Download Supplemental Figure legends

Supplemental Figure legends
Figure S1. Plants with HsCAT targeted to mitochondria do not accumulate HsCAT in
chloroplasts.
Total, mitochondrial and chloroplast proteins, respectively, were isolated from acd2 and
acd2/m-CAT plants. Human Catalase (CAT) proteins were detected by Western blot
analysis. Note the Human Catalase protein was absent in the chloroplast fraction. As
expected Human Catalase was absent in acd2 plants. Signals for CAT for all samples are
from one exposure of one continuous membrane. This experiment was done twice with
similar results.
Figure S2. Superoxide levels in acd2 and wild-type protoplasts.
The MFI of MitoSox dye coming from the light-exposed protoplasts of WT and acd2
leaves as measured by flow cytometry. Light-treated protoplasts were incubated with 5
µM of MitoSox at room temperature in the dark for 30 min, washed twice and subjected
to flow analysis. Unstained protoplasts were used as the autofluorescence control. Note
that there was no difference in MFI of MitoSox after different times of light exposures.
This experiment was repeated twice with similar results and the error bars represent SD
(n=2).
Figure S3. Singlet oxygen (1O2) production by red chlorophyll catabolite (RCC).
RCC produces 1O2 after light exposure; as detected by spin-trap EPR measurement using
TEMP (a trap for 1O2). The size of the EPR signal indicative of the amount of TEMPOformation (TEMPO was formed by the reaction of TEMP and 1O2) was plotted against
time. PPIX was used as a positive control. Note the EPR signal in light-exposed
Buffer+TEMP samples remained unchanged. Results are from a single analysis,
representative of two independent experiments that showed similar results.
Figure S4. Mutations in Executers or photoreceptors do not affect the survival of lighttreated acd2 protoplasts.
Protoplasts isolated from the indicated plant genotypes were exposed to light for 15 h and
their viability was determined by FDA staining. This experiment was repeated three
times with similar results and the error bars represent SD (n=3).
(a) Exexuter mutations did not affect the acd2 cell viability.
(b) and (c) The viability of protoplasts isolated from acd2cry1cry2 (b) or acd2phyAphyB
(c) leaves was not different than that of acd2 protoplasts. Bars with the same letters had
values that were not different as determined by Fisher’s PLSD test (P>0.45).
Figure S5. ACD2 in mitochondria or chloroplasts significantly suppresses the ROS
production in acd2 protoplasts.
Transgenic acd2 carrying ACD2 targeting to mitochondria (acd2/m-ACD2) or
chloroplasts (acd2/c-ACD2) were generated.
(a) and (b) Protoplasts were exposed to light for 2 h and 0.5 h, respectively. Protoplasts
were stained with (a) CM-H2DCFDA (green) and (b) SOSG, (green) respectively. In both
the cases protoplasts were double stained with CMXRos (red). Images were assessed by
LSCM. (I) In WT protoplasts, there was no detectable green signal after staining with
either CM-H2DCFDA or SOSG. (II) acd2 protoplast showing the green signal (CMH2DCFDA or SOSG) after light exposure. Note that the localization of the CMH2DCFDA and SOSG signals matched that of the CMXRos signals. (III) and (IV) CMH2DCFDA or SOSG signals were significantly decreased in both acd2/c-ACD2 and
acd2/m-ACD2 protoplasts, respectively. This experiment was done twice with similar
result. (c) and (d) Protoplasts were exposed to light for 2 h and 0.5 h, respectively, and
the MFI of CM-H2DCFDA or SOSG were measured by flow cytometry. These
experiments (F and G) were done three times with similar results and the error bars
represent SD (n = 3). Letters indicate different values using Fisher’s PLSD test (P <
0.001).
Figure S6. Targeting of ACD2** either to chloroplasts or mitochondria does not reduce
the ROS in acd2 protoplasts.
(a) and (b) The MFI of CM-H2DCFDA dye (a) and the MFI of SOSG dye (b) coming
from the protoplasts of WT, acd2, acd2/c-ACD2**, and acd2/m-ACD2** leaves as
measured by flow cytometry. Note that there was no difference in MFI of CM-H2DCFDA
and SOSG in acd2 protoplasts and protoplasts isolated from acd2/c-ACD2** or acd2/mACD2** leaves. Results (a and b) are from a single analysis, representative of two
independent experiments that showed similar results. The error bars represent SD (n=3).
Bars with the same letters had values that were not different as determined by Fisher’s
PLSD test (P>0.1).
Figure S7. Cre-Lox construct and characterization of the acd2 transgenic plants.
(a) Schematic representation for the Cre-Lox construct
(b) Constitutive overexpression of ACD2 along with NLS-GFP complemented the acd2
cell death phenotype. Note that the leaf of the complemented plant shows GFP
expression.
Figure S8. Expression and purification of ACD2 from E.coli
SDS–PAGE (12%) showing the protein profile of pellet (insoluble) and soluble fractions
of E. coli cell lysate over-expressing ACD2 (40-319 amino acids; lacking the chloroplast
transit peptide). The soluble fraction was Ni-NTA-column purified (Elution 1 and Elution
2). M; molecular weight markers in kDa. The arrow marks the overexpressed ACD2 with
a molecular mass of 35 kDa.
Figure S9. PPIX is present in methanol extract of recombinant ACD240-319.
Ni-NTA-column purified ACD240-319 was extracted with methanol and the extract was
analyzed by HPLC. HPLC was performed under isocratic reverse-phase conditions:
column, Cosmosil C18-AR-II (Nacalai Tesque, Kyoto, Japan); eluent, 85:15:20 (v/v)
methanol/acetonitrile/aqueous 1 M ammonium acetate mixture; flow rate, 0.7 mL/min.
Left columns show HPLC profiles in which the absorption at 400 nm was plotted. Right
columns show UV-Vis absorption spectra at the positions marked as asterisks in HPLC
profiles. (a) Authentic PPIX. (b) Methanol extract from ACD240-319.
Figure S10. How and where does ACD2 function to protect cells?
The presence of ACD2 in chloroplasts controls the amount, reactivity and/or the mobility
of PPIX/RCC or some other tetrapyrrolic substrates (1). In some conditions, the
accumulated PPIX/RCC or some other tetrapyrrolic substrates may come out of
chloroplasts and enter into mitochondria (2). Inside mitochondria, these substrate
molecules can produce different kinds of reactive oxygen species like 1O2 and H2O2 (3)
resulting in mitochondrial oxidative bursts that ultimately lead to cell death (4). Under
stress/infection (5), ACD2 gets targeted to mitochondria and protects the latter by acting
upon PPIX/RCC or other tetrapyrrolic substrates (6). When we targeted ACD2 to either
chloroplasts or mitochondria (7), the ACD2 acts upon either the same mobile substrate
(fail safe model) or different substrates (threshold model) and could protect the
organelles. (8) Although H2O2 was previously found in acd2 chloroplasts, it did not
contribute to cell death. However, there may be other chloroplast ROS that contribute to
PCD induction.