Supplementary Materials for

Arabidopsis ATXR2 deposits H3K36me3 at the promoters of LBD genes

to facilitate cellular dedifferentiation

Kyounghee Lee, Ok-Sun Park, Pil Joon Seo*

*Corresponding author. Email: pjseo1@skku.edu

Published 28 November 2017, Sci. Signal. 10, eaan0316 (2017)

DOI: 10.1126/scisignal.aan0316

This PDF file includes:

Fig. S1. Callus formation in leaf explants from atxr1 and atxr4 mutants.

Fig. S2. ATXR2 expression in atxr2 mutants.

Fig. S3. ATXR2 expression in 35S:ATXR2-MYC transgenic plants.

Fig. S4. Transcript accumulation of LBDs in atxr2-3 mutant calli.

Fig. S5. Spatial expression of LBD16 in leaf explants and calli.

Fig. S6. Phenotype of leaf explant–derived callus at 7 DAC.

Fig. S7. Transcript accumulation of WOX5 in atxr2-1 calli.

Fig. S8. ChIP assays using antibody-free resin.

Fig. S9. Transient expression assays.

Fig. S10. Binding of ATXR2 to the LBD1 promoter.

Fig. S11. Accumulation of H3K4me3, H3K9me3, and H3K27me3 in atxr2-1

mutants during callus induction.

Fig. S12. Accumulation of H3K4me3 and H3K36me3 in 35S:ATXR2-MYC calli.

Fig. S13. H3K4me3 accumulation at the LBD promoters in atxr2-1.

Fig. S14. Interactions of ATXR2 with ARF7 and ARF19.

Fig. S15. Interactions of ATXR2 with deletion constructs of ARF7 and ARF19.

Fig. S16. Interactions of ATXR2 with other ARFs.

Fig. S17. Accumulation of H3K4me3 at LBD promoters in arf7-1arf19-2 mutant

calli.

Fig. S18. Protein accumulation of ATXR2 in 35S:ATXR2-MYC/Col-0 and

35S:ATXR2-MYC/arf7-1arf19-2.

Fig. S19. Callus formation of leaf explants from 35S:ATXR2-MYC x 35S:LBD16-

SRDX plants.

Table S1. Primers used for PCR.

www.sciencesignaling.org/cgi/content/full/10/507/eaan0316/DC1

Table S2. Primers used for ChIP assays.

Figure S1. Callus formation in leaf explants from atxr1 and atxr4 mutants.

Leaf explants from the third leaves of two-week-old plants were excised and

cultured on callus-inducing medium (CIM). Plates were incubated for 2 weeks

under continuous dark conditions. Calli were collected from leaves of each

genotype to measure fresh weight. Three independent measurements were

averaged. Bars indicate the standard error of the mean. N=30 calli of each

genotype.

Figure S2. ATXR2 expression in atxr2 mutants.

(A) Mapping of the T-DNA insertion sites of atxr2 mutants. Black boxes

indicate exons. White arrowheads indicate T-DNA insertion sites. (B)

Accumulation of ATXR2 transcripts in wild-type (Col-0), atxr2-1, and atxr2-3

plants. Ten-day-old seedlings grown under long-day conditions (LDs) were

harvested for total RNA isolation. Transcript accumulation was analyzed by

semi-quantitative RT-PCR. The EUKARYOTIC TRANSLATION INITIATION

FACTOR 4A1 (eIF4a) gene (At3g13920) was used as an internal control. N=3

biological replicates.

Figure S3. ATXR2 expression in 35S:ATXR2-MYC transgenic plants.

(A) Transcript accumulation of ATXR2 in wild-type (Col-0) and 35S:ATXR2-

MYC plants. Ten-day-old seedlings grown under long-day conditions were

harvested for total RNA isolation. Transcript accumulation was analyzed by

semi-quantitative RT- PCR. The eIF4A gene was used as an internal control.

