RESUMO
The anaphase-promoting complex/cyclosome (APC/C) marks key cell cycle proteins for proteasomal breakdown, thereby ensuring unidirectional progression through the cell cycle. Its target recognition is temporally regulated by activating subunits, one of which is called CELL CYCLE SWITCH 52 A2 (CCS52A2). We sought to expand the knowledge on the APC/C by using the severe growth phenotypes of CCS52A2-deficient Arabidopsis (Arabidopsis thaliana) plants as a readout in a suppressor mutagenesis screen, resulting in the identification of the previously undescribed gene called PIKMIN1 (PKN1). PKN1 deficiency rescues the disorganized root stem cell phenotype of the ccs52a2-1 mutant, whereas an excess of PKN1 inhibits the growth of ccs52a2-1 plants, indicating the need for control of PKN1 abundance for proper development. Accordingly, the lack of PKN1 in a wild-type background negatively impacts cell division, while its systemic overexpression promotes proliferation. PKN1 shows a cell cycle phase-dependent accumulation pattern, localizing to microtubular structures, including the preprophase band, the mitotic spindle, and the phragmoplast. PKN1 is conserved throughout the plant kingdom, with its function in cell division being evolutionarily conserved in the liverwort Marchantia polymorpha. Our data thus demonstrate that PKN1 represents a novel, plant-specific protein with a role in cell division that is likely proteolytically controlled by the CCS52A2-activated APC/C.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas de Arabidopsis/metabolismo , Divisão Celular/genética , Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Arabidopsis/metabolismo , Ciclossomo-Complexo Promotor de Anáfase/genética , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Proteínas de Plantas/metabolismo , MitoseRESUMO
The ataxia-telangiectasia mutated (ATM) and ATM and Rad3-related (ATR) kinases coordinate the DNA damage response. The roles described for Arabidopsis thaliana ATR and ATM are assumed to be conserved over other plant species, but molecular evidence is scarce. Here, we demonstrate that the functions of ATR and ATM are only partially conserved between Arabidopsis and maize (Zea mays). In both species, ATR and ATM play a key role in DNA repair and cell cycle checkpoint activation, but whereas Arabidopsis plants do not suffer from the absence of ATR under control growth conditions, maize mutant plants accumulate replication defects, likely due to their large genome size. Moreover, contrarily to Arabidopsis, maize ATM deficiency does not trigger meiotic defects, whereas the ATR kinase appears to be crucial for the maternal fertility. Strikingly, ATR is required to repress premature endocycle onset and cell death in the maize endosperm. Its absence results in a reduction of kernel size, protein and starch content, and a stochastic death of kernels, a process being counteracted by ATM. Additionally, while Arabidopsis atr atm double mutants are viable, no such mutants could be obtained for maize. Therefore, our data highlight that the mechanisms maintaining genome integrity may be more important for vegetative and reproductive development than previously anticipated.
Assuntos
Reparo do DNA/genética , Endosperma/genética , Proteínas de Plantas/genética , Zea mays/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas Mutadas de Ataxia Telangiectasia/genética , Sistemas CRISPR-Cas , Morte Celular/genética , Quebras de DNA de Cadeia Dupla , Replicação do DNA/genética , Endosperma/citologia , Instabilidade Genômica , Mutação , Células Vegetais , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Sementes/citologia , Sementes/genética , Sementes/crescimento & desenvolvimento , Zea mays/citologia , Zea mays/crescimento & desenvolvimentoRESUMO
The anaphase promoting complex/cyclosome (APC/C) controls unidirectional progression through the cell cycle by marking key cell cycle proteins for proteasomal turnover. Its activity is temporally regulated by the docking of different activating subunits, known in plants as CELL DIVISION PROTEIN20 (CDC20) and CELL CYCLE SWITCH52 (CCS52). Despite the importance of the APC/C during cell proliferation, the number of identified targets in the plant cell cycle is limited. Here, we used the growth and meristem phenotypes of Arabidopsis (Arabidopsis thaliana) CCS52A2-deficient plants in a suppressor mutagenesis screen to identify APC/CCCS52A2 substrates or regulators, resulting in the identification of a mutant cyclin CYCA3;4 allele. CYCA3;4 deficiency partially rescues the ccs52a2-1 phenotypes, whereas increased CYCA3;4 levels enhance the scored ccs52a2-1 phenotypes. Furthermore, whereas the CYCA3;4 protein is promptly broken down after prophase in wild-type plants, it remains present in later stages of mitosis in ccs52a2-1 mutant plants, marking it as a putative APC/CCCS52A2 substrate. Strikingly, increased CYCA3;4 levels result in aberrant root meristem and stomatal divisions, mimicking phenotypes of plants with reduced RETINOBLASTOMA-RELATED PROTEIN1 (RBR1) activity. Correspondingly, RBR1 hyperphosphorylation was observed in CYCA3;4 gain-of-function plants. Our data thus demonstrate that an inability to timely destroy CYCA3;4 contributes to disorganized formative divisions, possibly in part caused by the inactivation of RBR1.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , Proteínas de Ciclo Celular/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Ciclo Celular/genética , Diferenciação Celular/genética , Divisão Celular , Metanossulfonato de Etila/farmacologia , Regulação da Expressão Gênica de Plantas , Meristema/citologia , Meristema/genética , Mutação , Fosforilação , Células Vegetais/efeitos dos fármacos , Folhas de Planta/citologia , Folhas de Planta/genética , Raízes de Plantas/citologia , Raízes de Plantas/genética , Caules de Planta/citologia , Plantas Geneticamente Modificadas , Polimorfismo de Nucleotídeo ÚnicoRESUMO
The WEE1 and ATM AND RAD3-RELATED (ATR) kinases are important regulators of the plant intra-S-phase checkpoint; consequently, WEE1KO and ATRKO roots are hypersensitive to replication-inhibitory drugs. Here, we report on a loss-of-function mutant allele of the FASCIATA1 (FAS1) subunit of the chromatin assembly factor 1 (CAF-1) complex that suppresses the phenotype of WEE1- or ATR-deficient Arabidopsis (Arabidopsis thaliana) plants. We demonstrate that lack of FAS1 activity results in the activation of an ATAXIA TELANGIECTASIA MUTATED (ATM)- and SUPPRESSOR OF GAMMA-RESPONSE 1 (SOG1)-mediated G2/M-arrest that renders the ATR and WEE1 checkpoint regulators redundant. This ATM activation accounts for the telomere erosion and loss of ribosomal DNA that are described for fas1 plants. Knocking out SOG1 in the fas1 wee1 background restores replication stress sensitivity, demonstrating that SOG1 is an important secondary checkpoint regulator in plants that fail to activate the intra-S-phase checkpoint.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/fisiologia , Replicação do DNA , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-myb/genética , Transdução de Sinais , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Genoma de Planta , Instabilidade Genômica , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-myb/metabolismo , Estresse Fisiológico , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Whereas our knowledge about the diverse pathways aiding DNA repair upon genome damage is steadily increasing, little is known about the molecular players that adjust the plant cell cycle in response to DNA stress. By a meta-analysis of DNA stress microarray data sets, three family members of the SIAMESE/SIAMESE-RELATED (SIM/SMR) class of cyclin-dependent kinase inhibitors were discovered that react strongly to genotoxicity. Transcriptional reporter constructs corroborated specific and strong activation of the three SIM/SMR genes in the meristems upon DNA stress, whereas overexpression analysis confirmed their cell cycle inhibitory potential. In agreement with being checkpoint regulators, SMR5 and SMR7 knockout plants displayed an impaired checkpoint in leaf cells upon treatment with the replication inhibitory drug hydroxyurea (HU). Surprisingly, HU-induced SMR5/SMR7 expression depends on ATAXIA TELANGIECTASIA MUTATED (ATM) and SUPPRESSOR OF GAMMA RESPONSE1, rather than on the anticipated replication stress-activated ATM AND RAD3-RELATED kinase. This apparent discrepancy was explained by demonstrating that, in addition to its effect on replication, HU triggers the formation of reactive oxygen species (ROS). ROS-dependent transcriptional activation of the SMR genes was confirmed by different ROS-inducing conditions, including high-light treatment. We conclude that the identified SMR genes are part of a signaling cascade that induces a cell cycle checkpoint in response to ROS-induced DNA damage.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/genética , Proteínas de Ciclo Celular/fisiologia , Proteínas Inibidoras de Quinase Dependente de Ciclina/fisiologia , Dano ao DNA , Espécies Reativas de Oxigênio/farmacologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Pontos de Checagem do Ciclo Celular , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Inibidoras de Quinase Dependente de Ciclina/genética , Proteínas Inibidoras de Quinase Dependente de Ciclina/metabolismo , Regulação da Expressão Gênica de Plantas , Técnicas de Inativação de Genes , Hidroxiureia/farmacologia , Estresse Oxidativo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologiaRESUMO
Cell division in photosynthetic organisms is tightly regulated by light. Although the light dependency of the onset of the cell cycle has been well characterized in various phototrophs, little is known about the cellular signaling cascades connecting light perception to cell cycle activation and progression. Here, we demonstrate that diatom-specific cyclin 2 (dsCYC2) in Phaeodactylum tricornutum displays a transcriptional peak within 15 min after light exposure, long before the onset of cell division. The product of dsCYC2 binds to the cyclin-dependent kinase CDKA1 and can complement G1 cyclin-deficient yeast. Consistent with the role of dsCYC2 in controlling a G1-to-S light-dependent cell cycle checkpoint, dsCYC2 silencing decreases the rate of cell division in diatoms exposed to light-dark cycles but not to constant light. Transcriptional induction of dsCYC2 is triggered by blue light in a fluence rate-dependent manner. Consistent with this, dsCYC2 is a transcriptional target of the blue light sensor AUREOCHROME1a, which functions synergistically with the basic leucine zipper (bZIP) transcription factor bZIP10 to induce dsCYC2 transcription. The functional characterization of a cyclin whose transcription is controlled by light and whose activity connects light signaling to cell cycle progression contributes significantly to our understanding of the molecular mechanisms underlying light-dependent cell cycle onset in diatoms.
