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. 2017 Jun 1;68(14):3903-3913.
doi: 10.1093/jxb/erx197.

Pyrenoid loss in Chlamydomonas reinhardtii causes limitations in CO2 supply, but not thylakoid operating efficiency

Oliver D Caspari  1 , Moritz T Meyer  1 , Dimitri Tolleter  2 , Tyler M Wittkopp  2   3 , Nik J Cunniffe  1 , Tracy Lawson  4 , Arthur R Grossman  2 , Howard Griffiths  1
Affiliations

Pyrenoid loss in Chlamydomonas reinhardtii causes limitations in CO2 supply, but not thylakoid operating efficiency

Oliver D Caspari et al. J Exp Bot. .

Abstract

The pyrenoid of the unicellular green alga Chlamydomonas reinhardtii is a microcompartment situated in the centre of the cup-shaped chloroplast, containing up to 90% of cellular Rubisco. Traversed by a network of dense, knotted thylakoid tubules, the pyrenoid has been proposed to influence thylakoid biogenesis and ultrastructure. Mutants that are unable to assemble a pyrenoid matrix, due to expressing a vascular plant version of the Rubisco small subunit, exhibit severe growth and photosynthetic defects and have an ineffective carbon-concentrating mechanism (CCM). The present study set out to determine the cause of photosynthetic limitation in these pyrenoid-less lines. We tested whether electron transport and light use were compromised as a direct structural consequence of pyrenoid loss or as a metabolic effect downstream of lower CCM activity and resulting CO2 limitation. Thylakoid organization was unchanged in the mutants, including the retention of intrapyrenoid-type thylakoid tubules, and photosynthetic limitations associated with the absence of the pyrenoid were rescued by exposing cells to elevated CO2 levels. These results demonstrate that Rubisco aggregation in the pyrenoid functions as an essential element for CO2 delivery as part of the CCM, and does not play other roles in maintenance of photosynthetic membrane energetics.

Keywords: Carbon-concentrating mechanism; Chlamydomonas; Rubisco; chlorophyll fluorescence; chloroplast; electrochromic shift; electron transport rate; green algae; photosynthesis; pyrenoid; reinhardtii.

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Figures

Fig. 1.
Fig. 1.
Pyrenoid absence has no effect on thylakoid arrangement. TEM shows WT and pyr– (Spinacia RBCS) cells grown in contrasting physiological conditions in (A–F) with the canonical pyrenoid location circled. Low light (LL) was 10 µmol photons m−2 s−1 and standard light (SL) was 50 µmol photons m−2 s−1. A quantification of thylakoid stacking is provided in terms of the width of appressed regions (G) as mean ±SE across 20 cells per condition (five measurements per cell). Block face SEM (H) reveals that knotted thylakoid tubules are retained in pyr–.
Fig. 2.
Fig. 2.
Under CCM-inducing conditions, pyr– shows a reduction in CO2-limited photosynthesis (ETRmax). Light response curves were collected using a Technologica CFimager from algal colonies grown on agar plates in LL (low light, 10 µmol photons m−2 s−1) or SL (standard light, 50 µmol photons m−2 s−1) in air or 5% CO2, as indicated. The ETR is shown as the mean of six biological replicates ±SE, overlaid with 95% confidence intervals derived from fitting a light response model (Equation 2). Probability density plots of marginal posteriors for the fit parameters ETRmax and Ik are shown in the figure margins.
Fig. 3.
Fig. 3.
High-resolution ECS and Chl fluorescence measurements reveal feedback limitation by Rubisco catalysis. Panels show JTS-10 data of (A) ETR through PSII estimated on the basis of Chl fluorescence data, (B) TEF estimated on the basis of ECS data, and (C) NPQ. Data are the mean ±SE based on ≥3 biological replicates each, overlaid with 95% confidence intervals derived using a Bayesian approach to capture the underlying physiological dynamics in terms of model parameters. ETRmax, TEFmax, and NPQmax describe the maximum attainable levels of each photosynthetic indicator. Ik is the light saturation point, while I50 is the light half-saturation point. Finally, n is the Hill coefficient which controls the sigmoidicity of the NPQ curve, and can be indicative of allosteric regulation when n>1. Probability density plots of the marginal posteriors for fit parameters are shown in the figure margins. Cells were grown in liquid culture in the absence of aeration at 50 µmol photons m−2 s−1.
Fig. 4.
Fig. 4.
Addition of bicarbonate restores photosynthetic performance in air-acclimated pyr– cells. PSII operating efficiency (ϕ II) was measured in the JTS-10 after 5, 30, and 120 s at ~350 µmol photons m−2 s−1 (to ensure saturation; see Ik; Figs 2, 3) as detailed above the data panels. Cells were grown in air at a range of light intensities (LL, SL, and HL) as shown on the x-axis. Cultures supplemented with 10 mM sodium bicarbonate directly before measurements are shown by dashed lines. Data are the mean of three biological replicates ±SE.
Fig. 5.
Fig. 5.
Accumulation of pigments undergoes physiological acclimation independent of pyrenoid phenotype. Chl expression on a per cell basis (A) is shown as a scatterplot of individual measurements. Starting with the accumulation of carotenoids relative to Chl (B), data are plotted against growth light intensity and shown as the mean of three biological replicates ±SE. The functional PSI/PSII ratio (C) and the Chl a/b ratio (D), respectively, enable quantification of Chl allocation to PSII from spectroscopic data (E) and Chl extraction data (F). Fluorescence saturation kinetics in the presence of DCMU (t2/3) provide a proxy for functional PSII antenna size (G). Growth rates (H) are shown as percentage change in Chl ml−1 over 24 h as mean ±SE based on four measurements over the course of 6 d of three biological replicates per condition grown in quasi-continuous culture; three asterisks signify statistical significance at the level of P<0.01 estimated through ANOVA.
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