Figure 2.

The mutant plant of ER membrane protein HRD3A is sensitive to ER stress inducer, tunicamycin. (A) Schematic diagram of the HRD3A gene structure and primers for RT-PCR. Closed boxes represented exons; lines between closed boxes represent introns; and black boxes represent the 5′- and 3′-UTR. The two null mutants, hrd3a-1 and hrd3a-2, were identified. LP, forward primer; RP, reverse primer. (B) Growth of WT and mutant plants on MS medium containing a range of concentrations of TM (0, 0.15, 0.20, and 0.25 μg/ml). Seeds were germinated and grown for 8 days on plates with or without TM, and representative plants are shown. Percentages are means (n = 60 each) of three independent repeats. (C) Quantitative analysis of hrd3a mutants and WT seedlings with green true leaves on plates containing a range of concentrations (0, 0.15, 0.2, and 0.25 μg/ml) of TM. (D) Expression patterns of transcripts of ER chaperones in response to TM treatment. The 2-week-old seedlings were treated with MS media containing 5 μg/ml TM for different times. Total RNA (15 μg) from each sample was hybridized with ³²P-labeled BIP1/2, PDIL-1, and CNX1 probes. The bottom panel shows methylene blue-stained 28S rRNA as a loading control. (E) Cell fractionation assays of HRD3A. 35S:HRD3A-GFP was infiltrated into tobacco leaves and the samples were collected after 3 d. HRD3A-GFP was detected using an anti-GFP antibody (top panel). Ponceau S staining of the RbcS is shown as a loading control (bottom panel). B, buffer-extracted fraction; F, final membrane fraction; M, membrane fraction; S, soluble fraction; SD, SDS-extracted fraction; T, total extract. (F) Co-localization of HRD3A-GFP with HDEL-RFP. Tobacco leaves were co-infiltrated with 35S: HRD3A-GFP and 35S: HDEL-RFP. Signals were observed directly under a confocal microscope after 3 d. The photographs in the bottom panel are the enlargements of the nuclear area of corresponding frames as indicated by the arrows.