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With growing concern regarding the effects of ocean acidification (OA) on the marine environment (Guinotte & Fabry, 2008; Doney et al., 2009), there has been a renewed interest in how marine microalgae acquire and utilize inorganic carbon (iC) to support carbon (C) fixation within the Calvin–Benson cycle (CBC) (Rost et al., 2008; Tortell et al., 2010; Brading et al., 2011). OA alters the speciation of iC within seawater and this, in turn, may affect the productivity and growth of marine microalgae (Fu et al., 2008; Brading et al., 2011). Understanding the mechanisms of iC acquisition is especially relevant for algae belonging to the dinoflagellate genus Symbiodinium, which are found both free-living and in symbiosis with reef-building corals and other cnidarians. This symbiosis is essential for the viability of many cnidaria, and is key to the persistence of coral reef ecosystems under present day and future climates (e.g. Iglesias-Prieto et al., 2004; Berkelmans & van Oppen, 2006). However, our current understanding of the iC environment within the host tissue is limited (Gattuso et al., 1999; Venn et al., 2009), despite the fact that it may have a significant impact on the productivity and growth of the in hospite Symbiodinium population (Wooldridge, 2009, 2010). The Symbiodinium genus is taxonomically divided into numerous phylotypes, physiologically distinct types that are distinguishable by genetic variability in the second internal transcribed spacer region (ITS2) of their ribosomal DNA (LaJeunesse, 2001). Different phylotypes can exhibit a range of responses to both environmental acclimation (Hennige et al., 2009) and stress (e.g. Robison & Warner, 2006; Suggett et al., 2008; Ragni et al., 2010; Buxton et al., 2012), allowing their coral hosts to occupy a wide range of niches (e.g. Iglesias-Prieto et al., 2004).
Unlike inorganic macronutrients, such as nitrogen and phosphorus, iC within ocean surface waters is relatively abundant (and constant) at c. 2.2 mM. However, most of this iC is in the form of bicarbonate ions (), whereas aqueous carbon dioxide (CO2(aq)), the actual iC species fixed within the CBC, contributes < 1%. Rubisco, the enzyme employed by marine microalgae to catalyse iC fixation, has a relatively low CO2 affinity and specificity, particularly within an O2-rich environment. Consequently, this key enzyme is, in fact, considerably undersaturated with respect to current seawater concentrations of CO2(aq). This is particularly true of dinoflagellates, such as Symbiodinium, which possess a form II Rubisco (Morse et al., 1995; Whitney et al., 1995) that has a significantly lower specificity for CO2 than the form I Rubisco of other phytoplankton taxa (e.g. Tortell, 2000).
In order to overcome the inherent enzymatic limitations of Rubisco, nearly all marine microalgae have evolved strategies, termed ‘carbon-concentrating mechanisms’ (CCMs), to actively elevate CO2 at the active site of Rubisco (Giordano et al., 2005; Reinfelder, 2011); thus, the majority of marine primary producers are not considered to be C limited under present day conditions (Giordano et al., 2005). CCMs not only allow marine microalgae to efficiently utilize the small and variable pool of CO2(aq) for C fixation, but also take advantage of the much larger fraction. The functional role of CCMs has been extensively reviewed within the literature (see Giordano et al., 2005; Reinfelder, 2011), and primarily involves one or more of the following processes: (1) the active transport of CO2(aq) into the cell; (2) the dehydration of to CO2(aq) at the cell surface, which is then transported into the cell; and (3) the active transport of directly into the cell, where it is then converted to CO2(aq). Interconversion between and CO2(aq), both outside and inside the cell, is catalysed by external and internal carbonic anhydrases (eCA and iCA, respectively) (Reinfelder, 2011), and the dehydration of to CO2(aq) at the cell's boundary layer by eCA can be further aided by membrane-bound proton (H+) pumps that acidify the immediate external environment (Bertucci et al., 2010).
A number of CCM components have been identified for Symbiodinium, although no direct comparison of iC acquisition across different phylotypes has been performed within a single study. Both eCAs and iCAs have been shown to be expressed by Symbiodinium, as well as H+-ATPase (a proton pump) and Na+/ co-transporters (Yellowlees et al., 1993; Al-Moghrabi et al., 1996; Leggat et al., 1999; Bertucci et al., 2010). It is also apparent that the expression of these mechanisms differs depending on the host type (e.g. giant clam vs coral) and whether the Symbiodinium is in hospite or free-living (Al-Moghrabi et al., 1996; Leggat et al., 1999; Bertucci et al., 2010). Although these various studies have demonstrated that the environment can play an important role in the type of CCM expressed, few have unfortunately specified the phylotype studied, with Bertucci et al. (2010) being the only exception. This has made it impossible to infer whether differences in CCM type previously described for Symbiodinium are solely driven by environment (i.e. acclimation to different iC conditions/availability) or also by adaptive differences between phylotypes.
It is widely accepted that the changes in productivity and growth under OA conditions represent ‘downstream effects’ on iC acquisition and fixation (Rost et al., 2008; Hurd et al., 2009). Recently, we have demonstrated that the effect of OA on free-living Symbiodinium is phylotype specific, indicating that key differences in iC acquisition may also exist between phylotypes of Symbiodinium (Brading et al., 2011). In particular, phylotypes A20 (previously termed A2 in Brading et al., 2011) and A13 exhibit contrasting responses on exposure to an increase in the partial pressure of CO2 (pCO2) from c. 390 to 800 ppmv. Specifically, an increase in productivity, with no change in growth rate, was observed for A20, whereas the opposite was observed for A13. Based on these previous observations, we sought to examine these alternative physiological responses to elevated iC availability (productivity vs growth), and hypothesized that: (1) A20, but not A13, is carbon limited under present day conditions because of an inefficient CCM; and thus (2) A13 and A20 express different CCM types when grown in the same iC environment.
In order to evaluate the uptake and utilization of iC, we first determined the light-dependent rates of productivity using two approaches. Light-dependent rates of C fixation and photosynthetic electron transfer (PET) were measured so as to compare the efficiency with which energy (and reductant), derived from PET, is utilized for C fixation vs other essential metabolic processes, such as CCM/CA activity. We subsequently assessed iC affinity and species preference by quantifying the Rubisco concentration, measuring the total carbon dioxide (TCO2) dependence of photosynthetic C fixation and performing short-term 14C disequilibrium, which also estimated eCA activity (Martin & Tortell, 2006; Rost et al., 2007).