Photosynthetic efficiencies in evolutionarily distant species
Steady-state, nitrogen-limited growth rates for T. weissflogii ranged from 0.2 to 1.0 d−1, which is only slightly less than the 0.12–1.2 d−1 range used earlier for D. tertiolecta (Halsey et al., 2010). Thalassiosira weissflogii chlorophyll content (chlorophyll a : C) varied in direct proportion to the growth rate (r2 = 0.99; Table 2), implying precisely tuned, highly efficient photosynthesis at all levels of NO3– limitation. This same linear relationship is observed in T. weissflogii grown under NH4 or PO4 limitation (Laws & Bannister, 1980). Qualitatively similar responses are observed among chlorophytes, haptophytes and other diatoms (Laws & Wong, 1978; Herzig & Falkowski, 1989; Halsey et al., 2010). However, quantitative comparison of photosynthesis between evolutionarily distant groups requires an accounting for taxonomic differences in light-harvesting pigments. We therefore calculated photosynthetic light utilization efficiencies by normalizing oxygen and carbon production to spectral absorption for T. weissflogii and D. tertiolecta (data for D. tertiolecta were re-analyzed from Halsey et al., 2010).
Table 2. Steady-state characteristics of nitrate-limited chemostat cultures of Thalassiosira weissflogii (values in parentheses are SE)a
|μ (d−1)||Cells ml−1 (×105)||Chlorophyll (Chl) a per cell (pg Chla)||Chl : C (μg μg−1) × 10−3||Particulate organic carbon per cell (pg C)||C : N (μg μg−1)||Cell volume (μm3)|
|0.20||1.29 (0.11)||0.57 (0.15)||3.3 (0.8)||174 (1.9)||15.0 (1.3)||414 (13)|
|0.50||1.53 (0.11)||1.10 (0.18)||10.8 (1.3)||106 (17)||9.5 (1.5)||361 (11)|
|1.0||1.41 (0.08)||1.32 (0.14)||23.8 (2.0)||55.8 (6.5)||4.3 (0.2)||274 (10)|
Absorption-specific GPO2, GPC and NPC are invariant across growth rates and are remarkably consistent between the two species (Fig. 1). Light utilization efficiencies for gross oxygen evolution are tightly constrained between 0.047 and 0.050 mmol (mmol photon)−1. For both species, c. 25% of GPO2 is immediately consumed by LDR pathways and for reduction of nitrogen and sulfur (Fig. 1, light and dark gray areas, respectively). Thus, 75% of GPO2 is invested in gross carbon fixation (GPC). For both species, only c. 30% of GPO2 is ultimately retained as net carbon production (NPC) at all growth rates (Fig. 1, green areas). The remaining c. 45% of photosynthetic production represents a transient carbon pool that is soon consumed for either mitochondrial ATP production (O2 consuming) (yellow areas in Fig. 1) or catabolism that provides electrons for carbon reduction (blue areas in Fig. 1). In both species, these reductive pathways are greater at faster growth rates (Fig. 1). Thalassiosira weissflogii mobilizes a larger fraction of its transient carbon pool for mitochondrial respiration, whereas, in D. tertiolecta, the reductive pathways dominate (compare blue and yellow wedges between species in Fig. 1). The overall cellular reduction states of green algae and diatoms increase with growth rate (Smith & Geider, 1985; Jakob et al., 2007; Halsey et al., 2011), a phenomenon that is reflected by a higher fraction of lipid and protein carbon in fast-growing D. tertiolecta relative to slow-growing cells (Halsey et al., 2011).
Figure 1. Absorption-normalized photosynthetic efficiencies in Dunaliella tertiolecta and Thalassiosira weissflogii across a wide range of nitrate-limited specific growth rates. Measurements of gross O2 production (GPO2), light-dependent respiration (LDR), net O2 production (NPO2) and net carbon production (NPC) yielded data symbols and regression lines. Error bars are SE of measurements made from triplicate chemostat cultures. Colored sections show the allocation of photosynthetic electron flow to different metabolic sinks. Data for D. tertiolecta were reanalyzed from Halsey et al. (2010).
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Our findings for T. weissflogii and D. tertiolecta highlight multiple significant features of photosynthesis in microalgae. First, highly comparable photosynthetic efficiencies were observed in evolutionarily distinct species when differences in light-harvesting pigment compositions were taken into account (i.e. if T. weissflogii data were normalized to chlorophyll a alone, the apparent photosynthetic efficiencies would be 24% higher than chlorophyll a-normalized data for D. tertiolecta; Fig. S1). Second, steady-state nutrient limitation is not synonymous with diminished photosynthetic efficiencies. Third, the observed GPO2/NPC ratios of 3.3 and 3.5 provide a baseline for comparison with values assessed in the field. This ratio is critical to the assessment of ecosystem carbon cycling from incubation-free oxygen isotope measurements. A canonical value of 2.7 is typically used in these assessments to interconvert GPO2 and NPC (Marra, 2001). However, in practice, this ratio appears to vary between at least 1.5 and 4 (Wagner et al., 2006; Jakob et al., 2007; Langner et al., 2009), with some studies suggesting an even broader range (e.g. Luz & Barkan, 2009; but note that the recent study of Nicholson et al. (2012) suggests that these extremes may reflect an assessment error). If diverse phytoplankton behave similarly to T. weissflogii and D. tertiolecta, explanations other than nutrient limitation might be needed to explain deviations in GPO2/NPC.