Assuntos
Divisão Celular , Ciclinas/genética , Diatomáceas/genética , Regulação da Expressão Gênica , Transdução de Sinais , Proteínas de Algas/genética , Proteínas de Algas/metabolismo , Ciclinas/metabolismo , Escuridão , Diatomáceas/citologia , Diatomáceas/fisiologia , Diatomáceas/efeitos da radiação , Teste de Complementação Genética , Luz , Modelos Biológicos , Mutação , Fotossíntese , Regiões Promotoras Genéticas , Biossíntese de Proteínas , Transcrição GênicaRESUMO
A sessile lifestyle forces plants to respond promptly to factors that affect their genomic integrity. Therefore, plants have developed checkpoint mechanisms to arrest cell cycle progression upon the occurrence of DNA stress, allowing the DNA to be repaired before onset of division. Previously, the WEE1 kinase had been demonstrated to be essential for delaying progression through the cell cycle in the presence of replication-inhibitory drugs, such as hydroxyurea. To understand the severe growth arrest of WEE1-deficient plants treated with hydroxyurea, a transcriptomics analysis was performed, indicating prolonged S-phase duration. A role for WEE1 during S phase was substantiated by its specific accumulation in replicating nuclei that suffered from DNA stress. Besides an extended replication phase, WEE1 knockout plants accumulated dead cells that were associated with premature vascular differentiation. Correspondingly, plants without functional WEE1 ectopically expressed the vascular differentiation marker VND7, and their vascular development was aberrant. We conclude that the growth arrest of WEE1-deficient plants is due to an extended cell cycle duration in combination with a premature onset of vascular cell differentiation. The latter implies that the plant WEE1 kinase acquired an indirect developmental function that is important for meristem maintenance upon replication stress.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Diferenciação Celular , Replicação do DNA , Feixe Vascular de Plantas/citologia , Proteínas Serina-Treonina Quinases/metabolismo , Estresse Fisiológico , Arabidopsis/citologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Bleomicina/farmacologia , Morte Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Análise por Conglomerados , Reparo do DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Estabilidade Enzimática/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes de Plantas/genética , Hidroxiureia/farmacologia , Cinética , Meristema/citologia , Meristema/efeitos dos fármacos , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Feixe Vascular de Plantas/efeitos dos fármacos , Fase S/efeitos dos fármacos , Estresse Fisiológico/efeitos dos fármacos , Fatores de TempoRESUMO
The anaphase-promoting complex/cyclosome (APC/C) is a multisubunit ubiquitin ligase that regulates progression through the cell cycle by marking key cell division proteins for destruction. To ensure correct cell cycle progression, accurate timing of APC/C activity is important, which is obtained through its association with both activating and inhibitory subunits. However, although the APC/C is highly conserved among eukaryotes, no APC/C inhibitors are known in plants. Recently, we have identified ULTRAVIOLET-B-INSENSITIVE4 (UVI4) as a plant-specific component of the APC/C. Here, we demonstrate that UVI4 uses conserved APC/C interaction motifs to counteract the activity of the CELL CYCLE SWITCH52 A1 (CCS52A1) activator subunit, inhibiting the turnover of the A-type cyclin CYCA2;3. UVI4 is expressed in an S phase-dependent fashion, likely through the action of E2F transcription factors. Correspondingly, uvi4 mutant plants failed to accumulate CYCA2;3 during the S phase and prematurely exited the cell cycle, triggering the onset of the endocycle. We conclude that UVI4 regulates the temporal inactivation of APC/C during DNA replication, allowing CYCA2;3 to accumulate above the level required for entering mitosis, and thereby regulates the meristem size and plant growth rate.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Divisão Celular , Ciclina A2/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ciclossomo-Complexo Promotor de Anáfase , Arabidopsis/anatomia & histologia , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular , Imunoprecipitação da Cromatina , Ciclina A2/genética , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Fatores de Transcrição E2F/genética , Fatores de Transcrição E2F/metabolismo , Regulação da Expressão Gênica de Plantas , Teste de Complementação Genética , Meristema/crescimento & desenvolvimento , Meristema/metabolismo , Meristema/ultraestrutura , Microscopia Eletrônica de Varredura , Mutagênese Sítio-Dirigida , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Fase S , Relação Estrutura-Atividade , Ativação Transcricional , Transformação Genética , Técnicas do Sistema de Duplo-Híbrido , Complexos Ubiquitina-Proteína Ligase/genéticaRESUMO
