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Supplemental data
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Description
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Generation of Bntt16 RNAi plants
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In order to study the regulatory function of BnTT16 proteins in endothelial development, RNAi
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Brassica napus plants were generated previously using a hairpin (HP) RNAi construct (Deng et
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al. 2012; Figure S4, S5). Since all four BnTT16 homologs exhibit a high level of similarity and
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the corresponding proteins displayed a high degree of functional overlap in their ability to
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recover the phenotype of an Attt16 mutant (Figure 5, S3), silencing of one particular homolog in
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B. napus may not have yield a detectable phenotype. For this reason, the RNAi construct was
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designed to silence all four BnTT16 homologs simultaneously (Figure S4a)(Deng et al. 2012).
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Briefly, a 146-bp BnTT16 cDNA fragment encoding a portion of the variable K domain was
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amplified from a B. napus cDNA library and cloned into the pENTR/D-TOPO vector
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(Invitrogen, Burlington, ON). The pENTR/D-TOPO vector provides flanking attB1 and attB2
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sequences enabling directional cloning into the pHellsgate 12 vector. The Gateway LR
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recombination reaction was performed according to manufacturer’s instructions (Invitrogen) and
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the resulting vectors were verified by restriction analysis. Positive clones contained two identical
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BnTT16 fragments in opposite orientations, separated by a combination of Pdk and Cat introns
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(Figure S4a). The pHellsgate-BnTT16-146 cassette was then electroporated into Agrobacterium
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tumefaciens GV3101 (pMP90) cells and used in the genetic transformation of B. napus line
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DH12075 as described previously (Bondaruk et al. 2007).
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The RNAi method (HP) used in this study has been proven to be a reliable technique to
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decrease the level of target protein in agricultural plants (Liu et al. 2002, Stoutjesdijk et al. 2002,
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Mietkiewska et al. 2008, Chen et al. 2011). In Arabidopsis, only Attt16 knock-out mutants with
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an absence of TT16 protein, but not AtTT16 overexpression lines, showed abnormal endothelial
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development and decreased PA content (Nesi et al. 2002). We also over-expressed BnTT16 in
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wild-type Arabidopsis, and the seed color was reverted to that of wild-type. Therefore, the
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decreased PA content and abnormal endothelial development in the B. napus RNAi plants, as
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well as the normal seed coat morphology in the transgenic Arabidopsis lines carrying the
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35S::BnTT16 construct in the Attt16 background indicated that BnTT16 protein levels were
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decreased in the RNAi plants as expected.
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As shown in 4 representative independent transgenic lines, BnTT16 expression was
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diminished by approximately 47% to 72% compared to that in the wild-type plants (Figure S4b).
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Expression levels of each individual BnTT16 gene were also assayed in these transgenic lines,
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and it was found that all four genes were down-regulated in every case (Figure S5). To confirm
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that the RNAi silencing was specific to BnTT16 homologs, we analyzed our RNAi construct with
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a publicly available Web-based computational tool (siRNA Scan) and did not find any potential
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off-target-silenced genes (Xu et al. 2006). In addition, the expression level of B. napus
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AGAMOUS-LIKE 6 (BnAGL6), a gene sharing the highest cDNA sequence similarity to the
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BnTT16 homologs based on the B. napus genome database (http://compbio.dfci.harvard.edu/cgi-
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bin/tgi/gimain.pl?gudb=oilseed_rape) was tested by qRT-PCR. The transcriptional levels of the
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BnAGL6 gene exhibited no difference between wild-type and Bntt16 RNAi lines in either 2-DAP
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or 15-DAP samples (Figure S4 c).
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Microarray analysis and data processing
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In order to broadly identify genes regulated by the BnTT16 proteins, microarray data obtained
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previously (Deng et al. 2012) was analyzed to compare gene expression levels in 2-DAP siliques
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of Bntt16 RNAi and wild-type plants. In brief, microarray analysis was performed using an
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Agilent 4x44k Brassica Gene Expression Microarray chip. Total RNA was isolated from 2-DAP
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samples using the RNeasy Plant Mini Kit and was Cy3-labeled with the Quick Amp Labeling Kit
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(Agilent Technologies). Hybridized arrays were scanned with a GenePix 4000B scanner
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(Molecular Device, Sunnyvale, CA) at 5 µm resolution. Image processing was performed using
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the Feature Extraction Software 10.5.1.1 (Agilent Technologies).
