Supporting Information
Figure S1. Imaging protocol for chloroplast movement measurements by red-light reflectance.
(a) Workflow summarizing the steps followed for non-invasive measurement of chloroplast
movements from intact plants. (b) Imaging platform setup for chloroplast movement
measurements. (c) Sample representation of gray-scale image data (red-light reflectance)
captured with a CCD camera. The left panel shows raw images and the right panels show
representative images after processing using ImageJ 1.47 (right upper) or with an automated
image-processing protocol (right lower). The yellow circle represents a sample area selected on a
flat part of the leaf surface (upper right panel) for quantification to obtain the plotted reflectance
intensity values. (d) Schematic representation of a plot showing the reflectance intensity,
determined as described above, relative to the last value obtained during dark pre-treatment (0.00
on the y-axis) versus time in dark (D) and increasing intensities of white light (L1-L5).
Figure S2. Chloroplast movement measurements in 12- to 18-day-old Arabidopsis plants based
on red-light reflectance. Light treatments are as described in Figure 1a. For all data points, n = 3
and error bars represent SD. (a) Phototropin mutants and their corresponding gl1 parent line. (b)
Chloroplast division mutants and the corresponding Col-0 wild type.
Figure S3. Confocal images showing chloroplast arrangement in mesophyll cells on the adaxial
side of leaf tissue of the indicated ftsZ mutants. The red signal shows autofluorescence from the
chloroplasts. The numbers at the top indicate the light intensity in μmol photons m-2 s-1. Bars =
50 μm. Leaf samples were fixed for imaging at the end of the indicated exposure period.
Figure S4. Raw PSII quantum yield (ΦII) for the results shown in Figure 3. The open and closed
symbols on Days 3 and 5 correspond to alternating periods of ambient and fluctuating light,
respectively. Each data point represents the mean of 4-5 plants; error bars represent SD.
Figure S5. Heat map showing the statistical significance of differences in ΦII between WT and
arc6-5 or phot1-5 phot2-1 at each measurement time point for the data shown in Figures 3 and
S4, evaluated using the unpaired, two-tailed t-test. P values ≤ 0.05 were taken as statistically
significant (black stripes). The light regime is shown at top.
Figure S6. Chloroplast movement (a) and photosynthetic parameters (b-e) measured in threeweek-old Col-0 and gl1 plants subjected to the fluctuating light regime shown on Day 3 of
Figure 3 using the dual-camera imaging platform. The open and closed symbols correspond to
alternating periods of ambient and fluctuating light, respectively. For all data points, n = 4 and
error bars represent SD.
Figure S7. Reflectance data recorded in phot1-5 phot2-1 compared to data imaged from the
black foam masks covering the soil (background) throughout the same fluctuating light regime
described for Figures 4 and 5. The open and closed symbols correspond to alternating periods of
ambient and fluctuating light, respectively. For all data points, n = 4 and error bars represent SD.
Figure S8. Raw PSII quantum yield (ΦII) data for results shown in Figure 5b. The open and
closed symbols correspond to alternating periods of ambient and fluctuating light, respectively.
For all data points, n = 4 and error bars represent SD. The same data for Col-0 and phot1-5
phot2-1 are shown in the two plots, but the data for arc6-5 (a) and the ftsZ mutants (b) are
presented separately for visual clarity.
Figure S9. Heat maps showing the statistical significance of differences in photosynthetic
parameters at each time point for data shown in Figure 5. (a) ΦII, (b) NPQcorr, (c) qESVcorr and (d)
qIcorr values in the indicated genotypes relative to those in Col-0 (upper panels), phot1-5 phot2-1
(middle panels) and triple ftsZ (lower panels). Differences were evaluated using the unpaired,
two-tailed t-test. P values ≤ 0.05 were taken as statistically significant (black stripes).
Figure S10. Empirical determination of a reflectance-based correction factor. (a) Dependence of
relative FM”/ FM on the change in reflectance under fluctuating light for Col-0 and the indicated
mutants was plotted using all data points. Averaged fluorescence and reflectance values from
processed images of whole plants as described in Experimental Procedures were used for
calculations of data points. FM”/FM values were normalized to 1 at the first point of data
collection at the beginning of the illumination period. The open and closed symbols correspond
to alternating periods of ambient and fluctuating light, respectively. (b) Data points from the
illumination period at the beginning of the day where there was a linear relationship between
relative FM”/ FM and change in reflectance were replotted on an expanded scale and regression
lines were computed. The slopes (m) and R2 values for each line are indicated. The slope is used
in calculating the correction factor, as described in Results.
Figure S11. Raw NPQcorr (a), qESVcorr (b) and qIcorr (c) data for results shown in Figure 5c-e. The
open and closed symbols correspond to alternating periods of ambient and fluctuating light,
respectively. For all data points, n = 4 and error bars represent SD. The same data for Col-0 and
phot1-5 phot2-1 are shown in the left and right plots of each panel, but the data for arc6-5 (left)
and the ftsZ mutants (right) are presented separately for visual clarity.
Figure S12. Raw data for “apparent” (traditional) NPQ (a), qESV (b) and qI (c) values for the
experiment shown in Figure 5c-e, but uncorrected for chloroplast movements. The open and
closed symbols correspond to alternating periods of ambient and fluctuating light, respectively.
For all data points, n = 4 and error bars represent SD. The same data for Col-0 and phot1-5
phot2-1 are shown in the left and right plots of each panel, but the data for arc6-5 (left) and the
ftsZ mutants (right) are presented separately for visual clarity.
Method S1. Light conditions for the five-day long experiment shown in Figure 3. The numbers
in the table show the white light intensities (µmol m-2 s-1) during every interval of the 16-h light
period for the experiment shown in Figure 3.