Physical interaction of PDIL1-1 with OsCP1.

<p>(A) Phylogenetic tree of <i>Arabidopsis</i> cysteine protease 43 (AtCP43) amino acid sequences with its rice homologs. The sequences of the proteins were aligned using the CLUSTALW2 software program and a phylogenetic tree was generated using the MEGA5 software program. B) Map of conserved domains of OsCP1. The three conserved domains, inhibitor_I29, peptidase_C1A, and granulin, are indicated by boxes. (C) Partial <i>OsCP1</i> cDNA encoding peptidase_C1A and granulin, and full-length <i>PDIL1-1</i> cDNA were fused to sequences encoding the Gal4 activation domain (AD) and the Gal4 DNA-binding domain (BD) in pGADT7 and pGBKT7, respectively. Each number indicates yeast cells transformed with a combination of only pGADT7 or pGBKT7 vectors or recombinant plasmids. Combinations are described in the box. Transformants were plated onto minimal medium (Leu⁻/Trp⁻) and (Leu⁻/Trp/His⁻ (5 mM 3-AT)), incubated for 4 days, and then photographed. (D) Expression analysis of <i>OsCP1</i> and its homologs. Transcript levels of <i>OsCP1</i> and its homologs, Os05g0108600 and Os01g0971400, were examined by real-time RT-PCR with gene-specific primers. These experiments were repeated three times independently. The reported values of transcript levels of <i>OsCP1</i> homologs are normalized to numerical values relative to the transcript level of <i>OsCP1</i> in DAF5, which is set at a value of 1.00±0.00. DAF, day after flowering.</p

Comparison of seed proteins of WT and <i>PDIL1-1Δ</i> mutant.

<p>(A) Total seed proteins were extracted from the WT and <i>PDIL1-1Δ</i> mutant and separated by 10–27% gradient SDS-PAGE. (B) Total proteins were extracted from mature seeds of the WT and <i>PDIL1-1Δ</i> mutant and then analyzed by 2-DE.</p

Aleurone layers of <i>PDIL1-1Δ</i> mutant seeds and analysis of PDIL1-1 protein level in differently polished seeds.

<p>(A) Mature seeds of the WT and <i>PDIL1-1Δ</i> mutant were stained with methylene blue and then analyzed by light microscopy. Abbreviations: vs, ventral side; ds, dorsal side. ls, lateral side. Bar, 1 mm (left), and 0.5 mm (middle and right). (B) Thickness of aleurone layers was measured in both seeds of the WT and <i>PDIL1-1Δ</i> mutant. * and ** indicate a significant difference from the wild type at P<0.05 and P<0.01 by t-test, respectively. (C) Dried seeds were polished to different extents. (D The remaining seeds were ground thoroughly, and the PDIL1-1 level was examined by western blot with an anti-PDIL1-1 antibody (left). After detection, the membrane was stained with Coomassie brilliant blue (right). (E) PDIL1-1 levels were examined in the polished powder by western blot with an anti-PDIL1-1 antibody (left). After detection, the membrane was stained with Coomassie brilliant blue (right).</p

Isolation of the <i>PDIL1-1Δ</i> mutant and its seed phenotype.

<p> (A) Identification of the T-DNA insertion site in the <i>PDIL1-1</i> gene (locus number Os11g09280) by PCR. T-DNA insertion mutant PFG_1B-16041.R was provided by Dr. Gynheung An, POSTECH. Independent transgenic lines were analyzed by PCR using two sets of primers (described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044493#pone.0044493.s008" target="_blank">Table S1</a>); 1,229-bp fragments were amplified by PCR with LP and RP, but not by the primer set in transgenic homozygote lines. Approximately 700-bp fragments were amplified by PCR with BP and RP. (B) Identification of <i>PDIL1-1</i> mutant rice by western blot. Total seed proteins extracted from the lines described in (A) were separated by SDS-PAGE and examined by western blot using an anti-PDIL1-1 antibody. (C) Grains of wild type and <i>PDIL1-1Δ</i> mutants were collected, and palea and lemma of the grains of wild type and two <i>PDIL1-1Δ</i> mutant alleles (PFG_1B-16041.R and PFG_2B-80111.R) were opened and removed. The mutants showed a chalky and uneven phenotype. Bar, 0.3 cm.</p

Free sugar content in <i>PDIL1-1Δ</i> mutant seeds.

<p>Palea and lemma of the grains of the wild type and <i>PDIL1-1Δ</i> mutant (0% milled rice) were removed and used for analysis of free sugar content.</p><p>The numerical values represent the mean of three independent experiments, and the values are expressed as means ± standard deviations.</p>**<p>indicates a significant difference from the wild type at P<0.01 and NS indicates no significant difference by t-test.</p

Comparison of seed weight and protein amounts between WT and <i>PDIL1-1Δ</i> mutant.

