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. 2022 Aug 16;34(11):4366–4387. doi: 10.1093/plcell/koac250

Figure 6.

Figure 6

The EAR motif and its flanking sequences determine the regulatory activity of OsIAA1/9/21/31 on OsEIL1-activated OsTAR2 expression through affecting the histone acetylation/deacetylation. A, Generation of mutation in the EAR motifs of OsIAA1/9/21/31. Three conserved Leu (L) residues in the EAR motif of OsIAA1 (aa 24–31), OsIAA9 (aa 2–9), OsIAA21 (aa 23–30), and OsIAA31 (aa 8–15) were changed to Ala (A) residues, and mutant OsIAAs were designated m1, m9, m21, and m31, respectively. B, Effects of EAR motif mutations on OsIAA1/9/21/31-mediated regulation of OsEIL1 activity. Data are means ± sd (n = 3). C, Effects of HDAC inhibitor (TSA) on OsIAA21/31 inhibition of OsEIL1 activity. Data are means ± sd (n = 3). D, Effects of OsGCN5 inhibitor (MB-3) on OsIAA1/9-mediated promotion of OsEIL1 activity. Data are means ± sd (n = 3). E, Effects of repressing OsGCN5 expression by siRNA on OsIAA1/9-mediated promotion of OsEIL1 activity. Three biological replicates were performed in analyzing OsTAR2 promoter activity and OsGCN5 expression. Data are means ± sd. F, H3K27ac levels over the OsTAR2 promoter region in response to ethylene, as analyzed by ChIP. One-DAG etiolated seedlings were treated with 10 ppm ethylene for 8 h, and root tips were used for analysis. PCR fragments 1 and 2 are the same as in Figure 2F. Data are means ± sd (n = 3). G, Interaction between OsGCN5 and OsIAA1/9 or EAR motif-mutated OsIAA1/9, as analyzed by Co-IP. H, Ethylene response of root length in OsGCN5-OE seedlings. Etiolated seedlings were grown in air or 2 ppm ethylene for 1.5 DAG. Root length of kanamycin-resistant seedlings from segregating lines was measured. Dashed lines indicate the seams between images. Scale bar, 10 mm. Data are means ± se (n ≥ 9). I, Ethylene response of root length in Osgcn5 mutant seedlings. Etiolated seedlings were grown in air or 2 ppm ethylene for 2 DAG. Analysis was performed using the root length of seedlings from a segregating Osgcn5 line generated by CRISPR/Cas9. +/+, WT genotype of OsGCN5; +/–, heterozygous genotype; –/–, homozygous genotype. Scale bar, 10 mm. Data are means ± se (n ≥ 13). J, Artificial mutations in the EAR motif-flanking sequences in domain I of OsIAA1/9. Mutations (Ser to Ala) in OsIAA1/9 are indicated, and mutant OsIAAs were named OsIAA1mSer and OsIAA9mSer, respectively. K, Effects of the Ser-to-Ala substitution in domain I on OsIAA1/9 promotion of OsEIL1-activated OsTAR2 promoter activity. Data are means ± sd (n = 4). L, Reduced interaction between OsGCN5 and OsIAA1/9mSer as revealed by Co-IP. *P  < 0.05; **P < 0.01; ***P < 0.001, as determined by a two-tailed Student’s t test compared to the corresponding control.