RESUMO
Auxin response factors (ARFs) are a family of transcription factors that are responsible for regulating gene expression in response to changes in auxin level. The analysis of ARF sequence and activity indicates that there are 2 major groups: activators and repressors. One clade of ARFs, clade-D, is sister to clade-A activating ARFs, but are unique in that they lack a DNA-binding domain. Clade-D ARFs are present in lycophytes and bryophytes but absent in other plant lineages. The transcriptional activity of clade-D ARFs, as well as how they regulate gene expression, is not well understood. Here, we report that clade-D ARFs are transcriptional activators in the model bryophyte Physcomitrium patens and have a major role in the development of this species. Δarfddub protonemata exhibit a delay in filament branching, as well as a delay in the chloronema to caulonema transition. Additionally, leafy gametophore development in Δarfddub lines lags behind wild type. We present evidence that ARFd1 interacts with activating ARFs via their PB1 domains, but not with repressing ARFs. Based on these results, we propose a model in which clade-D ARFs enhance gene expression by interacting with DNA bound clade-A ARFs. Further, we show that ARFd1 must form oligomers for full activity.
Assuntos
Ácidos Indolacéticos , Proteínas de Plantas , Ácidos Indolacéticos/metabolismo , Proteínas de Plantas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transdução de Sinais , Regulação da Expressão Gênica de PlantasRESUMO
Modification of host hormone biology is a common strategy used by plant pathogens to promote disease. For example, the bacterial pathogen strain Pseudomonas syringae DC3000 (PtoDC3000) produces the plant hormone auxin (indole-3-acetic acid [IAA]) to promote PtoDC3000 growth in plant tissue. Previous studies suggest that auxin may promote PtoDC3000 pathogenesis through multiple mechanisms, including both suppression of salicylic acid (SA)-mediated host defenses and via an unknown mechanism that appears to be independent of SA. To test if host auxin signaling is important during pathogenesis, we took advantage of Arabidopsis thaliana lines impaired in either auxin signaling or perception. We found that disruption of auxin signaling in plants expressing an inducible dominant axr2-1 mutation resulted in decreased bacterial growth and that this phenotype was suppressed by introducing the sid2-2 mutation, which impairs SA synthesis. Thus, host auxin signaling is required for normal susceptibility to PtoDC3000 and is involved in suppressing SA-mediated defenses. Unexpectedly, tir1 afb1 afb4 afb5 quadruple-mutant plants lacking four of the six known auxin coreceptors that exhibit decreased auxin perception, supported increased levels of bacterial growth. This mutant exhibited elevated IAA levels and reduced SA-mediated defenses, providing additional evidence that auxin promotes disease by suppressing host defense. We also investigated the hypothesis that IAA promotes PtoDC3000 virulence through a direct effect on the pathogen and found that IAA modulates expression of virulence genes, both in culture and in planta. Thus, in addition to suppressing host defenses, IAA acts as a microbial signaling molecule that regulates bacterial virulence gene expression.
Assuntos
Arabidopsis/microbiologia , Ácidos Indolacéticos/metabolismo , Doenças das Plantas/microbiologia , Imunidade Vegetal , Pseudomonas syringae/patogenicidade , Virulência , Regulação da Expressão Gênica de Plantas , Mutação , Pseudomonas syringae/genética , Ácido Salicílico/metabolismo , Transdução de SinaisRESUMO
Auxin has a fundamental role throughout the life cycle of land plants. Previous studies showed that the tomato cyclophilin DIAGEOTROPICA (DGT) promotes auxin response, but its specific role in auxin signaling remains unknown. We sequenced candidate genes in auxin-insensitive mutants of Physcomitrella patens and identified mutations in highly conserved regions of the moss ortholog of tomato DGT. As P. patens and tomato diverged from a common ancestor more than 500 million years ago, this result suggests a conserved and central role for DGT in auxin signaling in land plants. In this study we characterize the P. patens dgt (Ppdgt) mutants and show that their response to auxin is altered, affecting the chloronema-to-caulonema transition and the development of rhizoids. To gain an understanding of PpDGT function we tested its interactions with the TIR1/AFB-dependent auxin signaling pathway. We did not observe a clear effect of the Ppdgt mutation on the degradation of Aux/IAA proteins. However, the induction of several auxin-regulated genes was reduced. Genetic analysis revealed that dgt can suppress the phenotype conferred by overexpression of an AFB auxin receptor. Our results indicate that the DGT protein affects auxin-induced transcription and has a conserved function in auxin regulation in land plants.
