The role of Rubisco kinetics and pyrenoid morphology in shaping the CCM of haptophyte microalgae

Abstract

The haptophyte algae are a cosmopolitan group of primary producers that contribute significantly to the marine carbon cycle and play a major role in paleo-climate studies. Despite their global importance, little is known about carbon assimilation in haptophytes, in particular the kinetics of their Form 1D CO2-fixing enzyme, Rubisco. Here we examine Rubisco properties of three haptophytes with a range of pyrenoid morphologies (Pleurochrysis carterae, Tisochrysis lutea, and Pavlova lutheri) and the diatom Phaeodactylum tricornutum that exhibit contrasting sensitivities to the trade-offs between substrate affinity (Km) and turnover rate (kcat) for both CO2 and O2. The pyrenoid-containing T. lutea and P. carterae showed lower Rubisco content and carboxylation properties (KC and kCcat) comparable with those of Form 1D-containing non-green algae. In contrast, the pyrenoid-lacking P. lutheri produced Rubisco in 3-fold higher amounts, and displayed a Form 1B Rubisco kCcat-KC relationship and increased CO2/O2 specificity that, when modeled in the context of a C3 leaf, supported equivalent rates of photosynthesis to higher plant Rubisco. Correlation between the differing Rubisco properties and the occurrence and localization of pyrenoids with differing intracellular CO2:O2 microenvironments has probably influenced the divergent evolution of Form 1B and 1D Rubisco kinetics.

Keywords: Algae; Haptophyta; Rubisco; carbon-concentrating mechanisms; pyrenoid.

Fig. 1.

Rubisco evolution and catalysis. Geological history of the past versus present atmospheric [CO₂] (gray) and percentage atmospheric O₂ (% v/v) (black; modified from Berner and Canfield, 1989; Badger et al., 2002; Whitney et al., 2011) highlighting the estimated appearance of key primary producers (horizontal lines) (Yoon et al., 2002, 2004; Liu et al., 2010) their differing Form 1B or ID Rubisco lineages they produce, and the predicted timing when algal carbon-concentrating mechanisms (CCMs; gray shading) evolved (Badger et al., 2002; Moritz and Griffiths, 2013).

Fig. 2.

Microalgae pyrenoid and CCM composition. (A) TEM images were compiled from the literature to represent the range of pyrenoids presented in this. To represent a pyrenoid lacking Pavlovale, we use Pavlova viridis from Bendif et al. (2011) (Protist, 162, Bendif EM, Probert I, Hervé A, Billard C, Goux D, Lelong C, Cadoret JP, Véron B. Integrative taxonomy of the Pavlovophyceae (Haptophyta): a reassessment, 738–761, ©2011, with permission from Elsevier) as the TEM image clearly represents the lack of pyrenoid. Pavlova lutheri is visualized in the same study; however, the TEM image does not show the chloroplast (Ch) lacking a pyrenoid as clearly. TEM image of P. carterae from Beech and Wetherbee (1988) [republished with permission of the International Phycological Society from Observations on the flagellar apparatus and peripheral endoplasmic reticulum of the coccolithophorid, Pleurochrysis carterae (Prymnesiophyceae), Beech PL, Wetherbee R, Phycologia 27, 1988; permission conveyed through Copyright Clearance Center, Inc.] illustrates pyrenoids (Py) bulging toward the center of the cell, and the two species T. lutea (Bendif et al., 2014) (Journal of Applied Phycology, Erratum to: On the description of Tisochrysis lutea gen. nov. sp. nov. and Isochrysis nuda sp. nov. in the Isochrysidales, and the transfer of Dicrateria to the Prymnesiales (Haptophyta), 26, 2014, 1617, Bendif EM, Probert I, Schroeder DC, de Vargas C. With permission of Springer) and P. tricornutum (Allen et al., 2011) (Allen AE, Moustafa A, Montsant A, Eckert A, Kroth PG, Bowler C. Evolution and functional diversification of fructose bisphosphate aldolase genes in photosynthetic marine diatoms. Molecular Biology and Evolution 2012, 29, 367–279, by permission of Oxford University Press) show pyrenoids immersed within the chloroplast. (B) Summary of published experimental evidence for the presence of a CCM in the species with a pyrenoid. Evidence for a CCM is detectable by: (i) inhibition of CO₂ assimilation by the impermeable acetazolamide (AZA) or membrane-permeable ethoxyzolamide (EZA) CA inhibitors (Burns and Beardall, 1987; Okazaki et al., 1992; Badger et al., 1998; Hopkinson et al., 2013); (ii) stimulation of CA activity following cell illumination (Badger et al., 1998); (iii) whether the intercellular C_i pool is higher than the external environment (Badger et al., 1998); or (iv) the preliminary detection of δ-CA using methods described in the Materials and methods.

Fig. 3.

The diversity in the kinetic properties of haptophyte Rubisco at 25 °C. Comparative relationships between the kinetic properties measured in this study for Rubisco from P. lutheri (Pl), P. carterae (Pc), T. lutea (Tl), the diatom P. tricornutum (Pt), and from tobacco (Tob) with those of other Form 1B and 1D Rubiscos (see key) as curated by Young et al. (2016). The plotted maximal carboxylation and oxygenation turnover rates (k^C_cat, k^O_cat), relative specificity for CO₂ over O₂ (S_C/O). and the Michaelis constants (K_m) for CO₂ and O₂ (K_C, K_O) are from Table 1. Linear regressions are shown for the differing (A) k^C_cat–K_C and (B) k^O_cat–K_O relationships displayed for Form 1B and 1D Rubiscos. No statistically significant relationships were evident among correlative analyses of (C) k^C_cat and k^O_catK_C or between (D) K_C and K_O. (E) An exponential relationship was apparent when comparing the kinetic trade-off between S_C/O with k^C_cat with the differing phylogenetic Rubisco groupings aggregated at differing positions along the gradient

Fig. 4.

The differential effect of O₂ on Rubisco carboxylation efficiency. Variation in the response of carboxylation efficiency (CE; k^C_cat/K_C) to O₂ levels (O) for Rubisco from tobacco (tob, vascular plant control), the diatom P. tricornutum (Pt, dotted line), and the haptophytes P. lutheri (Pl, solid gray line), P. carterae (Pt, solid black line), and T. lutea (Tl, dashed black line). Lines were fitted to the equation CE=k^C_cat/{K_C×[1+(O/K_O)]} using the parameters listed in Table 1. Arrows indicate the differing O₂ levels in fresh water and the ocean surface [assuming ~3.5% (w/v) salinity] at an atmospheric pressure of 1.013 bar.

Fig. 5.

Rubisco content is reduced in pyrenoid-containing phytoplankton. The Rubisco content (quantified by [¹⁴C]CABP binding and expressed as a percentage of the cellular soluble protein) in cells grown at 20 °C under saturating nutrients was higher (11.4 ± 1.2%) in the pyrenoid-lacking P. lutheri cells relative to that in P. carterae (3.0 ± 0.8%), T. lutea (3.5 ± 0.9%), and P. tricornutum (3.2 ± 0.6%).

Fig. 6.

The varying potential of phytoplankton Rubisco in a C₃ leaf. The influence of each Rubisco analyzed in Table 1 on CO₂ assimilation rates (A) at 25 °C in a C₃ leaf as a function of C_c was modeled according to Farquhar et al. (1980) as described in the Materials and methods. For the tobacco Rubisco, the photosynthetic rate became light limited (indicated by 1*) at C_c>320 μbar.