RESUMEN
The ALOG (Arabidopsis LIGHT-DEPENDENT SHORT HYPOCOTYLS 1 (LSH1) and Oryza G1) proteins are conserved plant-specific Transcription Factors (TFs). They play critical roles in the development of various plant organs (meristems, inflorescences, floral organs, and nodules) from bryophytes to higher flowering plants. Despite the fact that the first members of this family were originally discovered in Arabidopsis, their role in this model plant has remained poorly characterized. Moreover, how these transcriptional regulators work at the molecular level is unknown. Here, we study the redundant function of the ALOG proteins LSH1,3,4 from Arabidopsis. We uncover their role in the repression of bract development and position them within a gene regulatory network controlling this process and involving the floral regulators LEAFY, BLADE-ON-PETIOLE, and PUCHI. Next, using in vitro genome-wide studies, we identified the conserved DNA motif bound by ALOG proteins from evolutionarily distant species (the liverwort Marchantia polymorpha and the flowering plants Arabidopsis, tomato, and rice). Resolution of the crystallographic structure of the ALOG DNA-binding domain in complex with DNA revealed the domain is a four-helix bundle with a disordered NLS and a zinc ribbon insertion between helices 2 and 3. The majority of DNA interactions are mediated by specific contacts made by the third alpha helix and the NLS. Taken together, this work provides the biochemical and structural basis for DNA-binding specificity of an evolutionarily conserved TF family and reveals its role as a key player in Arabidopsis flower development.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Embryophyta , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Arabidopsis/metabolismo , Proteínas de Plantas/metabolismo , Plantas/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Embryophyta/genética , Inflorescencia/metabolismo , ADN/metabolismo , Regulación de la Expresión Génica de las Plantas , Flores , Proteínas Nucleares/metabolismoRESUMEN
Auxin is a major developmental regulator in plants and the acquisition of a transcriptional response to auxin likely contributed to developmental innovations at the time of water-to-land transition. Auxin Response Factors (ARFs) Transcription Factors (TFs) that mediate auxin-dependent transcriptional changes are divided into A, B and C evolutive classes in land plants. The origin and nature of the first ARF proteins in algae is still debated. Here, we identify the most 'ancient' ARF homologue to date in the early divergent charophyte algae Chlorokybus atmophyticus, CaARF. Structural modelling combined with biochemical studies showed that CaARF already shares many features with modern ARFs: it is capable of oligomerization, interacts with the TOPLESS co-repressor and specifically binds Auxin Response Elements as dimer. In addition, CaARF possesses a DNA-binding specificity that differs from class A and B ARFs and that was maintained in class C ARF along plants evolution. Phylogenetic evidence together with CaARF biochemical properties indicate that the different classes of ARFs likely arose from an ancestral proto-ARF protein with class C-like features. The foundation of auxin signalling would have thus happened from a pre-existing hormone-independent transcriptional regulation together with the emergence of a functional hormone perception complex.
Asunto(s)
Carofíceas/genética , Ácidos Indolacéticos/metabolismo , Proteínas de Plantas/genética , Receptores de Superficie Celular/genética , Proteínas de Unión al ADN/genética , Evolución Molecular , Regulación de la Expresión Génica de las Plantas/genética , Familia de Multigenes/genética , Filogenia , Reguladores del Crecimiento de las Plantas/genética , Elementos de Respuesta/genética , Factores de Transcripción/genéticaRESUMEN
How biological systems generate reproducible patterns with high precision is a central question in science. The shoot apical meristem (SAM), a specialized tissue producing plant aerial organs, is a developmental system of choice to address this question. Organs are periodically initiated at the SAM at specific spatial positions and this spatiotemporal pattern defines phyllotaxis. Accumulation of the plant hormone auxin triggers organ initiation, whereas auxin depletion around organs generates inhibitory fields that are thought to be sufficient to maintain these patterns and their dynamics. Here we show that another type of hormone-based inhibitory fields, generated directly downstream of auxin by intercellular movement of the cytokinin signalling inhibitor ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 6 (AHP6), is involved in regulating phyllotactic patterns. We demonstrate that AHP6-based fields establish patterns of cytokinin signalling in the meristem that contribute to the robustness of phyllotaxis by imposing a temporal sequence on organ initiation. Our findings indicate that not one but two distinct hormone-based fields may be required for achieving temporal precision during formation of reiterative structures at the SAM, thus indicating an original mechanism for providing robustness to a dynamic developmental system.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Transporte Biológico , Citocininas/antagonistas & inhibidores , Transducción de Señal , Arabidopsis/anatomía & histología , Arabidopsis/citología , Citocininas/metabolismo , Ácidos Indolacéticos/metabolismo , Meristema/metabolismo , Reguladores del Crecimiento de las Plantas/antagonistas & inhibidores , Reguladores del Crecimiento de las Plantas/metabolismo , Brotes de la Planta/metabolismoRESUMEN