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Microarray data were analyzed using the open-source R statistical programming language
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(http://www.r-project.org/) and Bioconductor packages (Gentleman et al. 2004). The raw data
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were imported into the R statistical environment, and their overall high quality was determined
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using the arrayQualityMetrics package (Kauffmann et al. 2009). Data were normalized using
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Variance Stabilization Normalization. Differential gene expression analysis was subsequently
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performed using an empirical Bayes method implemented in Linear Models for Microarray
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Analysis (Smyth 2004). A gene was considered differentially expressed in the Bntt16 RNAi line
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compared with wild-type if its B statistic (log odds of differential expression) was ≥ 2.0,
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corresponding to a ≥80% probability of differential expression. For homologous genes, each
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specific copy was defined by its sequence in the genome database on the microarray chip (for
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example, TC number from assembly (TC, Tentative Consensus sequence from assembly of
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ESTs)), and a unique probe was designed to measure the expression level of that particular copy
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(homolog). The functional annotations of differentially expressed genes (DEGs) in Brassica were
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updated by running BLAST analyses (E value ≤ 10-5) against TAIR 10, the latest Arabidopsis
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genome release at The Arabidopsis Information Resource (http://www.arabidopsis.org). PA- and
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seed coat-associated genes reported in other plants were also used to assist gene identification.
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The gene ontology (GO) annotation of Arabidopsis genes was used for GO analysis of the DEGs.
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As expected, global similarity analysis indicated that the four wild-type samples clustered
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together whereas the four Bntt16 RNAi samples formed a separate group, demonstrating the
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reliability of our microarray experiment (Figure S8). Subsequently, the expression of eighteen
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genes related to seed coat development and flavonoid biosynthesis was measured by qRT-PCR.
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In all cases, the transcript enrichment patterns of qRT-PCR analysis were consistent with
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microarray data (Figure S9). The consistency between qRT-PCR and microarray data was also
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obtained in another set of genes in the same experiment (Deng, et al. 2012). The microarray data
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have been submitted to the NCBI’s Gene Expression Omnibus and are accessible through GEO
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Series accession number GSE37449.
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The microarray results indicated that 1032 genes were up-regulated and 691 genes were
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down-regulated in the Bntt16 RNAi samples compared to WT samples (Table S2, p<0.01),
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which were subsequently summarized by gene ontology (GO) analysis. Genes with roles in
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biological processes, protein metabolism, transport, secondary metabolism, stress and defense
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processes, development, nucleotide metabolism, cell organization and biogenesis, and
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transcription were significantly affected by Bntt16 down-regulation (Figure S10a). In terms of
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molecular function, a number of these genes encoded proteins with binding properties (including
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DNA binding and protein binding), as well as enzymes including hydrolase, transferase, and
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kinase (Figure S10b). In terms of cellular localization, the majority of genes affected by Bntt16
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down-regulation encoded proteins that were localized within the plastid, followed by those
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localized to the nucleus and plasma membrane (Figure S10c). To a lesser degree, Bntt16 down-
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regulation also affected genes encoding proteins localized within the cell wall, ER, and
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mitochondria (Figure S10c). In summary, the BnTT16 proteins regulate the expression of genes
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involved in a variety of molecular functions and biological processes. As is the case for many
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genome-scale surveys, a large portion of the identified genes have functions that are unknown as
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of yet (Table S2) and therefore this data may result in the identification of novel genes regulated
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by BnTT16s.
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Legends
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Figure S1. Comparison of Brassica TT16 genomic sequences. Exons and introns were labelled
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with black and white backgrounds respectively. Bn, B. napus; AA, B. rapa; CC, B. oleracea. The
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TT16 genomic DNA sequences cloned in this study were submitted to GenBank as follows:
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BnTT16.1, JF970611; BnTT16.2, JF970612; BnTT16.3, JF970613; BnTT16.4, JF970614;
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BoTT16.1, JF970615; BoTT16.2, JF970616; BoTT16.3, JF970617; BoTT16.4, JF970618;
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BrTT16.1, JF970619; BrTT16.3, JF970620; BrTT16.4, JF970621.
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Figure S2. Schematic diagram of BnTT16 overexpression constructs used to complement the
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Arabidopsis tt16-6 mutant phenotype.
