Heatmaps showing peroxisomal gene expression under stress conditions.

<p>Expression values are log2 normalized fold changes from untreated plants. Genes discussed in the text are in red. (A) Gene expression under abiotic stresses. Genes subjected to mutant analysis are underscored. (B) Gene expression under biotic stresses.</p

Heatmap of transcript levels of peroxisomal genes in various developmental stages.

<p>Absolute gene expression values downloaded from the AtGenExpress database were used for heatmap generation. Genes discussed in the text are in red. Developmental stages include: 1. seedling_cotyledons; 2. seedling_hypocotyl; 3. seedling_leaves1+2; 4. adult_leaves; 5. senescing leaves; 6. flower; 7. silique_stage3; 8. silique_stage4; 9. silique_stage5; 10. seed_stage6; 11. seed_stage7; 12. seed_stage8; 13. seed_stage9; 14. seed_stage10.</p

Drought resistance phenotypes of <i>lon2</i> and <i>hpr1</i> mutants.

<p>(A) Images of plants (left) and color-coded chlorophyll fluorescence that indicates F_v/F_m values (right). (B) F_v/F_m comparison between mutants and wild type. Four biological replicates of each genotype were used. No significant difference in F_v/F_m was observed. (C) Plant images and color-coded chlorophyll fluorescence images that indicate F_v/F_m values. (D-E) F_v/F_m comparison between mutants and wild type. Two biological replicates were used for each genotype under each condition. Asterisk, p < 0.01 in Students’ <i>t</i> test. (F-H) Quantification of anthocyanin (F), chlorophyll (G), and relative water content (H) in mutants and wild type plants. Three biological replicates were used for each genotype under each condition. Asterisk indicates p < 0.01 in Students’ <i>t</i> test; N.D, not detectable; D, drought; W, watered.</p

F_v/F_m comparison between the selected peroxisomal mutants and wild type plants grown in the same pot.

<p>The <i>aba1</i> mutant (salk_059469) was used as a positive control. Two biological replicates were used for each genotype. Asterisk indicates p-value < 0.01 in Student’s <i>t</i> test.</p

PEX2 and HY5 interact in yeast two-hybrid assays.

<p>(A) Yeast two-hybrid assays to show interaction between PEX2 and HY5. Yeast transformants containing the indicated GAL4 DNA binding domain (BD) and GAL4 activation domain (AD) fusion constructs were grown overnight in liquid culture and spotted on selection media plates (lacking leucine and tryptophan; –LW) and interaction media plates (lacking adenine, leucine, tryptophan and histidine; –ALWH, or –ALWH supplemented with 25 mM 3-amino-1,2,4-triazole; –ALWH+25 mM 3-AT). Growth on –ALWH and –ALWH+25 mM 3-AT media indicates protein interaction. (B) Immunoblot analysis of BD fusion constructs. Proteins extracted from transformed yeast cells shown in (A) were subjected to immunoblotting using α-c-Myc antibody. Numbers on the left of the blot indicate protein molecular weight markers in kDa.</p

qRT-PCR analysis of the expression of some of HY5’s target genes.

<p>RNA was extracted from 4d dark-grown seedlings in different genetic backgrounds. Three biological replicates of qRT reaction were performed for individual primer sets. The transcript level of each gene in the mutant is represented as arbitrary unit relative to the transcript level of the same gene in the wild-type plant, which was set to 1.0. The transcript level (relative expression) is the ratio between the transcript abundance of the studied gene and the transcript abundance of <i>UBQ10</i>. Values correspond to the mean and s.d. of three biological replicates. The experiments were repeated twice with consistent results.</p

Immunoblot analysis of the HY5 protein in various genetic backgrounds.

<p>Proteins were extracted from 4d dark-grown seedlings exposed to 1 hr white light and detected with the α–HY5 antibody. Purified HY5–6xHis from our previous study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108473#pone.0108473-Desai1" target="_blank">[11]</a> was used as a control. Asterisks indicate non-specific bands. A cross-reacting band indicated by a double asterisk served as the loading control.</p

Overexpression of a peptide that contains the PEX2 RING domain suppresses <i>det1</i>.

<p>(A) Schematic of the Arabidopsis PEX2 protein, showing positions of the transmembrane (TM) and RING finger (RF) domains, and the region (indicated by horizontal bar) used as PEX2RF and as antigen for antibody generation. (B) RT-PCR analyses of the <i>PEX2RF</i> transcript in two transgenic lines (lines2 & 3) overexpressing PEX2RF in the <i>det1</i> background. <i>UBQ10</i> is the internal control. (C) Phenotype of 4d dark-grown seedlings grown on 0.5X MS supplemented with 0.5% sucrose. Scale bar = 0.5 cm. Two seedlings are shown for each genotype. (D) Hypocotyl length measurements of 4d dark-grown seedlings shown in (C). n>30 for each genotype. Student <i>t</i>-test, P<0.0001 for all lines vs. <i>det1</i>. Error bars indicate s.e.m. (E) Four-week plants. Scale bar = 3 cm.</p

PEX2RF and HY5 interact in the nucleus.

<p>(A–C) Epifluorescence micrographs of tobacco leaf epidermal cells infiltrated with the indicated gene constructs. Strong YFP signals (BiFC) in the nucleus, as indicated by arrows in (C), were observed only when HY5-YFPct and YFPnt-PEX2RF were co-expressed. Scale bars = 100 µm. (D–F) Confocal micrographs of tobacco leaf epidermal cells co-infiltrated with HY5-YFPct and YFPnt-PEX2RF constructs. DAPI stains the nucleus (D), and BiFC signals are indicated by YFP fluorescence (E). Arrows in the merged image (F) indicate the overlaps of DAPI and BiFC. Scale bars = 50 µm. (G–H) Immunoblot analyses showing expression of HY5-YFPct and YFPnt-PEX2RF proteins in tobacco tissue. In (G), tissues were from plants shown in (A) and (B) respectively and α-HY5 (left) and α-RF (right) antibodies were used. In (H), tissue was from plant shown in (C), and α-GFP was used. Molecular weight markers in kDa are shown to the left of the blots.</p

Nuclear localization of PEX2RF-GFP in transgenic plants.

<p>(A) RT-PCR analysis of <i>PEX2RF</i> mRNA and the <i>UBQ10</i> control in Col-0 and 35S::PEX2RF-GFP lines. (B) Immunoblot analyses of proteins from Col-0 and PEX2RF-GFP-expressing plants, using α–PEX2RF and α–GFP antibodies respectively. Asterisks indicate cross-reacting bands, and arrowheads point to the PEX2RF-GFP fusion protein. Numbers on the left indicate molecular weight markers in kDa. (C–H) Confocal images of transgenic plants from hypocotyl cells of 10d seedlings (C–E) and leaf mesophyll cells of two-week plants (F–H). DAPI stains the nucleus, green signals are from PEX2RF-GFP, and red signals are from chlorophyll autofluorescence. Arrows in the merged images indicate the nucleus. Scale bars = 10 µm in (C–E) and  = 20 µm in (F–H).</p