RESUMO
Proper cell-type identity relies on highly coordinated regulation of gene expression. Regulatory elements such as enhancers can produce cell type-specific expression patterns, but the mechanisms underlying specificity are not well understood. We previously identified an enhancer region capable of driving specific expression in giant cells, which are large, highly endoreduplicated cells in the Arabidopsis thaliana sepal epidermis. In this study, we use the giant cell enhancer as a model to understand the regulatory logic that promotes cell type-specific expression. Our dissection of the enhancer revealed that giant cell specificity is mediated primarily through the combination of two activators and one repressor. HD-ZIP and TCP transcription factors are involved in the activation of expression throughout the epidermis. High expression of HD-ZIP transcription factor genes in giant cells promoted higher expression driven by the enhancer in giant cells. Dof transcription factors repressed the activity of the enhancer such that only giant cells maintained enhancer activity. Thus, our data are consistent with a conceptual model whereby cell type-specific expression emerges from the combined activities of three transcription factor families activating and repressing expression in epidermal cells.
Assuntos
Arabidopsis , Fatores de Transcrição , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas de Homeodomínio/genética , Sequências Reguladoras de Ácido Nucleico , Regulação da Expressão Gênica , Arabidopsis/metabolismo , Células Gigantes/metabolismo , Elementos Facilitadores Genéticos/genéticaRESUMO
Transcriptional regulators of the general stress response (GSR) reprogram the expression of selected genes to transduce informational signals into cellular events, ultimately manifested in a plant's ability to cope with environmental challenges. Identification of the core GSR regulatory proteins will uncover the principal modules and their mode of action in the establishment of adaptive responses. To define the GSR regulatory components, we employed a yeast-one-hybrid assay to identify the protein(s) binding to the previously established functional GSR motif, termed the rapid stress response element (RSRE). This led to the isolation of octadecanoid-responsive AP2/ERF-domain transcription factor 47 (ORA47), a methyl jasmonate inducible protein. Subsequently, ORA47 transcriptional activity was confirmed using the RSRE-driven luciferase (LUC) activity assay performed in the ORA47 loss- and gain-of-function lines introgressed into the 4xRSRE::Luc background. In addition, the prime contribution of CALMODULIN-BINDING TRANSCRIPTIONAL ACTIVATOR3 (CAMTA3) protein in the induction of RSRE was reaffirmed by genetic studies. Moreover, exogenous application of methyl jasmonate led to enhanced levels of ORA47 and CAMTA3 transcripts, as well as the induction of RSRE::LUC activity. Metabolic analyses illustrated the reciprocal functional inputs of ORA47 and CAMTA3 in increasing JA levels. Lastly, transient assays identified JASMONATE ZIM-domain1 (JAZ1) as a repressor of RSRE::LUC activity. Collectively, the present study provides fresh insight into the initial features of the mechanism that transduces informational signals into adaptive responses. This mechanism involves the functional interplay between the JA biosynthesis/signaling cascade and the transcriptional reprogramming that potentiates GSR. Furthermore, these findings offer a window into the role of intraorganellar communication in the establishment of adaptive responses.