The transcription factor TRANSPARENT TESTA 16 (TT16) plays an important role in endothelial cell specification and proanthocyanidin (PA) accumulation. However, its precise regulatory function with regard to the expression of endothelial-associated genes in developing seeds, and especially in the PA-producing inner integument, remains largely unknown. Therefore, we endeavored to characterize four TT16 homologs from the allotetraploid oil crop species Brassica napus, and systematically explore their regulatory function in endothelial development. Our results indicated that all four BnTT16 genes were predominantly expressed in the early stages of seed development, but at distinct levels, and encoded functional proteins. Bntt16 RNA interference lines exhibited abnormal endothelial development and decreased PA content, while PA polymerization was not affected. In addition to the previously reported function of TT16 in the transcriptional regulation of anthocyanidin reductase (ANR) and dihydroflavonol reductase (TT3), we also determined that BnTT16 proteins played a significant role in the transcriptional regulation of five other genes involved in the PA biosynthetic pathway (P <0.01). Moreover, we identified two genes involved in inner integument development that were strongly regulated by the BnTT16 proteins (TT2 and δ-vacuolar processing enzyme). These results will better our understanding of the precise role of TT16 in endothelial development in Brassicaceae species, and could potentially be used for the future improvement of oilseed crops.
Plant seeds generally comprise three main components, the embryo, endosperm and seed coat (testa). While the embryo and endosperm are products of fertilization, the maternal seed coat makes up the outer protective integuments (Moise et al., 2005). In many plant species, the endothelium is the innermost layer of the integuments and is also the site of synthesis and accumulation of proanthocyanidins (PAs, also called ‘condensed tannins’), which have important physiological functions in plants and can influence the quality of plant products (Nesi et al., 2002; Andersen and Markham, 2006; Lepiniec et al., 2006; Auger et al., 2010). While substantial progress has been made towards the understanding of PA biosynthesis and seed coat development over the past decade, the precise mechanisms behind the regulation of these pathways still require further exploration (Figure 1; for reviews see Haughn and Chaudhury, 2005; Lepiniec et al., 2006; Arsovski et al., 2010).
Indeed, from a genetic engineering perspective, understanding the regulation of seed coat development and PA accumulation in crops is particularly attractive as they can both have detrimental effects on crop quality (Moise et al., 2005; Dixon and Pasinetti, 2010). For example, Brassica napus (rapeseed), which is an important seed oil crop that also supplies one of the most abundant protein supplements for animal feed, possesses a seed coat that represents approximately one-sixth of the total seed weight, and can thus negatively affect both oil and protein yield (Akhov et al., 2009). In addition, insoluble fiber is the major component of the B. napus seed coat and is not digestible by monogastric animals, while the endothelium of the popular black-seeded B. napus is rich in PAs, which remain in oil-extracted meal and are considered anti-nutritional in livestock feed (Akhov et al., 2009). Therefore, genetic analyses of the development of PA-producing endothelium are likely going to be imperative for the improvement of the quality of B. napus through modern breeding or genetic engineering.
To date, the majority of genes required for the differentiation of the endothelium in Arabidopsis have been identified through the isolation of mutants with altered seed coat color or PA content (Abrahams et al., 2002; Haughn and Chaudhury, 2005; Lepiniec et al., 2006). The biosynthesis of PA (see Figure 1a) and endothelial development in plants are largely controlled by the tissue-specific expression of transcription factors belonging to several protein families. Six transcription factor proteins have been identified from Arabidopsis mutants with altered seed coat colors, including TRANSPARENT TESTA 1(TT1, a WIP zinc-finger protein), TT2 (a MYB protein), TT8 [a basic helix-loop-helix (bHLH) protein], TT16 (a MADS-domain protein, also known as Arabidopsis Bsister protein, ABS), TRANSPARENT TESTA GLABRA 1(TTG1, a WD40 repeat protein) and TTG2 (a WRKY protein) (Zhang et al., 2003; Lepiniec et al., 2006; Gonzalez et al., 2009; Appelhagen et al., 2011a). Among these, TT16 is of particular interest for its dual regulatory functions in both PA accumulation and endothelial development (Becker et al., 2002; Nesi et al., 2002; de Folter et al., 2006; Chen et al., 2012; Mizzotti et al., 2012). Indeed, this transcription factor appears to function upstream of several other seed coat-related transcription factors, and thus may have a global effect on these processes (Haughn and Chaudhury, 2005; Dean et al., 2011).
Recently, TT16 has been reported to regulate several endothelial-related genes, such as TT3, TT2 and anthocyanidin reductase (ANR) in Arabidopsis (Debeaujon et al., 2003; Dean et al., 2011). In addition, its function in the development of other plant organs has also been reported. While the down-regulation of this transcription factor in B. napus caused abnormal embryo development and a reduction in silique and seed numbers (Deng et al., 2012), a double knock-out mutant of the MADS-box genes TT16 and SEEDSTICK in Arabidopsis resulted in a complete lack of endothelium and subsequent defects in the female gametophyte, reduced fertilization, impediments during seed formation and a severely reduced seed set (Mizzotti et al., 2012). Since the endothelium plays a fundamental role in the interaction between the maternally derived integuments and the next generation, functioning to protect and nourish the embryo, it seems likely that the reduction or absence of an endothelium may be largely responsible for these phenotypes (Stadler et al., 2005; Morley-Smith et al., 2008; Mizzotti et al., 2012).
Unfortunately, although information is beginning to accumulate with regard to the ability of TT16 to regulate a small selection of endothelial-related genes in Arabidopsis (Debeaujon et al., 2003; Dean et al., 2011), its precise regulatory function, especially within the PA-producing endothelium, remains largely unknown and additional target genes are still to be identified. Brassica napus would be a good choice of organism to clarify these issues since their seeds are relatively large and can thus be easily dissected into four different components including embryo, endosperm, inner integument and outer integument (Jiang and Deyholos, 2010). In addition, B. napus and Arabidopsis are closely related members of the Brassicaceae family, and have similar seed coat structures (Haughn and Chaudhury, 2005; Nesi et al., 2009; Auger et al., 2010); therefore any information gained in B. napus would be relevant to our understanding of TT16 function in the model plant Arabidopsis.
Although the four BnTT16 homologs have previously been found to play broad roles in regulating plant development (Deng et al., 2012), their function in seed coat development has not been fully characterized and it remains unclear whether all of the homologs are functional in B. napus. The objective of this study, therefore, was to: (i) characterize the expression patterns and genome origins of BnTT16 homologs from B. napus, as well as their role in endothelial development, PA biosynthesis and PA polymerization; and (ii) identify their regulatory function with regard to genes involved in endothelial development and PA biosynthesis at different seed developmental stages and in distinct subseed components. Results from this study will further our comprehension of endothelial development and regulation in plants, and have the potential to be utilized for the future improvement of crop quality.
Brassica napus contains four TT16 homologs with different genome origins
As shown in Table 1, all four BnTT16 cDNAs shared over 83% identity with AtTT16. The predicted BnTT16 amino acid sequences bore the canonical structure of MIKCc-type MADS box proteins, with a highly conserved MADS (M) domain at the N terminus, a less conserved intervening (I) domain, a keratin-like (K) domain characterized by three strings of heptad repeats (K1, K2 and K3) and an integral C-terminal (C) domain (Figure 2a). Phylogenetic analysis confirmed that the BnTT16 proteins belonged to the Bsister subfamily of MADS-domain proteins (Figure 2b).
Table 1. Genomic (g), cDNA and amino acid sequence identity (%) of Brassica napus BnTT16 homologs and Arabidopsis AtTT16
Since B. napus (AACC; n =19) originated from Brassica. rapa (AA; n =10) and Brassica oleracea (CC; n =9), TT16 genomic DNA sequences were isolated from all three Brassica species for comparison (Figure S1). While four distinct TT16 genes were isolated from B. oleracea, only three were isolated from B. rapa, which was consistent with a search of the recently released B. rapa genome database (http://brassicadb.org). All Brassica TT16 genomic sequences were composed of six highly conserved exons and five weakly conserved introns (Figures 3a and S1). Interestingly, the three B. rapa TT16 homologs were located on different chromosomes: BrTT16.1 on chromosome A03 (20,934,632…20,936,926), BrTT16.3 on chromosome A02 (25,596,484…25,598,445) and BrTT16.4 on chromosome A01 (8,943,167…8,947,169).
To determine the genome origins of the BnTT16 homologs, genomic DNA sequences were compared with a focus on intronic regions, revealing that BnTT16.2, −16.3 and −16.4 exhibited the highest sequence similarity with three B. oleracea TT16 genes, while BnTT16.1 was most similar to a B. rapa TT16 gene (Figures 3b and S1). A further phylogenetic analysis of the genomic DNA sequences confirmed the homology among Brassica TT16 genes (Figure 3c) and also indicated that BnTT16.1 is evolutionarily closely related to BnTT16.2, while BnTT16.3 is most closely related to BnTT16.4. The TT16 genes identified in B. rapa and/or B. oleracea but not in B. napus may have been lost during evolution. Gene losses in B. napus have also been reported among ribosomal protein gene paralogs (Whittle and Krochko, 2009); indeed, this type of gene loss could result from the accumulation of deleterious mutations, which may be especially prevalent shortly following polyploidy events (Lynch and Conery, 2000).
BnTT16 genes have similar transcript expression patterns but different expression levels in B. napus
The transcript levels of BnTT16 genes in leaves, roots, stems, floral buds, open flowers at 0 days after pollination (0 DAP), siliques at 2 DAP, and developing seeds at 15, 20 and 35 DAP were measured by quantitative (q)RT-PCR. As shown in Figure 4, all four BnTT16 homologs were expressed in a tissue-specific manner predominantly in the early stages of seed development. The expression of all BnTT16 genes was highest in siliques at 2 DAP and drastically decreased in developing seeds harvested after 20 DAP. Conversely, very little expression of the BnTT16 genes was observed in any of the vegetative tissues tested (Figure 4). Despite these similarities in transcript expression patterns, the level of expression exhibited by each homolog varied substantially. BnTT16.4 was the most highly expressed homolog in developing seeds, followed by BnTT16.2, BnTT16.1 and BnTT16.3. Indeed, the expression level of BnTT16.4 at 2 DAP was double that of BnTT16.2 and BnTT16.1, while the expression level of BnTT16.3 was relatively low in all tissues.
Complementation of the Arabidopsis tt16-6 mutant phenotype by heterologous expression of BnTT16 cDNAs
To determine whether the BnTT16 proteins function in a similar fashion to AtTT16, which has been found previously to control endothelial development and PA biosynthesis (Nesi et al., 2002; de Folter et al., 2006), each of the four BnTT16 cDNAs were expressed under the control of the CaMV 35S promoter (Figure S2) in the Arabidopsis tt16-6 T-DNA mutant line, whose seeds lack PA, have abnormal endothelial development and are yellow in appearance (de Folter et al., 2006). T2 seeds from at least five transgenic lines bearing each construct, respectively, were analyzed. In all cases, expression of each of the BnTT16 cDNAs was sufficient to restore the ability of the plants to produce mature seeds with a brown, wild-type appearance (Figure 5a,c–f). Staining of developing seeds (cotyledon stage) with vanillin confirmed that the brown coloration of the resulting mature seeds was due to an accumulation of PA (Figure 5g,i–l).
To provide further evidence that the expression of each of the four BnTT16 cDNAs in tt16-6 mutant Arabidopsis plants restored PA development and accumulation in the endothelium, we subjected developing seeds (cotyledon stage) to various other types of analyses. Both Nomarski optics (Figure 5m–r) and toluidine blue staining (Figure 5s–x) indicated that the endothelium of transgenic seeds bearing the BnTT16 overexpression cassettes developed normally, which contrasted with the abnormal endothelial development observed in untransformed tt16-6 seeds. In addition, p-dimethylaminocinnamaldehyde (DMACA) staining of developing seeds demonstrated that in the complemented mutant lines, PA accumulated mainly in the endothelium (Figure S3). Taken together, these results clearly indicate that all four BnTT16 homologs from B. napus are functional proteins involved in the regulation of endothelial development and PA biosynthesis.
BnTT16 proteins regulate endothelial development and PA biosynthesis, but not PA polymerization, in B. napus
To further explore the physiological functions of the BnTT16 homologs with regards to seed coat development, we analyzed the seed coat morphology of Bntt16 RNA interference (RNAi) B. napus lines, which were generated using a hairpin RNAi construct and previously confirmed to down-regulate the expression of all four BnTT16 genes simultaneously (Deng et al., 2012; Figures S4 and S5). In B. napus, the inner integument comprises the PA-accumulating endothelium (ii1) and parenchyma (ii2), while the outer integument is composed of epidermis (oi3), parenchyma (oi2) and palisade (oi1) layers (Nesi et al., 2009; Auger et al., 2010). As shown in Figure 6, toluidine blue staining of developing seeds at 8 and 24 DAP confirmed the presence of all five seed coat layers in wild-type B. napus. Although the seed coat in RNAi lines was similar to wild-type plants at 8 DAP (Figure 6a,b), the endothelium in RNAi lines at 24 DAP had become far thinner than that in wild-type plants at the same developmental stage (Figure 6c,d).
To determine the function of BnTT16 proteins in PA biosynthesis, developing seeds at different stages (8, 15, 24 and 31 DAP) were treated with DMACA, which specifically stains PA blue. The results indicated that PA accumulation was inhibited in transgenic RNAi plants compared with wild-type plants, which implies a function of BnTT16 proteins in PA biosynthesis (Figure 7). This result was confirmed via staining of both whole developing seeds and seed sections (14 DAP) with vanillin, which stains PA red (Figures S6 and S7). Decreased PA content in developing seeds at various developmental stages from Bntt16 RNAi plants compared with wild-type plants was also verified by HPLC analysis (Figure 8a) and results were consistent with those from the staining experiments. For example, PA content was found to be detectable at 15 DAP and increased past 30 DAP in both HPLC analysis and DMACA staining experiments (Figures 7 and 8). Conversely, the mean degree of polymerization (mDP) values of PA in developing seeds of wild-type and RNAi lines did not differ significantly from one another (P <0.05, Figure 8b), indicating that the BnTT16 proteins do not play a role in regulating the PA polymerization process.
Microarray identification of multiple seed coat-associated genes affected by the down-regulation of BnTT16
In an attempt to identify additional genes regulated by the BnTT16 proteins, microarray data obtained previously (Deng et al., 2012) were further analyzed to compare gene expression levels in 2-DAP siliques of Bntt16 RNAi and wild-type plants (Figures S8–S10). Results indicated that 1032 genes were up-regulated and 691 genes were down-regulated in the Bntt16 RNAi samples compared with wild-type samples (Table S2, P <0.01). Among them, 33 seed coat-associated genes affected by BnTT16 down-regulation were identified, where eight candidate genes related to inner integument development and PA biosynthesis [including TT3, TT18, TT6, ANR, TT10, two δ-vacuolar processing enzymes (δ-VPEs) and one retinoblastoma-related gene (RBR1)] were down-regulated in every case (Table 2). In contrast to the down-regulation of inner integument related genes in RNAi plants, the remaining 25 candidate genes, which could be involved in epidermal development and/or mucilage production, showed considerable variance regarding their transcript expression levels (Table 2, Figure S9). This consistent down-regulation of genes related to inner integument development, as well as the specific inhibition of endothelial development and PA synthesis in Bntt16 RNAi lines (Figures 6-8), indicate that the BnTT16 transcription factors have a specific role in endothelial development and PA synthesis in the seed coat of B. napus.
Table 2. Candidate seed coat-associated genes identified in the microarray assay
Analysis of endothelium- and PA-associated gene expression in developing seeds of Bntt16 RNAi plants by qRT-PCR
Although BnTT16 proteins were found to specifically regulate endothelial development and PA biosynthesis (Figures 6 and 7), a number of genes that have previously been found to be involved in the production of PA and/or endothelial development in other species were not detected in our microarray analysis. This may have been due to microarray chip design, low expression levels or relatively small differences in expression levels between wild-type and Bntt16 RNAi plants. Therefore, to better understand the regulatory function of the BnTT16 proteins, qRT-PCR analysis was performed to assess the expression levels of known PA- and seed coat-associated genes in both wild-type and Bntt16 RNAi plants. For this purpose, we utilized various gene sequences reported in other plants to query the B. napus gene index database (http://compbio.dfci.harvard.edu) for homologs. Based on these results, along with our microarray results, we selected 15 genes for qRT-PCR analysis and divided them into two groups: members of group A are directly involved in the PA biosynthetic pathway (TT4, TT5, TT6, TT7, TT3, TT18, ANR and TT10; Figure 1) while members of group B are involved in the regulation of endothelial development and PA biosynthesis (VPE, TT2, TTG1, TT8, TTG2, TT1, KAN4).
Since BnTT16 proteins may regulate these genes to different extents at different seed developmental stages, and the majority of inner integument development occurs prior to 22 DAP in B. napus, RNA extracted from both 2-DAP siliques and 15-DAP developing seeds (cotyledon stage) was utilized for qRT-PCR (Wan et al., 2002). As shown in Figure 9(a), the majority of group A genes tested, with the exception of TT7 at 15 DAP, were down-regulated in Bntt16 RNAi plants at both 2 and 15 DAP, (P <0.01). This indicates a strong regulatory function for BnTT16 proteins in the PA biosynthetic pathway at the transcriptional level. In addition, all group A genes exhibited higher expression levels at 15 DAP than 2 DAP (P <0.01) in both wild-type and RNAi plants.
With regards to the group B genes analyzed, no clear trend relating down-regulation of the BnTT16 genes and group B transcript abundance could be discerned (Figure 9b). There was also no consistent trend with regards to gene expression levels at 2 and 15 DAP. Therefore, the regulatory function of the BnTT16 proteins on the group B genes appeared to be case-specific. For example, the expression of δ-VPE was down-regulated in Bntt16 RNAi lines, which is consistent with our microarray data. In addition, although the expression of TT2 at 2 DAP was not affected, its expression level at 15 DAP was significantly down-regulated in the RNAi plants. On the contrary, Bntt16 RNAi lines did not exhibit any differences in expression levels of TTG1 at either developmental stage compared with wild-type plants.
Analysis of seed coat-associated gene expression in subseed tissues of Bntt16 RNAi plants by qRT-PCR
Since BnTT16 proteins appear to function in the development of PA-producing endothelium via the transcriptional regulation of a selection of genes (Figures 6-9, Table 2), an investigation of the expression profiles of these genes in different seed tissues, and especially the inner integument, would further clarify their regulatory function. To accomplish this, we dissected developing seeds at 15 DAP into four contiguous components (embryo, endosperm, inner integument and epidermis) prior to RNA extraction and qRT-PCR analysis (Figure S11).
As shown in Figure 10(a), all four BnTT16s have the highest expression in the inner integument compared to the embryo and other seed coat layers in both WT and RNAi samples. Similarly, all group A genes exhibited the highest transcript levels in the inner integument, which is consistent with the location of PA biosynthesis and accumulation (Figure 10b). Conversely, the expression profiles of group B genes varied widely: δ-VPE, TT2 and TT8 exhibited the highest expression levels in the inner integument, TTG1 showed similar expression levels in the inner integument and epidermis, TTG2 had the highest expression levels in the epidermis, and TT1 and KAN4 exhibited the highest levels of expression in the embryo (Figure 10c,d).
Similarly, our results also indicated that BnTT16 proteins possessed different regulatory patterns within the different seed tissues (Figure 10). All group A genes, with the exception of TT7, were significantly down-regulated in the inner integument fraction of Bntt16 RNAi lines compared with wild type (P <0.05). Conversely, much variation was noted in the case of group B genes. The expression of δ-VPE and TT2 genes was significantly down-regulated in all subseed fractions in Bntt16 RNAi plants. Furthermore, although the expression of TTG2 in inner and outer integuments were both down-regulated in RNAi plants, this gene exhibited very low expression levels in all subseed fractions, which may suggest that it provides only a minor contribution in the BnTT16 regulatory cascade. In addition, the BnTT16 proteins did not show any significant regulatory function on the transcript levels of TTG1, TT8, TT1 or KAN4 in the inner integument, which may indicate that these genes are not involved in the BnTT16 regulatory cascade to control endothelial development.
Although it is well-known that the Arabidopsis TT16 gene encodes a Bsister MADS-domain protein that is involved in endothelial development and PA accumulation, our knowledge concerning its regulatory function at the transcriptional level is very limited. We recently reported the isolation of four B. napus TT16 homologs (Deng et al., 2012), which provide an ideal tool for exploring the regulatory role of TT16 upon endothelial- and PA-associated genes at different seed developmental stages and in distinct subseed components. In this study, we characterized the structure, genomic origins and expression patterns of these four BnTT16 homologs, and also provided evidence for their function in endothelium development and PA biosynthesis via a broad regulatory function of endothelial- and PA-associated genes at the transcriptional level.
The four BnTT16 genes showed similar spatiotemporal expression patterns in B. napus, with expression occurring predominantly in developing seeds at 2 DAP, which is consistent with the expression of other plant Bsister MADS-box genes (Becker et al., 2002; Nesi et al., 2002; de Folter et al., 2006; Erdmann et al., 2010; Prasad et al., 2010). Despite this similarity in expression patterns, the four homologs exhibited very different levels of expression (Figures 4 and 10a). In general, redundant gene copies in polyploid plants have several possible fates: some may become pseudogenes or are physically lost from the genome (pseudogenization), some can develop new adaptive functions (neofunctionalization), some may subdivide their functions among the duplicated copies (subfunctionalization) and some may be subjected to gene dosage (the number of copies of a given gene present in the cell) balance, which is required to maintain functionality of certain molecular complexes as well as regulatory and signaling pathways (Wendel, 2000; Birchler and Veitia, 2007; Veitia et al., 2008; Whittle and Krochko, 2009). With regard to the four BnTT16 genes, we found that each encoded a functional protein that could fully recover the tt16-6 mutant phenotype in Arabidopsis endothelial development, which rules out pseudogenization and subfunctionalization. While we did not observe any novel functions in any of the BnTT16 proteins in comparison with the others, we cannot rule out the possibility of neofunctionalization. However, taken together, even though the expression domain of the ancestral TT16 gene has not yet been elucidated, the variation in BnTT16 expression levels could be, at least partially, due to a gene dosage balance effect.
Similar to phenotypes reported in Attt16 mutants (Nesi et al., 2002; de Folter et al., 2006), Bntt16 RNAi plants exhibited abnormal endothelial development and decreased PA content (Figures 6-8), which is consistent with the ability of all four BnTT16 cDNAs to return tt16-6 mutant Arabidopsis plants to a wild-type phenotype (Figure 5). Subsequent microarray and qRT-PCR analyses of RNA isolated from Bntt16 RNAi lines revealed that the expression of a number of genes related to endothelial development, including the majority of genes known to be involved in the PA biosynthetic pathway, as well as several related genes such as δ-VPE, TT2, and TTG2 was significantly reduced in Bntt16 RNAi lines compared with wild-type plants (Table 2, Figures 9 and 10; P <0.01). Taken together, these data suggest that BnTT16 is a global regulator of endothelial development that functions upstream of the aforementioned genes, which are transcriptionally regulated by the BnTT16 proteins.
In terms of genes known to be required in the PA biosynthetic pathway, it was reported previously that the Arabidopsis TT16 protein is involved in the transcriptional regulation of TT3 and ANR (Nesi et al., 2002; Dean et al., 2011). In this study, we demonstrated that BnTT16 also had a significant effect over the expression of almost all genes required in the PA biosynthetic pathway (group A genes; Figure 1a) in both whole developing seeds at different stages and the PA-producing inner integument (Figures 9 and 10). The effect of TT16 down-regulation on these additional genes has not been previously reported. Our result suggests that the decreased PA content observed in Bntt16 RNAi seeds may be explained by the systematic down-regulation of genes in its biosynthetic pathway. Unlike the other PA biosynthesis-related genes, the expression of TT7 was not significantly affected by BnTT16 down-regulation in either whole seeds or the inner integument, which may indicate that the production of dihydroquercetin from dihydrokaempferol in the PA biosynthetic pathway, which is catalyzed by TT7, is not regulated by any of the BnTT16 proteins (Figures 1, 9 and 10).
Interestingly, while knock-down of the BnTT16 genes had a significant effect on PA content within seeds, it did not affect PA polymerization (Figure 8b); a novel finding that has not been reported previously. As of yet, the mechanism behind PA polymerization remains largely unknown. However, AtTT10 has recently been proposed to be involved in the initial PA oligomerization process since Arabidopsis tt10 mutants exhibit a delay in seed coat browning, which is a process resulting mainly from the oxidation of PAs, and accumulate more epicatechin monomers and soluble PAs than wild-type seeds (Pourcel et al., 2005; Zhao et al., 2010). As AtTT10 transcript abundance increased during seed development, it was suggested that AtTT10 was responsible for the oxidization of PAs that had already formed during the later stages of seed maturation (Pourcel et al., 2005). In our study, the soluble PA content in Bntt16 knock-down lines was reduced compared with that in wild-type seeds at 15 DAP (Figures 7 and 8). Simultaneously, the expression of BnTT10 was also significantly down-regulated at this stage in the inner integument of Bntt16 RNAi plants compared with wild-type (Figure 10b). This consistency between soluble PA content and BnTT10 expression levels could provide an explanation for the observation that although PA contents were significantly different in Bntt16 RNAi lines and wild-type plants the degree of PA polymerization was very similar.
Among the seven group B genes related to endothelial development and PA accumulation that were tested, only TT2, TTG2 and δ-VPE were significantly regulated by BnTT16 proteins. TT2 is a MYB transcription factor with high expression levels and a major function in endothelial development in Arabidopsis (Nesi et al., 2001; Gonzalez et al., 2009). Our data indicate that the expression of TT2 was highest in the inner integument fraction of B. napus seeds and was significantly down-regulated in Bntt16 RNAi plants (Figure 10c). This strong regulation of TT2 expression by BnTT16 was consistent with yeast one-hybrid results using these two genes from Arabidopsis (Debeaujon et al., 2003). While TTG1, TT8 and TT2 have been found previously to function as a complex to regulate the differentiation of endothelium and PA accumulation in Arabidopsis (Baudry et al., 2004; Gonzalez et al., 2009), our data indicated that BnTT16 proteins had no significant influence on the expression of TTG1 and TT8 in the inner integument (Figure 10c). Therefore, the abnormal endothelial development observed in Bntt16 RNAi plants may be at least partially due to the down-regulation of the transcript expression of TT2, but not TTG1 or TT8. Nevertheless, since TTG1 works in a complex with TTG1 and TT8 at the protein level, TT16 may still affect the function of the endothelium TTG1 complex.
