Large variation in the Rubisco kinetics of diatoms reveals diversity among their carbon-concentrating mechanisms

Abstract

While marine phytoplankton rival plants in their contribution to global primary productivity, our understanding of their photosynthesis remains rudimentary. In particular, the kinetic diversity of the CO2-fixing enzyme, Rubisco, in phytoplankton remains unknown. Here we quantify the maximum rates of carboxylation (k cat (c)), oxygenation (k cat (o)), Michaelis constants (K m) for CO2 (K C) and O2 (K O), and specificity for CO2 over O2 (SC/O) for Form I Rubisco from 11 diatom species. Diatom Rubisco shows greater variation in K C (23-68 µM), SC/O (57-116mol mol(-1)), and K O (413-2032 µM) relative to plant and algal Rubisco. The broad range of K C values mostly exceed those of C4 plant Rubisco, suggesting that the strength of the carbon-concentrating mechanism (CCM) in diatoms is more diverse, and more effective than previously predicted. The measured k cat (c) for each diatom Rubisco showed less variation (2.1-3.7s(-1)), thus averting the canonical trade-off typically observed between K C and k cat (c) for plant Form I Rubisco. Uniquely, a negative relationship between K C and cellular Rubisco content was found, suggesting variation among diatom species in how they allocate their limited cellular resources between Rubisco synthesis and their CCM. The activation status of Rubisco in each diatom was low, indicating a requirement for Rubisco activase. This work highlights the need to better understand the correlative natural diversity between the Rubisco kinetics and CCM of diatoms and the underpinning mechanistic differences in catalytic chemistry among the Form I Rubisco superfamily.

Keywords: Algae; Rubisco.; carbon fixation; diatoms; kinetics; photosynthesis.

Fig. 1.

Measurement of Rubisco activation status, maximal activity, and stability in vitro at 25 °C. Soluble cellular protein rapidly extracted from tobacco and each phytoplankton in CO₂-free extraction buffer (containing 5mM MgCl₂) was used to measure changes in the Rubisco ¹⁴CO₂ fixation rate after activating the extract for 0–20min in buffer containing 15mM MgCl₂ and 15mM NaHCO₃. Gray shading indicates the time when protein extract was assayed to quantify k _cat ^c, K _C, and K _O (Table 1). Data represent measures from duplicate biological samples (± SD).

Fig. 2.

Rubisco kinetic parameters measured at 25 °C. Rubisco properties measured from diatoms (blue circles), tobacco and maize (green circles, see Table 1) compared with previously published values for red algae (red, maroon dashes), C₃ plants (dark green dashes), and C₄ plants (light green dashes) (Badger et al., 1998; Savir et al., 2010; Supplementary Table S1). Kinetic parameters include (A) the maximum rates of carboxylation (k _cat ^c), (B) oxygenation rate (k _cat ^o), the K _m for (C) CO₂ (K _C) and (D) O₂ (K _O), (E) the specificity for CO₂ over O₂ (S_C/O), and (F) the carboxylation efficiency (k _cat ^c /K _C, mM s⁻¹).

Fig. 3.

Comparing the catalytic relationships of diatom and other Form I Rubiscos. Comparison of (A) maximum carboxylation rate (k _cat ^c) versus the K _m for CO₂ (K _C), (B) K _C versus the K _m for O₂ (K _O), (C) the carboxylation efficiency (k _cat ^c/K _C) versus specificity for CO₂ over O₂ (S_C/O), and (D) the maximum oxygenation rate (k _cat ^o) and K _O. Measured for diatoms (black circles) and compared with the compilation of plants and eukaryotic algae from Savir et al. (2010), Badger et al. (1998), Whitney et al. (2011), and Galmes et al. (2014) (gray circles).

Fig. 4.

Relationships of Rubisco content to catalysis. (A) Rubisco content as a percentage of total cellular protein (% TP) is positively correlated with carboxylation efficiency in 21% O₂ (k _cat ^c/K _C ^21%O2). (B) This is largely driven by the negative correlation of Rubisco content with K _C ^21%O2. Rubisco content was taken from Losh et al. (2013). I. galbana (Ig), C. muelleri (Cm), P. tricornutum 642 (Pt642), T. weissflogii (Tw), T. oceanica (To), and S. marinoi (Sm).

Fig. 5.

Resource allocation hypothesis for the balance between Rubisco content and K _C values. Organisms that invest their energy resources heavily in the CCM (e.g. cyanobacteria) to maintain high intracellular CO₂ levels to saturate Rubisco and limit photorespiration are able to reduce their resource investment in Rubisco. At the other end of the spectrum, organisms without a CCM (e.g. C₃ plants) have Rubisco that is undersaturated with CO₂ and require large resource investments in Rubisco content to maintain adequate rates of carbon fixation for survival. Organisms with a CCM fall somewhere along the saturation curve, depending on the carboxylation speed of their Rubisco and their potential to balance the investment of resources in Rubisco biogenesis suitably or maintain elevated intercellular CO₂ levels around Rubisco.