Project description:The MADS domain transcriptional regulator AGAMOUS-Like 15 promotes somatic embryogenesis by binding DNA and controlling downstream gene expression. Chromatin immunoprecipitation (ChIP) has been used to identify DNA fragments with which AGL15 is associated in vivo and a low-throughput approach to identify these fragments and determine regulatory consequences has revealed a role for AGL15 in GA catabolism that is relevant to embryogenesis. However, to understand more globally the gene networks in which AGL15 is involved, higher throughput methods to identify direct and indirect targets are needed. Here we report mapping of AGL15 in vivo binding sites using a ChIP-chip approach with Affymetrix tiling arrays for Arabidopsis and find that ~2000 sites represented in three biological replicates of the experiment are annotated to nearby genes.
Project description:The very late-flowering behavior of Arabidopsis winter-annual ecotypes is conferred mainly by two genes, FRIGIDA (FRI) and FLOWERING LOCUS C (FLC). A MADS-domain gene, AGAMOUS-LIKE 20 (AGL20), was identified as a dominant FRI suppressor in activation tagging mutagenesis. Overexpression of AGL20 suppresses not only the late flowering of plants that have functional FRI and FLC alleles but also the delayed phase transitions during the vegetative stages of plant development. Interestingly, AGL20 expression is positively regulated not only by the redundant vernalization and autonomous pathways of flowering but also by the photoperiod pathway. Our results indicate that AGL20 is an important integrator of three pathways controlling flowering in Arabidopsis.
Project description:AGAMOUS-like 15 (AGL15) is a member of the MADS domain family of transcription factors (TFs) that can directly induce and repress target gene expression, and for which promotion of somatic embryogenesis (SE) is positively correlated with accumulation. An ethylene-responsive element binding factor-associated amphiphilic repression (EAR) motif of form LxLxL within the carboxyl-terminal domain of AGL15 was shown to be involved in repression of gene expression. Here, we examine whether AGL15's ability to repress gene expression is needed to promote SE. While a form of AGL15 where the LxLxL is changed to AxAxA can still promote SE, another form with a strong transcriptional activator at the carboxy-terminal end, does not promote SE and, in fact, is detrimental to SE development. Select target genes were examined for response to the different forms of AGL15.
Project description:BackgroundMADS domain proteins are transcription factors that coordinate several important developmental processes in plants. These proteins interact with other MADS domain proteins to form dimers, and it has been proposed that they are able to associate as tetrameric complexes that regulate transcription of target genes. Whether the formation of functional tetramers is a widespread property of plant MADS domain proteins, or it is specific to few of these transcriptional regulators remains unclear.ResultsWe analyzed the structure of the network of physical interactions among MADS domain proteins in Arabidopsis thaliana. We determined the abundance of subgraphs that represent the connection pattern expected for a MADS domain protein heterotetramer. These subgraphs were significantly more abundant in the MADS domain protein interaction network than in randomized analogous networks. Importantly, these subgraphs are not significantly frequent in a protein interaction network of TCP plant transcription factors, when compared to expectation by chance. In addition, we found that MADS domain proteins in tetramer-like subgraphs are more likely to be expressed jointly than proteins in other subgraphs. This effect is mainly due to proteins in the monophyletic MIKC clade, as there is no association between tetramer-like subgraphs and co-expression for proteins outside this clade.ConclusionsOur results support that the tendency to form functional tetramers is widespread in the MADS domain protein-protein interaction network. Our observations also suggest that this trend is prevalent, or perhaps exclusive, for proteins in the MIKC clade. Because it is possible to retrodict several experimental results from our analyses, our work can be an important aid to make new predictions and facilitates experimental research on plant MADS domain proteins.
Project description:Until recently, the roles of plant MADS-box genes have mainly been characterized during inflorescence and flower differentiation. In order to precise the roles of AGAMOUS-LIKE 12, one of the few MADS-box genes preferentially expressed in roots, we placed its cDNA under the control of the double 35S CaMV promoter to produce transgenic walnut tree and Arabidopsis plants. In Juglans sp., transgenic somatic embryos showed significantly higher germination rates but abnormal development of their shoot apex prevented their conversion into plants. In addition, a wide range of developmental abnormalities corresponding to ectopic root-like structures affected the transgenic lines suggesting partial reorientations of the embryonic program toward root differentiation. In Arabidopsis, AtAGL12 overexpression lead to the production of faster growing plants presenting dramatically wider and shorter root phenotypes linked to increased meristematic cell numbers within the root apex. In the upper part of the roots, abnormal cell divisions patterns within the pericycle layer generated large ectopic cell masses that did not prevent plants to grow. Taken together, our results confirm in both species that AGL12 positively regulates root meristem cell division and promotes overall root vascular tissue formation. Genetic engineering of AGL12 expression levels could be useful to modulate root architecture and development.
