A Histone H3 Lysine-27 Methyltransferase Complex Represses Lateral Root Formation in Arabidopsis thaliana  Gu Xiaofeng , Xu Tongda , He Yuehui   Molecular Plant  Volume 7, Issue 6, Pages 977-988 (June 2014) DOI: 10.1093/mp/ssu035 Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 1 EMF2 Knockdown Causes Increased Primary Root Growth and LR Formation. (A)EMF2 gene structure. Exons are represented by black boxes; the blue bar indicates the 204-bp region used to knock down EMF2 expression. (B) Phenotypes of EMF2-knockdown lines (single-locus T3 homozygotes). Shown are 10-day-old seedlings. (C) Primary root length of the indicated seedlings. 20–23 10-day-old seedlings were scored for each line; error bars indicate standard deviation (SD). (D) Relative EMF2 transcript levels in the roots of indicated seedlings. The transcripts were quantified by RT–qPCR, and normalized to the endogenous control TUBULIN2 (TUB2); relative expression to Col is presented; error bars for SD of three measurements. (E) LR number per primary root of the indicated seedlings. 20–23 10-day-old seedlings were scored for each line; bars for SD. (F, G) LRP number per primary root (F) and LRP density (G) of the indicated seedlings. 20–21 10-day-old seedlings were scored for each line; bars for SD. (C, E–G) Double asterisks indicate statistically significant differences (p < 0.01) in the means between Col and EMF2–RNAi-1 or EMF2–RNAi-2, as revealed by a two-tailed Student’s t-test. Molecular Plant 2014 7, 977-988DOI: (10.1093/mp/ssu035) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 2 CLF Inhibits Primary Root Growth and LR Formation. (A) Phenotypes of 10-day-old Col and clf-81 seedlings. (B) LR number per primary root of Col and clf-81 seedlings from 4–12 d after germination (DAG). 20 seedlings were scored for each line; bars for SD. One of the two biological replicates with similar results is presented. (C, D) LRP number per primary root (C) and LRP density (D) of Col and clf-81seedlings from 4–12 DAG. 15–16 seedlings were scored for each line; bars for SD. (E) Primary root length of Col and clf-81seedlings from 4–12 DAG. 20 seedlings were scored for each line; bars for SD. (F, G) Primary root length (F) and LR number per primary root (G) of Ws and clf-59 (a gain-of-function allele) seedlings at 10 DAG. 17–20 seedlings were scored for each line. (H) LR number per primary root of Ws and clf-1 seedlings at 10 DAG. 20 seedlings were scored for each line. (F–H) Asterisks indicate statistically significant differences (* for p < 0.05; ** for p < 0.01) in the means between Ws and a clf mutant, as revealed by two-tailed Student’s t-test; bars for SD. Molecular Plant 2014 7, 977-988DOI: (10.1093/mp/ssu035) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 3 Genetic Interactions of CLF with SLR, ARF7, and ARF19. (A, B) Phenotypes of the indicated seedlings at 10 DAG. Molecular Plant 2014 7, 977-988DOI: (10.1093/mp/ssu035) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 4 GUS Staining of Col and clf-81 Primary Roots Expressing DR5–GUS. Ten 8-day-old seedlings for each genotype were stained for 2h and examined. Arrows mark pre-branch sites with black arrows for LRPs and white ones for presumptive founder cells (FCs); EZ for elongation zone, MZ for meristematic zone, and white scale bars for 0.5mm. Molecular Plant 2014 7, 977-988DOI: (10.1093/mp/ssu035) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 5 CLF Spatial Expression Patterns in Roots. (A) Schematic drawings of pCLF–CLF:GUS. Black boxes indicate CLF exons, and the blue box represents GUS. Note that the 2.1-kb fragment upstream of the CLF TSS consists of a promoter region and part of an upstream gene. (B, C) Histochemical staining of GUS activity in the primary root (B) and lateral root primordial at various stages (C). The primary roots of 6-day-old CLF:GUS seedlings were stained for 2h except for the Stage I primordial stained for 8h, and nine independent lines were stained. (D) The CLF:GFP expression pattern in the fully-functional pCLF–CLF:GFP line (in the clf-81 background). Molecular Plant 2014 7, 977-988DOI: (10.1093/mp/ssu035) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 6 Analysis of the DR5–GFP Auxin Maximum Reporter in Col and clf Primary Roots. (A, B) Auxin maxima (A) and relative GFP intensity (B) in Col and clf root tips expressing DR5–GFP. Primary roots from 6-day-old seedlings were stained with propidium iodide, and the GFP fluorescence was imaged with a Carl Zeiss Exciter 5 Confocal microscope and its intensity was quantified by ImageJ. The fluorescence intensities of eight or nine primary roots were measured for each line, and relative intensity to the Col line expressing DR5–GFP is presented; error bars for SD. Scale bars denote 50 μm, whereas the broken line indicates GFP fluorescence along the central axis of root meristematic zone. Molecular Plant 2014 7, 977-988DOI: (10.1093/mp/ssu035) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 7 CLF directly Represses PIN1 Expression in Roots. (A, B) Relative PIN1 transcript levels in the roots of indicated seedlings at 6 DAG. The transcripts were quantified by RT–qPCR, and normalized to TUB2; bars for SD of triple measurements. (C, D) The abundance of PIN1:GFP protein in the primary roots (C) and LRPs (D) of 6-day-old Col and clf-81 seedlings expressing pPIN1–PIN1:GFP. (E) Relative GFP intensity in Col and clf root tips expressing PIN1:GFP. The fluorescence intensities of seven or eight primary roots were measured for each line, and relative intensity to the Col line expressing PIN1:GFP is presented; bars for SD. (F) ChIP analysis of CLF enrichment and H3K27me3 state at the PIN1 locus in roots. Immunoprecipitated genomic fragments were quantified by qPCR, and normalized to the endogenous control TUBULIN8 (TUB8). The fold enrichments of CLF:GFP protein in each examined PIN1 region over control (Col) are shown, with error bars for SD of three biological repeats. With regard to the H3K27me3 levels in each examined region in clf-1, they were normalized to Ws (wild-type), with error bars indicating SD of three measurements; a biological repeat is shown in Supplemental Figure 3. Molecular Plant 2014 7, 977-988DOI: (10.1093/mp/ssu035) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions