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At least 19 recordsLinked to original sources

PubMed2000

Two rice MADS domain proteins interact with OsMADS1.

OsMADS1 is a MADS box gene controlling flower development in rice. In order to learn more about the function of OsMADS1, we searched for cellular proteins interacting with OsMADS1 employing the yeast two-hybrid system. Two novel proteins with MADS domains, which were named OsMADS14 and OsMADS15, were isolated from a rice cDNA library. OsMADS14 and -15 are highly homologous to the maize MADS box gene ZAP1 which is an orthologue of the floral homeotic gene APETALA1 (AP1). Interactions among the three MADS domain proteins were confirmed by in vitro experiments using GST-fused OsMADS1 expressed in Escherichia coli and in vitro translated proteins of OsMADS14 and -15. We determined which domains in OsMADS1, -14, and -15 were required for protein-protein interaction employing the two-hybrid system and pull-down experiments. While the K domain was essential for protein-protein interaction, a region preceded by the K domain augmented this interaction. Interestingly, the C-terminal region of OsMADS1 functioned as a transcriptional activation domain in yeast and mammalian cells, while, on the other hand, the C domains of OsMADS14 and -15 exhibited only very weak transcriptional activator functionality, if any at all.

Amino Acid Sequence↗

PubMed1996

DNA binding properties of two Arabidopsis MADS domain proteins: binding consensus and dimer formation.

MADS domain proteins are members of a highly conserved family found in all eukaryotes. Genetic studies clearly indicate that many plant MADS domain proteins have different regulatory functions in flower development, yet they share a highly conserved DNA binding domain and can bind to very similar sequences. How, then, can these MADS box genes confer their specific functions? Here, we describe results from DNA binding studies of AGL1 and AGL2 (for AGAMOUS-like), two Arabidopsis MADS domain proteins that are preferentially expressed in flowers. We demonstrate that both proteins are sequence-specific DNA binding proteins and show that each binding consensus has distinct features, suggestion a mechanism for specificity. In addition, we show that the proteins with more similar amino acid sequences have more similar binding sequences. We also found that AGL2 binds to DNA in vitro as a dimer and determined the region of AGL2 that is sufficient for DNA binding and dimerization. Finally, we show that several plant MADS domain proteins can bind to DNA either as homodimers or as heterodimers, suggesting that the number of different regulators could be much greater than the number of MADS box genes.

Amino Acid Sequence↗

PubMed1997

MADS domain proteins in plant development.

The MADS domain (MCM1, AGAMOUS, DEFICIENS, and SRF, serum response factor) is a conserved DNA-binding/dimerization region present in a variety of transcription factors from different kingdoms. MADS box genes represent a large multigene family in vascular plants. In angiosperms, many of the genes of the MADS family are involved in different steps of flower development, most notably in the determination of floral meristem and organ identity. The roles that MADS box genes play, however, are not restricted to control the development of the plant reproductive structures. The genetic, molecular, and biochemical basis of the action of the MADS domain proteins in the plant life cycle are reviewed here.

Amino Acid Sequence↗

PubMed2005

MIKC-type MADS-domain proteins: structural modularity, protein interactions and network evolution in land plants.

MIKC-type proteins represent a class of MADS-domain transcription factors and are defined by a unique domain structure: in addition to the highly conserved DNA-binding MADS-domain, they have three other domains ('I', 'K' and 'C'), with the keratin-like K-domain being the most highly conserved and characteristic one. The number and functional diversity of MIKC-type proteins increased considerably during land plant evolution, culminating in higher flowering plants, where they dominate the control of reproductive development from early to late stages. We wonder how one special class of proteins became important in the control of essentially all stages of a morphogenetic process. All MADS-domain proteins appear to bind to DNA as homo- or heterodimers and may function as part of ternary transcription factor complexes involving non-MADS-domain proteins. Only MIKC-type proteins, however, generate complex intrafamily interaction networks. These are based on the special potential of MIKC-type proteins to form complexes involving more than two homologous proteins constituting transcriptional regulators. We speculate that the potential to form heteromultimers of homologous proteins was achieved by the acquisition of the K-domain during evolution. There is emerging evidence that organismal complexity arises from progressively more elaborate regulation of gene expression. We hypothesize that combinatorial multimer formation of MIKC-type MADS-domain proteins facilitated an unusually efficient and rapid functional diversification based on gene duplication, sequence divergence and fixation. This 'networking' may have enabled a more sophisticated transcriptional control of target genes which was recruited for controlling increasingly complex and diverse developmental pathways during the rapid origin and diversification of plant reproductive structures. Therefore, MIKC-type proteins may owe their evolutionary 'success' and present-day developmental importance in part to their modular domain structure. Investigating the evolution of MIKC-type genes may thus help to better understand origin and diversification of gene regulatory networks.