Higher plants continuously and iteratively produce new above-ground organs in the form of leaves, stems and flowers. These organs arise from shoot apical meristems whose homeostasis depends on coordination between self-renewal of stem cells and their differentiation into organ founder cells. This coordination is stringently controlled by the central transcription factor WUSCHEL (WUS), which is both necessary and sufficient for stem cell specification in Arabidopsis thaliana ULTRAPETALA1 (ULT1) was previously identified as a plant-specific, negative regulator of WUS expression. However, molecular mechanisms underlying this regulation remain unknown. ULT1 protein contains a SAND putative DNA-binding domain and a B-box, previously proposed as a protein interaction domain in eukaryotes. Here, we characterise a novel partner of ULT1, named ULT1 INTERACTING FACTOR 1 (UIF1), which contains a Myb domain and an EAR motif. UIF1 and ULT1 function in the same pathway for regulation of organ number in the flower. Moreover, UIF1 displays DNA-binding activity and specifically binds to WUS regulatory elements. We thus provide genetic and molecular evidence that UIF1 and ULT1 work together in floral meristem homeostasis, probably by direct repression of WUS expression.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crecimiento & desarrollo , Proteínas de Homeodominio/metabolismo , Meristema/crecimiento & desarrollo , Morfogénesis/fisiología , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Proteínas de Arabidopsis/genética , Ensayo de Cambio de Movilidad Electroforética , Flores/crecimiento & desarrollo , Regulación de la Expresión Génica de las Plantas , Datos de Secuencia Molecular , Hojas de la Planta/crecimiento & desarrollo , Tallos de la Planta/crecimiento & desarrollo , Dominios y Motivos de Interacción de Proteínas , Estructura Terciaria de Proteína , Alineación de Secuencia , Células Madre/citología , Factores de Transcripción/genética , Transcripción Genética/genéticaRESUMEN
The Arabidopsis LEAFY (LFY) transcription factor is a key regulator of floral meristem emergence and identity. LFY interacts genetically and physically with UNUSUAL FLORAL ORGANS, a substrate adaptor of CULLIN1-RING ubiquitin ligase complexes (CRL1). The functionally redundant genes BLADE ON PETIOLE1 (BOP1) and -2 (BOP2) are potential candidates to regulate LFY activity and have recently been shown to be substrate adaptors of CULLIN3 (CUL3)-RING ubiquitin ligases (CRL3). We tested the hypothesis that LFY activity is controlled by BOPs and CUL3s in plants and that LFY is a substrate for ubiquitination by BOP-containing CRL3 complexes. When constitutively expressed, LFY activity is fully dependent on BOP2 as well as on CUL3A and B to regulate target genes such as APETALA1 and to induce ectopic flower formation. We also show that LFY and BOP2 proteins interact physically and that LFY-dependent ubiquitinated species are produced in vitro in a reconstituted cell-free CRL3 system in the presence of LFY, BOP2 and CUL3. This new post-translational regulation of LFY activity by CRL3 complexes makes it a unique transcription factor subjected to a positive dual regulation by both CRL1 and CRL3 complexes and suggests a novel mechanism for promoting flower development.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas Cullin/metabolismo , Regulación de la Expresión Génica de las Plantas , Factores de Transcripción/metabolismo , Transcripción Genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Proteínas Cullin/genética , Genes de Plantas , Humanos , Mutación/genética , Fenotipo , Células Vegetales/metabolismo , Hojas de la Planta/crecimiento & desarrollo , Plantas Modificadas Genéticamente , Unión Proteica , UbiquitinaciónRESUMEN
Flowering plants evolved from an unidentified gymnosperm ancestor. Comparison of the mechanisms controlling development in angiosperm flowers and gymnosperm cones may help to elucidate the mysterious origin of the flower. We combined gene expression studies with protein behaviour characterization in Welwitschia mirabilis to test whether the known regulatory links between LEAFY and its MADS-box gene targets, central to flower development, might also contribute to gymnosperm reproductive development. We found that WelLFY, one of two LEAFY-like genes in Welwitschia, could be an upstream regulator of the MADS-box genes APETALA3/PISTILLATA-like (B-genes). We demonstrated that, even though their DNA-binding domains are extremely similar, WelLFY and its paralogue WelNDLY exhibit distinct DNA-binding specificities, and that, unlike WelNDLY, WelLFY shares with its angiosperm orthologue the capacity to bind promoters of Welwitschia B-genes. Finally, we identified several cis-elements mediating these interactions in Welwitschia and obtained evidence that the link between LFY homologues and B-genes is also conserved in two other gymnosperms, Pinus and Picea. Although functional approaches to investigate cone development in gymnosperms are limited, our state-of-the-art biophysical techniques, coupled with expression studies, provide evidence that crucial links, central to the control of floral development, may already have existed before the appearance of flowers.
