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. 2001 May;126(1):421-33.
doi: 10.1104/pp.126.1.421.

The light sensitivity of ATP synthase mutants of Chlamydomonas reinhardtii

W Majeran  1 , J Olive, D Drapier, O Vallon, F A Wollman
Affiliations

The light sensitivity of ATP synthase mutants of Chlamydomonas reinhardtii

W Majeran et al. Plant Physiol. 2001 May.

Abstract

Chlamydomonas reinhardtii mutants defective in the chloroplast ATP synthase are highly sensitive to light. The ac46 mutant is affected in the MDH1 gene, required for production or stability of the monocistronic atpH mRNA encoding CF(O)-III. In this and other ATP synthase mutants, we show that short-term exposure to moderate light intensities-a few minutes-induces an inhibition of electron transfer after the primary quinone acceptor of photosystem II (PSII), whereas longer exposure-several hours-leads to a progressive loss of PSII cores. An extensive swelling of thylakoids accompanies the initial inhibition of electron flow. Thylakoids deflate as PSII cores are lost. The slow process of PSII degradation involves the participation of ClpP, a chloroplast-encoded peptidase that is part of a major stromal protease Clp. In the light of the above findings, we discuss the photosensitivity of ATP synthase mutants with respect to the regular photoinhibition process that affects photosynthetic competent strains at much higher light intensities.

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Figures

Figure 1
Figure 1
Characterization of the ac46 mutant. A, Immunoblots of whole cell protein extracts of the ac46 and WT strains, probed with specific antibodies for ATP synthase subunits. OEE2 was included as a loading control. B, Accumulation of the atpH transcripts in the ac46 and WT strains. The psbA probe was included as a loading control.
Figure 2
Figure 2
Early changes in room temperature fluorescence. Fluorescence induction kinetics were recorded with or without DCMU either after incubation in low light (LL = 6 μE m−2 s−1) or after 4-min exposure to moderate light (ML = 70 μE m−2 s−1). A, ac46; B, wild type and cytochrome b6f mutant F18; C, evolution of F0, Fs, and Fm during exposure of ac46 to moderate light. Inset, Evolution of (Fi − F0)/F0.
Figure 3
Figure 3
Light-induced thylakoid swelling in ac46. Cells were fixed in low light (A), or after exposure to moderate light for 30 min (B) or 3 h (C). D, Control experiment, wild-type cells exposed for 3 h to moderate light.
Figure 4
Figure 4
Light-induced swelling is prevented by addition of uncouplers or the absence of cytochrome b6f. A, ac46 treated with uncouplers [200 μm Crown, 5 μm carbonylcyanide-4-tri(fluoromethoxy)phenylhydrazone], then exposed to moderate light for 3 h. B, F54-F18 exposed to moderate light for 3 h.
Figure 5
Figure 5
Effect of state transitions on thylakoid swelling in the absence of PSII-driven electron transfer; F54-F34 double mutant lacking ATP synthase and PSII (A and C) and ac46 incubated with DCMU (B and D). Cells were exposed to moderate light for 30 min, either without Glc/Glc oxidase, i.e. in state I conditions (A and B), or with Glc/Glc oxidase, i.e. in state II conditions (C and D).
Figure 6
Figure 6
Loss of thylakoid swelling upon long-term exposure to light. A, ac46 cells exposed to 70 μE m−2 s−1 light for 48 h. B, Evolution of the average thylakoid width (arbitrary units) during light treatment.
Figure 7
Figure 7
Flash-induced electrochromic signal recorded at 515 nm. PSII activity is inhibited by DCMU + hydroxylamine prior to the measurements. Curves are normalized to the electrochromic shift recorded 100 μs after the actinic flash (phase a, representing PSI charge separation). Times of exposition to moderate light are indicated in hours.
Figure 8
Figure 8
Changes at Fm and FO upon prolonged exposure of ac46 to moderate light.
Figure 9
Figure 9
Freeze fracture analysis of ac46 grown in low light (A) or after 48-h exposure to 70 μE m−2 s−1 light (B). Note the loss in most of the large EF particles, whereas protoplasmic faces (top right) remain unaltered.
Figure 10
Figure 10
Accumulation of photosynthetic complexes in ATP synthase mutants after exposure to moderate light. A, ac46 was exposed to moderate light for 0, 6, and 24 h. PSII, PSI subunits, and cytochrome b6 were detected by immunoblotting, cytochrome f by TMBZ staining. B, Strain ac46-AUU treated as in A. Only PSII subunits are shown. C, Strains F54 (white symbols) and F54-AUU (black symbols) were exposed to moderate light in the presence of lincomycin, an inhibitor of chloroplast protein synthesis. The levels of CP47 (squares) and CP43 (triangles) were measured by immunoblotting and phosphoimager quantification (ImageQuant software, Molecular Dynamics, Sunnyvale, CA).
Figure 11
Figure 11
Summary of the major events occurring upon illumination of an ATP synthase mutant at 70 μE m−2 s−1.
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