Assuntos
Bryopsida/genética , Ciclofilinas/metabolismo , Ácidos Indolacéticos/metabolismo , Proteínas de Plantas/metabolismo , Solanum lycopersicum/genética , Sequência de Bases , Bryopsida/embriologia , Ciclofilinas/genética , Evolução Molecular , Proteínas F-Box/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Receptores de Superfície Celular/metabolismo , Análise de Sequência de DNA , Transdução de Sinais , Transcrição GênicaRESUMO
The development of multicellular tissues requires both local and global coordination of cell polarization, however, the mechanisms underlying their interplay are poorly understood. In Arabidopsis, leaf epidermal pavement cells (PC) develop a puzzle-piece shape locally coordinated through apoplastic auxin signaling. Here we show auxin also globally coordinates interdigitation by activating the TIR1/AFB-dependent nuclear signaling pathway. This pathway promotes a transient maximum of auxin at the cotyledon tip, which then moves across the leaf activating local PC polarization, as demonstrated by locally uncaged auxin globally rescuing defects in tir1;afb1;afb2;afb4;afb5 mutant but not in tmk1;tmk2;tmk3;tmk4 mutants. Our findings show that hierarchically integrated global and local auxin signaling systems, which respectively depend on TIR1/AFB-dependent gene transcription in the nucleus and TMK-mediated rapid activation of ROP GTPases at the cell surface, control PC interdigitation patterns in Arabidopsis cotyledons, revealing a mechanism for coordinating a local cellular process with the development of whole tissues.
RESUMO
The moss Physcomitrella patens has recently emerged as a powerful genetically tractable model plant system. As a member of the bryophytes, P. patens provides a unique opportunity to study the evolution of a myriad of plant traits, such as polarized cell growth, gametophyte-to-sporophyte transitions, and sperm-to-pollen transition. The availability of a complete genome sequence, together with the ability to perform gene targeting efficiently in P. patens has spurred a flurry of elegant reverse genetic studies in this plant model that address a variety of key questions in plant developmental biology.
Assuntos
Bryopsida/genética , Biologia do Desenvolvimento , Evolução Molecular , Animais , Bryopsida/crescimento & desenvolvimento , Técnicas de Laboratório Clínico , Biologia do Desenvolvimento/métodos , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Especiação Genética , Masculino , Modelos Biológicos , Modelos Genéticos , Filogenia , Fenômenos Fisiológicos Vegetais/genéticaRESUMO
The phytohormone auxin triggers root growth inhibition within seconds via a non-transcriptional pathway. Among members of the TIR1/AFB auxin receptor family, AFB1 has a primary role in this rapid response. However, the unique features that confer this specific function have not been identified. Here we show that the N-terminal region of AFB1, including the F-box domain and residues that contribute to auxin binding, is essential and sufficient for its specific role in the rapid response. Substitution of the N-terminal region of AFB1 with that of TIR1 disrupts its distinct cytoplasm-enriched localization and activity in rapid root growth inhibition by auxin. Importantly, the N-terminal region of AFB1 is indispensable for auxin-triggered calcium influx, which is a prerequisite for rapid root growth inhibition. Furthermore, AFB1 negatively regulates lateral root formation and transcription of auxin-induced genes, suggesting that it plays an inhibitory role in canonical auxin signaling. These results suggest that AFB1 may buffer the transcriptional auxin response, whereas it regulates rapid changes in cell growth that contribute to root gravitropism.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas F-Box , Arabidopsis/metabolismo , Ácidos Indolacéticos/farmacologia , Ácidos Indolacéticos/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas F-Box/metabolismo , Raízes de Plantas/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Citosol/metabolismo , Regulação da Expressão Gênica de PlantasRESUMO
The phytohormone auxin triggers root growth inhibition within seconds via a non-transcriptional pathway. Among members of the TIR1/AFBs auxin receptor family, AFB1 has a primary role in this rapid response. However, the unique features that confer this specific function have not been identified. Here we show that the N-terminal region of AFB1, including the F-box domain and residues that contribute to auxin binding, are essential and sufficient for its specific role in the rapid response. Substitution of the N-terminal region of AFB1 with that of TIR1 disrupts its distinct cytoplasm-enriched localization and activity in rapid root growth inhibition. Importantly, the N-terminal region of AFB1 is indispensable for auxin-triggered calcium influx which is a prerequisite for rapid root growth inhibition. Furthermore, AFB1 negatively regulates lateral root formation and transcription of auxin-induced genes, suggesting that it plays an inhibitory role in canonical auxin signaling. These results suggest that AFB1 may buffer the transcriptional auxin response while it regulates rapid changes in cell growth that contribute to root gravitropism.