Asunto(s)
Flores/crecimiento & desarrollo , Genes de Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Streptophyta/anatomía & histología , Streptophyta/crecimiento & desarrollo , Arabidopsis/genética , Secuencia de Bases , Sitios de Unión/genética , Regulación de la Expresión Génica de las Plantas , Cinética , Filogenia , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Homología de Secuencia de Aminoácido , Streptophyta/genéticaRESUMEN
A conserved genetic toolkit underlies the development of diverse floral forms among angiosperms. However, the degree of conservation vs divergence in the configuration of these gene regulatory networks is less clear. We addressed this question in a parallel genetic study between the closely related species Arabidopsis thaliana and Cardamine hirsuta. We identified leafy (lfy) and apetala1 (ap1) alleles in a mutant screen for floral regulators in C. hirsuta. C. hirsuta lfy mutants showed a complete homeotic conversion of flowers to leafy shoots, mimicking lfy ap1 double mutants in A. thaliana. Through genetic and molecular experiments, we showed that AP1 activation is fully dependent on LFY in C. hirsuta, by contrast to A. thaliana. Additionally, we found that LFY influences heteroblasty in C. hirsuta, such that loss or gain of LFY function affects its progression. Overexpression of UNUSUAL FLORAL ORGANS also alters C. hirsuta leaf shape in an LFY-dependent manner. We found that LFY and AP1 are conserved floral regulators that act nonredundantly in C. hirsuta, such that LFY has more obvious roles in floral and leaf development in C. hirsuta than in A. thaliana.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Cardamine/metabolismo , Secuencia Conservada , Proteínas de Dominio MADS/metabolismo , Factores de Transcripción/metabolismo , Arabidopsis/genética , Cardamine/genética , Cardamine/ultraestructura , Flores/fisiología , Regulación de la Expresión Génica de las Plantas , Mutación/genética , Hojas de la Planta/anatomía & histología , Brotes de la Planta/fisiología , Especificidad de la EspecieRESUMEN
In Arabidopsis (Arabidopsis thaliana), transcriptional control of seed maturation involves three related regulators with a B3 domain, namely LEAFY COTYLEDON2 (LEC2), ABSCISIC ACID INSENSITIVE3 (ABI3), and FUSCA3 (ABI3/FUS3/LEC2 [AFLs]). Although genetic analyses have demonstrated partially overlapping functions of these regulators, the underlying molecular mechanisms remained elusive. The results presented here confirmed that the three proteins bind RY DNA elements (with a 5'-CATG-3' core sequence) but with different specificities for flanking nucleotides. In planta as in the moss Physcomitrella patens protoplasts, the presence of RY-like (RYL) elements is necessary but not sufficient for the regulation of the OLEOSIN1 (OLE1) promoter by the B3 AFLs. G box-like domains, located in the vicinity of the RYL elements, also are required for proper activation of the promoter, suggesting that several proteins are involved. Consistent with this idea, LEC2 and ABI3 showed synergistic effects on the activation of the OLE1 promoter. What is more, LEC1 (a homolog of the NF-YB subunit of the CCAAT-binding complex) further enhanced the activation of this target promoter in the presence of LEC2 and ABI3. Finally, recombinant LEC1 and LEC2 proteins produced in Arabidopsis protoplasts could form a ternary complex with NF-YC2 in vitro, providing a molecular explanation for their functional interactions. Taken together, these results allow us to propose a molecular model for the transcriptional regulation of seed genes by the L-AFL proteins, based on the formation of regulatory multiprotein complexes between NF-YBs, which carry a specific aspartate-55 residue, and B3 transcription factors.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Semillas/genética , Factores de Transcripción/metabolismo , Transcripción Genética , Secuencia de Bases , Sitios de Unión/genética , Briófitas/metabolismo , ADN de Plantas/metabolismo , Inmunoprecipitación , Modelos Biológicos , Regiones Promotoras Genéticas , Unión Proteica/genética , Protoplastos/metabolismoRESUMEN
In indeterminate inflorescences, floral meristems develop on the flanks of the shoot apical meristem, at positions determined by auxin maxima. The floral identity of these meristems is conferred by a handful of genes called floral meristem identity genes, among which the LEAFY (LFY) transcription factor plays a prominent role. However, the molecular mechanism controlling the early emergence of floral meristems remains unknown. A body of evidence indicates that LFY may contribute to this developmental shift, but a direct effect of LFY on meristem emergence has not been demonstrated. We have generated a LFY allele with reduced floral function and revealed its ability to stimulate axillary meristem growth. This role is barely detectable in the lfy single mutant but becomes obvious in several double mutant backgrounds and plants ectopically expressing LFY. We show that this role requires the ability of LFY to bind DNA, and is mediated by direct induction of REGULATOR OF AXILLARY MERISTEMS1 (RAX1) by LFY. We propose that this function unifies the diverse roles described for LFY in multiple angiosperm species, ranging from monocot inflorescence identity to legume leaf development, and that it probably pre-dates the origin of angiosperms.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Regulación del Desarrollo de la Expresión Génica , Meristema/genética , Factores de Transcripción/genética , Alelos , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Cristalografía , Proteínas de Unión al ADN , Flores/genética , Flores/crecimiento & desarrollo , Flores/metabolismo , Regulación de la Expresión Génica de las Plantas , Meristema/crecimiento & desarrollo , Meristema/metabolismo , Modelos Biológicos , Mutación , Motivos de Nucleótidos , Hojas de la Planta/genética , Hojas de la Planta/crecimiento & desarrollo , Hojas de la Planta/metabolismo , Plantas Modificadas Genéticamente , Multimerización de Proteína , Estructura Terciaria de Proteína , Plantones/genética , Plantones/crecimiento & desarrollo , Plantones/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
Despite great advances in sequencing technologies, generating functional information for nonmodel organisms remains a challenge. One solution lies in an improved ability to predict genetic circuits based on primary DNA sequence in combination with detailed knowledge of regulatory proteins that have been characterized in model species. Here, we focus on the LEAFY (LFY) transcription factor, a conserved master regulator of floral development. Starting with biochemical and structural information, we built a biophysical model describing LFY DNA binding specificity in vitro that accurately predicts in vivo LFY binding sites in the Arabidopsis thaliana genome. Applying the model to other plant species, we could follow the evolution of the regulatory relationship between LFY and the AGAMOUS (AG) subfamily of MADS box genes and show that this link predates the divergence between monocots and eudicots. Remarkably, our model succeeds in detecting the connection between LFY and AG homologs despite extensive variation in binding sites. This demonstrates that the cis-element fluidity recently observed in animals also exists in plants, but the challenges it poses can be overcome with predictions grounded in a biophysical model. Therefore, our work opens new avenues to deduce the structure of regulatory networks from mere inspection of genomic sequences.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Fenómenos Biofísicos , Regulación de la Expresión Génica de las Plantas , Genoma de Planta/genética , Modelos Genéticos , Factores de Transcripción/genética , Proteína AGAMOUS de Arabidopsis/genética , Proteína AGAMOUS de Arabidopsis/metabolismo , Secuencia de Bases , Sitios de Unión , Inmunoprecipitación de Cromatina , ADN de Plantas/genética , Evolución Molecular , Flores/genética , Flores/crecimiento & desarrollo , Genes de Plantas/genética , Intrones/genética , Datos de Secuencia Molecular , Unión Proteica , Secuencias Reguladoras de Ácidos Nucleicos/genética , Reproducibilidad de los ResultadosRESUMEN
In angiosperms, flower development requires the combined action of the transcription factor LEAFY (LFY) and the ubiquitin ligase adaptor F-box protein, UNUSUAL FLORAL ORGANS (UFO), but the molecular mechanism underlying this synergy has remained unknown. Here we show in transient assays and stable transgenic plants that the connection to ubiquitination pathways suggested by the UFO F-box domain is mostly dispensable. On the basis of biochemical and genome-wide studies, we establish that UFO instead acts by forming an active transcriptional complex with LFY at newly discovered regulatory elements. Structural characterization of the LFY-UFO-DNA complex by cryo-electron microscopy further demonstrates that UFO performs this function by directly interacting with both LFY and DNA. Finally, we propose that this complex might have a deep evolutionary origin, largely predating flowering plants. This work reveals a unique mechanism of an F-box protein directly modulating the DNA binding specificity of a master transcription factor.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Proteínas F-Box , Factores de Transcripción/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas F-Box/metabolismo , Microscopía por Crioelectrón , Regulación de la Expresión Génica de las Plantas , Flores/genéticaRESUMEN
The transition to flowering in Arabidopsis is characterized by the sharp and localized upregulation of APETALA1 (AP1) transcription in the newly formed floral primordia. Both the flower meristem-identity gene LEAFY (LFY) and the photoperiod pathway involving the FLOWERING LOCUS T (FT) and FD genes contribute to this upregulation. These pathways have been proposed to act independently but their respective contributions and mode of interaction have remained elusive. To address these questions, we studied the AP1 regulatory region. Combining in vitro and in vivo approaches, we identified which of the three putative LFY binding sites present in the AP1 promoter is essential for its activation by LFY. Interestingly, we found that this site is also important for the correct photoperiodic-dependent upregulation of AP1. In contrast, a previously proposed putative FD-binding site appears dispensable and unable to bind FD and we found no evidence for FD binding to other sites in the AP1 promoter, suggesting that the FT/FD-dependent activation of AP1 might be indirect. Altogether, our data give new insight into the interaction between the FT and LFY pathways in the upregulation of AP1 transcription under long-day conditions.