Amino Acid Sequence↗

PubMed2003

Binding site selection for the plant MADS domain protein AGL15: an in vitro and in vivo study.

AGL15 (for AGAMOUS-like 15) is currently the only reported member of the plant MADS domain family of transcriptional regulators that preferentially accumulates during embryo development. Additionally, AGL15 is one of the more divergent members of the MADS domain family, including within the DNA-binding domain. Previous studies have shown that MADS domain proteins bind to DNA sequences with an overall consensus of CC(A/T)6GG (called a CArG motif). Nonetheless, different MADS domain proteins exhibit similar yet distinct binding site preferences that may be critical for differential gene regulation. To determine the consensus sequence preferentially bound by AGL15 in vitro, PCR-assisted binding site selection assays were performed. AGL15 was observed to prefer a CArG motif with a longer A/T-rich core and is to date the only plant MADS domain protein having such a preference. Next, the Arabidopsis genome data base was searched for genes containing AGL15 binding sites as candidates for direct regulation by AGL15. One gene, DTA4 (for Downstream Target of AGL15-4), was identified by this method, and then confirmed as a direct target of AGL15 in vivo.

Amino Acid Motifs↗

PubMed1996

The MADS domain protein AGL15 localizes to the nucleus during early stages of seed development.

Little is known about regulatory factors that act during the earliest stages of plant embryogenesis. The MADS domain protein AGL15 (for AGAMOUS-like) is expressed preferentially during embryogenesis and accumulates during early seed development in monocotyledonous and dicotyledonous flowering plants. AGL15-specific antibodies and immunohistochemistry were used to demonstrate that AGL15 accumulates before fertilization in the cytoplasm in the cells of the egg apparatus and moves into the nucleus during early stages of development in the suspensor, embryo, and endosperms. Relatively high levels of AGL15 are present in the nuclei during embryo morphogenesis and until the seeds start to dry in Brassica, maize, and Arabidopsis. AGL15 is associated with the chromosomes during mitosis, and gel mobility shift assays were used to demonstrate that AGL15 binds DNA in a sequence-specific manner. To assess whether AGL15 is likely to play a role in specifying the seed or embryonic phase of development, AGL15 accumulation was examined in Arabidopsis mutants that prematurely exit embryogenesis. lec1-2 mutants show an embryo-specific loss of AGL15 at the transition stage, suggesting that AGL15 interacts with regulators in the leafy cotyledons pathway.

Arabidopsis↗

PubMed2002

The TRANSPARENT TESTA16 locus encodes the ARABIDOPSIS BSISTER MADS domain protein and is required for proper development and pigmentation of the seed coat.

Screening for seed pigmentation phenotypes in Arabidopsis led to the isolation of three allelic yellow-seeded mutants, which defined the novel TRANSPARENT TESTA16 (TT16) locus. Cloning of TT16 was performed by T-DNA tagging and confirmed by genetic complementation and sequencing of two mutant alleles. TT16 encodes the ARABIDOPSIS BSISTER (ABS) MADS domain protein. ABS belongs to the recently identified "B-sister" (B(S)) clade, which contains genes of unknown function that are expressed mainly in female organs. Phylogenetic analyses using a maximum parsimony approach confirmed that TT16/ABS and related proteins form a monophyletic group. TT16/ABS was expressed mainly in the ovule, as are the other members of the B(S) clade. TT16/ABS is necessary for BANYULS expression and proanthocyanidin accumulation in the endothelium of the seed coat, with the exception of the chalazal-micropylar area. In addition, mutant phenotype and ectopic expression analyses suggested that TT16/ABS also is involved in the specification of endothelial cells. Nevertheless, TT16/ABS apparently is not required for proper ovule function. We report the functional characterization of a member of the B(S) MADS box gene subfamily, demonstrating its involvement in endothelial cell specification as well as in the increasingly complex genetic control of flavonoid biosynthesis in the Arabidopsis seed coat.

Amino Acid Sequence↗

PubMed1999

FLOWERING LOCUS C encodes a novel MADS domain protein that acts as a repressor of flowering.

