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A role of Arabidopsis COP9 signalosome in multifaceted developmental processes revealed by the characterization of its subunit 3

Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06520-8104, USA

View larger version (80K): [in a new window]   Fig. 1. Sequence comparison of Arabidopsis CSN3 with its homologs from other organisms. (A) Protein sequence comparison of Arabidopsis CSN3 and its homologs from human and Drosophila. The underlined sequences are the N-terminal leucine zipper domain and (second) the C-terminal PCI domain. Numbers on the right indicate the positions of the amino acid residues. The GenBank Accession Numbers are AF361759 for Arabidopsis, AF098109 for human (Homo sapiens) and AF071313 for Drosophila. (B) Comparison of the cauliflower (Brassica oleracea var. Botrytis) peptide sequences with the deduced CSN3 protein sequences from Arabidopsis and tomato (Lycopersicon chilense).

View larger version (22K): [in a new window]   Fig. 2. Arabidopsis CSN3 is a subunit of the COP9 signalosome. (A) Arabidopsis CSN3 co-fractionates with the COP9 signalosome. CSN3 co-fractionated with other known COP9 signalosome subunits in a peak around 500 kDa in wild type but was absent in the fus11-U203 mutant. Total soluble protein extracts from 6-day-old light-grown wild-type seedlings (WT) and 8-day-old light-grown fus11-U203 seedlings were separated using a Superose 6 HR gel filtration column, followed by SDS-PAGE and protein blot analysis with antibodies against CSN1, CSN3 and CSN5 as indicated on the left. The plant materials used are indicated on the right. (B) Arabidopsis CSN3 co-immunoprecipitates with other known COP9 signalosome subunits. The antibodies used for immunoprecipitation from the total soluble proteins are indicated on the top. The antibodies used in the immunoblots are labeled on the right. ‘PI IP’ is the preimmune serum from the same rabbit before immunization and was used as negative control. The immunoprecipitates were separated using 10% SDS-PAGE and probed with antibodies against CSN1, CSN3 and CSN5.

View larger version (1K): [in a new window]   Fig. 3. CSN3 gene structure and the molecular lesion in the fus11-U203 mutant. (A) The genomic structure of the CSN3 gene. The 11 exons are numbered and the protein coding regions of the exons are black. (B) The molecular lesion of the CSN3 gene in the fus11-U203 mutant. The G to A mutation at the end of the intron 9 is indicated. This mutation results in utilization of a cryptic splicing site within intron 9, which results in in-frame termination of the reading frame after two novel amino acids. (C) Diagrams of the wild-type (WT) and mutant version of the CSN3 protein. The two inserted amino acids and the stop codon (asterisk) in the FUS11-U203 are indicated. The leucine zipper and the PCI domains are represented by striped and black boxes respectively. The amino acid positions are labeled on the top.

View larger version (50K): [in a new window]   Fig. 4. Effect of COP9 signalosome deficiency on the profiles of cellular ubiquitinated proteins and on the degradation of HY5 and phyA. (A) Comparison of the profiles of cellular ubiquitinated proteins of the wild type, fus11-U203, fus6-T236 and cop1-6 mutant. The total protein extracts were size fractionated using a Superose 6 HR gel filtration column with TB buffer. The collected fractions were concentrated with StrataClean beads (Stratagene) and further separated on 10% SDS-PAGE. Antibodies against ubiquitin were used in the western blots to detect the ubiquitinated proteins. The gel filtration fraction numbers corresponding to each lane and the molecular size markers are indicated on the top. The molecular size markers on the SDS-PAGE are indicated on the left side. 8-day-old seedlings were used in the experiment. The smearing high molecular mass bands in fractions 5-15 are the multi-ubiquitinated proteins. White triangles indicate bands reduced in mutants, while black triangles indicate bands increased in mutants. (B) The effect of the COP9 signalosome deficiency on proteasome-mediated protein degradation. The seedlings were 4.5 days old. The antibodies used for western blot are indicated on the right side and the sample identity is indicated on the left side. Left: phyA degradation profile in both wild-type and fus11-U203 seedlings. The three samples used were continuous dark-grown seedlings (lane D), continuous light-grown seedlings (lane L), and dark-grown seedlings shifted to red light for 2 hours (R2). Right: HY5 degradation profiles in both wild-type and fus11-U203 seedlings. The samples shown were continuous light grown seedlings (lane L), continuous dark-grown seedlings (lane D), and continuous light-grown seedlings shifted to the dark for 13 hours (lane D13).

View larger version (93K): [in a new window]   Fig. 5. Immunoblot analysis of the transgenic plants. (A) Levels of the COP9 signalosome subunits in the CSN3 transgenic plants. Proteins were extracted from the defective organs (as indicated on top) from various transgenic lines (TG). Antibodies against CSN3, CSN6 and CSN1 were used to detect the COP9 signalosome level (marked on the right). The corresponding wild-type tissues (WT) were used as a control. Rpn6 was used to control equal loading. (B) Levels of ubiquitinated proteins in the transgenic plants and fus11-U203 mutant. The total protein extracts were separated on 12% SDS page and detected with antibodies against ubiquitin. J. Leaves, juvenile leaves; A. Leaves, adult leaves. The juvenile leaves, adult leaves and flower I were from lines S3-C-A, S3-C-A and S3-C-C, respectively of the T2 generation in the Columbia background. The transgenic seedling and flower II were from line S3-L-D of the T3 generation in the Landsberg background. 8-day-old fus11-U203 seedlings were used as a control. Many other transgenic lines were examined and similar results were observed.

View larger version (89K): [in a new window]   Fig. 6. Representative vegetative growth phenotypes of the CSN3 co-suppression plants. (A) Leaf developmental phenotypes of the CSN3 transgenic plants. The plants in a, b and c were from the T2 generation in the Columbia background. The plant in d was from the T3 generation and in the Landsberg background. Scale bars: 5 mm. Arrows indicate defective leaves. (B) The developmental phenotypes of the CSN3 transgenic plants with similarity to auxin related mutants. The plants were from the T3 generation in the Columbia background except that the plant in d was from the T2 generation. Scale bars: 5 mm. Arrows indicate defective organs. (C) Top: seedlings producing no true leaves. Bottom: northern blot analysis of the STM gene in transgenic and wild-type plants. The seedling shown was from the T3 generation in the Columbia background.

View larger version (74K): [in a new window]   Fig. 7. Representative phenotypes of CSN3 co-suppression plants during reproductive growth. (A) Representative inflorescence phenotypes of the CSN3 transgenic plants. The inflorescences in a, b and c were from T2 plants in the Columbia background. The inflorescences in d, e, and f were from the T3 plants in the Landsberg background. The arrows indicate defective organs. (B) Representative flower phenotypes of the CSN3 transgenic plants. The flowers in a, b, d and e were from T3 plants in the Landsberg background. The flowers in c and f are from T2 plants in the Columbia background. The arrows indicate organs with marked developmental abnormality.