Project description:The transition from vegetative to reproductive development is one of the most important phase changes in the plant life cycle. This step is controlled by various environmental signals that are integrated at the molecular level by so-called floral integrators. One such floral integrator in Arabidopsis (Arabidopsis thaliana) is the MADS domain transcription factor SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1). Despite extensive genetic studies, little is known about the transcriptional control of SOC1, and we are just starting to explore the network of genes under the direct control of SOC1 transcription factor complexes. Here, we show that several MADS domain proteins, including SOC1 heterodimers, are able to bind SOC1 regulatory sequences. Genome-wide target gene analysis by ChIP-seq confirmed the binding of SOC1 to its own locus and shows that it also binds to a plethora of flowering-time regulatory and floral homeotic genes. In turn, the encoded floral homeotic MADS domain proteins appear to bind SOC1 regulatory sequences. Subsequent in planta analyses revealed SOC1 repression by several floral homeotic MADS domain proteins, and we show that, mechanistically, this depends on the presence of the SOC1 protein. Together, our data show that SOC1 constitutes a major hub in the regulatory networks underlying floral timing and flower development and that these networks are composed of many positive and negative autoregulatory and feedback loops. The latter seems to be crucial for the generation of a robust flower-inducing signal, followed shortly after by repression of the SOC1 floral integrator. A. thaliana SOC1 ChIP-seq w. control, 3 replicates
Project description:The transition from vegetative to reproductive development is one of the most important phase changes in the plant life cycle. This step is controlled by various environmental signals that are integrated at the molecular level by so-called floral integrators. One such floral integrator in Arabidopsis (Arabidopsis thaliana) is the MADS domain transcription factor SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1). Despite extensive genetic studies, little is known about the transcriptional control of SOC1, and we are just starting to explore the network of genes under the direct control of SOC1 transcription factor complexes. Here, we show that several MADS domain proteins, including SOC1 heterodimers, are able to bind SOC1 regulatory sequences. Genome-wide target gene analysis by ChIP-seq confirmed the binding of SOC1 to its own locus and shows that it also binds to a plethora of flowering-time regulatory and floral homeotic genes. In turn, the encoded floral homeotic MADS domain proteins appear to bind SOC1 regulatory sequences. Subsequent in planta analyses revealed SOC1 repression by several floral homeotic MADS domain proteins, and we show that, mechanistically, this depends on the presence of the SOC1 protein. Together, our data show that SOC1 constitutes a major hub in the regulatory networks underlying floral timing and flower development and that these networks are composed of many positive and negative autoregulatory and feedback loops. The latter seems to be crucial for the generation of a robust flower-inducing signal, followed shortly after by repression of the SOC1 floral integrator.
Project description:The floral transition in Arabidopsis is tightly controlled by complex genetic regulatory networks in response to endogenous and environmental flowering signals. SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) and SHORT VEGETATIVE PHASE (SVP), two key MADS-domain transcription factors, perceive these signals and function as antagonistic flowering regulators. To understand how they mediate the floral transition, we mapped in vivo binding sites of SOC1 and SVP using chromatin immunoprecipitation followed by hybridization to whole-genome tiling arrays (ChIP-chip). Genes encoding proteins with transcription regulator activity and transcription factor activity were the most enriched groups of genes bound by SOC1 and SVP, indicating their central roles in flowering regulatory networks. In combination with gene expression microarray studies, we further identified the genes whose expression was directly regulated by SOC1 or SVP. Among the common direct targets identified, APETALA2 (AP2)-like genes that repress FT and SOC1 expression were downregulated by SOC1, but upregulated by SVP, revealing a complex feedback regulation among key genes determining the integration of flowering signals. SOC1 regulatory regions were also accessed by SOC1 itself and SVP, suggesting that self-activation and repression by SVP contribute to the regulation of SOC1 expression. In addition, ChIP-chip analysis demonstrated that miR156e and miR172a, which are involved in the regulation of AP2-like genes, were direct targets of SOC1 and SVP, respectively. Taken together, these findings reveal that feedback regulatory loops mediated by SOC1 and SVP are essential components of the gene regulatory networks underpinning the integration of flowering signals during the floral transition. soc1-101D and 35S:SVP ChIPed with SOC1 or SVP polyclonal antibody respectively vs. soc1-2 or svp-41 in Arabidopsis 9-day-old whole seedlings
Project description:The floral transition in Arabidopsis is tightly controlled by complex genetic regulatory networks in response to endogenous and environmental flowering signals. SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) and SHORT VEGETATIVE PHASE (SVP), two key MADS-domain transcription factors, perceive these signals and function as antagonistic flowering regulators. To understand how they mediate the floral transition, we mapped in vivo binding sites of SOC1 and SVP using chromatin immunoprecipitation followed by hybridization to whole-genome tiling arrays (ChIP-chip). Genes encoding proteins with transcription regulator activity and transcription factor activity were the most enriched groups of genes bound by SOC1 and SVP, indicating their central roles in flowering regulatory networks. In combination with gene expression microarray studies, we further identified the genes whose expression was directly regulated by SOC1 or SVP. Among the common direct targets identified, APETALA2 (AP2)-like genes that repress FT and SOC1 expression were downregulated by SOC1, but upregulated by SVP, revealing a complex feedback regulation among key genes determining the integration of flowering signals. SOC1 regulatory regions were also accessed by SOC1 itself and SVP, suggesting that self-activation and repression by SVP contribute to the regulation of SOC1 expression. In addition, ChIP-chip analysis demonstrated that miR156e and miR172a, which are involved in the regulation of AP2-like genes, were direct targets of SOC1 and SVP, respectively. Taken together, these findings reveal that feedback regulatory loops mediated by SOC1 and SVP are essential components of the gene regulatory networks underpinning the integration of flowering signals during the floral transition.
Project description:MADS-domain transcription factors play pivotal roles in numerous developmental processes in Arabidopsis thaliana. While their involvement in flowering transition and floral development has been extensively examined, their functions in root development remain relatively unexplored. Here, we explored the function and genetic interaction of three MADS-box genes (XAL2, SOC1 and AGL24) in primary root development. Our findings revealed that SOC1 and AGL24, both critical components in flowering transition, redundantly act as repressors of primary root growth as the loss of function of either SOC1 or AGL24 partially recovers the primary root growth, meristem cell number, cell production rate, and the length of fully elongated cells of the short-root mutant xal2-2. Furthermore, we observed that the simultaneous overexpression of AGL24 and SOC1 leads to short-root phenotypes, affecting meristem cell number, cell production rate, fully elongated cell size, but only the overexpression of SOC1 affects distal root stem cell differentiation. Additionally, these genes exhibit distinct modes of transcriptional regulation in roots compared to what has been previously reported for aerial tissues. Moreover, our findings revealed that the expression of certain genes involved in cell differentiation, as well as stress responses, which are either upregulated or downregulated in the xal2-2 mutant, reverted to WT levels in the absence of SOC1 or AGL24.
Project description:Optimised flowering time is an important trait ensuring successful plant adaptation and crop productivity. SOC1-like genes encode MADS transcription factors known to play important roles in flowering control in many plants. This includes the best characterised eudicot model Arabidopsis thaliana (Arabidopsis) where SOC1 promotes flowering and functions as a floral integrator gene integrating signals from different flowering time regulatory pathways. Medicago truncatula (Medicago) is a temperate reference legume with strong genomic and genetic resources used to study flowering pathways in legumes. Interestingly, despite responding to the similar floral-inductive cues of extended cold (vernalisation) followed by warm long days, as winter annual Arabidopsis, Medicago lacks FLC and CO which are key regulators of flowering in Arabidopsis. Unlike Arabidopsis with one SOC1 gene, multiple gene duplication events have given rise to three MtSOC1 paralogs within the Medicago genus in legumes; one Fabaceae group A SOC1 gene, MtSOC1a, and two tandemly-repeated Fabaceae group B SOC1 genes, MtSOC1b and MtSOC1c. Previously, we showed that MtSOC1a has unique functions in floral promotion in Medicago. The Mtsoc1a Tnt1 retroelement insertion single mutant showed moderately delayed flowering in long and short day photoperiods, with and without prior vernalization, compared with wild type. On the other hand, Mtsoc1b Tnt1 single mutants did not have altered flowering time or flower development, indicating that it was redundant in an otherwise wild type background. Here, we describe the generation of Mtsoc1 triple mutant plants by CRISPR-Cas9 gene editing. Two independent Mtsoc1 homozygous triple mutants were non-flowering and bushy in floral inductive VLD. Phenotyping and gene expression analyses by RNA-seq and RT-qPCR indicate that the Mtsoc1 triple mutants remain vegetative. Thus overall, the Mtsoc1 triple mutants are dramatically different from the single Mtsoc1a mutant and the Arabidopsis soc1 mutant; implicating multiple MtSOC1 genes in critical overlapping roles in the transition to flowering in Medicago.
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.
Project description:Floral organ identities in plants are specified by the combinatorial action of homeotic master regulatory transcription factors (TFs). How these factors achieve their regulatory specificities is however still largely unclear. Genome-wide in vivo DNA binding data show that homeotic MADS-domain proteins recognize partly distinct genomic regions, suggesting that DNA binding specificity contributes to functional differences of homeotic protein complexes. We used in vitro systematic evolution of ligands by exponential enrichment followed by high throughput DNA sequencing (SELEX-seq) on several floral MADS-domain protein homo- and heterodimers to measure their DNA-binding specificities. We show that specification of reproductive organs is associated with distinct binding preferences of a complex formed by SEPALLATA3 (SEP3) and AGAMOUS (AG). Binding specificity is further modulated by different binding site (BS) spacing preferences. Combination of SELEX-seq and genome-wide DNA binding data allows to differentiate between targets in specification of reproductive versus perianth organs in the flower. We validate the importance of DNA-binding specificity for organ-specific gene regulation by modulating promoter activity through targeted mutagenesis. Our study shows that intrafamily protein interactions affect DNA-binding specificity of floral MADS-domain proteins. DNA-binding specificity of individual dimers, as well as DNA-binding preferences of higher-order complexes differ between floral homeotic protein complexes. Differential DNA-binding of MADS-domain protein complexes plays a role in the specificity of target gene regulation.
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.
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. Keywords: ChIP-chip