Winter-annual ecotypes of Arabidopsis are relatively late flowering, unless the flowering of these ecotypes is promoted by exposure to cold (vernalization). This vernalization-suppressible, late-flowering phenotype results from the presence of dominant, late-flowering alleles at two loci, FRIGIDA (FRI) and FLOWERING LOCUS C (FLC). In this study, we report that flc null mutations result in early flowering, demonstrating that the role of active FLC alleles is to repress flowering. FLC was isolated by positional cloning and found to encode a novel MADS domain protein. The levels of FLC mRNA are regulated positively by FRI and negatively by LUMINIDEPENDENS. FLC is also negatively regulated by vernalization. Overexpression of FLC from a heterologous promoter is sufficient to delay flowering in the absence of an active FRI allele. We propose that the level of FLC activity acts through a rheostat-like mechanism to control flowering time in Arabidopsis and that modulation of FLC expression is a component of the vernalization response.

Arabidopsis↗

PubMed2000

The AGAMOUS-LIKE 20 MADS domain protein integrates floral inductive pathways in Arabidopsis.

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.

AGAMOUS Protein, Arabidopsis↗

PubMed2002

A chromatin immunoprecipitation (ChIP) approach to isolate genes regulated by AGL15, a MADS domain protein that preferentially accumulates in embryos.

AGAMOUS-like-15 (AGL15) is a member of the MADS-domain family of DNA-binding regulatory factors that accumulates preferentially in tissue developing in an embryonic mode. To better understand how AGL15 functions, we developed a chromatin immunoprecipitation (ChIP) approach to isolate genes regulated directly by AGL15. ChIP allows purification of in vivo protein-DNA complexes. The co-purified DNA is recovered and used to isolate the putatively regulated gene. Several tests must be performed to show that the putative downstream target gene is truly regulated by the DNA-binding protein. The DNA-binding regulatory protein must interact with cis regulatory elements. The downstream gene expression pattern should respond to the level of the trans-acting regulatory factor. The cis element should be able to confer regulation in response to the trans-acting factor. We describe, in this report, our ChIP protocol, and discuss in detail, tests to confirm regulation by AGL15 for two targets identified by ChIP. These targets are referred to as Downstream Target of AGL15 (DTA1 and DTA2). Expression of DTA1, which encodes a protein with high similarity to GA-2 oxidase-like proteins, is induced by AGL15. DTA2 encodes a novel protein and expression of this target is repressed by AGL15.

Arabidopsis↗

PubMed1999

The MADS-domain protein AGAMOUS-like 15 accumulates in embryonic tissues with diverse origins.

AGL15 (AGAMOUS-like 15), a member of the MADS-domain family of regulatory factors, accumulates preferentially in the organs and tissues derived from double fertilization in flowering plants (i.e. the embryo, suspensor, and endosperm). The developmental role of AGL15 is still undefined. If it is involved in embryogenesis rather than some other aspect of seed biology, then AGL15 protein should accumulate whenever development proceeds in the embryonic mode, regardless of the origin of those embryos or their developmental context. To test this, we used AGL15-specific antibodies to analyze apomictic embryogenesis in dandelion (Taraxacum officinale), microspore embryogenesis in oilseed rape (Brassica napus), and somatic embryogenesis in alfalfa (Medicago sativa). In every case, AGL15 accumulated to relatively high levels in the nuclei of the embryos. AGL15 also accumulated in cotyledon-like organs produced by the xtc2 (extra cotyledon2) mutant of Arabidopsis and during precocious germination in oilseed rape. Furthermore, the subcellular localization of AGL15 appeared to be developmentally regulated in all embryogenic situations. AGL15 was initially present in the cytoplasm of cells and became nuclear localized before or soon after embryogenic cell divisions began. These results support the hypothesis that AGL15 participates in the regulation of programs active during the early stages of embryo development.

Animals↗

PubMed1995

AGL15, a MADS domain protein expressed in developing embryos.

