Figure 1.
Overexpression of a peptide that contains the PEX2 RING domain suppresses det1.
(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 PEX2RF transcript in two transgenic lines (lines2 & 3) overexpressing PEX2RF in the det1 background. UBQ10 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 t-test, P<0.0001 for all lines vs. det1. Error bars indicate s.e.m. (E) Four-week plants. Scale bar = 3 cm.
Figure 2.
Nuclear localization of PEX2RF-GFP in transgenic plants.
(A) RT-PCR analysis of PEX2RF mRNA and the UBQ10 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).
Figure 3.
PEX2RF and HY5 interact in the nucleus.
(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.
Figure 4.
PEX2 and HY5 interact in yeast two-hybrid assays.
(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.
Figure 5.
Immunoblot analysis of the HY5 protein in various genetic backgrounds.
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 [11] was used as a control. Asterisks indicate non-specific bands. A cross-reacting band indicated by a double asterisk served as the loading control.
Figure 6.
qRT-PCR analysis of the expression of some of HY5’s target genes.
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 UBQ10. Values correspond to the mean and s.d. of three biological replicates. The experiments were repeated twice with consistent results.