- Research article
- Open Access
Genome-wide survey and expression profiles of the AP2/ERF family in castor bean (Ricinus communis L.)
© Xu et al.; licensee BioMed Central Ltd. 2013
- Received: 17 June 2013
- Accepted: 8 November 2013
- Published: 13 November 2013
The AP2/ERF transcription factor, one of the largest gene families in plants, plays a crucial role in the regulation of growth and development, metabolism, and responses to biotic and abiotic stresses. Castor bean (Ricinus communis L., Euphobiaceae) is one of most important non-edible oilseed crops and its seed oil is broadly used for industrial applications. The available genome provides a great chance to identify and characterize the global information on AP2/ERF transcription factors in castor bean, which might provide insights in understanding the molecular basis of the AP2/ERF family in castor bean.
A total of 114 AP2/ERF transcription factors were identified based on the genome in castor bean. According to the number of the AP2/ERF domain, the conserved amino acid residues within AP2/ERF domain, the conserved motifs and gene organization in structure, and phylogenetical analysis, the identified 114 AP2/ERF transcription factors were characterized. Global expression profiles among different tissues using high-throughput sequencing of digital gene expression profiles (DGEs) displayed diverse expression patterns that may provide basic information in understanding the function of the AP2/ERF gene family in castor bean.
The current study is the first report on identification and characterization of the AP2/ERF transcription factors based on the genome of castor bean in the family Euphobiaceae. Results obtained from this study provide valuable information in understanding the molecular basis of the AP2/ERF family in castor bean.
- Castor Bean
- Digital Gene Expression
- Conserve Amino Acid Residue
- Dehydration Responsive Element Binding
- Global Expression Profile
The AP2/ERF (APETALA2/ETHYLENE) transcription factor (TF), one of the biggest gene families, contains a typical AP2 DNA-binding domain and exists extensively in plants [1, 2]. The AP2/ERF domain is characterized by approximately 60–70 amino acid residues that constitute a typical helix-turn-helix structure responsible for sequence-specific DNA binding to modulate the target gene expression. Based on the number of AP2/ERF domains and the structural features, the AP2/ERF family is usually divided into four subfamilies (AP2, ERF, DREB and RAV). The AP2 subfamily, containing two repeated AP2/ERF domains, is comprised of two groups, the AP2 group  and the AINTEGUMENTA group (ANT) [4, 5]. Their main function involves the regulation of organ-specific growth and development, such as flower development , ovule development  and the formation of seed size , by binding to target sequences gCAC(A/G)N(A/T)TcCC(a/g)ANG(c/t) . Both the ERF and DREB subfamilies contain a single AP2/ERF domain with a specific WLG motif . The ERF subfamily can recognize the conserved nucleotide consensus sequence AGCCGCC of the GCC-box  in the promoter regions of pathogenesis-related (PR) genes and modulate their expression in disease resistance signaling pathways , whereas the DREB subfamily typically binds to the cis-acting elements by the binding sequence CCGAC and is involved in gene expression responsive to abiotic stresses (drought, low-temperature and high salinity) and plant hormones such as ethylene and ABA [12, 13]. The RAV subfamily, containing a single AP2/ERF domain and a specific B3 motif [14–16], is involved in regulating gene expression in response to ethylene , Brassinosteroid , and biotic and abiotic stresses [19, 20]. In addition, other members containing a single AP2/ERF domain and lacking additional motifs are often named as Soloist. Little is known about their function. Though the identification of structural characterization and the expression profiles for AP2/ERF transcription factors has been extensively studied and documented in several plants such as Arabidopsis, poplar , grapevine , a holistic profile of the AP2/ERF family detailing its structure and function in a given species is limited.
Based on genomic sequences, the AP2/ERF family has been characterized in Arabidopsis, poplar , grapevine , rice , wheat  and peach . Castor bean (Ricinus communis L. Euphobiaceae) is one of most important non-edible oilseed crops and its seed oil is broadly used in industry. In particular, the main composition of its seed oil is ricinoleic acid, which is considered an ideal and unique feedstock for biodiesel production [26–28]. Due to the increased demand for production of castor bean seed oils in many countries, breeding and improvement of varieties are drawing great attention from breeders . Further efforts should be made to elucidate the molecular mechanism underlying the regulation of growth and development. The recent completion of the castor bean genome  provides an opportunity to identify and characterize the holistic profile of the AP2/ERF family, which could add insights into understanding the molecular mechanism of the AP2/ERF family that underlies the regulation of growth and development in castor bean.
A genome-wide survey and characterization of the AP2/ERF family was conducted based on the complete genomic sequences of castor bean in this study. The expression profiles of the AP2/ERF transcription factors among different tissues were examined using high-throughput sequencing for Digital Gene Expression Tag Profiling (DGE). Results obtained from this study provide global information in understanding the molecular basis of the AP2/ERF family in castor bean and other plants in the family Euphobiaceae as well.
Detection of AP2/ERF transcription factors in castor bean
Summary of the AP2/ERF gene family in Arabidopsis , rice, poplar, grapevine and castor bean
Compared with Arabidopsis (147 members), rice (164 members), grapevine (132 members) and poplar (200 members), the AP2/ERF family seems to have relatively fewer members in castor bean. It is obvious that the number of the AP2/ERF members within different subfamilies and groups are varied among species (Table 1). For instance, the number of members in the DREB subfamily ranges from 34 (in castor bean) to 77 (in poplar), and the number of ERF members ranges from 56 (in castor bean) to 91 (in poplar). In addition, one member (30217.m000254) was identified as Soloist, encoded by a single-copy gene with low similarity to the Arabidopsis Soloist AT4G13040.