To extend our knowledge of genes expressed during early embryogenesis, the differential display technique was used to identify and isolate mRNA sequences that accumulate preferentially in young Brassica napus embryos. One of these genes encodes a new member of the MADS domain family of regulatory proteins; it has been designated AGL15 (for AGAMOUS-like). AGL15 shows a novel pattern of expression that is distinct from those of previously characterized family members. RNA gel blot analyses and in situ hybridization techniques were used to demonstrate that AGL15 mRNA accumulated primarily in the embryo and was present in all embryonic tissues, beginning at least as early as late globular stage in B. napus. Genomic and cDNA clones corresponding to two AGL15 genes from B. napus and the homologous single-copy gene from Arabidopsis, which is located on chromosome 5, were isolated and analyzed. Antibodies prepared against overexpressed Brassica AGL15 lacking the conserved MADS domain were used to probe immunoblots, and AGL15-related proteins were found in embryos of a variety of angiosperms, including plants as distantly related as maize. Based on these data, we suggest that AGL15 is likely to be an important component of the regulatory circuitry directing seed-specific processes in the developing embryo.

Amino Acid Sequence↗

PubMed2004

The embryo MADS domain protein AGAMOUS-Like 15 directly regulates expression of a gene encoding an enzyme involved in gibberellin metabolism.

AGL15 (for AGAMOUS-Like 15) is a member of the MADS domain family of DNA binding transcriptional regulators that accumulates to its highest amounts during embryo development. To better understand how AGL15 functions, a chromatin immunoprecipitation approach was used to identify directly regulated genes. One DNA fragment that coprecipitated with AGL15 corresponded to a portion of the regulatory region of a gene named DTA1 (for Downstream Target of AGL15-1). The expression of DTA1 was positively correlated with AGL15 abundance during embryogenesis. In this report, a cis element for response to AGL15 was identified, and the activity of DTA1 as a gibberellin (GA) 2-oxidase was confirmed. DTA1 corresponds to AtGA2ox6 and was renamed to indicate this identity. Further experiments related the function of AtGA2ox6 to regulation by AGL15. Constitutive expression of AGL15 and of AtGA2ox6 altered endogenous GA amounts and caused GA-deficient phenotypes in Arabidopsis thaliana that could be at least partially rescued by application of biologically active GA. The phenotype of plants with decreased expression of AtGA2ox6 was the converse of plants overexpressing AtGA2ox6 in terms of seed germination attributes and effects on somatic embryo production.

Arabidopsis↗

PubMed1997

Specific interactions between the K domains of AG and AGLs, members of the MADS domain family of DNA binding proteins.

MADS domain (for MCM1, AG, DEFA and SRF) proteins are regulatory proteins found in all major eukaryotic kingdoms. Plant MADS domain regulatory proteins have a region of moderate sequence similarity that has been designated as the K domain, and its predicted coiled-coil structure suggests a role in establishing a protein-protein interaction. In vivo studies with the Arabidopsis AGAMOUS (AG) protein have indicated that the K domain is important for AG function. Using a bait fusion protein containing the K domain and the C-terminal region of AG in a yeast two-hybrid selection, 156 clones that encode potential AG-interacting proteins were identified. These clones each encode one of four highly related MADS domain proteins: AGL2, AGL4, AGL6 and AGL9. Additional analysis showed that the K domain of AG alone was able to bind the K domains of these AGLs. This binding was further confirmed by immunoprecipitation experiments using in vitro synthesized AG and AGL K domains. These results strongly suggest that AG interacts with AGL2, AGL4, AGL6 and AGL9 in vivo. Based on these results and previous observations, it is proposed that the AG function requires interaction with at least one of these AGL proteins, and such interactions contribute to the functional specificity of the AG protein.

AGAMOUS Protein, Arabidopsis↗

PubMed1996

DNA-binding properties of Arabidopsis MADS domain homeotic proteins APETALA1, APETALA3, PISTILLATA and AGAMOUS.

The MADS domain proteins APETALA1 (AP1), APETALA3 (AP3), PISTILLATA (PI), and AGAMOUS (AG) specify the identity of Arabidopsis floral organs. AP1 and AG homocomplexes and AP3-PI heterocomplexes bind to CArG-box sequences. The DNA-binding properties of these complexes were investigated. We find that AP1, AG and AP3-PI are all capable of recognizing the same DNA-binding sites, although with somewhat different affinities. In addition, the three complexes induce similar conformational changes on a CArG-box sequence. Phasing analysis reveals that the induced distortion is DNA bending, oriented toward the minor groove. The molecular dissection of AP1, AP3, PI and AG indicates that the boundaries of the dimerization domains of these proteins vary. The regions required to form a DNA-binding complex include, in addition to the MADS box, the entire L region (which follows the MADS box) and the first putative amphipathic helix of the K box in the case of AP3-PI, while for AP1 and AG only a part of the L region is needed. The similarity of the DNA-binding properties of AP1, AP3-PI and AG is discussed with regard to the biological specificity that these proteins exhibit.