Our investigation also leads to the conclusion that different arrangements of biochemical pathways and energetic allocation strategies can result in similar growth efficiencies. Photosynthetic light reactions give an ATP/NADPH ratio of c. 3 : 2, similar to the consumption ratio of the Calvin–Benson cycle. However, this energetic stoichiometry does not capture the overall cellular cost for net carbon accumulation. The oxygen-consuming pathways, LDR and mitochondrial respiration, together constitute 42–59% of GPO2 (light gray and yellow wedges in Fig. 1), consistent with reports that respiration can account for 21–89% of GPC (Geider, 1992; Langdon, 1993) and 41–69% of GPO2 (Kunath et al., 2012). Another 13–29% of GPO2 is used to reduce nitrogen, sulfur and organic compounds (Fig. 1 dark gray and blue wedges). Overall, we find investment ratios for mitochondrial respiration, nonrespiratory carbon reduction and NPC of 1.4 : 1.0 : 1.0 for D. tertiolecta and 2.1 : 0.5 : 1.0 for T. weissflogii. These ratios reflect the relatively high lipid content of D. tertiolecta, a characteristic that makes this species attractive for biodiesel production (Williams & Laurens, 2010). Viewed from the perspective of their relative investment ratios, these two species are distinct in their photosynthate investment strategies, despite their striking similarity in photosynthetic efficiencies (Fig. 1). Differences in photosynthate investments between species are also found among cryptophytes and cyanobacteria (Kunath et al., 2012) and diatoms (Su et al., 2012).
Linking transient carbon lifetimes to the cell cycle
For both D. tertiolecta and T. weissflogii, the transient carbon pool is 60–63% of gross carbon fixation (i.e. the area between GPc and NPc in Fig. 1). By conducting time-resolved measurements of 14C uptake, Halsey et al. (2011) showed that the average lifetime of this transient carbon increases significantly with increasing growth rate in D. tertiolecta. We find nearly identical results for T. weissflogii (Fig. 3). To explain these observations, we hypothesized that carbon is allocated to different end products during different phases of the cell cycle. Specifically, we envisioned the growth phase (G1) as a period when newly formed GAP is rapidly mobilized for ATP production and carbon reduction (or direct reductant utilization; see Halsey et al., 2011), whereas the division phase of the cell cycle (S, G2 and M) is a period when a significant fraction of GAP is first converted to a temporary storage polysaccharide (thus imparting a longer average lifetime). Given that the duration of the division phase is constant and independent of growth rate (Burbage & Binder, 2007; Matsumura et al., 2010), it follows that a rapidly growing population will have a greater fraction of cells in the division phase than a slower growing population, thus yielding longer average transient carbon lifetimes when growth rates increase. Because cell cycles of D. tertiolecta and T. weissflogii are not readily synchronized in culture, we tested this hypothesis using the related species, Dunaliella bioculata, which is easily synchronized (Marano et al., 1978).
For synchronized D. bioculata cultures growing at one division per day, the S phase spanned a 6-h period that overlapped with the G2-M phase, which spanned another 6-h period. The 20-min 14C uptake was similar for G2-M and G1 phases, but was notably higher during S phase (Fig. 2a). Furthermore, polysaccharide production roughly doubled during S phase compared with the other cell cycle stages and resulted in a rapid drop in cellular N : C stoichiometry (Fig. 2b). This storage carbon was later mobilized during the G2-M phase, thereby restoring the N : C ratio (Fig. 2b). These results implicate the S phase specifically as the period of enhanced GAP allocation to a storage pool. More broadly, we interpret our results as suggesting that the S phase is a period of lower energy requirements in which new photosynthate can be stored for the subsequent, energy-demanding cell division event.
Figure 2. Cell dynamics of Dunaliella bioculata during synchronized growth (μ = 0.69 d−1) across a complete cell cycle. Cells were synchronized using an 8.7 h : 15.3 h light : dark cycle (black bar). Data points are every 3 h over 24 h. (a) Cell number (X) doubles during G2-M phase (green X) and 14C-based photosynthesis–irradiance parameters peak during S phase. Error bars are SE of the estimate for P* (closed triangles) or alpha (open triangles). (b) Polysaccharide synthesis (closed squares) peaks during S phase. Polysaccharide metabolism strongly impacts the ratio of N and C (open circles).
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Earlier investigations of gene expression during the cell cycle showed that synthesis and degradation genes for storage polysaccharides are up-regulated just before S phase and G2-M phase, respectively (Mohr et al., 2010). Taken together with our cell cycle-resolved data on carbon pools, these gene expression results imply a temporal disjunction between gene expression and protein activity.