Conserved residues in the AP2/ERF domain
Phylogenetic and conserved motif analyses
To dissect the evolutionary relationships of AP2/ERF transcription factors between castor bean and Arabidopsis, another unrooted phylogenetic tree was constructed based on the amino acid sequence similarity of 112 AP2/ERF family members in castor bean (excluding 30006.m000282 and 30170.m013669 due to their low similarity) and 120 AP2/ERF family members obtained from Arabidopsis in previous study . The phylogenetic tree generated four major clades (designated I to IV, see Additional file 6). Clade I was composed of the AP2 and RAV members; Clade II covered all DREB members (except for the clade II-1clustered by B6 members); Clade III, Clade IV and subclade II-1 included all ERF members; and the Soloist was clustered in Clade II. Further, it was also observed that subclades clustered by groups A1-A6 members of castor bean and Arabidopsis within the DREB subfamily and by groups B1-B5 members of castor bean and Arabidopsis within the ERF subfamily were substantially identified, but the group B6 members were split in subclade II-1 and other subclades within Clade III. In particular, all major clades and subclades were clustered by interspecies members, indicating that the AP2/ERF transcription factors are homologous between castor bean and Arabidopsis.
Structural analysis of AP2/ERF genes
Structural analyses of genes revealed that all members in the AP2 subfamily genes had diverse introns ranging from three to nine, whereas 40 of 56 members in the ERF subfamily, 33 of 34 members in the DREB subfamily and all the four members in the RAV subfamily were intronless. Only one was exceptional with a single intron in the DREB subfamily. The 16 members in the ERF subfamily contained just one intron whereas the Solosist gene contained four introns. Further we inspected the pattern of intron positions for those genes containing introns. We found that the positions occurring introns were conserved in both the AP2/ERF domain and the outside AP2/ERF domain regions in the AP2 subfamily, though the number of intron was varied. Similarly, most of genes shared same or similar intron patterns in the ERF subfamily with most introns occurring in the AP2/ERF domain regions (see Figure 3C). The pattern of exon/intron splicing phase usually provided useful information in understanding of the emergence and evolution of gene family. We checked the pattern of exon/intron splicing phase for each intron in the AP2/ERF family. The splicing phases were designated as three splicing phases: phase 0, splicing occurred after the third nucleotide of the codon; phase 1, splicing occurred after the first nucleotide of the codon; and phase 2, splicing occurred after the second nucleotide. Results showed that most members in the AP2 subfamily shared same or similar pattern of exon/intron splicing phase, and the pattern of exon/intron splicing phase also was conserved in the ERF subfamily (see Figure 3C).
Gene structure analyses could provide additional evidence to support the phylogenetic groupings in a given gene family. Our results provided strong evidence to validate our previous phylogenetic groupings. For instance, the five genes (28320.m001139, 29841.m002846, 29848.m004632, 29588.m000873 and 29848.m004566) categorized in the ERF-B6 subgroup shared the same gene structure including patterns of intron position and exon/intron splicing phase (see Figure 3C); the four genes (30174.m008755, 30174.m008756, 30174.m008757 and 30174.m008759) clustered in ERF-B3 subgroup were nearly identical in gene length and structure.
Expression profiles of the AP2/ERF gene family
To investigate the expression levels of AP2/ERF genes in different organs, high throughput Tag-seq analysis was performed using five tissues leaf, root, seed 1, seed 2 and endosperm (see Methods). The raw sequence data of the five tag libraries obtained from Illumina Genome Analyzer were submitted to the Sequence Read Archive (SRA) under accession SRX343933. In total, 4,574,301, 4,660,289, 4,543,329, 4,650,533 and 4,828,665 clean sequence tags for leaf, root, seed 1, seed 2 and endosperm libraries were obtained (see Additional file 9A). To estimate our sequence quality and sequencing depth, the tag coverage and saturation was analyzed for each library (see Additional file 9). As showed in Additional file 9B, when the sequencing counts reached 2 million tags, the number of detected genes tended towards saturation, meaning that our sequencing depth was sufficient to detect the expression of AP2/ERF genes in each library.
Further, we compared the expression profiles of AP2/ERF genes among different organs between castor bean and Arabidopsis. Although some AP2/ERF genes and their orthologs (such as 29983.m003227/AT2G20880.1, 28976.m000163/AT4G37750.1, 29584.m000234/AT3G23240.1, 28049.m000300/AT3G61630.1, 29635.m000461/AT5G19790.1, 30084.m000185/AT5G19790.1, 29680.m001737/AT5G18450.1) displayed different expression patterns among tissues, most of AP2/ERF genes and their orthologs presented similar expression profiles among organs between castor bean and Arabidopsis (see Additional file 10). Eight genes and their orthologs (29908.m006005/AT1G78080.1, 28752.m000339/AT2G28550.3, 29169.m000017/AT4G36920.1, 27904.m000217/AT3G15210.1, 29640.m000403/AT1G50640.1, 27585.m000144/AT1G53910.1, 28192.m000255/AT4G17500.1, 29738.m001050/AT1G25560.1), for instance, were highly expressed in all organs tested in both castor bean and Arabidopsis. In particular, most of genes and their orthologs exhibited a tissue/organ-specific expression profile. For instance, 30069.m000440/AT3G54320.1, 30170.m013668/AT4G37750.1 and 29726.m004094/AT1G72360.1, were preferentially expressed in developing seeds and 29841.m002846/AT1G15360.1 was specifically expressed in flower in both castor bean and Arabidopsis (see Additional file 10).
To identify potential transcription factors involved in regulating lipid biosynthesis in developing seeds of castor bean, we purposely analyzed the expressional differences of all transcription factors identified in castor bean (PlantTFDB: http://plntfdb.bio.uni-potsdam.de/v3.0/) between seed 1 (at the initial stage) and seed 2 (at the fast oil accumulation stage) libraries. As shown in Additional file 11, 23 transcription factors significantly up-regulated at the fast oil accumulation stage were identified. In particular, some key regulators of fatty acid biosynthesis, such as LEC1, LEC2, ABI3 and WRINKLED1 were significantly up-regulated, consistent with Brown et al.’s observation . For AP2/ERF genes, 18 genes were significantly down-regulated, and only two genes (30069.m000440 and 29726.m004094) were significantly up-regulated at the fast oil accumulation stage (p < 0.001 and fold-change > 2) (see Additional file 12).
Although the AP2/ERF family has been broadly studied in diverse plants, the current study is the first report on identification and characterization of the AP2/ERF transcription factors based on the genome in the family Euphobiaceae, one important group of resource plants. In total, 114 putative AP2/ERF family genes were identified based on the genome sequences of castor bean. Genome analyses showed that castor bean had undergone recent duplication events , which might contribute to the expansion of the AP2/ERF family in castor bean. Compared with Arabidopsis (genome size 125 Mb), rice (genome size 466 Mb), grapevine (genome size 490 Mb) and poplar (genome size 480 Mb), castor bean (genome size 310 Mb) harbored the minimum members in the AP2/ERF family (see Table 1). As mentioned above, the AP2/ERF family was extensively involved in regulating plant response to diverse biotic and abiotic stresses. Castor bean can easily grow in diverse habitats from template, subtropical to tropical areas. It appears that castor bean displays a strong tolerance or resistance to diverse environmental stresses. However, why castor bean harbors less members in the AP2/ERF family is yet unknown. The 114 members identified were unambiguously divided into four subfamilies, in consistence with the category of AP2/ERF family in other plants. In particular, Both ERF and DREB are dominant subfamilies containing a single AP2/ERF domain in structure, whereas both AP2 and RAV subfamilies were of minority exhibiting a more complex gene structure such as two AP2/ERF domains and more introns or a specific B3 motif in gene sequences. Probably, an early addition of introns or a second DNA binding domain in structure may have impaired the duplicative ability of the hypothesized ancestral HNH endonuclease in the early evolution of this family, or a longer piece of DNA might have made a transposition and duplication event less likely, resulting in the smaller number of members in the AP2 and RAV subfamilies . In addition, similar to other plants [24, 31], the AP2/ERF domain regions contained many highly conserved amino acid residues in castor bean.
In general, transcription factors functionally result from some important conserved motifs within and outside the DNA binding domain which are related to transcriptional activity, nuclear localization, and protein-protein interactions . Two conserved amino acid residues 14 V/A and 19E/D within the AP2/ERF domain have been proved to be critical for DNA-binding specificity . The identified divergence of amino acid residues 14 V and 19E in the DREB subfamily, or 14A and 19D in the ERF subfamily may be one of the important factors in the understanding of the functional divergence between the ERF and DREB subfamilies in castor bean. In particular, the two elements YRG and RAYD within the AP2/ERF domain had been reported to be critical in activating DNA binding to modulate the expression of target genes in Arabidopsis[3, 36]. The two elements YRG and RAYD were highly conserved and identified in most of the members of AP2/ERF family in castor bean, implying their structural and functional necessity. Outside the AP2/ERF domain regions 14 conserved motifs were identified in castor bean AP2/ERF family in this study. Most of these conserved motifs display a group-specific distribution pattern. Combining the structural differentiation of genes among subfamilies or groups, these observations strongly imply that the functional divergence exists among subfamilies or groups. The conserved motifs 13 and 18 may play important roles as transcriptional repressor in mediating plant growth and development . Both motifs 13 and 18 were the RAV subfamily specific, and shared by each member, implying that motifs 13 and 18 may be indispensable elements in structure of RAV subfamily in castor bean. Studies have showed that motifs 9 and 11 could form a long linker of the two β-sheets and these extruded residues or of AP2/ERF proteins and several linker residues in ANT lineage in Arabidopsis, which may participate in activating the function of transcription factors in the AP2 subfamily . Both motifs 9 and 11 were the AP2 subfamily specific in castor bean, shared by 10 and 15 members respectively, meaning that motifs 9 and 11 may provide a specific function for DNA binding in the AP2 subfamily in castor bean. The motif 10 was shared by 17 members from groups A1, A4 and A5 in the DREB subfamily, characterized by four blocks of conserved amino acid residues: LPRP, D[IV]QAA/DIR[RA], LRAA and [IHEYQAKS]LNFP (see Additional file 8C). These conserved amino acid residues have been identified to be essential signatures in Arabidopsis for CBL-interacting serine/threonine-proteins kinase-12 , Ethylene-responsive transcription factor ERF037 , dehydration responsive element binding proteins-1C and proteins-G , auxin response factor-19 , and disease resistance , respectively. The motif 16 containing a unique ‘EDLL’ residue was the group B3 specific in ERF subfamily (see Additional file 8D). The ‘EDLL’ residue might participate in activating the function for the group B3 members in the ERF subfamily . However, the function of most conserved motifs identified in castor bean is uncertain. Compared those additional conserved motifs identified outside of the AP2/ERF domain regions in castor bean with other plants, eight motifs (including motifs 9, 10, 11, 13, 16, 17, 18 and 22) were shared by castor bean, Arabidopsis and rice, indicating most of additional motifs outside of AP2/ERF domain regions were conserved in plants. The newly identified seven motifs (12, 15, 19, 20, 23, 24 and 25) might be variable among species or species-specific in castor bean.
Phylogenetic analysis of the AP2/ERF transcription factors in castor bean showed that the four subfamilies, AP2, RAV, DREB, ERF, and the main groups A1-A6 within the DREB subfamily, and groups B1-B6 within the ERF subfamily were able to be substantially identified. Compared with the phylogenetical relationships of the AP2/ERF members in Arabidopsis and rice [21, 31], phylograms displayed similar clades to our results. The phylogenetical tree generated by the combined members between castor bean and Arabidopsis showed a major clade I shared by the AP2 and RAV members, indicating a phylogenetically close relationship between the AP2 and RAV subfamilies. In particular, all major clades and subclades were clustered by interspecies members, indicating that the AP2/ERF transcription factors are homologous between castor bean and Arabidopsis. Based on similar gene structure and conserved motifs of AP2/ERF gene in different species, it was indicated that the AP2/ERF transcription factors were highly conserved in angiosperm. These observations strongly support Magnani et al.’s assumption that the AP2/ERF transcription factors might have an ancient origin during angiosperm evolution .
As mentioned above, researches have demonstrated that the activities of several AP2 transcription factors are regulated during the development of organs by the microRNA miR172 in Arabidopsis[6, 32, 44]. Our current study identified four AP2 genes containing unambiguous targeted sites for binding Rc-miR172, which could provide a potential clue to dissect the mechanisms underlying the AP2 gene regulation in castor bean.
Although our sequencing depth was sufficient, high throughput Tag-seq data obtained from five libraries identified the expression of only 54 AP2/ERF genes in this study. Based on the deep RNA-seq data in previous study , the expressions of additional 34 AP2/ERF genes were supplemented. The expressional differences of AP2/ERF genes between our data and Brown et al’s data could be explained because of 1) the different sequencing strategy and depth (Brown et al’s data was based on RNA-seq strategy with more deeper sequencing, which was more sensitive for detecting genes expressed at the low level than our Tag-seq strategy ); 2) the different tissues tested (Brown et al’s data included flower and geminating seed tissues). The expression profiles of most AP2/ERF genes displayed spatial and temporal expression patterns among different tissues, implying their functional specificity. For example, the 29929.m004537 gene was specifically expressed in root tip tissues, and its homologs (AT5G17430 and AT3G20840) in Arabidopsis were functionally involved in regulating the growth and development of root tips . In addition, the expression of 26 of 114 AP2/ERF genes (including 16 members in ERF subfamily, eight members in DREB subfamily, one member in AP2 subfamily, and one member in RAV subfamily) were not detected (see Additional file 10). The possible reasons include: 1) the limited tissues or developmental stages were examined in our analysis, and 2) the tissues tested in both our current examination and Brown et al’s study were sampled from individuals with normal growth (lack of environmental stresses). It is understandable that the expression of some genes in DREB and ERF subfamilies would not be detected if their expressions were majorly involved in responding to biotic and abiotic stresses.
One of main objectives of this study is to identify potential AP2/ERF transcription factors involved in oil accumulation or seed development of castor bean. The analyses of expressional differences between seed 1 and seed 2 libraries revealed that 18 of 20 AP2/ERF genes were down-regulated from the initial developing stage to the oil fast accumulation stage. These genes might be negatively regulated in oil accumulation in the developing seeds of castor bean. For the two genes (29726.m004094 and 30069.m000440) strongly up-regulated at the oil fast accumulation stage, we inspected the function of their the homologs (AT1G72360 and AT3G54320) in Arabidopsis and found that AT1G72360 was a hypoxia-inducible ethylene response factor and significantly up-regulated in developing seeds [47–50], and AT3G54320 (WRINKLED1) was a master gene responsible for transcriptionally regulating carbon metabolism and lipid biosynthesis in developing seeds [51–56]. Potentially, the gene 30069.m000440 may be an important transcription factor responsible for regulating oil accumulation in developing seeds of castor bean. Studies focusing on the functional analysis of 30069.m000440 might reveal the mechanism underlying the regulation of oil accumulation in developing seeds of castor bean.
The current study is the first report on identification and characterization of the AP2/ERF transcription factors based on the castor bean genome in the family Euphobiaceae. In total, 114 putative AP2/ERF family genes were identified in castor bean, one of most important non-edible oilseed crops and its seed oil is broadly used for industrial applications. Further, the 114 AP2/ERF transcription factors were characterized according to the conserved amino acid residues within AP2/ERF domain, the conserved motifs and gene organization in structure, phylogenetical analysis, and global expression profiles among different tissues using high-throughput sequencing. Results obtained from this study provide global information in understanding the molecular basis of the AP2/ERF family in castor bean.
Identification of AP2/ERF transcription factors from castor bean genomic sequences
Based on the castor bean genome (http://castorbean.jcvi.org/index.php), an extensive search was performed to identify all members of the AP2/ERF transcription factors. The Arabidopsis AP2/ERF gene and amino acid sequences were downloaded from the DATF database (http://datf.cbi.pku.edu.cn). The characterized ERF sequences from the representative members for each group in Arabidopsis thaliana were used as query sequences against the castor bean complete genome using WU-BLAST 2.0 program with an e-value of le-3 and more than 80% coverage. According to the hit position of sequences targeted in castor bean genome, the corresponding gene sequences (including ORF sequences), gene model, position in scaffold, amino acid sequences and their annotations were extracted for further analyses. To obtain an exhaustive search for identifying all members of AP2/ERF family in castor bean, we further used the full length sequences of representative members in other subfamilies, such as AT1G25560.1 representing RAV subfamily, AT2G28550.1 representing AP2 subfamily, and the Soloist AT4G13040 as query sequences for comparing our previous searches. After removing redundant sequences and incomplete ORF sequences, SMART tools (http://smart.embl-heidelberg.de/) and InterProScan (http://www.ebi.ac.uk/Tools/pfa/iprscan/) were used to confirm the presence of the characterized AP2/ERF domain in the candidate sequences. Further, the putative members of AP2/ERF family and their gene sequences were identified and defined for further analyses.
Phylogenetic, MEME motif and gene structure analyses
Multiple alignments of amino acid sequences of the AP2/ERF domain in Arabidopsis and castor bean were carried out using Clustal W  and an un-rooted phylogenetic tree was generated with neighbor-joining criteria in MEGA 5.0  with 1000 bootstrap replicates. Conserved motifs in castor bean AP2/ERF transcription factors were identified using motif based sequence analysis tool MEME (Suite version 4.9.0) with the following parameters: optimum width 10–200 amino acids, any number of repetitions of a motif, and maximum number of motifs set at 25. The BLAST search for the resulting motifs in the NCBI and MS-Homology databases was carried out to determine their biological contexts. In addition, gene structure was investigated using the online Gene Structure Display Server (http://gsds.cbi.pku.edu.cn/) based on full-length mRNA alignments with corresponding genomic sequences, whereas introns are gaps between exons consisting entirely of genomic sequence.
Gene expression analyses
To examine the global expression profiles of 114 AP2/ERF transcription factors identified among different organs or developmental stages, high-throughput sequencing of digital gene expression tag profiles (DGEs) for five tissues leave, root tips, developing seeds at the initial stage (seed 1), developing seeds at the fast oil accumulation stage (seed 2), and endosperm were conducted. Seeds of castor bean var. ZB306 elite inbred line (provided kindly by Zibo Academy of Agricultural Sciences, Shandong, China) were germinated and grown in the conservatory under natural conditions (11 h light, 13 h dark; 25°C during the day and 18°C at night). Mature female flowers were hand pollinated and tagged. Leaf tissues were collected from fully expanded young leaf and root tips were dissected, washed and collected. Immature seeds at two different stages, i.e. seed 1 at the initial stage (developing seeds do not start to accumulate oil, ca. 15 days after pollination) and seed 2 at the fast oil accumulation stage (developing seeds start to fast accumulate oil, ca 35 days after pollination) were collected. Endosperm tissues were dissected from the immature seeds (ca. 40 days after pollination). Three biological replicates were collected for each tissue type. For all the tissues, three randomly chosen samples were pooled to form a biological replicate. Total RNA was isolated from the leaves, roots, seed 1, seed 2 and endosperms of castor bean using Trizol reagent (Invitrogen, Carlsbad, CA) and purified using an RNeasy Mini Kit (Qiagen, Valencia, CA) following the manufacturer’s protocol. The quality of total RNA samples was checked using the NanoDrop Spectrometer (ND-1000 Spectrophotometer, Peqlab) as well as agarose gel electrophoresis.
The high quality RNAs were used to constructed tag libraries respectively for deep-sequencing. Briefly, total ploy A RNA (about 6 μg) was enriched by Oligo(dT) magnetic beads and Oligo(dT) used as the primer to synthesize the first and second-strand cDNA. The cDNA was digested by two types of Endonuclease: NlaIII or DpnII, acquiring 17 bp tags with different adaptors of both ends to form a tag library. After 15 cycles of linear PCR amplification, 105 bp fragments were purified by 6% PAGE Gel electrophoresis. After denaturation, the single-chain molecules were fixed onto the Illumina Sequencing Chip (flowcell). Each molecule then grows into a single-molecule cluster sequencing template through in situ amplification. Four colored nucleotides were added for sequencing using the method of sequencing by synthesis (SBS). Millions of raw reads were generated with a sequencing length of 49 bp. Sequencing was performed using a Illumina Genome Analyzer at BGI ShenZhen (China).
The raw data from the five tagged libraries were preprocessed to filter out low quality reads and clipped adapter sequences. After that, all clean reads were mapped to the castor bean genome (http://castorbean.jcvi.org/index.php) to obtain unique reads and reads abundance using SOAP2 software . To compare the differential expression of genes among tissues, the expression level of each gene in the different tissues was normalized to the number of transcripts per million clean (TPM). Genes with significantly different expression were determined by P ≤ 0.001 and fold-change ≥2 in two samples. To visualize a global transcription profile of genes detected in each subfamily across the five tissues, the hierarchical clustering was performed using R software .
This work was supported by the “Hundreds of Talents” program of the Chinese Academy of Sciences (AL). Authors gave many thanks to Zibo Academy of Agricultural Sciences, Shandong, China for providing the seeds of castor bean var. ZB306 elite inbred line.
- Riechmann JL, Meyerowitz EM: The AP2/EREBP family of plant transcription factors. Biol Chem. 1998, 379: 633-646.PubMedGoogle Scholar
- Wessler SR: Homing into the origin of the AP2 DNA binding domain. Trends in Plant Sci. 2005, 10: 54-56.View ArticleGoogle Scholar
- Jofuku KD, den Boer BGW, van Montagu M, Okamuro JK: Control of Arabidopsis flower and seed development by the homeotic gene APETALA2. Plant Cell. 1994, 6: 1211-1225.PubMed CentralView ArticlePubMedGoogle Scholar
- Elliott RC, Betzner AS, Huttner E, Oakes MP, Tucker WQJ, Gerentes D, Perez P, Smyth DR: AINTEGUMENTA, an APETALA2-like gene of Arabidopsis with pleiotropic roles in ovule development and floral organ growth. Plant Cell. 1996, 8: 155-168.PubMed CentralView ArticlePubMedGoogle Scholar
- Shigyo M, Ito M: Analysis of gymnosperm two-AP2-domain-containing genes. Dev Genes Evol. 2004, 214 (3): 105-14. 10.1007/s00427-004-0385-5.View ArticlePubMedGoogle Scholar
- Aukerman MJ, Sakai H: Regulation of flowering time and floral organ identity by a microRNA and its APETALA2-like target genes. Plant Cell. 2003, 15: 2730-2741. 10.1105/tpc.016238.PubMed CentralView ArticlePubMedGoogle Scholar
- Jofuku KD, Omidyar PK, Gee Z, Okamuro JK: Control of seed mass and seed yield by the floral homeotic gene APETALA2. Proc Natl Acad Sci U S A. 2005, 102: 3117-3122. 10.1073/pnas.0409893102.PubMed CentralView ArticlePubMedGoogle Scholar
- Nole-Wilson S, Krizek BA: DNA binding properties of the Arabidopsis floral development protein AINTEGUMENTA. Nucleic Acids Res. 2000, 21: 4076-4082.View ArticleGoogle Scholar
- Sakuma Y, Liu Q, Dubouzet JG, Abe H, Shinozaki K, Yamaguchi-Shinozaki K: DNA-binding specificity of the ERF/AP2 domain of Arabidopsis DREBs, transcription factors involved in dehydration- and cold-inducible gene expression. Biochemical d Biophyisical Res Comm. 2002, 290: 998-1009. 10.1006/bbrc.2001.6299.View ArticleGoogle Scholar
- Ohme-Takagi M, Shinshi H: Ethylene-inducible DNA binding proteins that interact with an ethylene-responsive element. Plant Cell. 1995, 7: 173-182.PubMed CentralView ArticlePubMedGoogle Scholar
- Hao DY, Ohme-Takagi M, Sarai A: Unique mode of GCC box recognition by the DNA-binding domain of ethylene responsive element-binding factor (ERF domain) in plants. J Biol Chem. 1998, 273: 26857-26861. 10.1074/jbc.273.41.26857.View ArticlePubMedGoogle Scholar
- Yamaguchi-Shinozaki K, Shinozaki K: A novel cis-acting element in an Arabidopsis gene is involved in responsiveness to drought, low-temperature, or high-salt stress. Plant Cell. 1994, 6: 251-264.PubMed CentralView ArticlePubMedGoogle Scholar
- Jiang C, Iu B, Singh J: Requirement of a CCGAC cis-acting element for cold induction of the BN115 gene from winter Brassica napus. Plant Mol Biol. 1996, 30: 679-684. 10.1007/BF00049344.View ArticlePubMedGoogle Scholar
- Giraudat J, Hauge BM, Valon C, Smalle J, Parcy F, Goodman HM: Isolation of the Arabidopsis ABI3 gene by positional cloning. Plant Cell. 1992, 4: 1251-1261.PubMed CentralView ArticlePubMedGoogle Scholar
- Suzuki M, Kao CY, McCarty DR: The conserved B3 domain of VIVIPAROUS1 has a cooperative DNA binding activity. Plant Cell. 1997, 9: 799-807.PubMed CentralView ArticlePubMedGoogle Scholar
- Kagaya Y, Ohmiya K, Hattori T: RAV1, a novel DNA-binding protein, binds to bipartite recognition sequence through two distinct DNA-binding domains uniquely found in higher plants. Nucleic Acids Res. 1999, 27: 470-478. 10.1093/nar/27.2.470.PubMed CentralView ArticlePubMedGoogle Scholar
- Alonso JM, Stepanova AN, Leisse TJ, Kim CJ, Chen H, Shinn P, Stevenson DK, Zimmerman J, Barajas P, Cheuk R, Gadrinab C, Heller C, Jeske A, Koesema E, Meyers CC, Parker H, Prednis L, Ansari Y, Choy N, Deen H, Geralt M, Hazari N, Hom E, Karnes M, Mulholland C, Ndubaku R, Schmidt I, Guzman P, Aguilar-Henonin L, Schmid M, Weigel D, Carter DE, Marchand T, Risseeuw E, Brogden D, Zeko A, Crosby WL, Berry CC, Ecker JR: Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science. 2003, 30: 653-657.View ArticleGoogle Scholar
- Hu YX, Wang YX, Liu XF, Li JY: Arabidopsis RAV1 is down-regulated by brassinosteroid and may act as a negative regulator during plant development. Cell Res. 2004, 14: 8-15. 10.1038/sj.cr.7290197.View ArticlePubMedGoogle Scholar
- Sohn KH, Lee SC, Jung HW, Hong JK, Hwang BK: Expression and functional roles of the pepper pathogen-induced transcription factor RAV1 in bacterial disease resistance, and drought and salt stress tolerance. Plant Mol Biol. 2006, 61: 897-915. 10.1007/s11103-006-0057-0.View ArticlePubMedGoogle Scholar
- Li CW, Su RC, Cheng CP, Sanjay , You SJ, Hsieh TH, Chao TC, Chan MT: Tomato RAV transcription factor is a pivotal modulator involved in the AP2/EREBP-mediated defense pathway. Plant Physiol. 2011, 156: 213-227. 10.1104/pp.111.174268.PubMed CentralView ArticlePubMedGoogle Scholar
- Zhuang J, Cai B, Peng RH, Zhu B, Jin XF, Xue Y, Gao F, Fu XY, Tian YS, Zhao W, Qiao YS, Zhang Z, Xiong AS, Yao QH: Genome-wide analysis of the AP2/ERF gene family in populus trichocarpa. Biochem Biophys Res Commun. 2008, 371: 468-474. 10.1016/j.bbrc.2008.04.087.View ArticlePubMedGoogle Scholar
- Licausi F, Giorgi FM, Zenoni S, Osti F, Pezzotti M, Perata P: Genomic and transcriptomic analysis of the AP2/ERF superfamily in vitis vinifera. BMC Genomics. 2010, 11: 719-10.1186/1471-2164-11-719.PubMed CentralView ArticlePubMedGoogle Scholar
- Rashid M, Guangyuan H, Guangxiao Y, Hussain J, Xu Y: AP2/ERF transcription factor in rice: genome-wide canvas and syntenic relationships between monocots and eudicots. Evol Bioinform Online. 2012, 8: 321-355.PubMed CentralView ArticlePubMedGoogle Scholar
- Zhuang J, Chen JM, Yao QH, Xiong F, Sun CC, Zhou XR, Zhang J, Xiong AS: Discovery and expression profile analysis of AP2/ERF family genes from Triticum aestivum. Mol Biol Rep. 2011, 38: 745-753. 10.1007/s11033-010-0162-7.View ArticlePubMedGoogle Scholar
- Zhang CH, Shangguan LF, Ma RJ, Sun X, Tao R, Guo L, Korir NK, Yu ML: Genome-wide analysis of the AP2/ERF superfamily in peach (Prunus persica). Genet Mol Res. 2012, 11: 4789-4809.PubMedGoogle Scholar
- Akpan U, Jimoh A: Mohammed AExtraction: characterization and modification of castor seed oil. Leonardo J Sci. 2006, 8: 43-52.Google Scholar
- Ogunniyi DS: Castor oil: a vital industrial raw material. Bioresour Technol. 2006, 97: 1086-1091. 10.1016/j.biortech.2005.03.028.View ArticlePubMedGoogle Scholar
- Scholz V, da Silva JN: Prospects and risks of the use of castor oil as a fuel. Biomass Bioenergy. 2008, 32: 95-100. 10.1016/j.biombioe.2007.08.004.View ArticleGoogle Scholar
- Qiu L, Yang C, Tian B, Yang JB, Liu A: Exploiting EST databases for the development and characterization of EST-SSR markers in castor bean (Ricinus communis L.). BMC Plant Biol. 2010, 10: 278-10.1186/1471-2229-10-278.PubMed CentralView ArticlePubMedGoogle Scholar
- Chan AP, Crabtree J, Zhao Q, Lorenzi H, Orvis J, Puiu D, Melake-Berhan A, Jones KM, Redman J, Chen G, Cahoon EB, Gedil M, Stanke M, Haas BJ, Wortman JR, Fraser-Liggett CM, Ravel J, Rabinowicz PD: Draft genome sequence of the oilseed species Ricinus communis. Nat Biotechnol. 2010, 28: 951-956. 10.1038/nbt.1674.PubMed CentralView ArticlePubMedGoogle Scholar
- Nakano T, Suzuki K, Fujimura T, Shinshi H: Genome-wide analysis of the ERF gene family in Arabidopsis and rice. Plant Physiol. 2006, 140: 411-432. 10.1104/pp.105.073783.PubMed CentralView ArticlePubMedGoogle Scholar
- Chen X: A microRNA as a translational repressor of APETALA2 in Arabidopsis flower development. Science. 2004, 303: 2022-2025. 10.1126/science.1088060.View ArticlePubMedGoogle Scholar
- Wei X, Cui QH, Li F, Liu AZ: Transcriptome-wide identification and characterization of microRNAs from castor bean (Ricinus communis L.). PLoS ONE. 2013, 8: e69995-10.1371/journal.pone.0069995.View ArticleGoogle Scholar
- Brown AP, Kroon JTM, Swarbreck D, Febrer M, Larson TR, Graham IA, Caccamo M, Slabas AR: Tissue-specific whole transcriptome sequencing in Castor, directed at understanding triacylglycerol lipid biosynthetic pathways. PLoS ONE. 2012, 7: e30100-10.1371/journal.pone.0030100.PubMed CentralView ArticlePubMedGoogle Scholar
- Magnani E, Sjölander K, Hake S: From endonucleases to transcription factors: evolution of the AP2 DNA binding domain in plants. Plant Cell. 2004, 16: 2265-2277. 10.1105/tpc.104.023135.PubMed CentralView ArticlePubMedGoogle Scholar
- Okamuro JK, Caster B, Villarroel R, Van Montagu M, Jofuku KD: The AP2 domain of APETALA2 defines a large new family of DNA binding proteins in Arabidopsis. Proc Natl Acad Sci U S A. 1997, 94: 7076-7081. 10.1073/pnas.94.13.7076.PubMed CentralView ArticlePubMedGoogle Scholar
- Kim S, Soltis PS, Wall K, Soltis DE: Phylogeny and domain evolution in the APETALA2-like gene family. Mol Biol Evol. 2006, 23: 107-120.View ArticlePubMedGoogle Scholar
- Albrecht V, Ritz O, Linder S, Harter K, Kudla J: The NAF domain defines a novel protein-protein interaction module conserved in Ca2+-regulated kinases. EMBO J. 2001, 20: 1051-1063. 10.1093/emboj/20.5.1051.PubMed CentralView ArticlePubMedGoogle Scholar
- Qu LJ, Zhu YX: Transcription factor families in Arabidopsis: major progress and outstanding issues for future research. Curr Opin Plant Biol. 2006, 9: 544-549. 10.1016/j.pbi.2006.07.005.View ArticlePubMedGoogle Scholar
- Feng JX, Liu D, Pan Y, Gong W, Ma LG, Luo JC, Deng XW, Zhu YX: An annotation update via cDNA sequence analysis and comprehensive profiling of developmental, hormonal or environmental responsiveness of the Arabidopsis AP2/EREBP transcription factor gene family. Plant Mol Biol. 2005, 59: 853-868. 10.1007/s11103-005-1511-0.View ArticlePubMedGoogle Scholar
- Hagen G, Guilfoyle T: Auxin-responsive gene expression: genes, promoters and regulatory factors. Plant Mol Biol. 2002, 49: 373-385. 10.1023/A:1015207114117.View ArticlePubMedGoogle Scholar
- Manosalva PM, Davidson RM, Liu B, Zhu X, Hulbert SH, Leung H, Leach JE: A germin-like protein gene family functions as a complex quantitative trait locus conferring broad-spectrum disease resistance in rice. Plant Physiol. 2009, 149: 286-296. 10.1104/pp.108.128348.PubMed CentralView ArticlePubMedGoogle Scholar
- Tiwari SB, Belachew A, Ma SF, Young M, Ade J, Shen Y, Marion CM, Holtan HE, Bailey A, Stone JK, Edwards L, Wallace AD, Canales RD, Adam L, Ratcliffe OJ, Repetti PP: The EDLL motif: a potent plant transcriptional activation domain from AP2/ERF transcription factors. Plant J. 2012, 70: 855-865. 10.1111/j.1365-313X.2012.04935.x.View ArticlePubMedGoogle Scholar
- Wollmann H, Mica E, Todesco M, Long JA, Weigel D: On reconciling the interactions between APETALA2, miR172 and AGAMOUS with the ABC model of flower development. Development. 2010, 137: 3633-3642. 10.1242/dev.036673.PubMed CentralView ArticlePubMedGoogle Scholar
- Hong LZ, Li J, Schmidt-Küntzel A, Warren WC, Barsh GS: Digital gene expression for non-model organisms. Genome Res. 2011, 21: 1905-1915. 10.1101/gr.122135.111.PubMed CentralView ArticlePubMedGoogle Scholar
- Galinha C, Hofhuis H, Luijten M, Willemsen V, Blilou I, Heidstra R, Scheres B: PLETHORA proteins as dose-dependent master regulators of Arabidopsis root development. Nature. 2007, 449: 1053-1057. 10.1038/nature06206.View ArticlePubMedGoogle Scholar
- Licausi F, van Dongen JT, Giuntoli B, Novi G, Santaniello A, Geigenberger P, Perata P: HRE1 and HRE2, two hypoxia-inducible ethylene response factors, affect anaerobic responses in Arabidopsis thaliana. Plant J. 2010, 62: 302-315. 10.1111/j.1365-313X.2010.04149.x.View ArticlePubMedGoogle Scholar
- Licausi F, Weits DA, Pant BD, Scheible WR, Geigenberger P, van Dongen JT: Hypoxia responsive gene expression is mediated by various subsets of transcription factors and miRNAs that are determined by the actual oxygen availability. New Phytol. 2011, 190: 442-456. 10.1111/j.1469-8137.2010.03451.x.View ArticlePubMedGoogle Scholar
- Hess N, Klode M, Anders M, Sauter M: The hypoxia responsive transcription factor genes ERF71/HRE2 and ERF73/HRE1 of Arabidopsis are differentially regulated by ethylene. Physiol Plant. 2011, 143: 41-49. 10.1111/j.1399-3054.2011.01486.x.View ArticlePubMedGoogle Scholar
- Yang CY, Hsu FC, Li JP, Wang NN, Shih MC: The AP2/ERF transcription factor AtERF73/HRE1 modulates ethylene responses during hypoxia in Arabidopsis. Plant Physiol. 2011, 156: 202-212. 10.1104/pp.111.172486.PubMed CentralView ArticlePubMedGoogle Scholar
- Cernac A, Benning C: WRINKLED1 Encodes an AP2/EREB domain protein involved in the control of storage compound biosynthesis in Arabidopsis. Plant J. 2004, 40: 575-585. 10.1111/j.1365-313X.2004.02235.x.View ArticlePubMedGoogle Scholar
- Baud S, Wuillème S, To A, Rochat C, Lepiniec L: Role of WRINKLED1 in the transcriptional regulation of glycolytic and fatty acid biosynthetic genes in Arabidopsis. Plant J. 2009, 60: 933-947. 10.1111/j.1365-313X.2009.04011.x.View ArticlePubMedGoogle Scholar
- Maeo K, Tokuda T, Ayame A, Mitsui N, Kawai T, Tsukagoshi H, Ishiguro S, Nakamura K: An AP2-type transcription factor, WRINKLED1, of Arabidopsis thaliana binds to the AW-box sequence conserved among proximal upstream regions of genes involved in fatty acid synthesis. Plant J. 2009, 60: 476-487. 10.1111/j.1365-313X.2009.03967.x.View ArticlePubMedGoogle Scholar
- Liu J, Hua W, Zhan G, Wei F, Wang X, Liu G, Wang H: Increasing seed mass and oil content in transgenic Arabidopsis by the overexpression of wri1-like gene from Brassica napus. Plant Physiol Biochem. 2010, 48: 9-15. 10.1016/j.plaphy.2009.09.007.View ArticlePubMedGoogle Scholar
- Pouvreau B, Baud S, Vernoud V, Morin V, Py C, Gendrot G, Pichon JP, Rouster J, Paul W, Rogowsky PM: Duplicate maize Wrinkled1 transcription factors activate target genes involved in seed oil biosynthesis. Plant Physiol. 2011, 156: 674-686. 10.1104/pp.111.173641.PubMed CentralView ArticlePubMedGoogle Scholar
- To A, Joubès J, Barthole G, Lécureuil A, Scagnelli A, Jasinski S, Lepiniec L, Baud S: WRINKLED transcription factors orchestrate tissue-specific regulation of fatty acid biosynthesis in Arabidopsis. Plant Cell. 2012, 24: 5007-5023. 10.1105/tpc.112.106120.PubMed CentralView ArticlePubMedGoogle Scholar
- Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG: Clustal W and clustal X version 2.0. Bioinformatics. 2007, 23: 2947-1948. 10.1093/bioinformatics/btm404.View ArticlePubMedGoogle Scholar
- Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S: MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol. 2011, 28: 2731-2739. 10.1093/molbev/msr121.PubMed CentralView ArticlePubMedGoogle Scholar
- Li R, Yu C, Li Y, Lam TW, Yiu SM, Kristiansen K, Wang J: SOAP2: an improved ultrafast tool for short read alignment. Bioinformatics. 2009, 25: 1966-1967. 10.1093/bioinformatics/btp336.View ArticlePubMedGoogle Scholar
- Gentleman RC, Carey VJ, Bates DM, Bolstad B, Dettling M, Dudoit S, Ellis B, Gautier L, Ge Y, Gentry J, Hornik K, Hothorn T, Huber W, Iacus S, Irizarry R, Leisch F, Li C, Maechler M, Rossini AJ, Sawitzki G, Smith C, Smyth G, Tierney L, Yang JY, Zhang J: Bioconductor: open software development for computational biology and bioinformatics. Genome Biol. 2004, 5: R80-10.1186/gb-2004-5-10-r80.PubMed CentralView ArticlePubMedGoogle Scholar
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