Legends to Supporting Figures
Figure S1. Inheritance of spectinomyinc resistance in a self-cross of a RpoA:HA
line
To demonstrate homoplastomy of transformed lines, plants were grown under selective
conditions. No bleached seedlings were found among > 1000 seedlings investigated. As
control, wt seedlings were also germinated on selective medium, which resulted in
exclusively white seedlings.
Figure S2. Verification of DNase I treatment of membrane fractions by agarose gel
electrophoresis and PCR
(a) DNaseI activity was verified by extracting the DNA from insoluble membrane and
supernatant after DNaseI treatment and analyzing it by agarose gel electrophoresis.
Signal for DNA only occurs in the untreated membrane samples.
(b) Validation of the success of DNase I treatment by PCR analysis on DNA extracted
from supernatant and insoluble membranes. A 376 bp psbA fragment was amplified
from preparations of chloroplast DNA (cpDNA 1), but not from the DNase I treated
samples, indicating successful DNA degradation.
Figure S3. Immunoprecipitation of tagged RpoA during light and dark periods
Chloroplasts were isolated from plants grown under standard conditions and from darkadapted plants, respectively. RpoA:HA was immunoprecipitated from the solubilized
membrane extracts. For immunological detection, 1/10 volume of the input, 1/25
volume of the supernatant and 1/10 volume of the pellet fractions from transplastomic
and wt plants were analyzed. P, pellet; S, supernatant; HC, heavy chain of the HA-
1
antibody; LC, light chain of the HA-antibody. Ponceau S staining of the same membrane
is shown as a loading control.
Figure S4. Identification of DNA fragments associated with HA-tagged RpoA in membranes
of chloroplasts by ChIP-on-chip analysis
Identification of DNA fragments associated with HA-tagged RpoA in membranes of
chloroplasts by ChIP-on-Chip analysis. Enrichment ratios of DNAs from the light IPs
described in figure 5 are divided by the ratios from the dark IPs, also shown in figure 5,
and plotted against their genome position.
Figure S5. Fragment length of sheared DNA after sonication
Chloroplast membranes were extracted, treated with NP-40 and sonicated as described
in Experimental Procedures. DNA was isolated, RNA contaminants
were RNaseA digested and seperated on an agarose gel.
Table S1. List of Primers used in the present study
Table S2. Top 25 hits of ChIP-on-chip data from light and dark experiments
Table S3. ChIP-on-Chip data for Figure 5 and Figure S4
2