Project description:The MADS transcription factors (TF) constitute an ancient family of TF found in all eukaryotes that bind DNA as obligate dimers. Plants have dramatically expanded the functional diversity of the MADS family during evolution by adding protein-protein interaction domains to the core DNA-binding domain, allowing the formation of heterotetrameric complexes. Tetramerization of plant MADS TFs is believed to play a central role in the evolution of higher plants by acting as one of the main determinants of flower formation and floral organ specification. The MADS TF, SEPALLATA3 (SEP3), functions as a central protein-protein interaction hub, driving tetramerization with other MADS TFs. Here, we use a SEP3 splice variant, SEP3?tet, which has dramatically abrogated tetramerization capacity to decouple SEP3 tetramerization and DNA-binding activities. We unexpectedly demonstrate that SEP3 heterotetramer formation is required for correct termination of the floral meristem, but plays a lesser role in floral organogenesis. The heterotetramer formed by SEP3 and the MADS protein, AGAMOUS, is necessary to activate two target genes, KNUCKLES and CRABSCLAW, which are required for meristem determinacy. These studies reveal unique and highly specific roles of tetramerization in flower development and suggest tetramerization may be required to activate only a subset of target genes in closed chromatin regions.
Project description:Floral organs are specified by the combinatorial action of MADS-domain transcription factors, yet the mechanisms by which MADS-domain proteins activate or repress the expression of their target genes and the nature of their cofactors are still largely unknown. Here, we show using affinity purification and mass spectrometry that five major floral homeotic MADS-domain proteins (AP1, AP3, PI, AG, and SEP3) interact in floral tissues as proposed in the "floral quartet" model. In vitro studies confirmed a flexible composition of MADS-domain protein complexes depending on relative protein concentrations and DNA sequence. In situ bimolecular fluorescent complementation assays demonstrate that MADS-domain proteins interact during meristematic stages of flower development. By applying a targeted proteomics approach we were able to establish a MADS-domain protein interactome that strongly supports a mechanistic link between MADS-domain proteins and chromatin remodeling factors. Furthermore, members of other transcription factor families were identified as interaction partners of floral MADS-domain proteins suggesting various specific combinatorial modes of action.
Project description:BACKGROUND:Correct flower formation requires highly specific temporal and spatial regulation of gene expression. In Arabidopsis thaliana the majority of the master regulators that determine flower organ identity belong to the MADS-domain transcription factor family. The canonical DNA binding motif for this transcription factor family is the CArG-box, which has the consensus CC(A/T)6GG. However, so far, a comprehensive analysis of MADS-domain binding patterns has not yet been performed. RESULTS:Eight publicly available ChIP-seq datasets of MADS-domain proteins that regulate the floral transition and flower formation were analyzed. Surprisingly, the preferred DNA binding motif of each protein was a CArG-box with an NAA extension. Furthermore, motifs of other transcription factors were found in the vicinity of binding sites of MADS-domain transcription factors, suggesting that interaction of MADS-domain proteins with other transcription factors is important for target gene regulation. Finally, conservation of CArG-boxes between Arabidopsis ecotypes was assessed to obtain information about their evolutionary importance. CArG-boxes that fully matched the consensus were more conserved than other CArG-boxes, suggesting that the perfect CArG-box is evolutionary more important than other CArG-box variants. CONCLUSION:Our analysis provides detailed insight into MADS-domain protein binding patterns. The results underline the importance of an extended version of the CArG-box and provide a first view on evolutionary conservation of MADS-domain protein binding sites in Arabidopsis ecotypes.
Project description:The MADS-box family of genes has been shown to play a significant role in the development of reproductive organs, including dry and fleshy fruits. In this study, the molecular properties of an AGAMOUS like MADS box transcription factor in banana cultivar Giant governor (Musa sp, AAA group, subgroup Cavendish) has been elucidated. We have detected a CArG-box sequence binding AGAMOUS MADS-box protein in banana flower and fruit nuclear extracts in DNA-protein interaction assays. The protein fraction in the DNA-protein complex was analyzed by mass spectrometry and using this information we have obtained the full length cDNA of the corresponding protein. The deduced protein sequence showed ~95% amino acid sequence homology with MA-MADS5, a MADS-box protein described previously from banana. We have characterized the domains of the identified AGAMOUS MADS-box protein involved in DNA binding and homodimer formation in vitro using full-length and truncated versions of affinity purified recombinant proteins. Furthermore, in order to gain insight about how DNA bending is achieved by this MADS-box factor, we performed circular permutation and phasing analysis using the wild type recombinant protein. The AGAMOUS MADS-box protein identified in this study has been found to predominantly accumulate in the climacteric fruit pulp and also in female flower ovary. In vivo and in vitro assays have revealed specific binding of the identified AGAMOUS MADS-box protein to CArG-box sequence in the promoters of major ripening genes in banana fruit. Overall, the expression patterns of this MADS-box protein in banana female flower ovary and during various phases of fruit ripening along with the interaction of the protein to the CArG-box sequence in the promoters of major ripening genes lead to interesting assumption about the possible involvement of this AGAMOUS MADS-box factor in banana fruit ripening and floral reproductive organ development.