Fig. 3.

EPYC1 is essential for Rubisco aggregation in the pyrenoid. (A) Representative TEMs of WT and epyc1 cells grown at low CO₂. (B) Quantification of the pyrenoid area as a percentage of cell area of WT and epyc1 cells grown at low CO₂. Data are from TEM images as represented in A. epyc1: n = 37; WT: n = 79. P < 10⁻¹⁹, Welch’s t test. (C) QFDEEM of the pyrenoid of WT and epyc1 cells grown at low CO₂. M, pyrenoid matrix; St, stroma; Th, thylakoids; SS, starch sheath. (Inset) Four hundred percent zoom view of the pyrenoid matrix. (D) Rubisco protein levels in WT and epyc1 cells grown at low and high CO₂ were probed by Western blot analysis. (E) Localization of Rubisco was determined by microscopy of WT and the epyc1 mutant containing RBCS1-mCherry. The sum of the fluorescence signals from Z stacks was used for quantitation. (F) The fraction of RBCS1-mCherry signal from outside the pyrenoid region (inner dotted line in E) was quantified in WT and epyc1 cells at low CO₂. epyc1: n = 27; WT: n = 27. ***P < 10⁻¹⁵, Student’s t test. (G) Representative images of anti-Rubisco immunogold labeling of WT and epyc1 cells grown at low CO₂. Gold particles were enlarged 10× for visibility. (H) The fraction of immunogold particles outside the pyrenoid was quantified. WT: n = 26 cells, 8,123 gold particles; epyc1: n = 27 cells, 2,708 gold particles. ***P < 10⁻¹⁵, Student’s t test. In F and H, data are mean values, with error bars indicating SEM. Yellow arrows indicate pyrenoids. (Scale bars: 1 µm.)