AGAMOUS Protein, Arabidopsis↗

PubMed1996

Dimerization specificity of Arabidopsis MADS domain homeotic proteins APETALA1, APETALA3, PISTILLATA, and AGAMOUS.

The MADS domain homeotic proteins APETALA1 (AP1), APETALA3 (AP3), PISTILLATA (PI), and AGAMOUS (AG) act in a combinatorial manner to specify the identity of Arabidopsis floral organs. The molecular basis for this combinatorial mode of action was investigated. Immunoprecipitation experiments indicate that all four proteins are capable of interacting with each other. However, these proteins exhibit "partner-specificity" for the formation of DNA-binding dimers; only AP1 homodimers, AG homodimers, and AP3/PI heterodimers are capable of binding to CArG-box sequences. Both the AP3/PI heterodimer and the AP1 or AG homodimers are formed when the three corresponding proteins are present together. The use of chimeric proteins formed by domain swapping indicates that the L region (which follows the MADS box) constitutes a key molecular determinant for the selective formation of DNA-binding dimers. The implications of these results for the ABC genetic model of flower development are discussed.

AGAMOUS Protein, Arabidopsis↗

PubMed2004

Defining subdomains of the K domain important for protein-protein interactions of plant MADS proteins.

The MADS proteins APETALA3 (AP3), PISTILLATA (PI), SEPALLATAI (SEPI), SEP2, SEP3, AGAMOUS, and APETALA are required for proper floral organ identity in Arabidopsis flowers. All of these floral MADS proteins conserve two domains: the MADS domain that mediates DNA binding and dimerization, and the K domain that mediates protein protein interaction. The K domain is postulated to form a several amphipathic c-helices referred to as K1, K2, and K3. The K1 and K2 helicies are located entirely within the K domain while the K3 helix spans the K domain-C domain boundary. Here we report on our studies on the interactions of the B class MADS proteins AP3 and PI with the E class MADS proteins SEP1, SEP2, and SEP3. A comparative analysis of mutants in the K domain reveals that the subdomains mediating the PI/AP3 interaction are different from the subdomains mediating the PI/SEP3 (or PI/SEP1) interaction. The strong PI/SEP3 (or PI/SEP1) interaction requires K2, part of K3, and the interhelical region between K1 and K2. By contrast, K1, K2 and the region between K1 and K2 are important for strong AP3/PI interaction. Most of the K3 helix does not appear to be important for either the PI/AP3 or the PI/SEP3 (or PI/SEP1) interaction. Conserved hydrophobic positions are most important for the strength of both PI/AP3 and PI/SEP3 dimerization, though ionic and/or polar interactions appear to play a secondary role.

AGAMOUS Protein, Arabidopsis↗

PubMed1996

Functional domains of the floral regulator AGAMOUS: characterization of the DNA binding domain and analysis of dominant negative mutations.

The Arabidopsis MADS box gene AGAMOUS (AG) controls reproductive organ identity and floral meristem determinacy. The AG protein binds in vitro to DNA sequences similar to the targets of known MADS domain transcription factors. Whereas most plant MADS domain proteins begin with the MADS domain, AG and its orthologs contain a region N-terminal to the MADS domain. All plant MADS domain proteins share another region with moderate sequence similarity called the K domain. Neither the region (I region) that lies between the MADS and K domains nor the C-terminal region is conserved. We show here that the AG MADS domain and the I region are necessary and sufficient for DNA binding in vitro and that AG binds to DNA as a dimer. To investigate the in vivo function of the regions of AG not required for in vitro DNA binding, we introduced several AG constructs into wild-type plants and characterized their floral phenotypes. We show that transgenic Arabidopsis plants with a 35S-AG construct encoding an AG protein lacking the N-terminal region produced apetala 2 (ap2)-like flowers similar to those ectopically expressing AG proteins retaining the N-terminal region. This result suggests that the N-terminal region is not required to produce the ap2-like phenotype. In addition, transformants with a 35S-AG construct encoding an AG protein lacking the C-terminal region produced ag-like flowers, indicating that this truncated AG protein inhibits normal AG function. Finally, transformants with a 35S-AG construct encoding an AG protein lacking both K and C regions produced flowers with more stamens and carpels. The phenotypes of the AG transformants demonstrate that both the K domain and the C-terminal region have important and distinct in vivo functions. We discuss possible mechanisms through which AG may regulate downstream genes.

Arabidopsis↗