The caspase family protease, separase, is required at anaphase onset to cleave the cohesin complex, which joins sister chromatids. However, among eukaryotes, separases have acquired novel functions. Here, we show that Arabidopsis thaliana radially swollen 4 (rsw4), a temperature-sensitive mutant isolated previously on the basis of root swelling, harbors a mutation in At4g22970, the A. thaliana separase. Loss of separase function in rsw4 at the restrictive temperature is indicated by the widespread failure of replicated chromosomes to disjoin. Surprisingly, rsw4 has neither pronounced cell cycle arrest nor anomalous spindle formation, which occur in other eukaryotes upon loss of separase activity. However, rsw4 roots have disorganized cortical microtubules and accumulate the mitosis-specific cyclin, cyclin B1;1, excessive levels of which have been associated with altered microtubules and morphology. Cyclin B1;1 also accumulates in certain backgrounds in response to DNA damage, but we find no evidence for aberrant responses to DNA damage in rsw4. Our characterization of rsw4 leads us to hypothesize that plant separase, in addition to cleaving cohesin, regulates cyclin B1;1, with profound ramifications for morphogenesis.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/genética , Proteínas de Ciclo Celular/genética , Endopeptidases/genética , Morfogênese/genética , Não Disjunção Genética/genética , Fatores de Transcrição/metabolismo , Arabidopsis/enzimologia , Proteínas de Arabidopsis/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/fisiologia , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos de Plantas/genética , Clonagem Molecular , Ciclina B/genética , Ciclina B/metabolismo , Endopeptidases/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Mutação/fisiologia , Raízes de Plantas/anatomia & histologia , Raízes de Plantas/genética , Plantas Geneticamente Modificadas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estabilidade Proteica , Separase , Temperatura , Fatores de Transcrição/genética , CoesinasRESUMO
As in other eukaryotes, cell division in plants is highly conserved and regulated by cyclin-dependent kinases (CDKs) that are themselves predominantly regulated at the posttranscriptional level by their association with proteins such as cyclins. Although over the last years the knowledge of the plant cell cycle has considerably increased, little is known on the assembly and regulation of the different CDK complexes. To map protein-protein interactions between core cell cycle proteins of Arabidopsis thaliana, a binary protein-protein interactome network was generated using two complementary high-throughput interaction assays, yeast two-hybrid and bimolecular fluorescence complementation. Pairwise interactions among 58 core cell cycle proteins were tested, resulting in 357 interactions, of which 293 have not been reported before. Integration of the binary interaction results with cell cycle phase-dependent expression information and localization data allowed the construction of a dynamic interaction network. The obtained interaction map constitutes a framework for further in-depth analysis of the cell cycle machinery.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Ciclo Celular , Quinases Ciclina-Dependentes/metabolismo , Mapeamento de Interação de Proteínas , Bases de Dados de Proteínas , Análise de Sequência com Séries de Oligonucleotídeos , Técnicas do Sistema de Duplo-HíbridoRESUMO
The plant hormone auxin is thought to provide positional information for patterning during development. It is still unclear, however, precisely how auxin is distributed across tissues and how the hormone is sensed in space and time. The control of gene expression in response to auxin involves a complex network of over 50 potentially interacting transcriptional activators and repressors, the auxin response factors (ARFs) and Aux/IAAs. Here, we perform a large-scale analysis of the Aux/IAA-ARF pathway in the shoot apex of Arabidopsis, where dynamic auxin-based patterning controls organogenesis. A comprehensive expression map and full interactome uncovered an unexpectedly simple distribution and structure of this pathway in the shoot apex. A mathematical model of the Aux/IAA-ARF network predicted a strong buffering capacity along with spatial differences in auxin sensitivity. We then tested and confirmed these predictions using a novel auxin signalling sensor that reports input into the signalling pathway, in conjunction with the published DR5 transcriptional output reporter. Our results provide evidence that the auxin signalling network is essential to create robust patterns at the shoot apex.
Assuntos
Arabidopsis/crescimento & desenvolvimento , Ácidos Indolacéticos/farmacologia , Reguladores de Crescimento de Plantas/farmacologia , Brotos de Planta/crescimento & desenvolvimento , Transdução de Sinais/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Análise por Conglomerados , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Hibridização in Situ Fluorescente , Meristema/química , Meristema/metabolismo , Microscopia Confocal , Modelos Teóricos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Organogênese , Brotos de Planta/genética , Brotos de Planta/metabolismo , Plantas Geneticamente Modificadas , Transcrição GênicaRESUMO
Endoreduplication represents a variation on the cell cycle in which multiple rounds of DNA replication occur without subsequent chromosome separation and cytokinesis, thereby increasing the cellular DNA content. It is known that the DNA ploidy level of cells is controlled by external stimuli such as light; however, limited knowledge is available on how environmental signals regulate the endoreduplication cycle at the molecular level. Previously, we had demonstrated that the conversion from a mitotic cell cycle into an endoreduplication cycle is controlled by the atypical E2F transcription factor, DP-E2F-LIKE1 (DEL1), that represses the endocycle onset. Here, the Arabidopsis (Arabidopsis thaliana) DEL1 gene was identified as a transcriptional target of the classical E2Fb and E2Fc transcription factors that antagonistically control its transcript levels through competition for a single E2F cis-acting binding site. In accordance with the reported opposite effects of light on the protein levels of E2Fb and E2Fc, DEL1 transcription depended on the light regime. Strikingly, modified DEL1 expression levels uncoupled the link between light and endoreduplication in hypocotyls, implying that DEL1 acts as a regulatory connection between endocycle control and the photomorphogenic response.
Assuntos
Proteínas de Arabidopsis/antagonistas & inibidores , Arabidopsis/genética , Arabidopsis/efeitos da radiação , Fatores de Transcrição E2F/antagonistas & inibidores , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Luz , Fatores de Transcrição/genética , Proteínas de Arabidopsis/metabolismo , Sequência de Bases , Fatores de Transcrição E2F/metabolismo , Hipocótilo/genética , Hipocótilo/efeitos da radiação , Modelos Biológicos , Dados de Sequência Molecular , Mutação/genética , Plantas Geneticamente Modificadas , Ploidias , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos da radiação , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Synchronized cell cultures are an indispensable tool for the identification and understanding of key regulators of the cell cycle. Nevertheless, the use of cell cultures has its disadvantages, because it represents an artificial system that does not completely mimic the endogenous conditions that occur in organized meristems. Here, we present a new and easy method for Arabidopsis thaliana root tip synchronization by hydroxyurea treatment. A major advantage of the method is the possibility of investigating available Arabidopsis cell-cycle mutants without the need to generate cell cultures. As a proof of concept, the effects of over-expression of a dominant negative allele of the B-type cyclin-dependent kinase CDKB1;1 gene on cell-cycle progression were tested. The previously observed prolonged G2 phase was confirmed, but was found to be compensated for by a reduced G1 phase. Furthermore, altered S-phase kinetics indicated a functional role for CDKB1;1 during the replication process.