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Flores/metabolismo , Proteínas de Dominio MADS/metabolismo , Factores de Transcripción/metabolismo , Proteínas de Arabidopsis/genética , Sitios de Unión , Regulación de la Expresión Génica de las Plantas , Proteínas de Dominio MADS/genética , Fotoperiodo , Plantas Modificadas Genéticamente , Regiones Promotoras Genéticas , Transducción de Señal , Factores de Transcripción/genética , Regulación hacia ArribaRESUMEN
The LEAFY (LFY) protein is a key regulator of flower development in angiosperms. Its gradually increased expression governs the sharp floral transition, and LFY subsequently controls the patterning of flower meristems by inducing the expression of floral homeotic genes. Despite a wealth of genetic data, how LFY functions at the molecular level is poorly understood. Here, we report crystal structures for the DNA-binding domain of Arabidopsis thaliana LFY bound to two target promoter elements. LFY adopts a novel seven-helix fold that binds DNA as a cooperative dimer, forming base-specific contacts in both the major and minor grooves. Cooperativity is mediated by two basic residues and plausibly accounts for LFY's effectiveness in triggering sharp developmental transitions. Our structure reveals an unexpected similarity between LFY and helix-turn-helix proteins, including homeodomain proteins known to regulate morphogenesis in higher eukaryotes. The appearance of flowering plants has been linked to the molecular evolution of LFY. Our study provides a unique framework to elucidate the molecular mechanisms underlying floral development and the evolutionary history of flowering plants.
Asunto(s)
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Flores/fisiología , Secuencias Hélice-Giro-Hélice , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Arabidopsis/anatomía & histología , Proteínas de Arabidopsis/genética , Cristalografía por Rayos X , ADN/metabolismo , Dimerización , Sustancias Macromoleculares/química , Sustancias Macromoleculares/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Regiones Promotoras Genéticas , Unión Proteica , Conformación Proteica , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Factores de Transcripción/genéticaRESUMEN
Pioneer transcription factors (TFs) are a special category of TFs with the capacity to bind to closed chromatin regions in which DNA is wrapped around histones and may be highly methylated. Subsequently, pioneer TFs are able to modify the chromatin state to initiate gene expression. In plants, LEAFY (LFY) is a master floral regulator and has been suggested to act as a pioneer TF in Arabidopsis. Here, we demonstrate that LFY is able to bind both methylated and non-methylated DNA using a combination of in vitro genome-wide binding experiments and structural modeling. Comparisons between regions bound by LFY in vivo and chromatin accessibility data suggest that a subset of LFY bound regions is occupied by nucleosomes. We confirm that LFY is able to bind nucleosomal DNA in vitro using reconstituted nucleosomes. Finally, we show that constitutive LFY expression in seedling tissues is sufficient to induce chromatin accessibility in the LFY direct target genes APETALA1 and AGAMOUS. Taken together, our study suggests that LFY possesses key pioneer TF features that contribute to launching the floral gene expression program.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citología , Flores/citología , Plantones/genética , Factores de Transcripción/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Cromatina/metabolismo , Flores/genética , Regulación de la Expresión Génica de las Plantas , Histonas/metabolismo , Nucleosomas/metabolismo , Plantas Modificadas Genéticamente , Factores de Transcripción/genéticaRESUMEN
Deciphering the mechanisms directing transcription factors (TFs) to specific genome regions is essential to understand and predict transcriptional regulation. TFs recognize short DNA motifs primarily through their DNA-binding domain. Some TFs also possess an oligomerization domain suspected to potentiate DNA binding but for which the genome-wide influence remains poorly understood. Here we focus on the LEAFY transcription factor, a master regulator of flower development in angiosperms. We have determined the crystal structure of its conserved amino-terminal domain, revealing an unanticipated Sterile Alpha Motif oligomerization domain. We show that this domain is essential to LEAFY floral function. Moreover, combined biochemical and genome-wide assays suggest that oligomerization is required for LEAFY to access regions with low-affinity binding sites or closed chromatin. This finding shows that domains that do not directly contact DNA can nevertheless have a profound impact on the DNA binding landscape of a TF.
