SUPPORTING INFORMATION FULL LEGENDS
Figure S1. Phylogeny of Arabidopsis thaliana MPH1 protein sequences and its homologs
in other land plants.
Percentages indicate similarities among amino acid sequences of eudicots poplar (Populus
trichocarpa), castor bean (Ricinus communis), tomato (Solanum lycopersicum), Arabidopsis
thaliana, and soybean (Glycine max), and monocots rice (Oryza sativa), maize (Zea mays),
and sorghum (sorghum bicolor), and moss (Physcomitrella patens). DNAMAN program was
used for the analysis using default settings.
Figure S2. Molecular characterization of homozygous mph1-1 and mph1-2 mutants.
(a) Confirmation of homozygosity of mph1 mutant lines. Genomic PCR was performed with
primers specific for the MPH1 coding region (RP, right border primer; LP, left border primer)
and with T-DNA-specific primers (LBb1.3, T-DNA left border primer 1 for SALK, and LB3
for SAIL lines). For primer sequences, see Table S2.
(b) Analysis of the At5g07020 transcript in the wild type and mph1 mutants. RT-PCR was
performed with primers specific for MPH1 and ACTIN2 (as a control) and the transcript of
At5g07020 was undetectable in the homozygous mutant.
(c) Analysis of the MPH1 protein detected by immunoblotting with anti-MPH1 antibody in
the wild type and mph1 mutant plants. Mature MPH1 protein has a predicted molecular mass
of 25 kDa and was present in the total leaf proteins of the wild-type plant but absent in mph1
mutants. C.B.B., Coomassie brilliant blue.
Figure S3. Lack of MPH1 caused consistent susceptibility of PSII to photoinhibition
under high light.
False-color images representing Fv/Fm of multiple wild-type and mph1 mutant plants grown
at different times under growth light (top) and after 3-h high-light treatment (bottom). (a-e)
Biological replicates, with experiments performed at different times; GL, growth light (top
row); HL, high light (bottom row).
Figure S4. Relative abundance of chloroplast photosynthetic proteins in the wild-type
plants and mph1 mutants.
Proteins detected as in Figure 5 were analyzed with Image LabTM Software (Bio-Rad). The
average values (mean ± SE, n = 3 independent replications) are calculated as the ratio of the
protein content of the wild type grown under growth light conditions. '#' indicates that the
value is significantly different from the wild-type value under growth light conditions
(Student’s t test, P< 0.05). Asterisks indicate that the value is significantly different from the
wild-type value after 3-h high-light treatment (Student’s t test; *, P< 0.05; **, P< 0.01). GL,
growth light; HL, after 3-h high-light treatment.
Figure S5. Analysis of photosynthesis-associated transcript abundance by RT-PCR in
the high light-treated wild type and mph1 mutant plants.
RT-PCR analysis for plastid-encoding genes (left panel) and nucleus-encoding genes (right
panel). For plastid-encoding genes, PsbA, PsbB, PsbC, PsbD, PsaA, PetA, and RbcL were
selected as NEP-dependent genes (class I); ClpP, AtpB, and NdhB were selected as both PEPand NEP-dependent genes (class II); and AccD, RpoB, and Ycf2 were selected as PEPdependent genes (class III). For nucleus-encoding genes, PsbO1, Lhcb2.1, Lhca2, and PetE2
were selected. The Actin2 gene was used as a control.
Figure S6. In vivo phosphorylation levels of the wild-type and mph1 mutant thylakoid
proteins were examined by phospho-threonine antibody.
Thylakoid membranes were isolated in phosphatase inhibitory conditions from leaves of
plants treated in the dark (D), grown at growth light (GL), or illuminated under high light
(HL) for 3 h. Equivalent chlorophyll (1.0 μg) was loaded to each well for immunoblotting
with p-thr antibody. Shown below is the reference gel stained with Coomassie Brilliant Blue
(C.B.B.). P, phosphorylated form of the protein.
Figure S7. Analysis of stability of chloroplast photosynthetic proteins in photoinhibitory
assays.
Detached leaves were incubated in water or lincomycin (chloroplast protein synthesis
inhibitor) solution in the dark overnight and subsequently illuminated under high light (1000
μmol photons m−2 s−1) for 2, or 4 h, and degradation of representative protein components of
each photosynthetic complexes was probed by specific antibodies as indicated to the left.
Images of SDS-PAGE gels stained with Coomassie Brilliant Blue (C.B.B.) are shown at the
bottom, indicating that equal amounts of total proteins were loaded in each lane.