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. 2012 Aug;159(4):1428-39.
doi: 10.1104/pp.112.199042. Epub 2012 Jun 14.

Cooperative D1 degradation in the photosystem II repair mediated by chloroplastic proteases in Arabidopsis

Yusuke Kato  1 , Xuwu Sun, Lixin Zhang, Wataru Sakamoto
Affiliations

Cooperative D1 degradation in the photosystem II repair mediated by chloroplastic proteases in Arabidopsis

Yusuke Kato et al. Plant Physiol. 2012 Aug.

Abstract

Light energy constantly damages photosynthetic apparatuses, ultimately causing impaired growth. Particularly, the sessile nature of higher plants has allowed chloroplasts to develop unique mechanisms to alleviate the irreversible inactivation of photosynthesis. Photosystem II (PSII) is known as a primary target of photodamage. Photosynthetic organisms have evolved the so-called PSII repair cycle, in which a reaction center protein, D1, is degraded rapidly in a specific manner. Two proteases that perform processive or endopeptidic degradation, FtsH and Deg, respectively, participate in this cycle. To examine the cooperative D1 degradation by these proteases, we engaged Arabidopsis (Arabidopsis thaliana) mutants lacking FtsH2 (yellow variegated2 [var2]) and Deg5/Deg8 (deg5 deg8) in detecting D1 cleaved fragments. We detected several D1 fragments only under the var2 background, using amino-terminal or carboxyl-terminal specific antibodies of D1. The appearance of these D1 fragments was inhibited by a serine protease inhibitor and by deg5 deg8 mutations. Given the localization of Deg5/Deg8 on the luminal side of thylakoid membranes, we inferred that Deg5/Deg8 cleaves D1 at its luminal loop connecting the transmembrane helices C and D and that the cleaved products of D1 are the substrate for FtsH. These D1 fragments detected in var2 were associated with the PSII monomer, dimer, and partial disassembly complex but not with PSII supercomplexes. It is particularly interesting that another processive protease, Clp, was up-regulated and appeared to be recruited from stroma to the thylakoid membrane in var2, suggesting compensation for FtsH deficiency. Together, our data demonstrate in vivo cooperative degradation of D1, in which Deg cleavage assists FtsH processive degradation under photoinhibitory conditions.

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Figures

Figure 1.
Figure 1.
Immunoblot analysis of D1 protein in the deg5 deg8 mutant under three light conditions. Detached mature leaves of Col and deg5 deg8 (approximately 6-week-old plants grown under normal conditions) were preincubated with 5 mm lincomycin. The leaves were incubated for 2 h under high-light conditions (1,200 µmol photons m−2 s−1) or for 8 h under growth-light and low-light conditions (180 and 20 µmol photons m−2 s−1, respectively). Representative immunoblots (normalized by fresh weight) using anti-D1 (C-term) antibodies and the bands corresponding to Coomassie Brilliant Blue-stained LHCII are depicted. Signals of immunoblots were quantified using the ImageJ program and were normalized to the amount of Coomassie Brilliant Blue-stained LHCII (error bars indicate sd; n = 3). To compare D1 levels, ratios at 0 h were adjusted to 1.
Figure 2.
Figure 2.
Accumulation of the cleavage products of D1 protein in extreme high-light-treated var2 leaves. A, Immunoblot analysis of the cleavage products of D1 protein under normal-light and extreme high-light conditions. Mature leaves of Col and var2 (approximately 6-week-old plants grown under normal conditions) were illuminated in normal-light (180 µmol photons m−2 s−1) and extreme high-light (2,500 µmol photons m−2 s−1) conditions for 1 h. Representative immunoblots (normalized by chlorophyll content) using anti-D1 (N-term) and anti-D1 (C-term) antibodies and the bands corresponding to Coomassie Brilliant Blue-stained LHCII are depicted. A selective detection of the areas indicated by the brackets at right is shown in the second panels from top. B, Immunoblot analysis of AEBSF-treated var2 leaves. Detached leaves were pretreated with Ser protease inhibitor (AEBSF) and subsequently incubated under extreme high-light conditions for 1 h. White and black arrowheads indicate specific and nonspecific signals, respectively, under high-light irradiation.
Figure 3.
Figure 3.
Phenotype of the var2 deg5 deg8 triple mutant and its photosensitivity. A, Photographs of 3-week-old Col, var2-1, deg5 deg8, and var2 deg5 deg8 plants. Bars = 5 mm. B, Fv/Fm in the mutants. Fv/Fm was measured in detached leaves of Col (circles), var2-1 (diamonds), deg5 deg8 (triangles), and var2 deg5 deg8 (squares). Values are means ± sd (n = 3). The asterisk indicates a value of P < 0.05 for a comparison between var2 and var2 deg5 deg8.
Figure 4.
Figure 4.
Immunoblot analysis of the cleavage products of D1 protein in the var2 deg5 deg8 mutant. Mature leaves of Col, var2, deg5 deg8, and var2 deg5 deg8 (approximately 6-week-old plants grown under normal conditions) were incubated in extreme high-light conditions (2,500 µmol photons m−2 s−1) for 1 h. Representative immunoblots (normalized by chlorophyll content) using anti-D1 (N-term) and anti-D1 (C-term) antibodies and the bands corresponding to Coomassie Brilliant Blue-stained LHCII are depicted. A selective detection of the cleavage products of D1 protein is shown in the second panels from the top.
Figure 5.
Figure 5.
Immunoblot analysis of the D1 cleavage products separated by BN/SDS-PAGE. A, Thylakoid protein complexes were solubilized with 0.5% n-dodecyl-β-d-maltoside and separated on 4% to 16% BN/PAGE gels (10 µg of chlorophyll per lane). Thylakoid membrane proteins were separated further using 14% SDS-PAGE and were silver stained. CBB, Coomassie Brilliant Blue. B, Proteins separated by BN/SDS-PAGE were immunodetected by anti-D1 (N-term) and anti-D1 (C-term) antibodies. Spots of the cleaved D1 products are indicated by white arrowheads. Positions corresponding to PSII supercomplexes, PSII dimer, PSII monomer, and the RC47 complex are shown at the bottom.
Figure 6.
Figure 6.
Steady-state accumulation and localization of chloroplast proteases. Chloroplasts were purified from mature leaves of Col and var2 using a Percoll step gradient. Intact chloroplasts were fractionated into stroma and membrane fractions. Proteins were separated using SDS-PAGE and probed against specific antibodies. D1 and Rubisco large subunits were used as markers of membranes and stroma, respectively. Samples were equally loaded based on chlorophyll content.
Figure 7.
Figure 7.
Proposed model of cooperative D1 degradation in the PSII repair cycle. Photodamaged PSII generated at all light intensities migrates to stroma thylakoids from grana stacks, and PSII monomerization occurs. In PSII repair, processive D1 degradation is conducted predominantly by FtsH irrespective of the light intensity (fundamental degradation). In contrast, under photoinhibitory conditions, an endopeptidic cleavage by Deg increases the rate of D1 degradation because smaller D1 cleavage fragments facilitate effective degradation (escape pathway). D1 cleavage by Deg most likely occurs in PSII dimer, PSII monomer, and the RC47 complex. Cleaved D1 fragments are subjected to further degradation by FtsH, but other proteases such as SppA and Clp proteases participate in this pathway with a mechanism that remains unclear.
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