RESUMO
Flowering phenology is important in the adaptation of many plants to their local environment, but its adaptive value has not been extensively studied in herbaceous perennials. We used Arabis alpina as a model system to determine the importance of flowering phenology to fitness of a herbaceous perennial with a wide geographical range. Individual plants representative of local genetic diversity (accessions) were collected across Europe, including in Spain, the Alps and Scandinavia. The flowering behaviour of these accessions was documented in controlled conditions, in common-garden experiments at native sites and in situ in natural populations. Accessions from the Alps and Scandinavia varied in whether they required exposure to cold (vernalization) to induce flowering, and in the timing and duration of flowering. By contrast, all Spanish accessions obligately required vernalization and had a short duration of flowering. Using experimental gardens at native sites, we show that an obligate requirement for vernalization increases survival in Spain. Based on our analyses of genetic diversity and flowering behaviour across Europe, we propose that in the model herbaceous perennial A. alpina, an obligate requirement for vernalization, which is correlated with short duration of flowering, is favoured by selection in Spain where the plants experience a long growing season.
Assuntos
Arabis , Arabis/genética , Flores/genética , Geografia , Países Escandinavos e Nórdicos , Europa (Continente)RESUMO
Information about the incidence and magnitude of local adaptation can help to predict the response of natural populations to a changing environment, and should be of particular interest in arctic and alpine environments where the effects of climate change are expected to be severe. To quantify adaptive differentiation in the arctic-alpine perennial herb Arabis alpina, we conducted reciprocal transplant experiments for 3 yr between Spanish and Scandinavian populations. At the sites of one Spanish and one Scandinavian population, we planted seedlings representing two Spanish and four Scandinavian populations, and recorded survival, flowering propensity and fecundity. The experiment was replicated in two subsequent years. The results demonstrate strong adaptive differentiation between A. alpina populations from the two regions. At the field site in Spain, survival and fruit production of Spanish populations were higher than those of Scandinavian populations, while the opposite was true at the site in Scandinavia, and these differences were consistent across years. By comparison, fitness varied little among populations from the same region. The results suggest that the magnitude and geographical scale of local adaptation need to be considered in predictions of the effects of global change on the dynamics of arctic and alpine plant populations.
Assuntos
Adaptação Fisiológica , Arabis/fisiologia , Regiões Árticas , Mudança Climática , Meio Ambiente , Geografia , Reprodução , Países Escandinavos e Nórdicos , EspanhaRESUMO
The CONSTITUTIVE EXPRESSOR OF PATHOGENESIS-RELATED GENES5 (CPR5) gene of Arabidopsis (Arabidopsis thaliana) encodes a putative membrane protein of unknown biochemical function and displays highly pleiotropic functions, particularly in pathogen responses, cell proliferation, cell expansion, and cell death. Here, we demonstrate a link between CPR5 and the GLABRA1 ENHANCER BINDING PROTEIN (GeBP) family of transcription factors. We investigated the primary role of the GeBP/GeBP-like (GPL) genes using transcriptomic analysis of the quadruple gebp gpl1,2,3 mutant and one overexpressing line that displays several cpr5-like phenotypes including dwarfism, spontaneous necrotic lesions, and increased pathogen resistance. We found that GeBP/GPLs regulate a set of genes that represents a subset of the CPR5 pathway. This subset includes genes involved in response to stress as well as cell wall metabolism. Analysis of the quintuple gebp gpl1,2,3 cpr5 mutant indicates that GeBP/GPLs are involved in the control of cell expansion in a CPR5-dependent manner but not in the control of cell proliferation. In addition, to our knowledge, we provide the first evidence that the CPR5 protein is localized in the nucleus of plant cells and that a truncated version of the protein with no transmembrane domain can trigger cpr5-like processes when fused to the VP16 constitutive transcriptional activation domain. Our results provide clues on how CPR5 and GeBP/GPLs play opposite roles in the control of cell expansion and suggest that the CPR5 protein is involved in transcription.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Membrana/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Afidicolina/farmacologia , Arabidopsis/citologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Tamanho Celular/efeitos dos fármacos , Epistasia Genética/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes de Plantas/genética , Mutação/genética , Proteínas Nucleares/metabolismo , Fenótipo , Epiderme Vegetal/citologia , Epiderme Vegetal/efeitos dos fármacos , Epiderme Vegetal/genética , Transporte Proteico/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fatores de Transcrição/genética , Transcrição Gênica/efeitos dos fármacosRESUMO