TTG2, on the other hand, is a WRKY transcription factor that functions downstream of the TTG1–TT8–TT2 complex and regulates the accumulation of PA and mucilage production in the Arabidopsis seed coat (Johnson et al., 2002). The high expression of TTG2 in both inner and outer integuments in B. napus seeds suggests that its encoded protein provides a similar function in this species (Figure 10d). Since the expression of TTG2 was down-regulated in Bntt16 RNAi plants in the inner integument, it is possible that the BnTT16 proteins may be involved in the transcriptional regulation of TTG2. However, TTG2 was expressed at very low levels in developing B. napus seeds compared with the other genes tested, and exhibited higher relative expression levels in the epidermis than the inner integument, which implies that the expression of TTG2 might not be tightly regulated by BnTT16 proteins in terms of endothelial development (Figure 10).
As was the case for TTG1 and TT8 in B. napus, TT1, which encodes a WIP zinc-finger protein, was not significantly affected by knocking down BnTT16 expression (Figures 9 and 10). These results indicate that TT1 may not be directly involved in the BnTT16 regulatory cascade in B. napus. Consistent with this, AtTT1 has been found to have only a very weak interaction with the Arabidopsis TT16 in vitro (Appelhagen et al., 2011b), providing further evidence that TT16 and TT1 may independently regulate endothelial development in Brassicaceae species.
In addition, δ-VPE, which is a Cys proteinase that is expressed in both layers of the inner integument and is involved in their programmed death by controlling the degradation of nuclei in Arabidopsis (Nakaune et al., 2005), was strongly down-regulated in Bntt16 RNAi plants (Figure 10). In this study, we found that δ-VPE was expressed in all subseed fractions of B. napus with the highest levels observed in the inner integument (Figure 10c). Indeed, cells derived from all constituents of the inner integument were affected in the Attt16 mutant (Nesi et al., 2002; Debeaujon et al., 2003). Therefore, the strong down-regulation of δ-VPE in Bntt16 RNAi plants may indicate that BnTT16 might also have a regulatory function in the development of inner integument other than endothelium.
In conclusion, we characterized four BnTT16 homologs in the allotetraploid oil crop species B. napus, and explored their regulatory function in endothelial development. All four BnTT16 genes were predominantly expressed in the early stages of seed development at distinct levels, and in every case encoded a functional protein. Bntt16 RNA interference lines exhibited decreased PA content and abnormal endothelial development, while PA polymerization was not affected. In addition to the reported regulatory function of AtTT16 on DFR (TT3), ANR and TT2, we revealed that BnTT16 broadly regulated the expression of almost all genes in the PA synthetic pathway (A group genes) in the inner integument, including CHS (TT4), CHI (TT5), F3H (TT6), LDOX (TT18) and PPO (TT10), but not F3′H (TT7). On the contrary, among the five previously reported endothelium-related transcription factor genes (TT1, TT2, TTG1, TTG2, TT8), BnTT16 proteins only strongly regulated TT2. We also demonstrated that BnTT16 regulated the expression of δ-VPE, which is actively involved in the development of the inner integument. This information not only expands our knowledge of the role of TT16 in Brassicaceae species, but it may also be applicable in identifying target genes for use breeding programs focusing on genetic improvement of Brassica oilseed species. Since the presence of antinutritional factors, including seed-coat-associated fiber and oxidized PAs, are undesirable in seed meal (Naczk et al., 1998; Lindeboom and Wanasundara, 2007), and the down-regulation of BnTT16 genes effectively inhibited both endothelial development and PA accumulation, a measurement of seed/seed meal quality and feeding experiments using Bntt16 RNAi seeds would be a useful next step in the investigation of the value of BnTT16 in enhancing the quality of canola.
Brassica napus (DH12075) and Arabidopsis thaliana (Ws-3 and tt16-6) were grown in a greenhouse or growth chamber with the following parameters: 16-h day/8-h night cycle, 25/20°C day/night temperature, 60% relative humidity and 250 μmol m−2 sec−1 light intensity.