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Ciclopentanos/metabolismo , Regulação da Expressão Gênica de Plantas , Oxilipinas/metabolismo , Transdução de Sinais/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Shade avoidance syndrome (SAS) is a strategy of major adaptive significance and typically includes elongation of the stem and petiole, leaf hyponasty, reduced branching and phototropic orientation of the plant shoot toward canopy gaps. Both cryptochrome 1 and phytochrome B (phyB) are the major photoreceptors that sense the reduction in the blue light fluence rate and the low red:far-red ratio, respectively, and both light signals are associated with plant density and the resource reallocation when SAS responses are triggered. The B-box (BBX)-containing zinc finger transcription factor BBX24 has been implicated in the SAS as a regulator of DELLA activity, but this interaction does not explain all the observed BBX24-dependent regulation in shade light. Here, through a combination of transcriptional meta-analysis and large-scale identification of BBX24-interacting transcription factors, we found that JAZ3, a jasmonic acid signaling component, is a direct target of BBX24. Furthermore, we demonstrated that joint loss of BBX24 and JAZ3 function causes insensitivity to DELLA accumulation, and the defective shade-induced elongation in this mutant is rescued by loss of DELLA or phyB function. Therefore, we propose that JAZ3 is part of the regulatory network that controls the plant growth in response to shade, through a mechanism in which BBX24 and JAZ3 jointly regulate DELLA activity. Our results provide new insights into the participation of BBX24 and JA signaling in the hypocotyl shade avoidance response in Arabidopsis.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Luz , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fitocromo B/metabolismo , Regulação da Expressão Gênica de PlantasRESUMO
B-Box-containing zinc finger transcription factors (BBX) are involved in light-mediated growth, affecting processes such as hypocotyl elongation in Arabidopsis thaliana. However, the molecular and hormonal framework that regulates plant growth through BBX proteins is incomplete. Here, we demonstrate that BBX21 inhibits the hypocotyl elongation through the brassinosteroid (BR) pathway. BBX21 reduces the sensitivity to 24-epiBL, a synthetic active BR, principally at very-low concentrations in simulated shade. The biosynthesis profile of BRs showed that two active BR -brassinolide (BL) and 28-homobrassinolide (28-homoBL)- and 8 of 11 intermediates can be repressed by BBX21 under white light (WL) or simulated shade. Furthermore, BBX21 represses the expression of CYTOCHROME P450 90B1 (DWF4/CYP90B1), BRASSINOSTEROID-6-OXIDASE 1 (BR6OX1, CYP85A1) and BR6OX2 (CYP85A2) genes involved in the BR biosynthesis in WL while specifically promoting DWF4 and PHYB ACTIVATION TAGGED SUPPRESSOR 1 (CYP2B1/BAS1) expression in WL supplemented with far-red (WL+FR), a treatment that simulates shade. In addition, BBX21 represses BR signalling genes such as PACLOBUTRAZOL RESISTANCE1 (PRE1), PRE3 and ARABIDOPSIS MYB-LIKE 2 (MYBL2), and auxin-related and expansin genes, such as INDOLE-3-ACETIC ACID INDUCIBLE 1 (IAA1), IAA4 and EXPANSIN 11 (EXP11) in short-term shade. By a genetic approach we found that BBX21 acts genetically upstream of BRASSINAZOLE-RESISTANT 1 (BZR1) for the promotion of DWF4 and BAS1 gene expression in shade. We propose that BBX21 integrates the BR homeostasis and shade-light signalling allowing the fine-tuning of hypocotyl elongation in Arabidopsis.
RESUMO
Circadian clocks play important roles in regulating cellular metabolism, but the reciprocal effect that metabolism has on the clock is largely unknown in plants. Here, we show that the central glycerolipid metabolite and lipid mediator phosphatidic acid (PA) interacts with and modulates the function of the core clock regulators LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED1 (CCA1) in Arabidopsis (Arabidopsis thaliana). PA reduced the ability of LHY and CCA1 to bind the promoter of their target gene TIMING OF CAB EXPRESSION1 Increased PA accumulation and inhibition of PA-producing enzymes had opposite effects on circadian clock outputs. Diurnal change in levels of several membrane phospholipid species, including PA, observed in wild type was lost in the LHY and CCA1 double knockout mutant. Storage lipid accumulation was also affected in the clock mutants. These results indicate that the interaction of PA with the clock regulator may function as a cellular conduit to integrate the circadian clock with lipid metabolism.
