Project description:Floral organs, whose identity is determined by specific combinations of homeotic genes, originate from a group of undifferentiated cells called the floral meristem. In Arabidopsis, the homeotic gene AGAMOUS (AG) terminates meristem activity and promotes development of stamens and carpels. To understand the program of gene expression activated by AG, we followed genome-wide expression during early stamen and carpel development. Keywords: Developmental time course
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:Regulation of homeotic gene expression is critical for proper developmental patterns in plants. The Arabidopsis thaliana floral homeotic gene AGAMOUS is regulated by LEUNIG (LUG). Mutations in LUG result in homeotic transformations of floral organ identity. LUG mutants also exhibit other defects that are independent of AG, including abnormal carpel and ovule development, reduced female and male fertility, and narrower leaves and floral organs, suggesting that LUG has a wider role in development. LUG is structurally similar to the transcription co-repressors Tup1 (S. cerevisiae) and Groucho (Drosophila), suggesting developmental pathways regulated by LUG may be controlled by a conserved eukaryotic repression mechanism. We aim to determine novel target genes regulated by LUG using transcriptome analysis. A mutant lug-3 line has been obtained from Dr. Zhongchi Liu. This line was used in the initial studies of LUG in the Meyerowitz lab. Lug-3 is a strong allele in the Lansberg erecta background, caused by a nonsense mutation that results in early termination of the protein and is likely null. The Lug-3 mutant exhibits narrow floral organs with staminoid petals and carpelloid sepals, abnormal carpel and ovule development, reduced plant height, increased lateral branching and narrow and smaller leaves. We proposed to use the lug-3 line in elucidating the role of LUG as a potential general regulator of transcription in Arabidopsis. mRNA populations will be isolated from wild type (Ler) and lug-3 plants that have produced inflorescence. This stage of development has been selected for several reasons. Firstly, mRNA populations from the widest range of tissue types from a single plant can be collected. Secondly, as AGAMOUS, which is involved in the regulation of floral development, is present in the Affymetrix gene chip, it will provided an excellent internal control to monitor between chips. Results will demonstrate whether LUG is likely to have a more global role in regulating transcription, and will identify pathways under the regulation of this co-repressor-like protein. 12 samples were used in this experiment.
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:Regulation of homeotic gene expression is critical for proper developmental patterns in plants. The Arabidopsis thaliana floral homeotic gene AGAMOUS is regulated by LEUNIG (LUG). Mutations in LUG result in homeotic transformations of floral organ identity. LUG mutants also exhibit other defects that are independent of AG, including abnormal carpel and ovule development, reduced female and male fertility, and narrower leaves and floral organs, suggesting that LUG has a wider role in development. LUG is structurally similar to the transcription co-repressors Tup1 (S. cerevisiae) and Groucho (Drosophila), suggesting developmental pathways regulated by LUG may be controlled by a conserved eukaryotic repression mechanism. We aim to determine novel target genes regulated by LUG using transcriptome analysis. A mutant lug-3 line has been obtained from Dr. Zhongchi Liu. This line was used in the initial studies of LUG in the Meyerowitz lab. Lug-3 is a strong allele in the Lansberg erecta background, caused by a nonsense mutation that results in early termination of the protein and is likely null. The Lug-3 mutant exhibits narrow floral organs with staminoid petals and carpelloid sepals, abnormal carpel and ovule development, reduced plant height, increased lateral branching and narrow and smaller leaves. We proposed to use the lug-3 line in elucidating the role of LUG as a potential general regulator of transcription in Arabidopsis. mRNA populations will be isolated from wild type (Ler) and lug-3 plants that have produced inflorescence. This stage of development has been selected for several reasons. Firstly, mRNA populations from the widest range of tissue types from a single plant can be collected. Secondly, as AGAMOUS, which is involved in the regulation of floral development, is present in the Affymetrix gene chip, it will provided an excellent internal control to monitor between chips. Results will demonstrate whether LUG is likely to have a more global role in regulating transcription, and will identify pathways under the regulation of this co-repressor-like protein.
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 gene expression profiles of inflorescences of the floral homeotic mutants apetala1, apetala2, apetala3, pistillata, and agamous were compared to wild-type inflorescences using a flower-specific cDNA array (GSM21001-21015) and a "whole-genome" oligonucleotide array (Operon) (GSM21016-21035). All experiments were performed at least in triplicate. This SuperSeries is composed of the SubSeries listed below.
Project description:Floral organs, whose identity is determined by specific combinations of homeotic genes, originate from a group of undifferentiated cells called the floral meristem. In Arabidopsis, the homeotic gene AGAMOUS (AG) terminates meristem activity and promotes development of stamens and carpels. To understand the program of gene expression activated by AG, we followed genome-wide expression during early stamen and carpel development. Experiment Overall Design: Described in Gomez-Mena et al, 2005, Development 132: 429-438. Briefly, synchronous development of stamens and carpels was initiated by steroid treatment of plants homozygous for the ap1-1 and cal-1 mutations and expressing a fusion between AGAMOUS and the rat glucocorticoid receptor (35S:AGGR). RNA was extracted one, three and seven days after steroid treatment; two independent steroid-treated samples and two independent untreated controls were used for each time point.
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.