RNA extraction and generation of cDNA
Plant tissues were harvested and stored at −80°C. In the case of subseed fractions, B. napus developing seeds were harvested at 15 DAP and separated into four components, embryos, endosperm, inner integument and epidermis, prior to storage (Jiang and Deyholos, 2010). Total RNA was extracted using the RNeasy Plant Mini Kit according to the manufacturer's instructions (Qiagen, http://www.qiagen.com/). Subsequently, 1 μg of total RNA was used to synthesize first-strand cDNA in a 20-μl reaction volume using an optimized blend of oligo-dT and random primers with the QuantiTech Reverse Transcription Kit (Qiagen).
Sequence and phylogenetic analyses
Specific primer pairs were designed to amplify full-length genomic DNA sequences from B. napus, B. rapa and B. oleracea (Table S1). Full-length amino acid sequences were aligned using ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/) and phylogenetic trees were generated according to Erdmann et al. (2010). The proteins used for phylogenetic analysis were listed in Table S3.
Generation of constructs for the expression of BnTT16 homologs in the Arabidopsis tt16-6 mutant line
The coding regions of BnTT16.1, -16.2, -16.3 and -16.4 were individually amplified from B. napus cDNA with primers bearing restriction sites at their 5′ ends (see Table S1 for primer sequences) and inserted downstream of the CaMV 35S promoter and upstream of the CaMV 35S transcriptional terminator in the binary vector pDH51 (Pietrzak et al., 1986). The resulting BnTT16 vectors were then digested with EcoRI (partial digestion was utilized in the case of the BnTT16.3 vector due to the presence of an EcoRI site within the TT16.3 coding region) to isolate the 35S-p::BnTT16::35S-t cassettes, which were subsequently inserted into the pGreen 0029 vector (Figure S2) (Hellens et al., 2000). All vectors were subsequently confirmed via sequencing, introduced into Agrobacterium tumefaciens strain GV3101 using electroporation and transformed into the Arabidopsis tt16-6 T-DNA mutant line with the floral dip method (Zhang et al., 2006).
Whole-mount vanillin staining was performed on developing seeds (cotyledon stage) as described by Nesi et al. (2002). For staining of seed sections, developing seeds were fixed, embedded in paraffin and sectioned to a thickness of 8 μm according to Zifkin et al. (2012). For the observation of seed coat development, seed sections were de-waxed with toluene and stained with toluidine blue O (0.01% in water) for 1 min (Zifkin et al., 2012). For PA localization, the de-waxed sections were strained with 1% vanillin solution (prepared by adding vanillin in 10 ml 100% ethanol and 5 ml 37% HCl) for 10 min under room temperature (21°C) or 2% DMACA solution (in 0.5 m H2SO4 in 1-butanol) at 60°C for 15 min (Zifkin et al., 2012). Analyses using Nomarski optics were performed on developing seeds (cotyledon stage) using a Leica DMRXA microscope (Leica, http://www.leica.com/) equipped with Nomarski optics.
Quantification of PA and calculation of mean degree of polymerization
Soluble PAs were extracted from developing B. napus seeds (15, 25, 35 and 45 DAP) with 80% methanol and quantified on an Agilent 1200 HPLC system (Agilent Technologies, http://www.agilent.com/) equipped with two Chromolith RP-18e (4.6 × 100 mm) columns and an Agilent G1315B photodiode array detector, as described previously (Zifkin et al., 2012). The mean degree of polymerization was calculated as described previously (Kennedy and Jones, 2001).
Quantitative RT-PCR analysis was performed with primers listed in Table S1 as reported previously (Chen et al., 2010). In the case of experiments used to validate our microarray data (Figure S9), unique qRT-PCR primers were designed based on sequences utilized in the microarray. Results are presented as mean relative transcript levels ± standard error of three biological replicates.
The authors would like to thank the following colleagues for their help and suggestions: Dr Lihua Jin and Arlene Oatway for microscopy work and proanthocyanidin quantification, Troy Locke for the microarray assay, Drs Yuanqing Jiang and Michael K. Deyholos for developing seed dissection, Dr Richard G. H. Immink for providing the homozygous tt16-6 Arabidopsis seeds and Dr Xue Chen for providing genomic DNA of B. napus, B. rapa and B. oleracea, and critical review of this manuscript. This work was supported by Alberta Enterprise and Advanced Education, Alberta Innovates Bio Solutions, the Canada Foundation for Innovation, the Canada Research Chairs Program, Genome Alberta, and Genome Canada.