Supporting Information Legends
Figure S1. Chlorophyll cycle. Chlorophyll b is synthesized from chlorophyll a by chlorophyllide a
oxygenase (CAO) and re-converted to chlorophyll a by chlorophyll b reductase (CBR) and 7hydroxymethyl chlorophyll a reductase (HCAR).
Figure S2. HCAR and NOL levels in plants. The band intensities in the immunoblotting analysis of
Figure 1 were emphasized to compare each band between the plants. The NOL and HCAR levels in
the plants with only endogenous NOL and HCAR, respectively, were similar in those plants.
Figure S3. Characteristics of chlorophyll, chlorophyll fluorescence and NYC1 protein in CAO-overexpressing plants during dark incubation. (a) Image of detached leaves and whole plants from 5week old WT, BCG and PhCAO plants. The detached leaves from WT, BCG and PhCAO plants were
placed on wet filter paper in a plastic rectangular plate, which was wrapped in dual tinfoil and
incubated in complete darkness for up to 3 days. (b) Analysis of the chlorophyll content. Chlorophyll
was extracted from the detached leaves of WT, BCG and PhCAO plants and analyzed by HPLC. Error
bars represent ±SD (n=3). (c) Measurement of chlorophyll fluorescence. The minimal fluorescence
parameter (Fo) was measured by PAM-2000 after placing the detached leaves under dark conditions
for 15 minutes. Error bars represent ±SD (n=7). (d) Measurement of the NYC1 protein level. The
membrane proteins extracted from the leaves incubated in darkness were subjected to
immunoblotting analysis, and the NYC1 protein was detected. All of the detached leaves were
collected from fully expanded rosettes.
Figure S4. Characteristics of chlorophyll and NYC1 protein in ch1 plants during dark incubation. (a)
Image of detached leaves and whole plants from 5-week old ch1-1, ch1-2, and ch1-3 plants.
Detached leaves from ch1-1, ch1-2, and ch1-3 were placed on wet filter paper in a plastic rectangular
plate which was wrapped in dual tinfoil and incubated in complete darkness for up to 3 days. (b)
Analysis of chlorophyll content in ch1 plants. Chlorophyll was extracted from the detached leaves of
ch1-1, ch1-2, and ch1-3 plants and analyzed by HPLC. Error bars represent ±SD (n=3). (c)
Measurement of non-specific band in ch1 mutant plants. Membrane proteins were extracted from
detached leaves and subjecting to immunoblotting analysis. Anti-NYC1 antibody was used to detect
NYC1 protein. Lower bands detected in ch1 plants were also found in ch1-1/nyc1 plants but were not
found in nyc1 deficient plants, suggesting that these lower bands are not NYC1 and specifically
accumulated in chlorophyll b less or deficient mutant plants. All of the detached leaves were
collected from fully expanded rosettes.
Figure S5. Phenotypes of 4, 5, and 6-week old whole plants and detached leaves. (a) Visible
phenotype of WT, BCG and ch1-1 plants. (b) Visible phenotype of detached leaves from WT, BCG and
ch1-1 plants before and after dark incubation. Detached leaves before dark senescence from 4, 5,
and 6-week old WT, BCG, and ch1-1 plants were placed on wet filter paper in a plastic rectangular
plate, which was wrapped in dual tinfoil and incubated in complete darkness for up to 3 days. (c)
Analysis of the chlorophyll a and chlorophyll b content in 4, 5, and 6-week old WT, BCG and ch1-1
plants during dark incubation. Chlorophyll was extracted from the detached leaves of WT, BCG and
ch1-1 plants before and after 3 days of dark senescence and analyzed by HPLC. Error bars represent
±SD (n=3). All of detached leaves before and after dark treatment were collected from fully
expanded rosettes.
Figure S6. Difference spectra of the low temperature chlorophyll fluorescence. The fluorescence
spectra of leaves from WT plants and BCG plants before and after a 3-day dark treatment at 77K
were measured. The excitation wavelength was 470 nm. The fluorescence intensities of each sample
were normalized to the emission intensity at 670 nm and 687 nm and subtracted from the
fluorescence spectrum of the WT plants before dark treatment.