The carbonic anhydrase CAH1 is an essential component of the carbon-concentrating mechanism in Nannochloropsis oceanica

Abstract

Aquatic photosynthetic organisms cope with low environmental CO₂ concentrations through the action of carbon-concentrating mechanisms (CCMs). Known eukaryotic CCMs consist of inorganic carbon transporters and carbonic anhydrases (and other supporting components) that culminate in elevated [CO₂] inside a chloroplastic Rubisco-containing structure called a pyrenoid. We set out to determine the molecular mechanisms underlying the CCM in the emerging model photosynthetic stramenopile, Nannochloropsis oceanica, a unicellular picoplanktonic alga that lacks a pyrenoid. We characterized CARBONIC ANHYDRASE 1 (CAH1) as an essential component of the CCM in N. oceanica CCMP1779. We generated insertions in this gene by directed homologous recombination and found that the cah1 mutant has severe defects in growth and photosynthesis at ambient CO₂ We identified CAH1 as an α-type carbonic anhydrase, providing a biochemical role in CCM function. CAH1 was found to localize to the lumen of the epiplastid endoplasmic reticulum, with its expression regulated by the external inorganic carbon concentration at both the transcript and protein levels. Taken together, these findings show that CAH1 is an indispensable component of what may be a simple but effective and dynamic CCM in N. oceanica.

Keywords: algae; carbon-concentrating mechanism; carbonic anhydrase; heterokont; photosynthesis.

Fig. 1.

CAH1 is required for normal growth and photosynthesis at low CO₂. (A) Spot growth phenotype of WT, cah1 mutant, and tagged complementation lines. High CO₂, 3% CO₂; low CO₂, 0.04% CO₂; HygR, control line expressing the hygromycin resistance cassette; CAH1:FLAG or CAH1:Venus, cah1 (#5) complemented with CAH1 CDS with a C-terminal tag. Two independent lines are shown for cah1 and complemented lines. (B) Whole-cell photosynthetic DIC affinity assay. Cells were acclimated to low CO₂ for 24 h, after which O₂ evolution was measured in response to increasing concentrations of DIC. Chl, chlorophyll. (C) Calculated [DIC] for half maximal rate = K_0.5 (DIC). Bar height and numbers above bars indicate the mean. Individual data points are plotted as white circles. *P < Bonferroni-corrected α (0.05/3 = 0.0167), Welch’s t test (n = 4). n.s., not significant. (D) Quantum yield of ΦPSII and photoprotection (NPQ) of WT and cah1 cells at two [DIC]. Data markers indicate the mean. Error bars indicate SD (n = 9). Two-factor ANOVA yielded significant genotype × [DIC] interaction terms (P < 0.001).

Fig. 2.

CAH1 is an α-type carbonic anhydrase. (A) Multiple sequence alignment of α-type carbonic anhydrases. Catalytic zinc-binding histidine residues are denoted by triangles; the red triangle indicates the target of the His to Ala site-directed mutagenesis to produce the mCAH1 lines. (B) Spot growth assay of H177A mCAH1 lines. The assay was performed as in Fig. 1A. High CO₂, 3%; low CO₂, 0.04%. (C) The carbonic anhydrase inhibitor ethoxyzolamide reduces DIC affinity in WT cells, but not in cah1 cells. WT and cah1 liquid cultures were incubated with 100 μM ethoxyzolamide before assessment of photosynthetic DIC affinity by oxygen evolution. *P < 0.05/2, Welch’s t test. n.s., not significant.

Fig. 3.

Subcellular localization of CAH1:Venus fusion protein by fluorescence microscopy. (A) WT cell showing baseline plastid autofluorescence in the CFP and YFP channels. (B) A CAH1:Venus fusion protein (YFP channel) was coexpressed with a cytoplasmic mCerulean marker (CFP channel). (C) Similar to B, but with an ER-luminal mCerulean marker. Cells were grown in liquid culture in 3% CO₂ and then transferred to ambient air for 24 h before imaging. (Scale bar: 5 µm.)

Fig. 4.

Increased DIC affinity and CAH1 expression are associated with low CO₂. (A) K_0.5 (DIC) calculated from whole-cell O₂ evolution assays of WT and cah1 cells after acclimation to low CO₂ for 24 h. (B) CAH1 protein expression was assessed by immunoblot analysis of CAH:FLAG complementation lines (three independent lines shown). Protein samples were taken from cells placed at low CO₂ for 24 h or kept at high CO₂. (C) Fluorescent reporter lines for CAH1 transcription and protein level were grown at high CO₂ (3%) and transferred to low CO₂ (0.04%). The transcriptional reporter consisted of the native 1-kb promoter region driving Venus (CAH1_pro:Venus). The CAH1 protein reporter was similar but included the CAH1 CDS in frame (CAH1:Venus). Normalized fluorescence, in relative fluorescence units (RFU), divided by absorbance at 750 nm, was quantified at the indicated time points for three independent lines (colored lines). Error bars show SD of n = 6 wells per line. Replicate plates were kept at 3% CO₂ as controls (gray lines).

Fig. 5.

A proposed model for the CCM of N. oceanica. The plastid is separated from the cytoplasm by a total of four membranes, the outermost of which is contiguous with the ER and outer nuclear envelope (called the epiplastid ER). Transporters pump bicarbonate into the cytoplasm and then into the lumen of the epiplastid ER, where the carbonic anhydrase CAH1 also accumulates. CAH1 catalyzes the formation of CO₂, which diffuses (dotted line) either into the chloroplast stroma to be fixed by Rubisco in the Calvin–Benson–Bassham cycle, or back out into the environment. Although leaky, this CCM is necessary for growth and photosynthesis at ambient (400 ppm CO₂) conditions, likely by enhancing the carboxylation rate and suppressing photorespiration. CBB, Calvin–Benson–Bassham cycle.