Figure S1. Microhomology-Mediated Break-Induced Replication (MMBIR) Leads to DNA Rearrangements

SUPPLEMENTAL DATA

Figure S1. Microhomology-Mediated Break-Induced Replication (MMBIR) Leads to DNA Rearrangements.

A) Scheme presenting the repair of a DNA double-strand break by MMBIR. Dotted lines represent newly synthesized DNA. Yellow and green boxes represent micohomologous repeat sequences.

B) Scheme presenting the detection of MMBIR-associated DNA rearrangements by outward-facing PCR. Arrows represent the position of the PCR primers.

Figure S2. The why1why3polIb-1 Mutant Develops Distinct Yellow and Green Sectors on Its Leaves.

Photographs of a representative 28-day-old why1why3polIb-1 yellow variegated plant. From top to bottom, the images represent the use of a green filter, a yellow filter, a merge of the two filters and the original picture. Color filters were applied using Photoshop CS4 color range function.

Figure S3. Some why1why3polIb-1Plants Exhibit Yellow and White Variegation Simultaneously.

Photograph of a representative 28-day-old why1why3polIb-1 yellow and white variegated plant.

Figure S4. Ciprofloxacin Negatively Impacts Photosynthetic Efficiency in Arabidopsis.

Fv/Fm ratios of 15-day-old plants treatedfrom days 10 to 15 to50 µM NOVO, 10 µM CIP or no drug (MS). The error bars represent the standard deviation. Two asterisks indicate a significant difference of a Student’s t-test P-value ≤ 0.01 with MS.Measurements were performed independently on at least five biological samples.

Figure S5. why1why3and why1why3polIb-1 Plants Are Hypersensitive to Paraquat.

Representative photograph of 3-week-old plants grown under normal light conditions on MS basal media containing 0, 250 or 500 nm paraquat (PAR).

Figure S6.The Abundance of MMBIR-Associated ptDNA Rearrangements Is LittleAffected by the Light-Regimen in why1why3polIb-1.

Representative outward-facing PCR reactions showing the abundance of MMBIR-associated plastid DNA rearrangements of 3-week-old why1why3polIb-1 plants grown either under normal (NL) or low-light conditions (LL). PCR reactions were carried out using the indicated primer pairs. The amount of DNA loaded in each reaction was equilibrated relative to the amplification of aYCF2 fragment. Every reaction was performed on three different biological samples and a representative duplicate is presented.

Figure S7. Paraquat and Atrazine do not induce MMBIR-Associated ptDNA Rearrangements.

Representative outward-facing PCR reactions showing the abundance of MMBIR-associated plastid DNA rearrangements of 3-week-old Arabidopsis plants grown in the presence of the ROS-inducing agents paraquat (PAR) and atrazine (ATR). PCR reactions were carried out using the indicated primer pairs. The amount of DNA loaded in each reaction was equilibrated relative to the amplification of aYCF2 fragment. Every reaction was performed on three different biological samples and a representative reactionis presented. The results indicate that MMBIR-associated ptDNA rearrangements are not the consequence of superoxide, singlet oxygen or hydrogen peroxide oxidation.

Figure S8. Elevated ROS Levels Do not Lead to Cell Death in why1why3polIb-1.

Representative photographs of 4-week-old plants grown under normal light conditions and stained with trypan blue.

Figure S9. The why1why3polIb-1 Nuclear Genetic Reprogramming Is Not Due to the PolIb-1 and why1why3 Genetic Backgrounds.

Histograms presenting the real-time PCR measurements of NUDT4, UPOX (AT2G21640), ACS6, AT3G06530,ELIP1 and ELIP2 expression in wild type,polIb-1 andwhy1why3plants. The error bars represent the standard error of the mean (SEM) of the biological triplicates.

Supplemental Table S2.Primer Pairs Used for the Specific Amplification of MMBIR-Associated ptDNA Rearrangements and Described Previously (Cappadocia et al., 2010).

Supplemental Table S3.Primer Pairs Used for Quantitative RT-PCR experiments.

SUPPLEMENTAL MATERIALS AND METHODS

Plant Material and Growth Conditions

Arabidopsis (Arabidopsis thaliana; ecotype Columbia-0) mutant lines why1why3 and why1why3polIb-1 were reported previously (Maréchal et al., 2009; Parent et al., 2011). Seeds were sown on soil, vernalised for 3 days at 4oC and grown either under normal light (100 µmol m-2 s-1) or low light (20 µmol m-2 s-1) at 22oC on a 16 h day/8 h dark cycle. When grown at low light, plants were germinated at 10 µmol m-2 s-1 for one week prior to their transfer at 20 µmol m-2 s-1. For drug hypersensitivity experiments, seeds were sterilized and sown on Murashige and Skoog basal media (MS medium, Sigma-Aldrich) supplemented with 1 % sucrose, 0.8 % agar and the indicated amount of drug.

Measurement of PSII Efficiency

For each condition, Fv/Fm measurements were performed on 15-day-old plants treatedfrom days 10 to 15 with 50 µM NOVO, 10 µM CIP or no drug with an Open FluorCam (Photon Systems Instruments) according to the manufacturer’s instructions.

Detection of ptDNA Rearrangements

Total DNA was isolated for each plant sample using a cetyltrimethylammonium bromide DNA extraction protocol (Weigel and Glazebrook, 2002). For every DNA samples, ptDNA amount was equilibrated with a low-cycle amplification of aYCF2 DNA fragment. Primers and approaches for rearrangements detection were described previously (Cappadocia et al., 2010) and PCR products were visualized after migration on GelRed (Biotium) stained agarose gels. Every reaction shown is representative of the eight primer pairs presented in Supplemental Table S2.

Trypan blue staining

The trypan blue staining of dead cells was conducted as described previously (Weigel and Glazebrook, 2002) with 4-week-old Arabidopsis leaves.

Quantitative RT-PCR Analysis

cDNA synthesis was carried out using a first-strand cDNA synthesis kit (Fermentas) according to manufacturer’s instructions. cDNAs were diluted 20-fold prior to qRT-PCR experiments. Primers used for qRT-PCR are listed in Supplemental Table S3. Every reaction was carried out on biological and technical triplicates and normalised relative to UBQ5. Real-time qPCR reactions were done using the SYBR green master mix(SABiosciences, according to the manufacturer’sinstructions. Melting curves confirmed the amplification of a unique PCR product for each of theqRT-PCR primer pairs used. LightCycler480 (Roche) was used for qRT-PCR experiments and data analyzed using the Light-Cycler480 software version 1.5.

SUPPLEMENTAL REFERENCES

Cappadocia, L., Maréchal, A., Parent, J.S., Lepage, E., Sygusch, J., and Brisson, N. (2010). Crystal structures of DNA-Whirly complexes and their role in Arabidopsis organelle genome repair. Plant Cell 22: 1849-1867.

Maréchal, A., Parent, J.S., Veronneau-Lafortune, F., Joyeux, A., Lang, B.F., and Brisson, N. (2009). Whirly proteins maintain plastid genome stability in Arabidopsis. Proc. Natl. Acad. Sci. U. S. A. 106: 14693-14698.

Parent, J.S., Lepage, E., and Brisson, N. (2011). Divergent roles for the two PolI-like organelle DNA polymerases of Arabidopsis. Plant Physiol. 156: 254-262.

Weigel, D., and Glazebrook, J. (2002). Arabidopsis: A Laboratory Manual (Cold Spring Harbor Lab Press, Cold Spring Harbor, NY).