The requirement for carotenoids in the assembly and function of the photosynthetic complexes in Chlamydomonas reinhardtii

Abstract

We have investigated the importance of carotenoids on the accumulation and function of the photosynthetic apparatus using a mutant of the green alga Chlamydomonas reinhardtii lacking carotenoids. The FN68 mutant is deficient in phytoene synthase, the first enzyme of the carotenoid biosynthesis pathway, and therefore is unable to synthesize any carotenes and xanthophylls. We find that FN68 is unable to accumulate the light-harvesting complexes associated with both photosystems as well as the RC subunits of photosystem II. The accumulation of the cytochrome b₆f complex is also strongly reduced to a level approximately 10% that of the wild type. However, the residual fraction of assembled cytochrome b₆f complexes exhibits single-turnover electron transfer kinetics comparable to those observed in the wild-type strain. Surprisingly, photosystem I is assembled to significant levels in the absence of carotenoids in FN68 and possesses functional properties that are very similar to those of the wild-type complex.

Figure 1.

Growth phenotypes on solid medium of the wild type (WT), FN68, and FN68 transformed with wild-type PSY (T1–T4). Colonies were grown under heterotrophic (acetate, no light), mixotrophic (acetate, light), and phototrophic (no acetate, light) conditions. [See online article for color version of this figure.]

Figure 2.

A and B, Absorption spectra of whole cells of C. reinhardtii at room temperature (A) and fluorescence emission spectrum at 77 K upon excitation at 460 nm (B). Solid lines, the wild type; dashed lines, Fud7-P71 strain; dashed-dotted lines, FN68. The spectra are normalized to maximal absorption and fluorescence emission, respectively. a.u., Arbitrary units. C, Deriphat-PAGE of thylakoid membranes (35,000g fractions) from the wild type (WT) and FN68. PSI/PSII-sc, Nondissociated PSI/PSII supercomplexes; LHC-m, monomeric LHC; FP, free pigment. Lanes were loaded based on equal Chl (5 µg Chl a+b) concentration. D, Immunoblot analysis of whole cell extracts using antibodies raised to subunits of PSI (PsaD, PsaF, PsaN), PSII (D1), Cyt b₆f complex (Cyt. f), and LHC of PSI (p11) and of PSII (p15). Lanes were loaded based on equal cell numbers. [See online article for color version of this figure.]

Figure 3.

Biochemical characterization of the Cyt b₆f complex in the FN68 mutant. Immunoblot (A) and specific heme staining for Cyt f and Cyt b (B) are shown for membrane protein extracts probed with antibodies against Cyt f, Cyt b₆, subunit IV, the Rieske iron-sulfur center (FeS), and subunit β of the ATP synthase. Loading was normalized based on an equal Chl basis. wt, Wild type.

Figure 4.

A and B, Kinetics of difference absorption induced by saturating laser excitation in whole cells of strains Fud7-P71 (A) and FN68 (B) at room temperature in the 0.5- to 100-μs time range. Detection wavelengths were as follows: 540 nm (squares), 545 nm (circles), 550 nm (triangles), 555 nm (stars), 560 nm (diamonds), and 565 nm (hexagons). Continuous lines are the fit to the experimental data with a sum of exponential function. Each kinetic trace is normalized to the intensity of the ΔI/I signal at 10-ns delay. C and D, DAS obtained from global fitting of the kinetic transient in the Fud7-P71 (C) and the FN68 (D) strains. For DAS, τ = 77 μs (black diamonds), τ = 357 μs (white squares), offset (black circles). The insets show the details of the DAS in the α- and β-bands of Cyt b₆f absorption for 77 and 357 μs. The DAS are normalized to the value of extrapolated P₇₀₀⁺ absorption bleaching at 430 nm, extrapolated to t_→0 (for time that tends to zero). a.u., Arbitrary units.

Figure 5.

DAS obtained from global fitting of the kinetic transient in the near UV and visible regions of the absorption spectrum. Black squares/solid lines, 20-ns component; white circles/solid lines, 250-ns component; white triangles/dashed-dotted lines, 6-μs component (for precise values, see Table I). The continuous line is the initial spectrum calculated at t_→0 (for time that tends to zero) after subtraction of nondecaying signal within the sampled time window. The DAS are normalized to the value of the t_→0 spectrum at 430 nm. A, FuD7-P71 (control). B, FN68.