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Supplementary Material
Methods
Chlorophyll fluorescence and pigment analysis
In-vivo Chl a fluorescence of single leaves was measured using a PAM (pulse amplitude
modulation) 101/103 fluorometer (Walz). Pulses (0.8 s) of white light (6000 µmol photons·m-2·s1
) were used to determine the maximum fluorescence (FM) and the ratio (FM - F0)/FM = FV/FM. A
15-min illumination with actinic light (80 µmol photons·m-2·s-1) served to drive electron transport
before II and qP were measured. State transitions were followed by measuring maximum PSII
fluorescence signals in state 1 (FM1) and state 2 (FM2), after irradiating plants at wavelengths that
target PSII and PSI, respectively. The ratio (FM1-FM2)/FM2 was taken as a measure of state
transitions as described1. To measure PSII activity under high light and its recovery under low
light, leaf discs were illuminated for 4 to 8 h at high light intensity (PFD of 2000 µmol
photons·m-2·s-1) and subsequently for 4 h at low light intensity (20 µmol photons·m-2·s-1), and
FV/FM was recorded as described2. Pigment analysis was performed by reverse-phase HPLC3.
For measurement of Chl fluorescence to long-term changes in light quality, in-vivo Chl a
fluorescence was recorded by video imaging (Fluorcam 700MF; Photon System Instruments)4.
Immediately after dark acclimation (15 min) the measuring light was turned on, and minimal
fluorescence (F0) was determined. Then leaves were exposed to a 1600-ms flash of saturating
white light (3000 µE) to determine maximal fluorescence (FM). Subsequently, leaves were
illuminated with 90 µmol of photons·m-2·s-1 of actinic red light of 620 nm for 10 min.
Fluorescence was recorded until a stable level (FT) was reached. Actinic light was then switched
off to determine minimal fluorescence F0’ in the light-acclimated state. The steady-state
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fluorescence FS was calculated as FT – F0' = FS. A normal acclimation response to PSI or PSII
light is characterised by a significant change in the FS/FM value as shown earlier5.
For the measurement of Chl content to changes in light quality, total chlorophyll was
determined spectroscopically after grinding leaves in liquid nitrogen and extracting chlorophylls
with 80% (v/v) buffered acetone. Concentrations of chlorophylls a and b were calculated using
the extinction coefficients from previous studies6.
Immunoblot analysis of D1 protein upon exposure to high-intensity light
For experiments on the effects of high light exposure (PFD of 2000 µmol photons·m-2·s-1) to the
accumulation of the D1 protein, leaf discs were vacuum-infiltrated either with water as control, or
with lincomycin to inhibit protein synthesis in the plastids, before measurements7. Thylakoid
proteins were extracted, fractionated on a SDS-PA gradient gel and transferred to PVDF
membranes2. Filters were then probed with an antibody specific for D1 (provided by D. Godde,
Bochum, Germany). After stripping, the same filters were immuno-labelled with a Lhcb2-specific
antibody to control the loading. Signals were detected by chemiluminescence (Amersham
Biosciences).
mRNA expression profiling
Generation and use of a 3292-GST nylon array enriched for nuclear chloroplast genes have been
described8. At least three experiments with cDNA probes from independent plant pools were
carried out for each condition. cDNA synthesis was primed with an oligonucleotide mixture
matching the 3292 genes in antisense orientation, and hybridised to the array as described8,9.
Hybridisation images were read with a phosphorimager (Typhoon, Amersham Biosciences), data
were imported into ArrayVision (version 6.0; Imaging Research Inc.), and statistically evaluated
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using ArrayStat (version 1.0 Rev. 2.0; Imaging Research Inc.) as described8,10. Data were
normalised with reference to all spots on the array9 and average expression ratios derived from at
least three independent experiments were analysed.
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