Assuntos
Relógios Circadianos/fisiologia , Ácidos Fosfatídicos/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Hipocótilo/metabolismo , Metabolismo dos Lipídeos/genética , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/metabolismoRESUMO
Plant stem cells in the shoot apical meristem (SAM) and root apical meristem are necessary for postembryonic development of aboveground tissues and roots, respectively, while secondary vascular stem cells sustain vascular development. WUSCHEL (WUS), a homeodomain transcription factor expressed in the rib meristem of the Arabidopsis SAM, is a key regulatory factor controlling SAM stem cell populations, and is thought to establish the shoot stem cell niche through a feedback circuit involving the CLAVATA3 (CLV3) peptide signalling pathway. WUSCHEL-RELATED HOMEOBOX 5 (WOX5), which is specifically expressed in the root quiescent centre, defines quiescent centre identity and functions interchangeably with WUS in the control of shoot and root stem cell niches. WOX4, expressed in Arabidopsis procambial cells, defines the vascular stem cell niche. WUS/WOX family proteins are evolutionarily and functionally conserved throughout the plant kingdom and emerge as key actors in the specification and maintenance of stem cells within all meristems. However, the nature of the genetic regime in stem cell niches that centre on WOX gene function has been elusive, and molecular links underlying conserved WUS/WOX function in stem cell niches remain unknown. Here we demonstrate that the Arabidopsis HAIRY MERISTEM (HAM) family of transcription regulators act as conserved interacting cofactors with WUS/WOX proteins. HAM and WUS share common targets in vivo and their physical interaction is important in driving downstream transcriptional programs and in promoting shoot stem cell proliferation. Differences in the overlapping expression patterns of WOX and HAM family members underlie the formation of diverse stem cell niche locations, and the HAM family is essential for all of these stem cell niches. These findings establish a new framework for the control of stem cell production during plant development.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Células-Tronco/citologia , Células-Tronco/metabolismo , Transcrição Gênica , Arabidopsis/genética , Proliferação de Células , Histona Acetiltransferases/metabolismo , Proteínas de Homeodomínio/metabolismo , Brotos de Planta/citologia , Brotos de Planta/genética , Ligação Proteica , Nicho de Células-TroncoRESUMO
Integration of environmental signals and interactions among photoreceptors and transcriptional regulators is key in shaping plant development. TANDEM ZINC-FINGER PLUS3 (TZP) is an integrator of light and photoperiodic signaling that promotes flowering in Arabidopsis thaliana Here we elucidate the molecular role of TZP as a positive regulator of hypocotyl elongation. We identify an interacting partner for TZP, the transcription factor ZINC-FINGER HOMEODOMAIN 10 (ZFHD10), and characterize its function in coregulating the expression of blue-light-dependent transcriptional regulators and growth-promoting genes. By employing a genome-wide approach, we reveal that ZFHD10 and TZP coassociate with promoter targets enriched in light-regulated elements. Furthermore, using a targeted approach, we show that ZFHD10 recruits TZP to the promoters of key coregulated genes. Our findings not only unveil the mechanism of TZP action in promoting hypocotyl elongation at the transcriptional level but also assign a function to an uncharacterized member of the ZFHD transcription factor family in promoting plant growth.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas , Hipocótilo/crescimento & desenvolvimento , Regiões Promotoras Genéticas , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Hipocótilo/genética , Fotoperíodo , Transativadores/genética , Fatores de Transcrição/genética , Transcrição Gênica , Dedos de ZincoRESUMO
Photoperiod is one of the most reliable environmental cues for plants to regulate flowering timing. In Arabidopsis thaliana, CONSTANS (CO) transcription factor plays a central role in regulating photoperiodic flowering. In contrast to posttranslational regulation of CO protein, still little was known about CO transcriptional regulation. Here we show that the CINCINNATA (CIN) clade of class II TEOSINTE BRANCHED 1/ CYCLOIDEA/ PROLIFERATING CELL NUCLEAR ANTIGEN FACTOR (TCP) proteins act as CO activators. Our yeast one-hybrid analysis revealed that class II CIN-TCPs, including TCP4, bind to the CO promoter. TCP4 induces CO expression around dusk by directly associating with the CO promoter in vivo. In addition, TCP4 binds to another flowering regulator, GIGANTEA (GI), in the nucleus, and induces CO expression in a GI-dependent manner. The physical association of TCP4 with the CO promoter was reduced in the gi mutant, suggesting that GI may enhance the DNA-binding ability of TCP4. Our tandem affinity purification coupled with mass spectrometry (TAP-MS) analysis identified all class II CIN-TCPs as the components of the in vivo TCP4 complex, and the gi mutant did not alter the composition of the TCP4 complex. Taken together, our results demonstrate a novel function of CIN-TCPs as photoperiodic flowering regulators, which may contribute to coordinating plant development with flowering regulation.
Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Ligação a DNA/genética , Flores/genética , Fatores de Transcrição/genética , Transcrição Gênica , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Ritmo Circadiano/genética , Flores/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas , Mutação , Fotoperíodo , Desenvolvimento Vegetal/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Regiões Promotoras GenéticasRESUMO
Plants sense neighbor proximity as a decrease in the ratio of red to far-red light, which triggers a series of developmental responses. In Arabidopsis, phytochrome B (PHYB) is the major sensor of shade, but PHYB excitation has not been linked directly to a growth response. We show that the basic helix-loop-helix (bHLH) transcription factor PIF7 (phytochrome-interacting factor 7), an interactor of PHYB, accumulates in its dephosphorylated form in shade, allowing it to bind auxin biosynthetic genes and increase their expression. New auxin synthesized through a PIF7-regulated pathway is required for shade-induced growth, linking directly the perception of a light quality signal to a rapid growth response.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Proteínas de Ligação a DNA/metabolismo , Ácidos Indolacéticos/metabolismo , Arabidopsis/anatomia & histologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Ligação a DNA/genética , Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas , Ácidos Indolacéticos/farmacologia , Luz , Mutação , Fitocromo B/metabolismoRESUMO
The water stress-associated hormone abscisic acid (ABA) acts through a well-defined signal transduction cascade to mediate downstream transcriptional events important for acclimation to stress. Although ABA signaling is known to function in specific tissues to regulate root growth, little is understood regarding the spatial pattern of ABA-mediated transcriptional regulation. Here, we describe the construction and evaluation of an ABSCISIC ACID RESPONSIVE ELEMENT (ABRE)-based synthetic promoter reporter that reveals the transcriptional response of tissues to different levels of exogenous ABA and stresses. Genome-scale yeast one-hybrid screens complemented these approaches and revealed how promoter sequence and architecture affect the recruitment of diverse transcription factors (TFs) to the ABRE. Our analysis also revealed ABA-independent activity of the ABRE-reporter under nonstress conditions, with expression being enriched at the quiescent center and stem cell niche. We show that the WUSCHEL RELATED HOMEOBOX5 and NAC DOMAIN PROTEIN13 TFs regulate QC/SCN expression of the ABRE reporter, which highlights the convergence of developmental and DNA-damage signaling pathways onto this cis-element in the absence of water stress. This work establishes a tool to study the spatial pattern of ABA-mediated transcriptional regulation and a repertoire of TF-ABRE interactions that contribute to the developmental and environmental control of gene expression in roots.
Assuntos
Ácido Abscísico/genética , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Genes Reporter , Regiões Promotoras Genéticas , Ácido Abscísico/metabolismo , Ácido Abscísico/farmacologia , Arabidopsis/efeitos dos fármacos , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Dano ao DNA , Redes Reguladoras de Genes , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Plantas Geneticamente Modificadas , Elementos de Resposta , Transdução de Sinais/genética , Análise Espaço-Temporal , Estresse Fisiológico/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Leveduras/genéticaRESUMO
Eukaryotic circadian clocks utilize the ubiquitin proteasome system to precisely degrade clock proteins. In plants, the F-box-type E3 ubiquitin ligases ZEITLUPE (ZTL), FLAVIN-BINDING, KELCH REPEAT, F-BOX1 (FKF1), and LOV KELCH PROTEIN2 (LKP2) regulate clock period and couple the clock to photoperiodic flowering in response to end-of-day light conditions. To better understand their functions, we expressed decoy ZTL, FKF1, and LKP2 proteins that associate with target proteins but are unable to ubiquitylate their targets in Arabidopsis (Arabidopsis thaliana). These dominant-negative forms of the proteins inhibit the ubiquitylation of target proteins and allow for the study of ubiquitylation-independent and -dependent functions of ZTL, FKF1, and LKP2. We demonstrate the effects of expressing ZTL, FKF1, and LKP2 decoys on the circadian clock and flowering time. Furthermore, the decoy E3 ligases trap substrate interactions, and using immunoprecipitation-mass spectrometry, we identify interacting partners. We focus studies on the clock transcription factor CCA1 HIKING EXPEDITION (CHE) and show that ZTL interacts directly with CHE and can mediate CHE ubiquitylation. We also demonstrate that CHE protein is degraded in the dark and that degradation is reduced in a ztl mutant plant, showing that CHE is a bona fide ZTL target protein. This work increases our understanding of the genetic and biochemical roles for ZTL, FKF1, and LKP2 and also demonstrates an effective methodology for studying complicated genetic redundancy among E3 ubiquitin ligases.
