Project description:The debate on the origin and evolution of flowers has recently entered the field of developmental genetics, with focus on the design of the ancestral floral regulatory program. Flowers can differ dramatically among angiosperm lineages, but in general, sterile perianth organs surrounding stamens (male reproductive organs) and carpels (female reproductive organs) constitute the basic floral structure. However, the basal angiosperm lineages exhibit spectacular diversity in the number, arrangement, and structure, of floral organs, while the evolutionarily derived monocot and eudicot lineages share a far more uniform floral ground plan. As such, regulatory mechanisms underlying the archetypal floral plan, for instance that of the eudicot genetic model Arabidopsis thaliana, are unlikely to apply to the original flowers. Here we show that broadly overlapping transcriptional programs characterise the floral transcriptome of the basal angiosperm Persea americana (avocado), while floral gene expression domains are typically organ-specific in Arabidopsis. Our findings extend the “fading borders” model for basal angiosperms from organ identity genes to the downstream floral transcriptome, and suggest that the combinatorial mechanism for organ identity may not operate in basal angiosperms as it does in Arabidopsis. Furthermore, fading expression of components of the stamen transcriptome in central and peripheral regions of Persea flowers resembles the developmental program of the hypothesized gymnosperm “floral progenitor”. Accordingly, in contrast to the canalized organ-specific regulatory apparatus of Arabidopsis, floral development may have been originally regulated by overlapping transcriptional cascades with fading gradients of influence from focal to bordering organs.
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.
Project description:The debate on the origin and evolution of flowers has recently entered the field of developmental genetics, with focus on the design of the ancestral floral regulatory program. Flowers can differ dramatically among angiosperm lineages, but in general, sterile perianth organs surrounding stamens (male reproductive organs) and carpels (female reproductive organs) constitute the basic floral structure. However, the basal angiosperm lineages exhibit spectacular diversity in the number, arrangement, and structure, of floral organs, while the evolutionarily derived monocot and eudicot lineages share a far more uniform floral ground plan. As such, regulatory mechanisms underlying the archetypal floral plan, for instance that of the eudicot genetic model Arabidopsis thaliana, are unlikely to apply to the original flowers. Here we show that broadly overlapping transcriptional programs characterise the floral transcriptome of the basal angiosperm Persea americana (avocado), while floral gene expression domains are typically organ-specific in Arabidopsis. Our findings extend the âfading bordersâ model for basal angiosperms from organ identity genes to the downstream floral transcriptome, and suggest that the combinatorial mechanism for organ identity may not operate in basal angiosperms as it does in Arabidopsis. Furthermore, fading expression of components of the stamen transcriptome in central and peripheral regions of Persea flowers resembles the developmental program of the hypothesized gymnosperm âfloral progenitorâ. Accordingly, in contrast to the canalized organ-specific regulatory apparatus of Arabidopsis, floral development may have been originally regulated by overlapping transcriptional cascades with fading gradients of influence from focal to bordering organs. Expression profiles of inflorescence buds, pre-meiotic floral buds, inner and outer tepals, stamens, carpels, initiating fruit, and leaves were assessed in an interwoven double loop design for eight samples with 16 arrays. Sample materials were collected from two individuals (biological replicates) cultivated on the University of Floridaâs Gainesville campus, and RNA was isolated twice for technical replication. Thus, four RNA extractions from each of the eight tissue types listed above were individually labeled with Cy3 (twice) or Cy5 (twice) and hybridized with four other Cy3 or Cy5 labeled samples as a dual channel array system.
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: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: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 Polycomb Repressive Complex 2 (PRC2) represses the transcriptional activity of target genes through trimethylation of Lysine 27 of Histone H3. The functions of the plant PRC2 have been chiefly described in Arabidopsis but specific PRC2 functions in other plant species, especially the cereals, are still largely unknown. Here we characterize mutants in the rice EMF2B gene, an ortholog of the Arabidopsis PRC2 gene EMBRYONIC FLOWER2 (EMF2). Loss of EMF2B in rice results in complete sterility, and mutant flowers have severe floral organ defects and indeterminacy that resemble loss of function mutants in rice E-function floral organ specification genes. Transcriptome analysis identified the E-function genes OsMADS1, OsMADS6 and OsMADS34 that are involved in floral development as differentially expressed in the emf2b mutant compared to wild type. OsMADS1 and OsMADS6 are known to be required for meristem determinacy in rice, and are down-regulated in the emf2b mutant, whereas OsMADS34 which interacts genetically with OsMADS1 was up-regulated. Chromatin immunoprecipitation for H3K27me3 followed by deep sequencing showed that all three genes are amongst the presumptive targets of PRC2 in the meristem. We propose that the PRC2 operates through a mechanism that involves regulation of E-function genes to play a major role in floral organ specification and floral meristem determinacy in rice, and possibly in other cereals. RNA-seq: The transcriptome of WT and emf2b rice panicles were compared via RNA-seq.
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.