Asunto(s)
Proteínas de Arabidopsis/química , Arabidopsis/genética , Flores/genética , Regulación de la Expresión Génica de las Plantas , Genoma de Planta , Oryza/genética , Factores de Transcripción/química , Secuencia de Aminoácidos , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sitios de Unión , Cromatina/química , Cromatina/metabolismo , Clonación Molecular , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Flores/crecimiento & desarrollo , Flores/metabolismo , Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Modelos Moleculares , Datos de Secuencia Molecular , Oryza/crecimiento & desarrollo , Oryza/metabolismo , Unión Proteica , Multimerización de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
Brunkard et al. propose that the identification of novel LEAFY sequences contradicts our model of evolution through promiscuous intermediates. Based on the debate surrounding land plant phylogeny and on our analysis of these interesting novel sequences, we explain why there is no solid evidence to disprove our model.
Asunto(s)
ADN de Plantas/química , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Evolución Molecular , Proteínas de Plantas/química , Proteínas de Plantas/genéticaRESUMEN
Transcription factors (TFs) are key players in evolution. Changes affecting their function can yield novel life forms but may also have deleterious effects. Consequently, gene duplication events that release one gene copy from selective pressure are thought to be the common mechanism by which TFs acquire new activities. Here, we show that LEAFY, a major regulator of flower development and cell division in land plants, underwent changes to its DNA binding specificity, even though plant genomes generally contain a single copy of the LEAFY gene. We examined how these changes occurred at the structural level and identify an intermediate LEAFY form in hornworts that appears to adopt all different specificities. This promiscuous intermediate could have smoothed the evolutionary transitions, thereby allowing LEAFY to evolve new binding specificities while remaining a single-copy gene.
Asunto(s)
ADN de Plantas/química , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Evolución Molecular , Proteínas de Plantas/química , Proteínas de Plantas/genética , Secuencia de Aminoácidos , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/clasificación , Proteínas de Arabidopsis/genética , Proteínas de Unión al ADN/clasificación , Ensayo de Cambio de Movilidad Electroforética , Dosificación de Gen , Datos de Secuencia Molecular , Mutación , Filogenia , Proteínas de Plantas/clasificación , Unión Proteica/genética , Estructura Terciaria de Proteína , Especificidad de la Especie , Factores de Transcripción/química , Factores de Transcripción/clasificación , Factores de Transcripción/genéticaRESUMEN
The plant hormone auxin is a key morphogenetic regulator acting from embryogenesis onwards. Transcriptional events in response to auxin are mediated by the auxin response factor (ARF) transcription factors and the Aux/IAA (IAA) transcriptional repressors. At low auxin concentrations, IAA repressors associate with ARF proteins and recruit corepressors that prevent auxin-induced gene expression. At higher auxin concentrations, IAAs are degraded and ARFs become free to regulate auxin-responsive genes. The interaction between ARFs and IAAs is thus central to auxin signalling and occurs through the highly conserved domain III/IV present in both types of proteins. Here, we report the crystal structure of ARF5 domain III/IV and reveal the molecular determinants of ARF-IAA interactions. We further provide evidence that ARFs have the potential to oligomerize, a property that could be important for gene regulation in response to auxin.