Assuntos
Proteínas de Arabidopsis/metabolismo , Relógios Circadianos/fisiologia , Proteínas Repressoras/metabolismo , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Flores/fisiologia , Regulação da Expressão Gênica de Plantas , Plantas Geneticamente Modificadas , Domínios Proteicos , Mapas de Interação de Proteínas , Proteínas Repressoras/genética , UbiquitinaçãoRESUMO
Gene-centered yeast one-hybrid (Y1H) screens provide a powerful and effective strategy to identify transcription factor (TF)-promoter interactions. While genome-wide TF ORFeome clone collections are increasingly available, screening protocols have limitations inherent to the properties of the enzymatic reaction used to identify interactions and to the procedure required to perform the assay in a high-throughput format. Here, we present the development and validation of a streamlined strategy for quantitative and fully automated gene-centered Y1H screens using a novel cell surface Gaussia luciferase reporter.
Assuntos
Genes Reporter , Ensaios de Triagem em Larga Escala/métodos , Luciferases/genética , Técnicas do Sistema de Duplo-Híbrido , Automação Laboratorial , Sítios de Ligação/genética , Regulação da Expressão Gênica/genética , Técnicas de Transferência de Genes , Organismos Geneticamente Modificados , Regiões Promotoras Genéticas , Ligação Proteica , Elementos Reguladores de Transcrição/genética , Saccharomyces cerevisiae , Fatores de Transcrição/metabolismoRESUMO
The plant hormone salicylic acid (SA) is essential for local defense and systemic acquired resistance (SAR). When plants, such as Arabidopsis, are challenged by different pathogens, an increase in SA biosynthesis generally occurs through transcriptional induction of the key synthetic enzyme isochorismate synthase 1 (ICS1). However, the regulatory mechanism for this induction is poorly understood. Using a yeast one-hybrid screen, we identified two transcription factors (TFs), NTM1-like 9 (NTL9) and CCA1 hiking expedition (CHE), as activators of ICS1 during specific immune responses. NTL9 is essential for inducing ICS1 and two other SA synthesis-related genes, phytoalexin-deficient 4 (PAD4) and enhanced disease susceptibility 1 (EDS1), in guard cells that form stomata. Stomata can quickly close upon challenge to block pathogen entry. This stomatal immunity requires ICS1 and the SA signaling pathway. In the ntl9 mutant, this response is defective and can be rescued by exogenous application of SA, indicating that NTL9-mediated SA synthesis is essential for stomatal immunity. CHE, the second identified TF, is a central circadian clock oscillator and is required not only for the daily oscillation in SA levels but also for the pathogen-induced SA synthesis in systemic tissues during SAR. CHE may also regulate ICS1 through the known transcription activators calmodulin binding protein 60g (CBP60g) and systemic acquired resistance deficient 1 (SARD1) because induction of these TF genes is compromised in the che-2 mutant. Our study shows that SA biosynthesis is regulated by multiple TFs in a spatial and temporal manner and therefore fills a gap in the signal transduction pathway between pathogen recognition and SA production.
Assuntos
Arabidopsis/imunologia , Resistência a Medicamentos , Regulação da Expressão Gênica de Plantas , Nicotiana/imunologia , Imunidade Vegetal , Ácido Salicílico/química , Arabidopsis/genética , Ritmo Circadiano , Microscopia Confocal , Mutação , Oscilometria , Fenótipo , Doenças das Plantas/imunologia , Folhas de Planta , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Estômatos de Plantas/metabolismo , Regiões Promotoras Genéticas , Transdução de Sinais , Fatores de Tempo , Nicotiana/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
The circadian clock in Arabidopsis exerts a critical role in timing multiple biological processes and stress responses through the regulation of up to 80% of the transcriptome. As a key component of the clock, the Myb-like transcription factor CIRCADIAN CLOCK ASSOCIATED1 (CCA1) is able to initiate and set the phase of clock-controlled rhythms and has been shown to regulate gene expression by binding directly to the evening element (EE) motif found in target gene promoters. However, the precise molecular mechanisms underlying clock regulation of the rhythmic transcriptome, specifically how clock components connect to clock output pathways, is poorly understood. In this study, using ChIP followed by deep sequencing of CCA1 in constant light (LL) and diel (LD) conditions, more than 1,000 genomic regions occupied by CCA1 were identified. CCA1 targets are enriched for a myriad of biological processes and stress responses, providing direct links to clock-controlled pathways and suggesting that CCA1 plays an important role in regulating a large subset of the rhythmic transcriptome. Although many of these target genes are evening expressed and contain the EE motif, a significant subset is morning phased and enriched for previously unrecognized motifs associated with CCA1 function. Furthermore, this work revealed several CCA1 targets that do not cycle in either LL or LD conditions. Together, our results emphasize an expanded role for the clock in regulating a diverse category of genes and key pathways in Arabidopsis and provide a comprehensive resource for future functional studies.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Genoma de Planta , Fatores de Transcrição/genética , TranscriptomaRESUMO
In Arabidopsis, the circadian clock allows the plant to coordinate daily external signals with internal processes, conferring enhanced fitness and growth vigor. Although external cues such as temperature can entrain the clock, an important feature of the clock is the ability to maintain a relatively constant period over a range of physiological temperatures; this ability is referred to as "temperature compensation." However, how temperature actually is perceived and integrated into the clock molecular circuitry remains largely unknown. In an effort to identify additional regulators of the circadian clock, including putative components that could modulate the clock response to changes in environmental signals, we identified in a previous large-scale screen a transcription factor that interacts with and regulates the promoter activity of a core clock gene. In this report, we characterized this transcription factor, flowering basic helix-loop-helix 1 (FBH1) that binds in vivo to the promoter of the key clock gene circadian clock-associated 1 (CCA1) and regulates its expression. We found that upon temperature changes, overexpression of FBH1 alters the pace of CCA1 expression by causing a period shortening and thus preventing the clock from buffering against this change in temperature. Furthermore, as is consistent with the current mechanistic model of feedback loops observed in the clock regulatory network, we also determined that CCA1 binds in vivo to the FBH1 promoter and regulates its expression. Together these results establish a role for FBH1 as a transcriptional modulator of warm temperature signals and clock responses in Arabidopsis.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Relógios Circadianos/genética , Regulação da Expressão Gênica de Plantas , Temperatura , Adaptação Fisiológica/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Retroalimentação Fisiológica , Genótipo , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas/genética , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The circadian clock perceives environmental signals to reset to local time, but the underlying molecular mechanisms are not well understood. Here we present data revealing that a member of the heat shock factor (Hsf) family is involved in the input pathway to the plant circadian clock. Using the yeast one-hybrid approach, we isolated several Hsfs, including Heat Shock Factor B2b (HsfB2b), a transcriptional repressor that binds the promoter of Pseudo Response Regulator 7 (PRR7) at a conserved binding site. The constitutive expression of HsfB2b leads to severely reduced levels of the PRR7 transcript and late flowering and elongated hypocotyls. HsfB2b function is important during heat and salt stress because HsfB2b overexpression sustains circadian rhythms, and the hsfB2b mutant has a short circadian period under these conditions. HsfB2b is also involved in the regulation of hypocotyl growth under warm, short days. Our findings highlight the role of the circadian clock as an integrator of ambient abiotic stress signals important for the growth and fitness of plants.
Assuntos
Proteínas de Arabidopsis/biossíntese , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Relógios Circadianos/fisiologia , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica de Plantas/fisiologia , Proteínas de Choque Térmico/metabolismo , Proteínas de Plantas/metabolismo , Proteínas Repressoras/biossíntese , Proteínas Repressoras/metabolismo , Estresse Fisiológico/fisiologia , Fatores de Transcrição/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Ligação a DNA/genética , Flores/genética , Flores/metabolismo , Fatores de Transcrição de Choque Térmico , Proteínas de Choque Térmico/genética , Hipocótilo/genética , Hipocótilo/metabolismo , Proteínas de Plantas/genética , Proteínas Repressoras/genética , Fatores de Transcrição/genéticaRESUMO
To compete for nutrients in diverse soil microenvironments, plants proliferate lateral roots preferentially in nutrient-rich zones. For nitrate, root foraging involves local and systemic signaling; however, little is known about the genes that function in the systemic signaling pathway. By using nitrate enhancer DNA to screen a library of Arabidopsis transcription factors in the yeast one-hybrid system, the transcription factor gene TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR1-20 (TCP20) was identified. TCP20, which belongs to an ancient, plant-specific gene family that regulates shoot, flower, and embryo development, was implicated in nitrate signaling by its ability to bind DNA in more than 100 nitrate-regulated genes. Analysis of insertion mutants of TCP20 showed that they had normal primary and lateral root growth on homogenous nitrate media but were impaired in preferential lateral root growth (root foraging) on heterogeneous media in split-root plates. Inhibition of preferential lateral root growth was still evident in the mutants even when ammonium was uniformly present in the media, indicating that the TCP20 response was to nitrate. Comparison of tcp20 mutants with those of nlp7 mutants, which are defective in local control of root growth but not in the root-foraging response, indicated that TCP20 function is independent of and distinct from NLP7 function. Further analysis showed that tcp20 mutants lack systemic control of root growth regardless of the local nitrate concentrations. These results indicate that TCP20 plays a key role in the systemic signaling pathway that directs nitrate foraging by Arabidopsis roots.