<p>Palea and lemma of the grains of the wild type and <i>PDIL1-1Δ</i> mutant (0% milled rice) were removed and then used for analysis of seed weight, and protein amounts.</p><p>The numerical values represent the mean of three independent experiments, and the values are expressed as means ± standard deviations.</p>*<p>and **indicate significant difference from the wild type at P<0.05 and P<0.01 by t-test, respectively.</p

List of proteins for which spot intensity changed over two-fold in <i>PDIL1-1Δ</i> seeds compared to WT.

<p>This experiment was repeated three times using different WT and the <i>PDIL1-1Δ</i> mutant seeds. Here, two results are shown.</p><p>Accession numbers indicate GenBank accession number.</p><p>ND indicate ‘not detectable’.</p

Expression pattern of <i>PDIL1-1</i> gene.

<p>(A) Expression profile of <i>PDIL1-1 I</i> gene during development. Total RNA was isolated from developing rice organs at the indicated time points and used for real-time RT-PCR. DAG, day after germination; DAF, days after flowering. (B) Total RNA was isolated from developing seeds at the indicated time points and separated on formaldehyde-agarose gels. After electrophoresis, total RNA was transferred onto a nylon membrane. The membrane was hybridized with ³²P-labeled <i>PDIL1-1</i> cDNA and exposed on X-ray film. DAF, days after flowering. (C) Examination of the level of PDIL1-1 protein during seed development. Total proteins were extracted from the wild type at the indicated time points. After 12% SDS-PAGE, proteins were transferred onto a nitrocellulose membrane and treated with an anti-PDIL1-1 antibody (left). After blotting, the membrane was stained with Coomassie brilliant blue (right). (D) Proteomic analysis of rice seed proteins. Total proteins were extracted from mature seeds of WT and then separated by 2-DE. Each protein spot was identified by MALDI-TOF MS (left). The boxed region was enlarged (right). Arrowheads indicate PDIL1-1 protein spots.</p

AtBBD1 interacts with AtJAZ proteins.

<p>(A) Y2H assay between AtBBD1 and each of 12 AtJAZs. Full length CDS of AtBBD1 was fused to GAL4 DNA binding domain (BD) and each full length CDSs of 12 AtJAZs was fused to AD. (B) The pull-down assay between AtBBD1 and AtJAZ1. <i>35S:6xmyc-AtBBD1</i> plant extract (input) was incubated with amylose resin bound recombinant MBP-AtJAZ1 protein. Pulled-down protein complex was detected by immunoblotting using anti-MYC antibody (left). MBP protein was used as a pull-down control. The panel on the right shows input recombinant MBP and MBP-AtJAZ1 proteins in the pull-down assay. (C) Immunodetection of the AtBBD1 and AtJAZ1 complex <i>in vivo.</i> 35S:6xMYC-AtBBD1 and 35S:3xHA-AtJAZ1 constructs were transiently coexpressed in tobacco leaves by agroinfiltration. The expressed proteins were immunoprecipitated (IP) using anti-HA antibody (+/+) and immunoblotting was carried out with anti-myc antibody. Left lane (−/−) is control leaf extract that was not agroinfiltrated. MYC-AtBBD1 and HA-AtJAZ1 proteins were detected in input coexpressed leaf extracts by each antibody (right). (D) Each truncated AtBBD1 protein was fused to AD as a prey for Y2H assay with AtJAZ1. AtJAZ1 was fused to BD as bait. Numbers indicates amino acid residues, and putative domains were represented. (E) Each truncated AtJAZ1 protein was fused to AD as a prey for Y2H assay with AtBBD1 protein. AtBBD1 was fused to BD as bait.</p

Proposed model for transcription repression by AtBBD1.

<p>In the absence of signal, AtBBD1 represses <i>AtJMT</i> gene expression by recruiting corepressor or HDAc through AtJAZ. In the presence of signal, JA-Ile is released and the SCF^COI1 complex degrades JAZ proteins. A putative activator (+) that binds to the JARE competes with AtBBD1 (repressor). In knockout plants, the putative activator dominantly occupies the JARE and <i>AtJMT</i> gene expression is activated higher than wild type. In the AtBBD1-overexpressing plant, AtBBD1(repressor) competes with the putative activator and dominantly occupies the JARE; therefore, <i>AtJMT</i> gene expression is repressed more than in wild type. Size of each circle represents relative abundance.</p