-100 [250/yl-100mf1] *RuBP stock (store frozen): 23mM; 5mg Na4RuBP-1.5 H 2 0 [429g-mol"1] in 0.5ml of QH 2 0 *PEP stock (store frozen): 50mM; 19mg Na 3 PEP [378gmol"1] in 1ml of QH 2 0 59 

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