Our results for D. bioculata provide a mechanistic explanation for the observed growth rate dependence of 14C uptake data. Drawing from these results, we developed a cell model of carbon utilization for D. tertiolecta and T. weissflogii (see the 'Materials and Methods' section for details). In the model, newly fixed carbon is divided into a pool of short-lived (‘fast-burn’) GAP pathways that dominate during G1 and G2-M phases (note that, in diatoms, inorganic carbon can pass through a C4 form which may provide a small fraction of carbon backbones) and a longer lived (‘slow-burn’) pool, representing the temporary polysaccharide storage of S phase. The allocation of carbon between these two pools was described as a function of growth rate (i.e. fraction of the population in division). The application of this model to 14C uptake data collected across 24-h time courses for D. tertiolecta and T. weissflogii yields an excellent description (r2 = 0.94 for both species, Fig. S3) of the observed photosynthetic efficiencies across all growth rates and incubation time scales (Figs 3, S3, S4). Moreover, this performance is achieved with model parameters that are very similar for the two species, where optimized half-lives for the ‘fast-burn’ and ‘slow-burn’ carbon pools were 26 s and 1.9 h, respectively for D. tertiolecta and 34 s and 3.8 h, respectively for T. weissflogii. These parameters again highlight the remarkable similarity of photosynthetic and catabolic processes in these two evolutionarily distinct species.
Figure 3. Growth rate-dependent changes in lifetimes of newly fixed carbon (C) cause 14C-based production to decline over time. Comparison of data (symbols) collected from 14C time courses using nitrogen (N)-limited steady-state cultures grown under constant light, and a new model of C utilization describing C fixation and allocation to two pools with different mobilization rates (lines). Allocation of newly fixed C is a function of growth rate, with a greater fraction of C allocated to a temporary storage polysaccharide (long-half-life pool) in faster growing populations. (a) Data points (from Halsey et al., 2011) and lines are for Dunaliella tertiolecta grown at 1.2 d−1 (green triangles and line), 0.5 d−1 (red squares and line) and 0.12 d−1 (blue circles and line). (b) Thalassiosira weissflogii grown at 1.0 d−1 (green triangles and line), 0.5 d−1 (red squares and line) and 0.20 d−1 (blue circles and line). Data points are averages of duplicate measurements from two independent experiments. Bars show the range of values for each point. For all growth rates of D. tertiolecta, the model was implemented using optimized half-life parameters of 26 s for C allocated to the ‘fast-burn’ pool and 1.9 h for C allocated to the ‘slow-burn’ polysaccharide pool. For all growth rates of T. weissflogii, the optimized half-life parameters of the short- and long-half-life pools were 34 s and 3.8 h, respectively.
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This investigation aimed to identify the mechanisms underlying growth rate-dependent carbon metabolism in nutrient-limited, steady-state cultures grown under constant light. For this, we relied on information learned using a different species grown under different conditions (D. bioculata grown in a light–dark cycle and nutrient-replete conditions). Despite these differences, the strong correlation between the model and laboratory data suggests that changes in carbon metabolism across the cell cycle are key factors causing average lifetimes in newly fixed carbon to vary with nutrient-limited growth rate. Future studies using cultures synchronized under nutrient-limited growth conditions could reveal additional complexities involving metabolic shifts associated with cell cycle phases.
Generalities for photoautotrophy
Gross photosynthesis is related to net oxygen evolution and organic carbon production through the partitioning of photosynthate among diverse metabolic pathways. The results presented here indicate that this partitioning can be highly conserved between phytoplankton taxonomic groups and independent of the steady-state growth rate. Additional studies are required to generalize our findings to other phytoplankton groups and nutrient conditions. For example, cyanobacteria (Kunath et al., 2012) or highly motile picoeukaryotic species (Goulbourne & Greenberg, 1980; Raven & Richardson, 1984; Kaneda & Furuya, 1986) may have sufficiently different ATP : reductant requirements to significantly alter GPO2 : GPC : NPC ratios from those reported here. Furthermore, growth under high macronutrient levels and limiting iron supply appears to result in over-expressed photosynthetic pigments and lower absorption-normalized photosynthetic efficiencies relative to macronutrient-limited growth (Schrader et al., 2011). Nevertheless, the similarities in photosynthate partitioning and absorption-normalized photosynthetic efficiencies observed during our study suggest that a common optimization solution for photoautotrophic growth has evolved for at least two evolutionarily distinct phytoplankton species. Deeper evaluation of this energetic optimization is clearly warranted.
For both D. tertiolecta and T. weissflogii, we show that the measured 14C uptake rates register gross carbon fixation only during very short incubations (minute time scale) and then approach net carbon fixation with increasing incubation duration. Short-term (c. 30 min to 2 h) field measurements of photosynthesis–irradiance relationships are commonly used to parameterize global satellite-based models of ocean production. Our results indicate that the correct interpretation of these data requires consideration of the incubation duration and a knowledge of the population growth rates. Conversely, it may be possible to infer information about phytoplankton growth rates in the field by conducting 14C uptake measurements over a range of incubation times. Our results also indicate that growth rate-dependent differences in transient carbon lifetimes are linked to cell cycle-related changes in photosynthate storage and mobilization, thus contributing new insight towards a systems-level understanding of cellular carbon flow.