N=3 biological replicates. (B) Callus formation in wild-type (Col-0) and

35S:ATXR2-MYC plants. Third leaves of two-week-old plants were explanted

and cultured for 2 weeks under continuous dark conditions on CIM to induce

calli. Scale bar, 5 mm. N>30 plants of each genotype. (C) Fresh weight

measurement of calli from Col-0 and 35S:ATXR2-MYC plants. Calli were

collected to measure fresh weight. Three independent measurements were

averaged. Statistically significant differences between wild-type and transgenic

calli are indicated by asterisks (Student’s t-test, *P < 0.05). Bars indicate the

standard error of the mean. N=30 calli of each genotype.

Figure S4. Transcript accumulation of LBDs in atxr2-3 mutant calli.

(A) Expression of LBD genes in wild-type (Col-0) and atxr2 mutant calli after

7 days of culture on callus-inducing medium (DAC). (B) Kinetics of LBD16

expression in Col-0 and atxr2-3 calli at the indicated time points. Transcript

accumulation was analyzed by RT-qPCR and normalized to gene expression

at time 2 in wild-type (Col-0) plants. Bars indicate the standard error of the

mean. *P < 0.05 (Student’s t-test). N=3 biological replicates.

Figure S5. Spatial expression of LBD16 in leaf explants and calli.

Third leaves from two-week-old pLBD16:GUS transgenic plants, in which the

LBD16 promoter sequence covering an approximately 1-kb region upstream of

the LBD16 transcription start site was fused to the GUS coding sequence, were

cultured on callus-inducing medium (CIM) for 4 days and subjected to

histochemical staining. The arrow indicates emerging callus tissues. Scale bar,

0.5 mm. N>30 leaf explants.

Figure S6. Phenotype of leaf explant–derived callus at 7 DAC.

Third leaves of two-week-old wild-type (Col-0) and atxr2-1 plants were

explanted and cultured on callus-inducing medium (CIM) for 7 days under

continuous dark conditions. Scale bar, 5 mm. N>30 calli of each genotype.

Figure S7. Transcript accumulation of WOX5 in atxr2-1 calli.

Transcript accumulation in wild-type (Col-0) and atxr2-1 calli was analyzed

by RT-qPCR and normalized to gene expression at time 0 in Col-0 plants.

Bars indicate the standard error of the mean. N=3 biological replicates. DAC,

days after incubation on CIM.

Figure S8. ChIP assays using antibody-free resin.

Enrichment of the putative binding regions of the LBD promoters was

analyzed by qPCR after ChIP with resin alone with no antibody.

Enrichment was quantified relative to the levels of EV at each gene

promoter. Biological triplicates were averaged. Bars indicate standard

error of the mean. N=3 biological replicates.

Figure S9. Transient expression assays. The core cis-elements of the LBD promoters were cloned into the reporter

plasmid. The recombinant reporter and effector constructs were coexpressed

transiently in Arabidopsis protoplasts, and GUS activities were determined by

fluorimetry with 4-methylumbelliferyl-glucuronide as substrate. Luciferase gene

expression from the CaMV 35S promoter-luciferase construct was used to

normalize the GUS activities. The normalized values in control protoplasts were

set to 1 and represented as relative activation. Different letters represent a

significant difference at P < 0.05 (one-way anova with Fisher’s post hoc test).

Bars indicate the standard error of the mean. N=3 biological replicates.

Figure S10. Binding of ATXR2 to the LBD1 promoter.

The positions of potential binding sites for ATXR2 are indicated by arrowheads.

Black lines indicate the regions amplified by PCR following ChIP. Enrichment

of ATXR2 at the putative binding regions in the LBD1 promoter was analyzed

by ChIP-qPCR. Enrichment was quantified relative to that at each region in

control (EV) plants. Bars indicate the standard error of the mean. N=3 biological

replicates.

Figure S11. Accumulation of H3K4me3, H3K9me3, and H3K27me3 in

atxr2-1 mutants during callus induction.

Third leaves of two-week-old wild-type (Col-0) and atxr2-1 plants were

explanted and incubated on callus-inducing medium (CIM) for 2 or 4 days

(DAC). Immature calli (N>30) were collected for immunoblot analysis and

quantification. (A) H3K4me3, (B) H3K9me3, and (C) H3K27me3

(arrowheads) were detected immunologically using the corresponding

antibodies. Part of a Coomassie blue–stained gel is shown as a loading

control (left panels). Bands from three blots were quantified using Image J

software. Biological triplicates were averaged and statistically analyzed by

Student’s t-test (*P < 0.05). Bars indicate the standard error of the mean

(right panels). DAC, days after incubation on CIM. N=3 biological

replicates.