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Nitratos/metabolismo , Raízes de Plantas/metabolismo , Fatores de Transcrição/metabolismo , Ecossistema , Elementos Facilitadores Genéticos , Escherichia coli/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Homozigoto , Mutação , Nitrogênio/metabolismo , Fenótipo , Fenômenos Fisiológicos Vegetais , Brotos de Planta/metabolismo , Regiões Promotoras Genéticas , Transdução de Sinais/efeitos dos fármacos , Microbiologia do SoloRESUMO
Plant architecture shows a large degree of developmental plasticity. Some of the key determinants are the timing of the floral transition induced by a systemic flowering signal (florigen) and the branching pattern regulated by key factors such as BRANCHED1 (BRC1). Here, we report that BRC1 interacts with the florigen proteins FLOWERING LOCUS T (FT) and TWIN SISTER OF FT (TSF) but not with TERMINAL FLOWER1, a floral repressor. FT protein induced in leaves moves into the subtended bud, suggesting that FT protein also plays a role in promotion of the floral transition in the axillary meristem (AM). The brc1-2 mutant shows an earlier floral transition in the axillary shoots compared with the wild type, suggesting that BRC1 plays a role in delaying the floral transition of the AMs. Genetic and gene expression analyses suggest that BRC1 interferes with florigen (FT and TSF) function in the AMs. Consistent with this, BRC1 ectopically expressed in the shoot apical meristem delays the floral transition in the main shoot. These results taken together suggest that BRC1 protein interacts with FT and TSF proteins and modulates florigen activity in the axillary buds to prevent premature floral transition of the AMs.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Flores/genética , Meristema/genética , Fatores de Transcrição/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Florígeno/metabolismo , Flores/crescimento & desenvolvimento , Flores/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Meristema/crescimento & desenvolvimento , Meristema/metabolismo , Mutação , Proteína de Ligação a Fosfatidiletanolamina/genética , Proteína de Ligação a Fosfatidiletanolamina/metabolismo , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas , Ligação Proteica , Transporte Proteico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Fatores de Transcrição/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
The first described feedback loop of the Arabidopsis circadian clock is based on reciprocal regulation between Timing of CAB Expression 1 (TOC1) and Circadian Clock-associated 1 (CCA1)/late elongated hypocotyl (LHY). CCA1 and LHY are Myb transcription factors that bind directly to the TOC1 promoter to negatively regulate its expression. Conversely, the activity of TOC1 has remained less well characterized. Genetic data support that TOC1 is necessary for the reactivation of CCA1/LHY, but there is little description of its biochemical function. Here we show that TOC1 occupies specific genomic regions in the CCA1 and LHY promoters. Purified TOC1 binds directly to DNA through its CCT domain, which is similar to known DNA-binding domains. Chemical induction and transient overexpression of TOC1 in Arabidopsis seedlings cause repression of CCA1/LHY expression, demonstrating that TOC1 can repress direct targets, and mutation or deletion of the CCT domain prevents this repression showing that DNA-binding is necessary for TOC1 action. Furthermore, we use the Gal4/UAS system in Arabidopsis to show that TOC1 acts as a general transcriptional repressor, and that repression activity is in the pseudoreceiver domain of the protein. To identify the genes regulated by TOC1 on a genomic scale, we couple TOC1 chemical induction with microarray analysis and identify previously unexplored potential TOC1 targets and output pathways. Taken together, these results define a biochemical action for the core clock protein TOC1 and refine our perspective on how plant clocks function.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Relógios Circadianos , DNA de Plantas/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Sequência de Bases , Relógios Circadianos/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Repressoras/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
We present an Arabidopsis thaliana full-length transcription factor resource of 92% of root stele-expressed transcription factors and 74.5% of root-expressed transcription factors. We demonstrate its use with enhanced yeast one-hybrid (eY1H) screening for rapid, systematic mapping of plant transcription factor-promoter interactions. We identified 158 interactions with 13 stele-expressed promoters, many of which occur physically or are regulatory in planta.