Fig 1.
Increased hypocotyl elongation and auxin accumulation in the sob3-6 mutant.
(A) Hypocotyl growth of light-grown mutant seedlings. Seeds were germinated and grown for 7 days on vertical plates under long-day (LD) conditions (left panel). Hypocotyl lengths (n > 30 in each genotype) were measured using Image J applications (http://rsb.info.nih.gov/ij/). Three biological replicates were averaged and statistically analyzed by two-tailed Student's t-test assuming unequal variance. Statistically significant differences between wild-type and mutants are indicated by asterisks (*P < 0.05). Bars indicate standard error of the mean (right panel). (B) Transcript accumulation of YUCs in sob3-6. Transcript accumulation was analyzed by RT–qPCR. The eIF4a (At3g13920) gene was used as an internal control. Three biological replicates were averaged and statistically analyzed by two-tailed Student's t-test assuming unequal variance (*P < 0.05). (C) Enhanced auxin signaling in sob3-6. The pDR5:GUS construct was introduced into wild-type and the sob3-6 mutant. Seven-day-old seedlings grown under LD conditions were subjected to GUS staining. (D) Effects of NPA on hypocotyl elongation of sob3-6. Seeds were germinated on MS medium supplemented with 1 uM NPA and incubated for 7 days under LD conditions. Hypocotyl lengths (n > 30 in each genotype) were measured using Image J applications (http://rsb.info.nih.gov/ij/). Scale bars, 1 mm.
Fig 2.
Binding of ESC and SOB3 to the YUC9 promoter.
(A) Putative S/MAR region in the YUC9 promoter. The predicted S/MAR region is marked with an arrowhead. Underbars represent the amplified genomic regions. (B and C) ChIP assays. Total protein extracts from 35S:ESC-MYC (B) and 35S:SOB3-MYC (C) transgenic plants grown for 9 days under LD conditions were immunoprecipitated with an anti-MYC antibody. Fragmented DNA was eluted from the protein-DNA complexes and used for qPCR analysis. Three independent biological replicates were averaged, and the statistical significance of the measurements was determined by two-tailed Student's t-test assuming unequal variance (*P < 0.05). Bars indicate the standard error of the mean.
Fig 3.
Interaction of SOB3 with the SWR1 component.
(A) Y2H analysis. Y2H assays were performed with the SOB3 protein fused to the DNA-binding domain (BD) of GAL4 and ARP4 fused with the transcriptional activation domain (AD) of GAL4 for analysis of interactions. Interactions were examined by cell growth on selective media. -LWHA indicates Leu, Trp, His, and Ade drop-out plates. -LW indicates Leu and Trp drop-out plates. GAL4 was used as a positive control. (B) BiFC assays. Partial fragments of YFP protein were fused with SOB3 and ARP4. IDD14-RFP was used as a nuclear marker.
Fig 4.
Regulation of YUC9 expression by the SWR1 complex.
(A) Binding of SEF to the YUC9 promoter. Two-week-old 35S:SEF-MYC transgenic plants grown under LD conditions were used to conduct ChIP assays. (B) H2A.Z deposition in the YUC9 promoter. Two-week-old pHTA11::HTA11-GFP transgenic plants grown under LD conditions were used for ChIP analysis with anti-GFP antibody. Eluted DNA was subject to qPCR analysis. (C and D) Transcript accumulation of YUC9 in the genetic mutants of H2A.Z exchange. Nine-day-old hta9-1 hta11-2 (C) and arp6-3 (D) mutants grown under LD conditions were used to examine transcript accumulation. Three biological replicates were averaged and statistically analyzed by two-tailed Student's t-test assuming unequal variance (*P < 0.05). (E) Recruitment of Pol II at the YUC9 promoter in arp6-3. Two-week-old plants grown under LD conditions were used to conduct ChIP assays with an anti-N-terminus of Arabidopsis Pol II antibody. (F) H2A.Z deposition at the YUC9 promoter in sob3-6. Two-week-old plants grown under LD conditions were used to conduct ChIP assays with an anti-H2A.Z antibody. (G) Recruitment of Pol II at the YUC9 promoter in sob3-6. Two-week-old plants grown under LD conditions were used to conduct ChIP assays with an anti-N-terminus of Arabidopsis Pol II antibody.
Fig 5.
Genetic hierarchy of S/MAR-assisted H2A.Z exchange and auxin biosynthesis.
(A) Effects of yucasin on hypocotyl elongation of sob3-6, arp6-3 and sef-1. Seeds were germinated on MS medium supplemented with 250 uM yucasin, and incubated for 7 days under LD conditions. Scale bar, 1 mm. (B) Quantification of hypocotyl length. Hypocotyl lengths (n > 30 in each genotype) were measured using Image J applications (http://rsb.info.nih.gov/ij/). Three independent biological replicates were averaged, and the statistical significance of the measurements was determined by two-tailed Student's t-test assuming unequal variance (*P < 0.05). Bars indicate the standard error of the mean. (C and D) Proposed working diagram. Light-inducible ESC and SOB3 bind to the YUC9 promoter and recruit the SWR1 complex that catalyzes H2A.Z exchange in order to suppress its expression. As a consequence, auxin biosynthesis is inhibited, and thus hypocotyl elongation is suppressed in light.