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Figure S3. Localization of proanthocyanidins using p-dimethylaminocinnamaldehyde staining in
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developing seeds of complemented Arabidopsis lines. (a) wild-type Arabidopsis Ws-3 as a
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positive control; (b) Arabidopsis transparent testa 16-6 (Attt16-6) mutant as a negative control;
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(c) representative complemented line expressing Brassica napus BnTT16.1 (Attt16-
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6/35S::BnTT16.1); (d) representative complemented line expressing BnTT16.2 (Attt16-
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6/35S::BnTT16.2); (e) representative complemented line expressing BnTT16.3 (Attt16-
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6/35S::BnTT16.3); (f) representative complemented line expressing BnTT16.4 (Attt16-
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6/35S::BnTT16.4). Scale bar = 100 µm. The arrows indicate the endothelium containing PA.
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Figure S4. RNAi-mediated silencing of BnTT16 expression. (a) Schematic diagram of the
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construct utilized for RNAi silencing of BnTT16 homologs in B. napus. (b) The overall level of
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expression of BnTT16 genes in open flowers (0-DAP) was down-regulated in Bntt16 RNAi lines.
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(c) The expression level of B. napus AGAMOUS-LIKE 6 (BnAGL6) exhibited no significant
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difference between wild-type (WT) and Bntt16 RNAi lines (RNAi) (p < 0.05). The data in (b)
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and (c) were presented as the relative expression levels (2-ΔCT) of target genes compared with
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reference genes (Table S1). 4-1, 10-3, 12-4, and 23-4 are 4 representative Bntt16 RNAi lines.
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Figure S5. Transcriptional expression (2-ΔCT) of each individual BnTT16 gene in open flowers (0
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day after pollination) was down-regulated in RNAi plants. (a) BnTT16.1; (b) BnTT16.2; (c)
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BnTT16.3; (d) BnTT16.4. 4-1, 10-3, 12-4, and 23-4 are 4 representative Bntt16 RNAi lines. The
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data were presented as the relative expression levels of each BnTT16 gene compared with
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reference genes listed in Table S1.
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Figure S6. Vanillin staining of whole developing seeds (14 day after pollination) indicated that
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proanthocyanidin content was less in Bntt16 RNAi plants than wild-type plants. (a) Wild-type
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whole seeds; (b) halved wild-type seeds; (c) whole seeds from RNAi plants; (d) halved seeds
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from RNAi plants. Bars = 1 mm.
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Figure S7. Localization of proanthocyanidins with vanillin in developing seeds of B. napus
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wild-type (WT) and RNAi lines. Arrows indicate PAs that have accumulated in the endothelium.
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(a) WT 15-DAP; (b) RNAi 15-DAP; (c) WT 20-DAP; (d) RNAi 20-DAP; (e) WT 24-DAP; (f)
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RNAi 24-DAP; (g) WT 31-DAP; (h) RNAi 31-DAP. RNAi, Bntt16 RNAi plants; DAP, days
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after pollination. Scale bar = 100 µm.
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Figure S8. Global similarity of the eight microarray samples for wild-type B. napus and RNAi
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plants.
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Sample clustering was used to assess the overall similarity of the eight samples. The four
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replicates of wild-type plants (sample 1-4) clustered with one another, while the four replicates
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of RNAi plants formed a separate group (sample 5-8).
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Figure S9. Validation of microarray data (fold-difference) using qRT-PCR. (a) Candidate genes
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involved in flavonoid biosynthesis and regulation. (b) Candidate genes involved in seed coat
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development. Data include the mean relative transcript levels ± standard error of four biological
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replicates and four technical replicates.
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Figure S10. Effects of Bntt16 silencing on the gene expression profile of B. napus (n=4). (a)
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Biological process. (b) Molecular function. (c) Cellular component. GO, gene ontology.
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Unknown and ambiguous genetic programming categories were not included.
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Figure S11. Vanillin staining of representative dissected developing seeds. Samples were
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harvested at 15 day after pollination (15-DAP, cotyledon stage) from wild-type B. napus plants.
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(a) embryo; (b) endosperm; (c) inner integument; (d) epidermis. Bar = 1 mm. Briefly, we
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dissected 15-DAP developing seed coats into three contiguous components: inner fraction
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composed primarily of developing endosperm, middle fraction containing inner integument and
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palisade cells, and outer fraction composed of epidermal and sub-epidermal cells (Jiang and
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Deyholos 2010). To detect which fraction contained the endothelium, we stained these three
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fractions and embryos with vanillin. The middle fraction highly stained to red, and the inner
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fraction was only slightly stained. Since almost all PA accumulated in the endothelium at 15-
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DAP (Figure 7), we confirmed that the middle fraction contained virtually all the endothelium.