Figure S12. Accumulation of H3K4me3 and H3K36me3 in 35S:ATXR2-

MYC calli.

Third leaves of two-week-old wild-type (Col-0) and 35S:ATXR2-MYC plants

were incubated on callus-inducing medium (CIM) for 7 days. Calli (N>30

plants of each genotype) were collected for immunoblot analysis. H3K4me3,

H3K36me3, and H3K27me3 were detected immunologically using the

corresponding antibodies. Part of a Coomassie blue–stained gel is shown as a

loading control (A). Bands from three independent blots were quantified

using Image J software and averaged (B). *P < 0.05 (Student’s t-test). N=3

biological replicates.

Figure S13. H3K4me3 accumulation at the LBD promoters in atxr2-1.

ChIP assays were performed on extracts from wild-type (Col-0) and

atrx2-1 calli following 7 days of leaf explant culture in callus-inducing

medium (CIM) with an antibody recognizing H3K4me3. Enrichment of

specific regions in the LBD promoters was analyzed by qPCR. Enrichment

was quantified relative to that at each region in Col-0 plants. Bars indicate

the standard error of the mean. N=3 biological replicates.

Figure S14. Interactions of ATXR2 with ARF7 and ARF19.

Partial YFP fusion constructs containing either ATXR2, ARF7 or ARF19 were

transiently co-expressed as indicated in Arabidopsis protoplasts. Chloroplasts

appear in red. Scale bar, 20 μm. N=3 biological replicates.

Figure S15. Interactions of ATXR2 with deletion constructs of ARF7

and ARF19.

Partial YFP fusion constructs containing either ATXR2 or fragments of

ARF7 and ARF19 were transiently co-expressed as indicated in Arabidopsis

protoplasts. Chloroplasts appear in red. Scale bar, 20 μm. N=5 biological

replicates.

Figure S16. Interactions of ATXR2 with other ARFs.

Partial YFP fusion constructs containing either ATXR2, ARF5, ARF6, or

ARF8 were transiently co-expressed as indicated in Arabidopsis

protoplasts. Chloroplasts appear in red. Scale bar, 20 μm. N=3 biological

replicates.

Figure S17. Accumulation of H3K4me3 at LBD promoters in arf7-

1arf19-2 mutant calli.

ChIP assays were performed on extracts from wild-type (Col-0) and arf7-

1arf9-2 double-mutant calli following 7 days of leaf explant culture in callus-

inducing medium (CIM) with an antibody recognizing H3K4me3.

Enrichment of specific regions in the LBD promoters was analyzed by

qPCR. Enrichment was quantified relative to that at each region in Col-0.

Bars indicate the standard error of the mean. N=3 biological replicates.

Figure S18. Protein accumulation of ATXR2 in 35S:ATXR2-

MYC/Col-0 and 35S:ATXR2-MYC/arf7-1arf19-2.

Ten-day-old seedlings grown under long-day (LD) conditions were harvested for

total protein isolation. The ATXR2 proteins (arrowheads) were detected

immunologically. Part of a Coomassie blue–stained gel is shown as a loading

control. N=3 biological replicates.

Figure S19. Callus formation of leaf explants from 35S:ATXR2-MYC

x 35S:LBD16-SRDX plants.

Third leaves from two-week-old plants were used to induce calli on CIM

(N>30 plants of each genotype). Plates were incubated for 10 days under

continuous dark conditions and photographed (left panel). Thirty calli

were collected to measure fresh weight (right panel). Three independent

measurements were averaged. Different letters represent a significant

difference at P < 0.05 (one-way anova with Fisher’s post hoc test). Bars

indicate the standard error of the mean. N=30 calli of each genotype.

Scale bar, 5 mm.

Table S1. Primers used for PCR.

The sizes of PCR products ranged from 80 to 300 nucleotides in length. F,

forward primer; R, reverse primer.

Table S2. Primers used for ChIP assays.

F, forward primer; R, reverse primer.