ABSTRACT
The gynoecium is critical for the reproduction of flowering plants as it contains the ovules and the tissues that foster pollen germination, growth, and guidance. These tissues, known as the reproductive tract (ReT), comprise the stigma, style, and transmitting tract (TT). The ReT and ovules originate from the carpel margin meristem (CMM) within the pistil. SHOOT MERISTEMLESS (STM) is a key transcription factor for meristem formation and maintenance. In all above-ground meristems, including the CMM, local STM downregulation is required for organ formation. However, how this downregulation is achieved in the CMM is unknown. Here, we have studied the role of HISTONE DEACETYLASE 19 (HDA19) in Arabidopsis (Arabidopsis thaliana) during ovule and ReT differentiation based on the observation that the hda19-3 mutant displays a reduced ovule number and fails to differentiate the TT properly. Fluorescence-activated cell sorting coupled with RNA-sequencing revealed that in the CMM of hda19-3 mutants, genes promoting organ development are downregulated while meristematic markers, including STM, are upregulated. HDA19 was essential to downregulate STM in the CMM, thereby allowing ovule formation and TT differentiation. STM is ectopically expressed in hda19-3 at intermediate stages of pistil development, and its downregulation by RNA interference alleviated the hda19-3 phenotype. Chromatin immunoprecipitation assays indicated that STM is a direct target of HDA19 during pistil development and that the transcription factor SEEDSTICK is also required to regulate STM via histone acetylation. Thus, we identified factors required for the downregulation of STM in the CMM, which is necessary for organogenesis and tissue differentiation.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Histones/genetics , Ovule/genetics , Ovule/metabolism , Arabidopsis/physiology , Transcription Factors/metabolism , Meristem , Gene Expression Regulation, Plant , MADS Domain Proteins/genetics , Histone Deacetylases/metabolismABSTRACT
Tissue patterning in multicellular organisms is the output of precise spatio-temporal regulation of gene expression coupled with changes in hormone dynamics. In plants, the hormone auxin regulates growth and development at every stage of a plant's life cycle. Auxin signaling occurs through binding of the auxin molecule to a TIR1/AFB F-box ubiquitin ligase, allowing interaction with Aux/IAA transcriptional repressor proteins. These are subsequently ubiquitinated and degraded via the 26S proteasome, leading to derepression of auxin response factors (ARFs). How auxin is able to elicit such a diverse range of developmental responses through a single signaling module has not yet been resolved. Here we present an alternative auxin-sensing mechanism in which the ARF ARF3/ETTIN controls gene expression through interactions with process-specific transcription factors. This noncanonical hormone-sensing mechanism exhibits strong preference for the naturally occurring auxin indole 3-acetic acid (IAA) and is important for coordinating growth and patterning in diverse developmental contexts such as gynoecium morphogenesis, lateral root emergence, ovule development, and primary branch formation. Disrupting this IAA-sensing ability induces morphological aberrations with consequences for plant fitness. Therefore, our findings introduce a novel transcription factor-based mechanism of hormone perception in plants.
Subject(s)
Arabidopsis/growth & development , Arabidopsis/genetics , Gene Expression Regulation, Plant/genetics , Indoleacetic Acids/metabolism , Morphogenesis/genetics , Arabidopsis/enzymology , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , F-Box Proteins/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Protein Binding , Protein Serine-Threonine Kinases/metabolism , Receptors, Cell Surface/metabolism , Transcription Factors/metabolismABSTRACT
mRNA methylation at the N6-position of adenosine (m6A) enables multiple layers of post-transcriptional gene control, often via RNA-binding proteins that use a YT521-B homology (YTH) domain for specific m6A recognition. In Arabidopsis, normal leaf morphogenesis and rate of leaf formation require m6A and the YTH-domain proteins ECT2, ECT3 and ECT4. In this study, we show that ect2/ect3 and ect2/ect3/ect4 mutants also exhibit slow root and stem growth, slow flower formation, defective directionality of root growth, and aberrant flower and fruit morphology. In all cases, the m6A-binding site of ECT proteins is required for in vivo function. We also demonstrate that both m6A methyltransferase mutants and ect2/ect3/ect4 exhibit aberrant floral phyllotaxis. Consistent with the delayed organogenesis phenotypes, we observe particularly high expression of ECT2, ECT3 and ECT4 in rapidly dividing cells of organ primordia. Accordingly, ect2/ect3/ect4 mutants exhibit decreased rates of cell division in leaf and vascular primordia. Thus, the m6A-ECT2/ECT3/ECT4 axis is employed as a recurrent module to stimulate plant organogenesis, at least in part by enabling rapid cellular proliferation.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Organogenesis, Plant/genetics , Adenosine/metabolism , Arabidopsis/growth & development , Arabidopsis Proteins/antagonists & inhibitors , Arabidopsis Proteins/genetics , Binding Sites , Cell Proliferation , Flowers/growth & development , Flowers/metabolism , Gene Expression Regulation, Plant , Intracellular Signaling Peptides and Proteins/genetics , Methylation , Methyltransferases/genetics , Methyltransferases/metabolism , Mutagenesis, Site-Directed , Plant Leaves/cytology , Plant Leaves/growth & development , Plant Leaves/metabolism , Plant Roots/growth & development , Plant Roots/metabolism , Plant Stems/growth & development , Plant Stems/metabolism , Plants, Genetically Modified/growth & development , Plants, Genetically Modified/metabolism , Protein Binding , RNA Interference , RNA, Small Interfering/metabolism , RNA-Binding Proteins/antagonists & inhibitors , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolismABSTRACT
Organ formation in multicellular organisms depends on the coordinated activities of regulatory components that integrate developmental and hormonal cues to control gene expression and mediate cell-type specification. For example, development of the Arabidopsis gynoecium is tightly controlled by distribution and synthesis of the plant hormone auxin. The functions of several transcription factors (TFs) have been linked with auxin dynamics during gynoecium development; yet how their activities are coordinated is not known. Here, we show that five such TFs function together to ensure polarity establishment at the gynoecium apex. The auxin response factor ETTIN (ARF3; herein, ETT) is a central component of this framework. Interaction of ETT with TF partners is sensitive to the presence of auxin and our results suggest that ETT forms part of a repressive gene-regulatory complex. We show that this function is conserved between members of the Brassicaceae family and that variation in an ETT subdomain affects interaction strengths and gynoecium morphology. These results suggest that variation in affinities between conserved TFs can lead to morphological differences and thus contribute to the evolution of diverse organ shapes.
Subject(s)
Brassicaceae/growth & development , Brassicaceae/genetics , Flowers/growth & development , Flowers/genetics , Plant Development/genetics , Flowers/anatomy & histology , Gene Expression Regulation, Developmental/drug effects , Gene Expression Regulation, Plant/drug effects , Indoleacetic Acids/pharmacology , Phenotype , Plant Development/drug effects , Plant Growth Regulators/pharmacology , Plants, Genetically Modified , Transcription Factors/physiologyABSTRACT
The phytohormone auxin governs crucial developmental decisions throughout the plant life cycle. Auxin signaling is effectuated by auxin response factors (ARFs) whose activity is repressed by Aux/IAA proteins under low auxin levels, but relieved from repression when cellular auxin concentrations increase. ARF3/ETTIN (ETT) is a conserved noncanonical Arabidopsis thaliana ARF that adopts an alternative auxin-sensing mode of translating auxin levels into multiple transcriptional outcomes. However, a mechanistic model for how this auxin-dependent modulation of ETT activity regulates gene expression has not yet been elucidated. Here, we take a genome-wide approach to show how ETT controls developmental processes in the Arabidopsis shoot through its auxin-sensing property. Moreover, analysis of direct ETT targets suggests that ETT functions as a central node in coordinating auxin dynamics and plant development and reveals tight feedback regulation at both the transcriptional and protein-interaction levels. Finally, we present an example to demonstrate how auxin sensitivity of ETT-protein interactions can shape the composition of downstream transcriptomes to ensure specific developmental outcomes. These results show that direct effects of auxin on protein factors, such as ETT-TF complexes, comprise an important part of auxin biology and likely contribute to the vast number of biological processes affected by this simple molecule.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/genetics , DNA-Binding Proteins/metabolism , Gene Expression Regulation, Plant/drug effects , Indoleacetic Acids/pharmacology , Nuclear Proteins/metabolism , Arabidopsis/drug effects , Arabidopsis/growth & development , Arabidopsis Proteins/genetics , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/metabolism , DNA-Binding Proteins/genetics , Gene Expression Profiling , Genes, Plant , Genetic Loci , Introns/genetics , Models, Biological , Mutation/genetics , Nuclear Proteins/genetics , Plant Leaves/physiology , Protein Binding/drug effects , Protein Multimerization , Repressor Proteins/metabolism , Transcription, Genetic/drug effectsABSTRACT
Hormones, such as auxin and cytokinin, are involved in the complex molecular network that regulates the coordinated development of plant organs. Genes controlling ovule patterning have been identified and studied in detail; however, the roles of auxin and cytokinin in ovule development are largely unknown. Here we show that key cytokinin pathway genes, such as isopentenyltransferase and cytokinin receptors, are expressed during ovule development. Also, in a cre1-12 ahk2-2 ahk3-3 triple mutant with severely reduced cytokinin perception, expression of the auxin efflux facilitator PIN-FORMED 1 (PIN1) was severely reduced. In sporocyteless/nozzle (spl/nzz) mutants, which show a similar phenotype to the cre1-12 ahk2-2 ahk3-3 triple mutant, PIN1 expression is also reduced. Treatment with the exogenous cytokinin N(6)-benzylaminopurine also altered both auxin distribution and patterning of the ovule; this process required the homeodomain transcription factor BELL1 (BEL1). Thus, this article shows that cytokinin regulates ovule development through the regulation of PIN1. Furthermore, the transcription factors BEL1 and SPL/NZZ, previously described as key regulators of ovule development, are needed for the auxin and cytokinin signaling pathways for the correct patterning of the ovule.
Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis/physiology , Homeodomain Proteins/genetics , Membrane Transport Proteins/genetics , Nuclear Proteins/genetics , Plant Growth Regulators/metabolism , Repressor Proteins/genetics , Signal Transduction/physiology , Transcription Factors/genetics , Arabidopsis/cytology , Arabidopsis/drug effects , Arabidopsis/genetics , Arabidopsis Proteins/metabolism , Biological Transport , Body Patterning , Cytokinins/metabolism , Cytokinins/pharmacology , Gene Expression Regulation, Plant/genetics , Genotype , Homeodomain Proteins/metabolism , Indoleacetic Acids/metabolism , Indoleacetic Acids/pharmacology , Membrane Transport Proteins/metabolism , Meristem/cytology , Meristem/drug effects , Meristem/genetics , Meristem/physiology , Mutation , Nuclear Proteins/metabolism , Ovule/cytology , Ovule/drug effects , Ovule/genetics , Ovule/physiology , Phenotype , Plant Growth Regulators/pharmacology , Plant Roots/cytology , Plant Roots/drug effects , Plant Roots/genetics , Plant Roots/physiology , Plant Shoots/cytology , Plant Shoots/drug effects , Plant Shoots/genetics , Plant Shoots/physiology , Plants, Genetically Modified , Repressor Proteins/metabolism , Transcription Factors/metabolismABSTRACT
Basic pentacysteine (BPC) transcription factors have been identified in a large variety of plant species. In Arabidopsis thaliana there are seven BPC genes, which, except for BPC5, are expressed ubiquitously. BPC genes are functionally redundant in a wide range of developmental processes. Recently, we reported that BPC1 binds to guanine and adenine (GA)-rich consensus sequences in the seedstick (STK) promoter in vitro and induces conformational changes. Here we show by chromatin immunoprecipitation experiments that in vivo BPCs also bind to the consensus boxes, and when these were mutated, expression from the STK promoter was derepressed, resulting in ectopic expression in the inflorescence. We also reveal that short vegetative phase (SVP) is a direct regulator of STK. SVP is a floral meristem identity gene belonging to the MADS box gene family. The SVP-APETALA1 (AP1) dimer recruits the SEUSS (SEU)-LEUNIG (LUG) transcriptional cosuppressor to repress floral homeotic gene expression in the floral meristem. Interestingly, we found that GA consensus sequences in the STK promoter to which BPCs bind are essential for recruitment of the corepressor complex to this promoter. Our data suggest that we have identified a new regulatory mechanism controlling plant gene expression that is probably generally used, when considering BPCs' wide expression profile and the frequent presence of consensus binding sites in plant promoters.
Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis/genetics , Gene Expression Regulation, Plant , MADS Domain Proteins/metabolism , Arabidopsis/metabolism , Arabidopsis Proteins/chemistry , Arabidopsis Proteins/metabolism , Arabidopsis Proteins/physiology , Binding Sites , Chromatin Immunoprecipitation , Consensus Sequence , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/physiology , Flowers/genetics , Flowers/metabolism , Genes, Homeobox , MADS Domain Proteins/chemistry , MADS Domain Proteins/genetics , MADS Domain Proteins/physiology , Meristem/genetics , Meristem/metabolism , Promoter Regions, Genetic , Transcription Factors/genetics , Transcription Factors/metabolism , Transcription Factors/physiologyABSTRACT
TBP-Associated Factors (TAFs) are components of complexes like TFIID, TFTC, SAGA/STAGA and SMAT that are important for the activation of transcription, either by establishing the basic transcription machinery or by facilitating histone acetylation. However, in Drosophila embryos several TAFs were shown to be associated with the Polycomb Repressive Complex 1 (PRC1), even though the role of this interaction remains unclear. Here we show that in Arabidopsis TAF13 interacts with MEDEA and SWINGER, both members of a plant variant of Polycomb Repressive Complex 2 (PRC2). PRC2 variants play important roles during the plant life cycle, including seed development. The taf13 mutation causes seed defects, showing embryo arrest at the 8-16 cell stage and over-proliferation of the endosperm in the chalazal region, which is typical for Arabidopsis PRC2 mutants. Our data suggest that TAF13 functions together with PRC2 in transcriptional regulation during seed development.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/embryology , Gene Expression Regulation, Plant , Repressor Proteins/metabolism , Seeds/growth & development , Agrobacterium tumefaciens/genetics , Agrobacterium tumefaciens/metabolism , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Cell Proliferation , Genes, Plant , Genetic Complementation Test , Mutation , Polycomb Repressive Complex 2 , Protein Interaction Mapping , Repressor Proteins/genetics , Seeds/genetics , Seeds/metabolism , Nicotiana/genetics , Nicotiana/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Transcription, GeneticABSTRACT
Upon hormonal signaling, ovules develop as lateral organs from the placenta. Ovule numbers ultimately determine the number of seeds that develop, and thereby contribute to the final seed yield in crop plants. We demonstrate here that CUP-SHAPED COTYLEDON 1 (CUC1), CUC2 and AINTEGUMENTA (ANT) have additive effects on ovule primordia formation. We show that expression of the CUC1 and CUC2 genes is required to redundantly regulate expression of PINFORMED1 (PIN1), which in turn is required for ovule primordia formation. Furthermore, our results suggest that the auxin response factor MONOPTEROS (MP/ARF5) may directly bind ANT, CUC1 and CUC2 and promote their transcription. Based on our findings, we propose an integrative model to describe the molecular mechanisms of the early stages of ovule development.
Subject(s)
Arabidopsis Proteins/physiology , Arabidopsis/embryology , Ovule/embryology , Transcription Factors/physiology , Arabidopsis/metabolism , Arabidopsis Proteins/metabolism , DNA-Binding Proteins/metabolism , Green Fluorescent Proteins , Membrane Transport Proteins/metabolism , Models, Biological , Promoter Regions, Genetic , Transcription Factors/metabolismABSTRACT
The BASIC PENTACYSTEINE (BCP) family is a poorly characterized plant transcription factor family of GAGA BINDING PROTEINS. In Arabidopsis, there are seven members (BPC1-7) that are broadly expressed, and they can potentially bind more than 3000 Arabidopsis GAGA-repeat-containing genes. To date, BPCs are known to be direct regulators of the INNER NO OUTER (INO), SEEDSTICK (STK), and LEAFY COTYLEDON 2 (LEC2) genes. Because of the high functional redundancy, neither single knockout nor double bpc mutant combinations cause aberrant phenotypes. The bpc1-2 bpc2 bpc3 triple mutant shows several pleiotropic developmental defects, including enlargement of the inflorescence meristem and flowers with supernumerary floral organs. Here, we demonstrated through expression analysis and chromatin immunoprecipitation assays that this phenotype is probably due to deregulation of the expression of the SHOOTMERISTEMLESS (STM) and BREVIPEDICELLUS/KNAT1 (BP) genes, which are both direct targets of BPCs. Moreover, we assigned a role to BPCs in the fine regulation of the cytokinin content in the meristem, as both ISOPENTENYLTRANSFERASE 7 (IPT7) and ARABIDOPSIS RESPONSE REGULATOR 7 (ARR7) genes were shown to be overexpressed in the bpc1-2 bpc2 bpc3 triple mutant.
Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis/genetics , Cytokinins/metabolism , Gene Expression Regulation, Plant , Meristem/genetics , Plant Growth Regulators/metabolism , Arabidopsis/cytology , Arabidopsis/growth & development , Arabidopsis/metabolism , Arabidopsis Proteins/metabolism , Gene Expression Regulation, Developmental , Genes, Reporter , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Inflorescence/cytology , Inflorescence/genetics , Inflorescence/growth & development , Inflorescence/metabolism , Meristem/cytology , Meristem/growth & development , Meristem/metabolism , Mutation , Phenotype , Plants, Genetically Modified , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
In multicellular organisms, sexual reproduction relies on the formation of highly differentiated cells, the gametes, which await fertilization in a quiescent state. Upon fertilization, the cell cycle resumes. Successful development requires that male and female gametes are in the same phase of the cell cycle. The molecular mechanisms that reinstate cell division in a fertilization-dependent manner are poorly understood in both animals and plants. Using Arabidopsis, we show that a sperm-derived signal induces the proliferation of a female gamete, the central cell, precisely upon fertilization. The central cell is arrested in S phase by the activity of the RETINOBLASTOMA RELATED1 (RBR1) protein. Upon fertilization, delivery of the core cell cycle component CYCD7;1 causes RBR1 degradation and thus S phase progression, ensuring the formation of functional endosperm and, consequently, viable seeds.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Endosperm , Gametogenesis, Plant , Paternal Inheritance , Arabidopsis/cytology , Arabidopsis/growth & development , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Cell Division , Endosperm/cytology , Endosperm/physiologyABSTRACT
Establishing the embryonic body plan of multicellular organisms relies on precisely orchestrated cell divisions coupled with pattern formation, which, in animals, are regulated by Polycomb group (PcG) proteins. The conserved Polycomb Repressive Complex 2 (PRC2) mediates H3K27 trimethylation and comes in different flavors in Arabidopsis. The PRC2 catalytic subunit MEDEA is required for seed development; however, a role for PRC2 in embryonic patterning has been dismissed. Here, we demonstrate that embryos derived from medea eggs abort because MEDEA is required for patterning and cell lineage determination in the early embryo. Similar to PcG proteins in mammals, MEDEA regulates embryonic patterning and growth by controlling cell-cycle progression through repression of CYCD1;1, which encodes a core cell-cycle component. Thus, Arabidopsis embryogenesis is epigenetically regulated by PcG proteins, revealing that the PRC2-dependent modulation of cell-cycle progression was independently recruited to control embryonic cell proliferation and patterning in animals and plants.
Subject(s)
Arabidopsis Proteins/genetics , Cyclin D3/genetics , Plant Development/genetics , Polycomb-Group Proteins/genetics , Arabidopsis/genetics , Arabidopsis/growth & development , Body Patterning/genetics , Cell Proliferation/genetics , Flowers/genetics , Flowers/growth & development , Gene Expression Regulation, Plant/genetics , Histones/genetics , Methylation , Polycomb Repressive Complex 2/genetics , Seeds/genetics , Seeds/growth & developmentABSTRACT
Multicellular organisms, such as plants, fungi, and animals, develop organs with specialized functions. Major challenges in developing such structures include establishment of polarity along three axes (apical-basal, medio-lateral, and dorso-ventral/abaxial-adaxial), specification of tissue types and their coordinated growth, and maintenance of communication between the organ and the entire organism. The gynoecium of the model plant Arabidopsis thaliana embodies the female reproductive organ and has proven an excellent model system for studying organ establishment and development, given its division into different regions with distinct symmetries and highly diverse tissue types. Upon pollination, the gynoecium undergoes dramatic changes in morphology and developmental programming to form the seed-containing fruit. In this review, we wish to provide a detailed overview of the molecular and genetic mechanisms that are known to guide gynoecium and fruit development in A. thaliana. We describe networks of key genetic regulators and their interactions with hormonal dynamics in driving these developmental processes. The discoveries made to date clearly demonstrate that conclusions drawn from studying gynoecium and fruit development in flowering plants can be used to further our general understanding of organ formation across the plant kingdom. Importantly, this acquired knowledge is increasingly being used to improve fruit and seed crops, facilitated by the recent profound advances in genomics, cloning, and gene-editing technologies.
Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis/growth & development , Flowers/growth & development , Fruit/growth & development , Magnoliopsida/growth & development , Arabidopsis/genetics , Flowers/genetics , Fruit/genetics , Gene Expression Regulation, Plant , Magnoliopsida/geneticsABSTRACT
Specification of new organs from transit amplifying cells is critical for higher eukaryote development. In plants, a central stem cell pool maintained by the pluripotency factor SHOOTMERISTEMLESS (STM), is surrounded by transit amplifying cells competent to respond to auxin hormone maxima by giving rise to new organs. Auxin triggers flower initiation through Auxin Response Factor (ARF) MONOPTEROS (MP) and recruitment of chromatin remodelers to activate genes promoting floral fate. The contribution of gene repression to reproductive primordium initiation is poorly understood. Here we show that downregulation of the STM pluripotency gene promotes initiation of flowers and uncover the mechanism for STM silencing. The ARFs ETTIN (ETT) and ARF4 promote organogenesis at the reproductive shoot apex in parallel with MP via histone-deacetylation mediated transcriptional silencing of STM. ETT and ARF4 directly repress STM, while MP acts indirectly, through its target FILAMENTOUS FLOWER (FIL). Our data suggest that - as in animals- downregulation of the pluripotency program is important for organogenesis in plants.
Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis/growth & development , Arabidopsis/genetics , Homeodomain Proteins/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/metabolism , Chromatin Assembly and Disassembly/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Flowers/genetics , Flowers/growth & development , Flowers/metabolism , Gene Expression Regulation, Developmental , Gene Expression Regulation, Plant , Gene Silencing , Genes, Plant , Homeodomain Proteins/metabolism , Indoleacetic Acids/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Organogenesis, Plant/genetics , Plant Growth Regulators/metabolism , Plants, Genetically Modified , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
The plant hormone auxin regulates numerous aspects of the plant life cycle. Auxin signalling is mediated by auxin response factors (ARFs) that dimerise with modulating Aux/IAA repressors. ARF3 (ETTIN or ETT) is atypical as it does not interact with Aux/IAA repressors. It is proposed to be a non-canonical auxin sensor, regulating diverse functions essential for development. This sensing ability relies on a unique C-terminal ETT specific domain (ES domain). Alignments of ETT orthologues across the angiosperm phylum revealed that the length and sequence identities of ES domains are poorly conserved. Computational predictors suggested the ES domains to be intrinsically disordered, explaining their tolerance of insertions, deletions and mutations during evolution. Nevertheless, five highly conserved short linear motifs were identified suggesting functional significance. High-throughput library screening identified an almost full-length soluble ES domain that did not bind auxin directly, but exhibited a dose-dependent response in a yeast two-hybrid system against the Arabidopsis INDEHISCENT (IND) transcription factor. Circular dichroism confirmed the domain was disordered. The identification and purification of this domain opens the way to the future characterisation of the ETT auxin-sensing mechanism in planta and an improved understanding of auxin-mediated regulation.
Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Basic Helix-Loop-Helix Transcription Factors/metabolism , DNA-Binding Proteins/genetics , Indoleacetic Acids/metabolism , Intrinsically Disordered Proteins/genetics , Nuclear Proteins/genetics , Protein Interaction Domains and Motifs/genetics , Arabidopsis Proteins/chemistry , Arabidopsis Proteins/isolation & purification , Circular Dichroism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/isolation & purification , DNA-Binding Proteins/metabolism , Gene Expression Regulation, Plant/physiology , High-Throughput Screening Assays , Intrinsically Disordered Proteins/metabolism , Nuclear Proteins/chemistry , Nuclear Proteins/isolation & purification , Nuclear Proteins/metabolism , Plants, Genetically Modified , Point Mutation , Protein Binding , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sequence Alignment , Two-Hybrid System TechniquesABSTRACT
BACKGROUND: MADS-domain transcription factors play important roles during plant development. The Arabidopsis MADS-box gene SHORT VEGETATIVE PHASE (SVP) is a key regulator of two developmental phases. It functions as a repressor of the floral transition during the vegetative phase and later it contributes to the specification of floral meristems. How these distinct activities are conferred by a single transcription factor is unclear, but interactions with other MADS domain proteins which specify binding to different genomic regions is likely one mechanism. RESULTS: To compare the genome-wide DNA binding profile of SVP during vegetative and reproductive development we performed ChIP-seq analyses. These ChIP-seq data were combined with tiling array expression analysis, induction experiments and qRT-PCR to identify biologically relevant binding sites. In addition, we compared genome-wide target genes of SVP with those published for the MADS domain transcription factors FLC and AP1, which interact with SVP during the vegetative and reproductive phases, respectively. CONCLUSIONS: Our analyses resulted in the identification of pathways that are regulated by SVP including those controlling meristem development during vegetative growth and flower development whereas floral transition pathways and hormonal signaling were regulated predominantly during the vegetative phase. Thus, SVP regulates many developmental pathways, some of which are common to both of its developmental roles whereas others are specific to only one of them.