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For convenience, we named the four sub-tissue fractions as embryo, endosperm, inner
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integument and epidermis in this study.
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Table S1. Primers utilized in this study.
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Table S2. Microarray data.
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REFERENCES
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Bondaruk, M., Johnson, S., Degafu, A., Boora, P., Bilodeau, P., Morris, J., Wiehler, W.,
182
Foroud, N., Weselake, R. and Shah, S. (2007) Expression of a cDNA encoding palmitoyl-
183
acyl carrier protein desaturase from cat's claw (Doxantha unguis-cati L.) in Arabidopsis
8
184
thaliana and Brassiea napus leads to accumulation of unusual unsaturated fatty acids and
185
increased stearic acid content in the seed oil. Plant Breed., 126, 186-194.
186
Chen, X., Truksa, M., Snyder, C.L., EI-Mezawy, A., Shah, S. and Weselake, R.J. (2011)
187
Three homologous genes encoding sn-glycerol-3-phosphate acyltransferase 4 exhibit different
188
expression patterns and functional divergence in Brassica napus. Plant Physiol., 155, 851-865.
189
Deng, W., Chen, G., Peng, F., Truksa, M., Snyder, C.L. and Weselake, R.J. (2012)
190
Transparent testa16 plays multiple roles in plant development and is involved in lipid
191
synthesis and embryo development in canola. Plant Physiol., 160, 978-989.
192
Gentleman, R.C., Carey, V.J., Bates, D.M., Bolstad, B., Dettling, M., Dudoit, S., Ellis, B.,
193
Gautier, L., Ge, Y.C., Gentry, J., Hornik, K., Hothorn, T., Huber, W., Iacus, S., Irizarry,
194
R., Leisch, F., Li, C., Maechler, M., Rossini, A.J., Sawitzki, G., Smith, C., Smyth, G.,
195
Tierney, L., Yang, J.Y.H. and Zhang, J.H. (2004) Bioconductor: open software development
196
for computational biology and bioinformatics. Genome Biol., 5, R80.
197
198
199
200
201
Jiang, Y. and Deyholos, M. (2010) Transcriptome analysis of secondary-wall-enriched seed
coat tissues of canola (Brassica napus L.). Plant Cell Rep., 29, 327-342.
Kauffmann, A., Gentleman, R. and Huber, W. (2009) arrayQualityMetrics-a bioconductor
package for quality assessment of microarray data. Bioinformatics, 25, 415-416.
Liu, Q., Singh, S.P. and Green, A.G. (2002) High-stearic and high-oleic cottonseed oils
202
produced by hairpin RNA-mediated post-transcriptional gene silencing. Plant Physiol., 129,
203
1732-1743.
204
205
Mietkiewska, E., Hoffman, T.L., Brost, J.M., Giblin, E.M., Barton, D.L., Francis, T., Zhang,
Y. and Taylor, D.C. (2008) Hairpin-RNA mediated silencing of endogenous FAD2 gene
9
206
combined with heterologous expression of Crambe abyssinica FAE gene causes an increase in
207
the level of erucic acid in transgenic Brassica carinata seeds. Mol Breeding, 22, 619-627.
208
Nesi, N., Debeaujon, I., Jond, C., Stewart, A.J., Jenkins, G.I., Caboche, M. and Lepiniec, L.
209
(2002) The TRANSPARENT TESTA16 locus encodes the ARABIDOPSIS BSISTER MADS
210
domain protein and is required for proper development and pigmentation of the seed coat.
211
Plant Cell, 14, 2463-2479.
212
213
Smyth, G.K. (2004) Linear models and empirical bayes methods for assessing differential
expression in microarray experiments. Stat. Appl. Genet. Mol. Biol., 3, Article3.
214
Stoutjesdijk, P.A., Singh, S.P., Liu, Q., Hurlstone, C.J., Waterhouse, P.A. and Green, A.G.
215
(2002) hpRNA-mediated targeting of the Arabidopsis FAD2 gene gives highly efficient and
216
stable silencing. Plant Physiol., 129, 1723-1731.
217
Xu, P., Zhang, Y., Kang, L., Roossinck, M.J. and Mysore, K.S. (2006) Computational
218
estimation and experimental verification of off-target silencing during posttranscriptional gene
219
silencing in plants. Plant Physiol., 142, 429-440.
220
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