Volume 5, Issue 3, Pages 669-677 (May 2012) A Dual Mechanism Controls Nuclear Localization in the Atypical Basic-Helix-Loop-Helix Protein PAR1 of Arabidopsis thaliana  Anahit Galstyan, Jordi Bou-Torrent, Irma Roig-Villanova, Jaime F. Martínez-García  Molecular Plant  Volume 5, Issue 3, Pages 669-677 (May 2012) DOI: 10.1093/mp/sss006 Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 1 Alignment of the Amino Acid Sequence (A) Adjacent to the HLH Region (‘Basic’ Domain) and (B) Corresponding to the Putative NLS Motifs of Several DNA- (PIF3, PIF4, PIF5, BIM1, and SPT) and Non-DNA-Binding (PAR1, PAR2, HFR1, KDR, IND, and NAN) bHLH Proteins. In panel (A), positions 5, 9, and 13 in the basic region (that correspond to the H-E-R conserved residues in the DNA-binding bHLHs) are shown in bold and underlined. In panel (B), those positions corresponding to basic clusters within the putative NLS motifs are shown in bold. Molecular Plant 2012 5, 669-677DOI: (10.1093/mp/sss006) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 2 Phenotypes of Plants Overexpressing Mutated Versions of PAR1 (PAR1N and PAR1B). (A) Scheme showing the truncated PAR1 derivatives overexpressed and the changes introduced in the amino acid sequence. G refers to GFP. Boxes corresponding to the mutated region are highlighted in gray. (B) The GFP fluorescence in roots from 10-day-old W-grown seedlings overexpressing the indicated GFP-derived proteins. Arrows indicate nuclear fluorescence. Panels are shown to the same scale. (C) Hypocotyl response to simulated shade of lines shown in (A) and (B). Seedlings were germinated and grown for 2 d under W and then either kept in W or transferred to W+FR for five more days. Columns represent the mean ± SE. Symbols indicate significant differences (* P < 0.01) relative to control (Col-0) growing under the same light conditions. (D) Yeast two-hybrid (Y2H) assay of PAR1-mutated derivative forms: PAR1N and PAR1B. As controls, wild-type PAR1, PAR1L66E, and PAR1L92K fused to GAL4 activation domain were used. SD-LT and SD-AHLT refer to the selective media indicative of transformed cells or protein–protein interaction, respectively. Numbers refer to the combinations of binding domain (BD) and activation domain (AD) constructs specified on the right panels. Molecular Plant 2012 5, 669-677DOI: (10.1093/mp/sss006) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 3 Phenotypes of Plants Overexpressing Truncated and Mutated Versions of PAR1 (AHCN and AHCB). (A) Scheme showing truncated PAR1 derivatives overexpressed. G refers to GFP. Boxes corresponding to the mutated region are highlighted in gray. (B) The GFP fluorescence in roots (lines G and PAR1-G) or hypocotyls (lines AHCN–G and AHCB–G) from 10-day-old W-grown seedlings overexpressing the indicated GFP-derived proteins. Bar = 50 μm. (C) Hypocotyl response to simulated shade of lines shown in (A). Seedlings were germinated and grown as indicated in Figure 2C. Columns represent the mean ± SE. (D) GFP and P1R1 expression in the indicated transgenic lines. RNA extracted from 7-day-old W-grown seedlings was used for RNA blot analysis of P1R1 and transgene GFP expression levels. Each RNA sample was extracted from a pool of homozygous seedlings. Normalized expression of the genes analyzed is presented relative to the expression levels in the control line (G.06). Values are means ± SE of three independent samples. 25S rRNA levels were used to normalize gene expression. Symbols indicate significant differences (°P < 0.05, * P < 0.01) relative to controls (Col-0 growing under the same light conditions as in (C), line G.06 in (D). Molecular Plant 2012 5, 669-677DOI: (10.1093/mp/sss006) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 4 Phenotypes of Plants Overexpressing Truncated and Mutated Versions of PAR1 (PAR1L92K, AHCL66E, and AHCL92K). (A) Scheme showing the truncated PAR1 derivatives overexpressed. G refers to GFP. Boxes corresponding to the mutated region are highlighted in gray. Symbols (open circle on top of a vertical line) represent the approximate position within the HLH region of the introduced point mutation. (B) The GFP fluorescence in roots from 10-day-old W-grown seedlings overexpressing the indicated GFP-derived proteins. Bar = 50 μm. (C) GFP and P1R1 expression in the indicated transgenic lines. RNA extracted from 7-day-old W-grown seedlings was used for RNA blot analysis of P1R1 and transgene GFP expression levels. Each RNA sample was extracted from a pool of homozygous seedlings. Normalized expression of the genes analyzed is presented relative to the expression levels in the control line (G.06). Values are means ± SE of three independent samples. 25S rRNA levels were used to normalize gene expression. (D) Hypocotyl response to simulated shade of lines shown in (A). Seedlings were germinated and grown as indicated in Figure 2C. Columns represent the mean ± SE. Symbols indicate significant differences (°P < 0.05, * P < 0.01) relative to controls (Col-0 growing under the same light conditions, hypocotyl length measurements, and line G.06 in expression analyses). Molecular Plant 2012 5, 669-677DOI: (10.1093/mp/sss006) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions