Identification of genes regulated by the MADS transcription factor (TF), SEPALLATA3, in the context of the double sep1sep2 and triple sep1sep2sep3 mutant background, by RNA-Seq analysis
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ABSTRACT:
ORGANISM(S): Arabidopsis thaliana
PROVIDER: GSE150605 | GEO | 2020/09/07
REPOSITORIES: GEO
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Project description:The MADS genes encode transcription factors (TF) that act as master regulators of plant reproduction and flower development. The SEPALLATA (SEP) MADS subfamily is not only absolutely required for the development of floral organs, but also plays roles in inflorescence architecture and determinacy of the floral meristem. The SEPs act as organizers of MADS complexes and are able to form both heterodimers and heterotetramers in vitro. To date, the MADS TF complexes characterized in angiosperm floral organ development contain at least one SEP TF. Whether DNA-binding by SEP-containing dimeric MADS complexes are sufficient for launching floral organ identity programs, however, is not clear as only defects in floral meristem determinacy were observed in tetramerization impaired SEP mutants. Here we used a combination of genome-wide binding studies, high resolution structural studies of the SEP3-AGAMOUS tetramerisation domain, structure-based mutagenesis and complementation experiments in sep1 sep2 sep3 and sep1 sep2 sep3 ag-4 plants transformed with versions of SEP3 encoding tetramerization mutants. We demonstrate that while SEP3 heterodimers are able to bind DNA both in vitro and in vivo and recognize the majority of SEP3 wild type binding sites genome-wide, tetramerisation is not only required for floral meristem determinacy, but also absolutely required for floral organ identity in the second, third and fourth whorls.
2024-04-30 | GSE227012 | GEO
Project description:The molecular mechanisms by which floral homeotic genes act as major developmental switches to specify the identity of floral organs, are still largely unknown. Floral homeotic genes encode transcription factors of the MADS-box family, which are supposed to assemble in a combinatorial fashion into organ-specific multimeric protein complexes. Major mediators of protein interactions are MADS-domain proteins of the SEPALLATA subfamily, which play a crucial role in the development of all types of floral organs. In order to characterize the roles of the SEPALLATA3 transcription factor complexes at the molecular level, we analyzed genome-wide the direct targets of SEPALLATA3. We used chromatin immunoprecipitation followed by ultrahigh-throughput sequencing or hybridization to whole-genome tiling arrays to obtain genome-wide DNA-binding patterns of SEPALLATA3. The results demonstrate that SEPALLATA3 binds to thousands of sites in the genome. Most potential target sites that were strongly bound in wild-type inflorescences, are also bound in the floral homeotic agamous mutant, which displays only the perianth organs, sepals and petals. Characterization of the target genes shows that SEPALLATA3 integrates and modulates different growth-related and hormonal pathways in a combinatorial fashion with other MADS-box proteins and possibly with non-MADS transcription factors. In particular, the results suggest multiple links between SEPALLATA3 and auxin signaling pathways. Our gene expression analyses link the genomic binding site data with the phenotype of plants expressing a dominant repressor version of SEPALLATA3, suggesting that it modulates auxin response to facilitate floral organ outgrowth and morphogenesis. Furthermore, the binding of the SEPALLATA3 protein to cis-regulatory elements of other MADS-box genes and expression analyses reveal that this protein is a key component in the regulatory transcriptional network underlying the formation of floral organs. ChIP experiments were performed on Arabidopsis wildtype and agamous mutant inflorescences using an antibody raised against a C-terminal peptide of SEP3. As control, ChIP experiments were performed on the sep3 mutant.
2009-04-20 | E-GEOD-14600 | biostudies-arrayexpress
Project description:The receptor-like protein kinases encoded by HAESA (HAE) and HAESA-LIKE 2 (HSL2) are essential for floral organ abscission in Arabidopsis thaliana and the double hae hsl2 mutant fails to abscise. Expression of HAE and HSL2 is specific to Abscission Zone (AZ) cells and is higher in stage 15 flowers than in earlier developmental stages. By stage 16 floral organs have begun to abscise, suggesting that HAE HSL2 are most active in stage 15 flowers. Samples were enriched for AZ RNA by isolating RNA from flower receptacles, the region from the base of the flower to slightly above the base of attachment of the sepals, petals, and stamen. RNA-seq was then used to analyze and compare the transcriptomes of wild type and hae-3 hsl2-3 mutants. 2034 genes were differentially expressed with a False Discovery Rate adjusted p < 0.05, of which 349 genes 2 fold or greater change. Of these 349, 277 were lower in the mutant and 72 were higher. Differentially expressed genes with lower expression were enriched for hydrolytic enzymes, cell-wall modifying enzymes, and defense related genes. This suggests that HAE HSL2 signaling regulates gene expression of enzymes necessary for abscission. 6 samples were sequenced, 3 biological replicates of Col-0 wild type and 3 biological replicates of the hae-3 hsl2-3 double mutant. Samples were barcoded and all 6 samples multiplexed and sequenced on 3 lanes, each lane on a separate flow cell, of an Illumina HiSeq 2000.
2013-01-14 | E-GEOD-35288 | biostudies-arrayexpress
Project description:In this study, we compare the DNA binding specifify and affinity of SEPALLATA3 and AGAMOUS complexes The MADS transcription factors, SEPALLATA3 (SEP3) and AGAMOUS (AG), are required for floral organ identity and determinacy of the floral meristem in Arabidopsis. Dimerization is obligatory for their DNA binding, however SEP3 and SEP3-AG also form tetrameric complexes. The goal of this study is to understand how homo and hetero-dimerization and tetramerization of MADS TFs affect genome-wide DNA-binding patterns. Using a modified sequential DNA affinity purification sequencing protocol (seq-DAP-seq), we selectively purified SEP3 homomeric and SEP3-AG heteromeric complexes, including the dimeric SEP3 tet-AG complex and the tetrameric SEP3-AG complex, and determined their genome-wide binding.
2020-09-07 | GSE150528 | GEO
Project description:The receptor-like protein kinases encoded by HAESA (HAE) and HAESA-LIKE 2 (HSL2) are essential for floral organ abscission in Arabidopsis thaliana and the double hae hsl2 mutant fails to abscise. Expression of HAE and HSL2 is specific to Abscission Zone (AZ) cells and is higher in stage 15 flowers than in earlier developmental stages. By stage 16 floral organs have begun to abscise, suggesting that HAE HSL2 are most active in stage 15 flowers. Samples were enriched for AZ RNA by isolating RNA from flower receptacles, the region from the base of the flower to slightly above the base of attachment of the sepals, petals, and stamen. RNA-seq was then used to analyze and compare the transcriptomes of wild type and hae-3 hsl2-3 mutants. 2034 genes were differentially expressed with a False Discovery Rate adjusted p < 0.05, of which 349 genes 2 fold or greater change. Of these 349, 277 were lower in the mutant and 72 were higher. Differentially expressed genes with lower expression were enriched for hydrolytic enzymes, cell-wall modifying enzymes, and defense related genes. This suggests that HAE HSL2 signaling regulates gene expression of enzymes necessary for abscission.
2013-01-14 | GSE35288 | GEO
Project description:Background: Vicia sativa (the common vetch) possesses a predominant zygomorphic flower and belongs to the subfamily Papilionoideae, which is related to Arabidopsis thaliana in the eurosid II clade of the core eudicots. Each vetch flower consists of 21 concentrically arranged organs: the outermost five sepals, then five petals and ten stamens, and a single carpel in the center. Methodology/Principal Findings: We explored the floral transcriptome to examine a genome-scale genetic model of the zygomorphic flower of vetch. mRNA was obtained from an equal mixture of six floral organs, leaves and roots. De novo assembly of the vetch transcriptome using Illumina paired-end technology produced 71,553 unigenes with an average length of 511 bp. We then compared the expression changes in the 71,553 unigenes in the eight independent organs through RNA-Seq Quantification analysis. We predominantly analyzed gene expression patterns specific to each floral organ and combinations of floral organs that corresponded to the traditional ABC model domains. Comparative analyses were performed in the floral transcriptomes of vetch and Arabidopsis, and genomes of vetch and Medicago truncatula. Conclusions/Significance: Our comparative analysis of vetch and Arabidopsis showed that the vetch flowers conform to a strict ABC model. We analyzed the evolution and expression of the TCP gene family in vetch at a whole-genome level, and several unigenes specific to three different vetch petals, which might offer some clues toward elucidating the molecular mechanisms underlying floral zygomorphy. Our results provide the first insights into the genome-scale molecular regulatory network that controls the evolution and development of the zygomorphic flower in Papilionoideae. Examination of the transcriptomic changes in the six floral organs, leaves and roots in common vetch.
2013-03-26 | E-GEOD-35437 | biostudies-arrayexpress
Project description:Background: Vicia sativa (the common vetch) possesses a predominant zygomorphic flower and belongs to the subfamily Papilionoideae, which is related to Arabidopsis thaliana in the eurosid II clade of the core eudicots. Each vetch flower consists of 21 concentrically arranged organs: the outermost five sepals, then five petals and ten stamens, and a single carpel in the center. Methodology/Principal Findings: We explored the floral transcriptome to examine a genome-scale genetic model of the zygomorphic flower of vetch. mRNA was obtained from an equal mixture of six floral organs, leaves and roots. De novo assembly of the vetch transcriptome using Illumina paired-end technology produced 71,553 unigenes with an average length of 511 bp. We then compared the expression changes in the 71,553 unigenes in the eight independent organs through RNA-Seq Quantification analysis. We predominantly analyzed gene expression patterns specific to each floral organ and combinations of floral organs that corresponded to the traditional ABC model domains. Comparative analyses were performed in the floral transcriptomes of vetch and Arabidopsis, and genomes of vetch and Medicago truncatula. Conclusions/Significance: Our comparative analysis of vetch and Arabidopsis showed that the vetch flowers conform to a strict ABC model. We analyzed the evolution and expression of the TCP gene family in vetch at a whole-genome level, and several unigenes specific to three different vetch petals, which might offer some clues toward elucidating the molecular mechanisms underlying floral zygomorphy. Our results provide the first insights into the genome-scale molecular regulatory network that controls the evolution and development of the zygomorphic flower in Papilionoideae.
2013-03-26 | GSE35437 | GEO
Project description:Plant inflorescence meristems and floral meristems possess specific boundary domains that result in floral organ separation, and in proper numbers of floral organs. HANABA TARANU (HAN) encodes a boundary-expressing GATA type zinc finger transcription factor that regulates shoot apical meristem organization, cell division and flower development in Arabidopsis, but the underlying mechanism remains unclear. Through time-course whole genome oligonucleotide microarray analyses following transient overexpression of HAN, we find that HAN represses hundreds of genes, especially genes involved in hormone responses and floral organ regulation. Transient overexpression of HAN also causes the repression of HAN itself and three other HAN family genes: HANL2 (HAN-LIKE2), GNC (GATA, NITRATE-INDUCIBLE, CARBON-METABOLISM-INVOLVED) and GNL (GNC LIKE), forming a negative regulatory feedback loop. Double- and triple-mutant strains of han with hanl2, gnc and gnl show synergistic effects on sepal fusion, petal number, and silique length, and embryo development, as well as carpelloid stamens. Transcripts of HANL2, GNC and GNL have similar accumulation patterns, specifically in petals, stamens, carpels and inflorescence meristems, which are partially overlapping with the expression pattern of HAN, suggesting that HAN and HAN family genes share redundant functions during flower development. We further show by yeast two hybrid assays that HAN can homodimerize as well as heterodimerize with other HAN family proteins. Chromatin-immunoprecipitation analyses indicate that HAN directly binds to its own promoter and the promoter of GNC in vivo. These findings, together with the fact that constitutive overexpression of HAN has an even stronger phenotype than a loss of function mutation, support the hypothesis that HAN may function as a key repressor that regulates floral development via intricate regulatory networks involving genes in the GATA3 family, hormone actions and floral organ specification. Time course induction experiment. Arabidopsis inflorescences containing flower buds from stages 1-9 were collected for microarray experiment 0h, 4h, 12h and 72h after 10M-BM-5M Dex treatment. RNA samples from mock- and Dex-treated plants at each time point were co-hybridized, and labeling dyes were swapped between replicates to reduce dye-related bias. Four biological replicates were used for the microarray hybridization.
2013-09-01 | E-GEOD-38658 | biostudies-arrayexpress
Project description:Flower development is a dynamics process in which floral organs are produced from pools of stem cells residing in meristems (Smyth et al., 1990). In order to obtain a high resolution map of the changes of gene expression during this process thus to provide insights into specific expression patterns and their underlying gene regulatory networks, an inducible system which allows us to obtain synchronized flowers (Wellmer et al., 2006) was used to collect stage-specific floral tissues at four stages (stages 0, 2, 4 and 8) for transcriptome profiling by RNA-seq . These stages represent the status of inflorescence meristem, floral meristem specification, floral organ specification and floral organ differentiation, respectively during Arabidopsis flower development.
2018-11-06 | GSE110500 | GEO
Project description:Plant inflorescence meristems and floral meristems possess specific boundary domains that result in floral organ separation, and in proper numbers of floral organs. HANABA TARANU (HAN) encodes a boundary-expressing GATA type zinc finger transcription factor that regulates shoot apical meristem organization, cell division and flower development in Arabidopsis, but the underlying mechanism remains unclear. Through time-course whole genome oligonucleotide microarray analyses following transient overexpression of HAN, we find that HAN represses hundreds of genes, especially genes involved in hormone responses and floral organ regulation. Transient overexpression of HAN also causes the repression of HAN itself and three other HAN family genes: HANL2 (HAN-LIKE2), GNC (GATA, NITRATE-INDUCIBLE, CARBON-METABOLISM-INVOLVED) and GNL (GNC LIKE), forming a negative regulatory feedback loop. Double- and triple-mutant strains of han with hanl2, gnc and gnl show synergistic effects on sepal fusion, petal number, and silique length, and embryo development, as well as carpelloid stamens. Transcripts of HANL2, GNC and GNL have similar accumulation patterns, specifically in petals, stamens, carpels and inflorescence meristems, which are partially overlapping with the expression pattern of HAN, suggesting that HAN and HAN family genes share redundant functions during flower development. We further show by yeast two hybrid assays that HAN can homodimerize as well as heterodimerize with other HAN family proteins. Chromatin-immunoprecipitation analyses indicate that HAN directly binds to its own promoter and the promoter of GNC in vivo. These findings, together with the fact that constitutive overexpression of HAN has an even stronger phenotype than a loss of function mutation, support the hypothesis that HAN may function as a key repressor that regulates floral development via intricate regulatory networks involving genes in the GATA3 family, hormone actions and floral organ specification.
2013-09-01 | GSE38658 | GEO