Tobacco (Nicotiana sylvestris) glandular trichomes make an attractive target for isoprenoid metabolic engineering because they produce large amounts of one type of diterpenoids, alpha- and beta-cembratrien-diols. This article describes the establishment of tools for metabolic engineering of tobacco trichomes, namely a transgenic line with strongly reduced levels of diterpenoids in the exudate and the characterization of a trichome specific promoter. The diterpene-free tobacco line was generated by silencing the major tobacco diterpene synthases, which were found to be encoded by a family of four highly similar genes (NsCBTS-2a, NsCBTS-2b, NsCBTS-3 and NsCBTS-4), one of which is a pseudogene. The promoter regions of all four CBTS genes were sequenced and found to share over 95% identity between them. Transgenic plants expressing uidA under the control of the NsCBTS-2a promoter displayed a specific pattern of GUS expression restricted exclusively to the glandular cells of the tall secretory trichomes. A series of sequential and internal deletions of the NsCBTS-2a promoter led to the identification of two cis-acting regions. The first, located between positions -589 to -479 from the transcription initiation site, conferred a broad transcriptional activation, not only in the glandular cells, but also in cells of the trichome stalk, as well as in the leaf epidermis and the root. The second region, located between positions -279 to -119, had broad repressor activity except in trichome glandular cells and is mainly responsible for the specific expression pattern of the NsCBTS-2a gene. These results establish the basis for the identification of trans-regulators required for the expression of the CBTS genes restricted to the secretory cells of the glandular trichomes.
Assuntos
Alquil e Aril Transferases/genética , Nicotiana/genética , Proteínas de Plantas/genética , Sequências Reguladoras de Ácido Nucleico/genética , Alquil e Aril Transferases/classificação , Alquil e Aril Transferases/metabolismo , Sequência de Bases , Diterpenos/análise , Diterpenos/química , Diterpenos/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Regulação da Expressão Gênica de Plantas , Glucuronidase/genética , Glucuronidase/metabolismo , Dados de Sequência Molecular , Estrutura Molecular , Família Multigênica , Filogenia , Epiderme Vegetal/citologia , Epiderme Vegetal/genética , Epiderme Vegetal/metabolismo , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência do Ácido Nucleico , Nicotiana/metabolismoRESUMO
N6-Methyladenosine is a ubiquitous modification identified in the mRNA of numerous eukaryotes, where it is present within both coding and noncoding regions. However, this base modification does not alter the coding capacity, and its biological significance remains unclear. We show that Arabidopsis thaliana mRNA contains N6-methyladenosine at levels similar to those previously reported for animal cells. We further show that inactivation of the Arabidopsis ortholog of the yeast and human mRNA adenosine methylase (MTA) results in failure of the developing embryo to progress past the globular stage. We also demonstrate that the arrested seeds are deficient in mRNAs containing N6-methyladenosine. Expression of MTA is strongly associated with dividing tissues, particularly reproductive organs, shoot meristems, and emerging lateral roots. Finally, we show that MTA interacts in vitro and in vivo with At FIP37, a homolog of the Drosophila protein FEMALE LETHAL2D and of human WILMS' TUMOUR1-ASSOCIATING PROTEIN. The results reported here provide direct evidence for an essential function for N6-methyladenosine in a multicellular eukaryote, and the interaction with At FIP37 suggests possible RNA processing events that might be regulated or altered by this base modification.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Proteínas de Transporte/metabolismo , Metiltransferases/metabolismo , Adenosina/análogos & derivados , Adenosina/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Cromatografia em Camada Fina , Flores/enzimologia , Flores/genética , Flores/metabolismo , Regulação da Expressão Gênica de Plantas , Teste de Complementação Genética , Imunoprecipitação , Metiltransferases/genética , Dados de Sequência Molecular , Folhas de Planta/enzimologia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Ligação Proteica , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA , Técnicas do Sistema de Duplo-HíbridoRESUMO
Understanding the role of transcription factors (TFs) is essential in reconstructing developmental regulatory networks. The plant-specific GeBP TF family of Arabidopsis thaliana (Arabidopsis) comprises 21 members, all of unknown function. A subset of four members, the founding member GeBP and GeBP-like proteins (GPL) 1, 2, and 3, shares a conserved C-terminal domain. Here we report that GeBP/GPL genes represent a newly defined class of leucine-zipper (Leu-zipper) TFs and that they play a redundant role in cytokinin hormone pathway regulation. Specifically, we demonstrate using yeast, in vitro, and split-yellow fluorescent protein in planta assays that GeBP/GPL proteins form homo- and heterodimers through a noncanonical Leu-zipper motif located in the C-terminal domain. A triple loss-of-function mutant of the three most closely related genes gebp gpl1 gpl2 shows a reduced sensitivity to exogenous cytokinins in a subset of cytokinin responses such as senescence and growth, whereas root inhibition is not affected. We find that transcript levels of type-A cytokinin response genes, which are involved in the negative feedback regulation of cytokinin signaling, are higher in the triple mutant. Using a GPL version that acts as a constitutive transcriptional activator, we show that the regulation of Arabidopsis response regulators (ARRs) is mediated by at least one additional, as yet unknown, repressor acting genetically downstream in the GeBP/GPL pathway. Our results indicate that GeBP/GPL genes encode a new class of unconventional Leu-zipper TF proteins and suggest that their role in the cytokinin pathway is to antagonize the negative feedback regulation on ARR genes to trigger the cytokinin response.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Citocininas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Ligação a DNA/genética , Dimerização , Retroalimentação Fisiológica/fisiologia , Regulação da Expressão Gênica de Plantas , Zíper de Leucina , Família Multigênica , Mutação , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação para CimaRESUMO
Leaf hairs (trichomes) of Arabidopsis thaliana are a model system for studying cell development, differentiation and cell cycle regulation. To exploit this model system with ultimate spatial resolution we applied single cell sampling, thus avoiding the averaging effect induced by complex tissue mixtures. In particular, we analysed gene expression profiles of two selected stages of the developing trichome: trichome initial cells and mature trichomes, as well as pavement cells. Ten single cells per sample were collected by glass microcapillaries and used for the generation of radioactive probes for subsequent hybridization to nylon filters representing approximately 8000 genes of A. thaliana. Functional categorization of genes transcribed in trichome initials, mature trichomes and pavement cells demonstrated involvement of these surface cells in the stress response. In silico promoter analysis of genes preferentially expressed in trichome initials revealed enrichment in MYB-binding sites and presence of elements involved in hormonal, metal, sulphur response and cell cycle regulation. Three candidate genes preferentially expressed in trichome initials were selected for further analysis: At3g16980 (putative RNA polymerase II), At5g15230 (GASA4) and At4g27260 (GH3.5, WES1). Promoter:GUS studies confirmed expression of the putative RNA polymerase II and the gibberellin responsive GASA4 in trichome initials and partially in mature trichomes. Functional implication of the three selected candidates in trichome development and hence in cell cycle regulation in A. thaliana is discussed. We suggest that these genes are involved in differentiation and initiation of endocycling during trichome development.
Assuntos
Arabidopsis/genética , Perfilação da Expressão Gênica/métodos , Epiderme Vegetal/genética , Folhas de Planta/genética , Arabidopsis/citologia , Biologia Computacional , Regulação da Expressão Gênica de Plantas , Epiderme Vegetal/citologia , Epiderme Vegetal/crescimento & desenvolvimento , Folhas de Planta/citologia , Regiões Promotoras Genéticas/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Recently, an alternative route to the proteasomal protein-degradation pathway was discovered that specifically targets transmembrane proteins marked with a single ubiquitin to the endosomal multivesicular body (MVB) and, subsequently, to the vacuole (yeast) or lysosome (animals), where they are degraded by proteases. Vps23p/TSG101 is a key component of the ESCRT I-III machinery in yeast and animals that recognizes mono-ubiquitylated proteins and sorts them into the MVB. Here, we report that the Arabidopsis ELCH (ELC) gene encodes a Vps23p/TSG101 homolog, and that homologs of all known ESCRT I-III components are present in the Arabidopsis genome. As with its animal and yeast counterparts, ELC binds ubiquitin and localizes to endosomes. Gel-filtration experiments indicate that ELC is a component of a high-molecular-weight complex. Yeast two-hybrid and immunoprecipitation assays showed that ELC interacts with Arabidopsis homologs of the ESCRT I complex. The elc mutant shows multiple nuclei in various cell types, indicating a role in cytokinesis. Double-mutant analysis with kaktus shows that increased ploidy levels do not influence the cytokinesis effect of elc mutants, suggesting that ELC is only important during the first endoreduplication cycle. Double mutants with tubulin folding cofactor a mutants show a synergistic phenotype, suggesting that ELC regulates cytokinesis through the microtubule cytoskeleton.
Assuntos
Arabidopsis/genética , Arabidopsis/metabolismo , Sequência de Aminoácidos , Arabidopsis/citologia , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sequência de Bases , Sequência Conservada , Citocinese/genética , Citocinese/fisiologia , Citoesqueleto/metabolismo , Primers do DNA/genética , DNA de Plantas/genética , Endossomos/metabolismo , Genes de Plantas , Microtúbulos/metabolismo , Dados de Sequência Molecular , Complexos Multiproteicos , Mutação , Fenótipo , Plantas Geneticamente Modificadas , Homologia de Sequência de Aminoácidos , Ubiquitina/metabolismoRESUMO
Embryonic regulators LEC2 (LEAFY COTYLEDON2) and FUS3 (FUSCA3) are involved in multiple aspects of Arabidopsis (Arabidopsis thaliana) seed development, including repression of leaf traits and premature germination and activation of seed storage protein genes. In this study, we show that gibberellin (GA) hormone biosynthesis is regulated by LEC2 and FUS3 pathways. The level of bioactive GAs is increased in immature seeds of lec2 and fus3 mutants relative to wild-type level. In addition, we show that the formation of ectopic trichome cells on lec2 and fus3 embryos is a GA-dependent process as in true leaves, suggesting that the GA pathway is misactivated in embryonic mutants. We next demonstrate that the GA-biosynthesis gene AtGA3ox2, which encodes the key enzyme AtGA3ox2 that catalyzes the conversion of inactive to bioactive GAs, is ectopically activated in embryos of the two mutants. Interestingly, both beta-glucuronidase reporter gene expression and in situ hybridization indicate that FUS3 represses AtGA3ox2 expression mainly in epidermal cells of embryo axis, which is distinct from AtGA3ox2 pattern at germination. Finally, we show that the FUS3 protein physically interacts with two RY elements (CATGCATG) present in the AtGA3ox2 promoter. This work suggests that GA biosynthesis is directly controlled by embryonic regulators during Arabidopsis embryonic development.
Assuntos
Arabidopsis/embriologia , Arabidopsis/genética , Genes de Plantas , Giberelinas/biossíntese , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Germinação , Mutação , Regiões Promotoras Genéticas , Plântula/embriologia , Plântula/genética , Plântula/metabolismoRESUMO
The FKBP12 (FK506-binding protein 12 kD) immunophilin interacts with several protein partners in mammals and is a physiological regulator of the cell cycle. In Arabidopsis, only one specific partner of AtFKBP12, namely AtFIP37 (FKBP12 interacting protein 37 kD), has been identified but its function in plant development is not known. We present here the functional analysis of AtFIP37 in Arabidopsis. Knockout mutants of AtFIP37 show an embryo-lethal phenotype that is caused by a strong delay in endosperm development and embryo arrest. AtFIP37 promoter::beta-glucuronidase reporter gene constructs show that the gene is expressed during embryogenesis and throughout plant development, in undifferentiating cells such as meristem or embryonic cells as well as highly differentiating cells such as trichomes. A translational fusion with the enhanced yellow fluorescent protein indicates that AtFIP37 is a nuclear protein localized in multiple subnuclear foci that show a speckled distribution pattern. Overexpression of AtFIP37 in transgenic lines induces the formation of large trichome cells with up to six branches. These large trichomes have a DNA content up to 256C, implying that these cells have undergone extra rounds of endoreduplication. Altogether, these data show that AtFIP37 is critical for life in Arabidopsis and implies a role for AtFIP37 in the regulation of the cell cycle as shown for FKBP12 and TOR (target of rapamycin) in mammals.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Transporte/genética , Extensões da Superfície Celular/genética , Imunofilinas/genética , Epiderme Vegetal/genética , Sequência de Aminoácidos , Arabidopsis/enzimologia , Proteínas de Arabidopsis/metabolismo , Proteínas de Transporte/metabolismo , Extensões da Superfície Celular/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Imunofilinas/metabolismo , Dados de Sequência Molecular , Mutação , Epiderme Vegetal/fisiologia , Mapeamento de Interação de Proteínas , Proteínas de Ligação a RNA , Homologia de Sequência de AminoácidosRESUMO
Trichomes of Arabidopsis are single-celled epidermal hair that are a useful model for studying plant cell fate determination. Trichome initiation requires the activity of the GLABROUS1 (GL1) gene whose expression in epidermal and trichome cells is dependent on the presence of a 3'-cis-regulatory element. Using a one-hybrid screen, we have isolated a cDNA, which encodes for a protein, GL1 enhancer binding protein (GeBP), that binds this regulatory element in yeast and in vitro. GeBP and its three homologues in Arabidopsis share two regions: a central region with no known motifs and a C-terminal region with a putative leucine-zipper motif. We show that both regions are necessary for trans-activation in yeast. A translational fusion with the Yellow Fluorescent Protein (YFP) indicates that GeBP is a nuclear protein whose localization is restricted to, on average, 3-5 subnuclear foci that might correspond to nucleoli. Transcriptional fusion with the GUS reporter indicates that GeBP is mainly expressed in vegetative meristematic tissues and in very young leaf primordia. We looked at GeBP expression in plants mutated in or misexpressing KNAT1, a KNOX gene, expressed in the shoot apical meristem and downregulated in leaf founder cells, and found that GeBP transcript level is regulated by KNAT1 suggesting that KNAT1 is a transcriptional activator of GeBP. This regulation suggests that GeBP is acting as a repressor of leaf cell fate.
Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Proteínas de Homeodomínio/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/química , Elementos Facilitadores Genéticos/genética , Proteínas de Homeodomínio/genética , Zíper de Leucina , Meristema/metabolismo , Dados de Sequência Molecular , Mutação/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fenótipo , Folhas de Planta/metabolismo , Ligação Proteica , Transporte Proteico , Fatores de Transcrição , Ativação Transcricional , Regulação para Cima , LevedurasRESUMO
The biogenesis of microtubules comprises several steps, including the correct folding of alpha- and beta-tubulin and heterodimer formation. In vitro studies and the genetic analysis in yeast revealed that, after translation, alpha- and beta-tubulin are processed by several chaperonins and microtubule-folding cofactors (TFCs) to produce assembly-competent alpha-/beta-tubulin heterodimers. One of the TFCs, TFC-C, does not exist in yeast, and a potential function of TFC-C is thus based only on the biochemical analysis. In this study and in a very recently published study by Steinborn and coworkers, the analysis of the Arabidopsis porcino (por) mutant has shown that TFC-C is important for microtubule function in vivo. The predicted POR protein shares weak amino acid similarity with the human TFC-C (hTFC-C). Our finding that hTFC-C under the control of the ubiquitously expressed 35S promoter can rescue the por mutant phenotype shows that the POR gene encodes the Arabidopsis ortholog of hTFC-C. The analysis of plants carrying a GFP:POR fusion construct showed that POR protein is localized in the cytoplasm and is not associated with microtubules. While, in por mutants, microtubule density was indistinguishable from wild-type, their organization was affected.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/metabolismo , Proteínas Associadas aos Microtúbulos/fisiologia , Tubulina (Proteína)/metabolismo , Alelos , Sequência de Aminoácidos , Arabidopsis/embriologia , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Divisão Celular , Clonagem Molecular , Sequência Consenso , Dimerização , Genes de Plantas , Teste de Complementação Genética , Humanos , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/ultraestrutura , Dados de Sequência Molecular , Peso Molecular , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/fisiologia , Reprodução , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
The control of the stoichiometric balance of alpha- and beta-tubulin is important during microtubule biogenesis. This process involves several tubulin-folding cofactors (TFCs), of which only TFC A is not essential in mammalian in vitro systems or in vivo in yeast. Here, we show that the TFC A gene is important in vivo in plants. The Arabidopsis gene KIESEL (KIS) shows sequence similarity to the TFC A gene. Expression of the mouse TFC A gene under the control of the 35S promoter rescues the kis mutation, indicating that KIS is the Arabidopsis ortholog of TFC A. kis plants exhibit a range of defects similar to the phenotypes associated with impaired microtubule function: plants are reduced in size and show meiotic defects, cell division is impaired, and trichomes are bulged and less branched. Microtubule density was indistinguishable from that of the wild type, but microtubule organization was affected in trichomes and hypocotyl cells of dark-grown kis plants. The kis phenotype was rescued by overexpression of an alpha-tubulin, indicating that KIS is involved in the control of the correct balance of alpha- and beta-tubulin monomers.