RESUMO
A major challenge in plant biology is to understand how the plant hormone auxin regulates diverse transcriptional responses throughout development, in different environments, and in different species. The answer may lie in the specific complement of auxin signaling components in each cell. The balance between activators (class-A AUXIN RESPONSE FACTORS) and repressors (class-B ARFs) is particularly important. It is unclear how this balance is achieved. Through comparative analysis of novel, dominant mutants in maize and the moss Physcomitrium patens , we have discovered a â¼500-million-year-old mechanism of class-B ARF protein level regulation, important in determining cell fate decisions across land plants. Thus, our results add a key piece to the puzzle of how auxin regulates plant development.
RESUMO
To facilitate genetic mapping of developmental mutants of Physcomitrium patens, we produced a genetic marker that combines recessive auxotrophy with dominant positive selection. We first identified the gene affected by the pabB4 auxotrophic mutation and then replaced it with a cassette that confers antibiotic resistance. This strain may be used to produce bi-parental somatic hybrids with nearly any other strain.
RESUMO
The TIR1/AFB auxin co-receptors mediate diverse responses to the plant hormone auxin. The Arabidopsis genome encodes six TIR1/AFB proteins representing three of the four clades that were established prior to angiosperm radiation. To determine the role of these proteins in plant development we performed an extensive genetic analysis involving the generation and characterization of all possible multiply-mutant lines. We find that loss of all six TIR1/AFB proteins results in early embryo defects and eventually seed abortion, and yet a single wild-type allele of TIR1 or AFB2 is sufficient to support growth throughout development. Our analysis reveals extensive functional overlap between even the most distantly related TIR1/AFB genes except for AFB1. Surprisingly, AFB1 has a specialized function in rapid auxin-dependent inhibition of root growth and early phase of root gravitropism. This activity may be related to a difference in subcellular localization compared to the other members of the family.
Assuntos
Proteínas de Arabidopsis/genética , Proteínas F-Box/genética , Receptores de Superfície Celular/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/fisiologia , Proteínas F-Box/fisiologia , Proteínas de Plantas/genética , Proteínas de Plantas/fisiologia , Receptores de Superfície Celular/fisiologiaRESUMO
MicroRNAs (miRNAs) are 21 nt RNAs that regulate many biological processes in plants by mediating translational inhibition or cleavage of target transcripts. Arabidopsis mutants defective in miRNA biogenesis have overlapping and highly pleiotropic phenotypes including serrated leaves and ABA hypersensitivity. Recent evidence indicates that miRNA genes are transcribed by RNA polymerase II (Pol II). Since Pol II transcripts are capped, we hypothesized that CBP (cap-binding protein) 20 and 80 may bind to capped primary miRNA (pri-miRNA) transcripts and play a role in their processing. Here, we show that cbp20 and cbp80 mutants have reduced miRNA levels and increased pri-miRNA levels. Co-immunoprecipitation experiments revealed that pri-miRNAs 159, 166, 168 and 172 could be associated with CBP20 and CBP80. We found that CBP20 and CBP80 are stabilized by ABA by a post-translational mechanism, and these proteins are needed for ABA induction of miR159 during seed germination. The lack of miR159 accumulation in ABA-treated seeds of cbp20/80 mutants leads to increased MYB33 and MYB101 transcript levels, and presumably higher levels of these positive regulators result in ABA hypersensitivity. Genetic and molecular analyses show that CBP20 and 80 have overlapping function in the same developmental pathway as SE and HYL1. Our results identify new components in miRNA biogenesis.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , MicroRNAs/metabolismo , Proteínas de Ligação ao Cap de RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ácido Abscísico/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Cicloeximida/farmacologia , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Genes de Plantas , Imunoprecipitação , Mutação , Fenótipo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Ligação Proteica , Proteínas de Ligação ao Cap de RNA/genética , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , RNA de Plantas/metabolismo , Proteínas de Ligação a RNA/genéticaRESUMO
The coordinated action of the auxin-sensitive Aux/IAA transcriptional repressors and ARF transcription factors produces complex gene-regulatory networks in plants. Despite their importance, our knowledge of these two protein families is largely based on analysis of stabilized forms of the Aux/IAAs, and studies of a subgroup of ARFs that function as transcriptional activators. To understand how auxin regulates gene expression we generated a Physcomitrella patens line that completely lacks Aux/IAAs. Loss of the repressors causes massive changes in transcription with misregulation of over a third of the annotated genes. Further, we find that the aux/iaa mutant is blind to auxin indicating that auxin regulation of transcription occurs exclusively through Aux/IAA function. We used the aux/iaa mutant as a simplified platform for studies of ARF function and demonstrate that repressing ARFs regulate auxin-induced genes and fine-tune their expression. Further the repressing ARFs coordinate gene induction jointly with activating ARFs and the Aux/IAAs.
Assuntos
Bryopsida/fisiologia , Regulação da Expressão Gênica de Plantas , Ácidos Indolacéticos/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Proteínas de Plantas/metabolismo , Fatores de Transcrição/metabolismoRESUMO
The plant hormone auxin is perceived by a family of F-box proteins called the TIR1/AFBs. Phylogenetic studies reveal that these proteins fall into four clades in flowering plants called TIR1, AFB2, AFB4, and AFB6. Genetic studies indicate that members of the TIR1 and AFB2 groups act as positive regulators of auxin signaling by promoting the degradation of the Aux/IAA transcriptional repressors. In this report, we demonstrate that both AFB4 and AFB5 also function as auxin receptors based on in vitro assays. We also provide genetic evidence that AFB4 and AFB5 are targets of the picloram family of auxinic herbicides in addition to indole-3-acetic acid. In contrast to previous studies we find that null afb4 alleles do not exhibit obvious defects in seedling morphology or auxin hypersensitivity. We conclude that AFB4 and AFB5 act in a similar fashion to other members of the family but exhibit a distinct auxin specificity.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/metabolismo , Proteínas F-Box/metabolismo , Herbicidas/farmacologia , Picloram/farmacologia , Receptores de Superfície Celular/metabolismo , Alelos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Resistência a Herbicidas/genética , Ácidos Indolacéticos/metabolismo , Mutação , Fenótipo , Plantas Geneticamente Modificadas , Ligação Proteica , Receptores de Superfície Celular/genética , Plântula/genética , Plântula/metabolismoRESUMO
Auxin regulates most aspects of flowering-plant growth and development, including key developmental innovations that evolved within the vascular plant lineage after diverging from a bryophyte-like ancestor nearly 500 million years ago. Recent studies in Arabidopsis indicate that auxin acts by directly binding the TIR1 subunit of the SCF(TIR1) ubiquitin ligase; this binding results in degradation of the Aux/IAA transcriptional repressors and de-repression of auxin-responsive genes. Little is known, however, about the mechanism of auxin action in other plants. To characterize auxin signaling in a nonflowering plant, we utilized the genetically tractable moss Physcomitrella patens. We used a candidate-gene approach to show that previously identified auxin-resistant mutants of P. patens harbor mutations in Aux/IAA genes. Furthermore, we show that the moss Aux/IAA proteins interact with Arabidopsis TIR1 moss homologs called PpAFB and that a reduction in PpAFB levels results in a phenotype similar to that of the auxin-resistant mutants. Our results indicate that the molecular mechanism of auxin perception is conserved in land plants despite vast differences in the role auxin plays in different plant lineages.
Assuntos
Bryopsida/metabolismo , Ácidos Indolacéticos/farmacologia , Mutação , Proteínas de Plantas/metabolismo , Transdução de Sinais , Motivos de Aminoácidos , Bryopsida/efeitos dos fármacos , Bryopsida/genética , Sequência Conservada , Fenótipo , Proteínas de Plantas/genética , Proteínas de Plantas/fisiologiaRESUMO
The plant hormone auxin plays an essential role in plant development. However, only a few auxin biosynthetic genes have been isolated and characterized. Here, we show that the TRANSPORT INHIBITOR RESPONSE2 (TIR2) gene is required for many growth processes. Our studies indicate that the tir2 mutant is hypersensitive to 5-methyl-tryptophan, an inhibitor of tryptophan synthesis. Further, treatment with the proposed auxin biosynthetic intermediate indole-3-pyruvic acid (IPA) and indole-3-acetic acid rescues the tir2 short hypocotyl phenotype, suggesting that tir2 may be affected in the IPA auxin biosynthetic pathway. Molecular characterization revealed that TIR2 is identical to the TAA1 gene encoding a tryptophan aminotransferase. We show that TIR2 is regulated by temperature and is required for temperature-dependent hypocotyl elongation. Further, we find that expression of TIR2 is induced on the lower side of a gravitropically responding root. We propose that TIR2 contributes to a positive regulatory loop required for root gravitropism.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas/fisiologia , Ácidos Indolacéticos/metabolismo , Triptofano Transaminase/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Clonagem Molecular , Temperatura Alta , Mutação , Ftalimidas/farmacologia , Filogenia , Plântula/citologia , Plântula/metabolismo , Triptofano Transaminase/genéticaRESUMO
Arabidopsis class III homeodomain-leucine zipper (HD-Zip III) proteins play overlapping, distinct, and antagonistic roles in key aspects of development that have evolved during land plant evolution. To better understand this gene family's role in plant evolution and development as well as to address broader questions of how duplicated genes functionally diversify, we investigated the evolutionary history of this gene family. Phylogenetic analyses including homologs from diverse land plants indicate that a gene duplication event before the angiosperm--gymnosperm split gave rise to two gene lineages that diversified during angiosperm plant radiation. Heterologous expression of an HD-Zip III gene from the nonvascular plant moss within the Arabidopsis HD-zip III revoluta mutant modified but did not complement the phenotype. Comparison of the expression domains of flowering and nonflowering plant homologs indicate an ancestral role in vascular development and organ initiation but not in specifying organ polarity, a prominent role for angiosperm homologs.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Evolução Molecular , Proteínas de Homeodomínio/genética , Zíper de Leucina/genética , Família Multigênica , Proteínas de Arabidopsis/classificação , Proteínas de Homeodomínio/classificação , Dados de Sequência MolecularRESUMO
Organogenesis at the shoot meristem requires a delicate balance between stem cell specification and differentiation. In Arabidopsis thaliana, WUSCHEL (WUS) is a key factor promoting stem cell identity, whereas the CLAVATA (CLV1, CLV2, and CLV3) loci appear to promote differentiation by repressing WUS expression. In a screen for mutations modifying clv1 mutants, we have identified a novel regulator of meristem development we term CORONA (CNA). Whereas cna single mutant plants exhibit subtle defects in meristem development, clv cna double mutants develop massively enlarged apices that display early loss of organogenesis, misexpression of WUS and CLV3, and eventual differentiation of the entire apex. The CNA gene was isolated by positional cloning and found to encode a class III homeodomain Leu zipper protein. A missense mutation resulting in the dominant-negative cna-1 allele was identified in a conserved domain of unknown function, and a likely null allele was shown to display a similar but weaker phenotype. CNA is expressed in developing vascular tissue, diffusely through shoot and flower meristems, and within developing stamens and carpels. Our analysis of WUS expression in wild-type, clv, and clv cna plants revealed that, contrary to current models, WUS is neither necessary nor sufficient for stem cell specification and that neither WUS nor CLV3 is a marker for stem cell identity. We propose that CNA functions in parallel to the CLV loci to promote organ formation.
Assuntos
Arabidopsis/crescimento & desenvolvimento , Arabidopsis/genética , Genes de Plantas , Alelos , Sequência de Aminoácidos , Arabidopsis/citologia , Proteínas de Arabidopsis/genética , Mapeamento Cromossômico , Epistasia Genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Genes Homeobox , Zíper de Leucina/genética , Meristema/citologia , Meristema/crescimento & desenvolvimento , Dados de Sequência Molecular , Família Multigênica , Mutação , Homologia de Sequência de Aminoácidos , Células-Tronco/citologiaRESUMO
The Arabidopsis thaliana genome contains five class III homeodomain-leucine zipper genes. We have isolated loss-of-function alleles for each family member for use in genetic analysis. This gene family regulates apical embryo patterning, embryonic shoot meristem formation, organ polarity, vascular development, and meristem function. Genetic analyses revealed a complex pattern of overlapping functions, some of which are not readily inferred by phylogenetic relationships or by gene expression patterns. The PHABULOSA and PHAVOLUTA genes perform overlapping functions with REVOLUTA, whereas the PHABULOSA, PHAVOLUTA, and CORONA/ATHB15 genes perform overlapping functions distinct from REVOLUTA. Furthermore, ATHB8 and CORONA encode functions that are both antagonistic to those of REVOLUTA within certain tissues and overlapping with REVOLUTA in other tissues. Differences in expression patterns explain some of these genetic interactions, whereas other interactions are likely attributable to differences in protein function as indicated by cross-complementation studies.