Assuntos
Arabidopsis/efeitos dos fármacos , Hidroxiureia/farmacologia , Meristema/efeitos dos fármacos , Mitose/efeitos dos fármacos , Inibidores da Síntese de Ácido Nucleico/farmacologia , Alelos , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Citometria de Fluxo , Perfilação da Expressão Gênica , Histonas/metabolismo , Nucleotídeos/metabolismo , Estresse FisiológicoRESUMO
Large-scale screening studies carried out to date for genetic variants that affect gene regulation are generally limited to descriptions of differences in allele-specific expression (ASE) detected in vivo. Allele-specific differences in gene expression provide evidence for a model whereby cis-acting genetic variation results in differential expression between alleles. Such gene surveys for regulatory variation are a first step in identifying the specific nucleotide changes that govern gene expression differences, but they leave the underlying mechanisms unexplored. Here, we propose a quantitative genetics approach to perform a genome-wide analysis of ASE differences (GASED). The GASED approach is based on a diallel design that is often used in plant breeding programs to estimate general combining abilities (GCA) of specific inbred lines and to identify high-yielding hybrid combinations of parents based on their specific combining abilities (SCAs). In a context of gene expression, the values of GCA and SCA parameters allow cis- and trans-regulatory changes to be distinguished and imbalances in gene expression to be ascribed to cis-regulatory variation. With this approach, a total of 715 genes could be identified that are likely to carry allelic polymorphisms responsible for at least a 1.5-fold allelic expression difference in a total of 10 diploid Arabidopsis thaliana hybrids. The major strength of the GASED approach, compared to other ASE detection methods, is that it is not restricted to genes with allelic transcript variants. Although a false-positive rate of 9/41 was observed, the GASED approach is a valuable pre-screening method that can accelerate systematic surveys of naturally occurring cis-regulatory variation among inbred lines for laboratory species, such as Arabidopsis, mouse, rat and fruitfly, and economically important crop species, such as corn.
Assuntos
Alelos , Regulação da Expressão Gênica , Genômica/métodos , Polimorfismo Genético , Arabidopsis/genética , Arabidopsis/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Locos de Características Quantitativas , Elementos Reguladores de TranscriçãoRESUMO
Regeneration of a tissue damaged by injury represents a physiological response for organ recovery1-3. Although this regeneration process is conserved across multicellular taxa, plants appear to display extremely high regenerative capacities, a feature widely used in tissue culture for clonal propagation and grafting4,5. Regenerated cells arise predominantly from pre-existing populations of division-competent cells6,7; however, the mechanisms by which these cells are triggered to divide in response to injury remain largely elusive8. Here, we demonstrate that the heterodimeric transcription factor complex ETHYLENE RESPONSE FACTOR115 (ERF115)-PHYTOCHROME A SIGNAL TRANSDUCTION1 (PAT1) sustains meristem function by promoting cell renewal after stem cell loss. High-resolution time-lapse imaging revealed that cell death promotes ERF115 activity in cells that are in direct contact with damaged cells, triggering divisions that replenish the collapsed stem cells. Correspondingly, the ERF115-PAT1 complex plays an important role in full stem cell niche recovery upon root tip excision, whereas its ectopic expression triggers neoplastic growth, correlated with activation of the putative target gene WOUND INDUCED DEDIFFERENTIATION1 (WIND1)9. We conclude that the ERF115-PAT1 complex accounts for the high regenerative potential of plants, granting them the ability to efficiently replace damaged cells with new ones.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/fisiologia , Meristema/fisiologia , Fitocromo/genética , Regeneração , Fatores de Transcrição/genética , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Fitocromo/metabolismo , Fatores de Transcrição/metabolismoRESUMO
The mitosis-to-endocycle transition requires the controlled inactivation of M phase-associated cyclin-dependent kinase (CDK) activity. Previously, the B-type CDKB1;1 was identified as an important negative regulator of endocycle onset. Here, we demonstrate that CDKB1;1 copurifies and associates with the A2-type cyclin CYCA2;3. Coexpression of CYCA2;3 with CDKB1;1 triggered ectopic cell divisions and inhibited endoreduplication. Moreover, the enhanced endoreduplication phenotype observed after overexpression of a dominant-negative allele of CDKB1;1 could be partially complemented by CYCA2;3 co-overexpression, illustrating that both subunits unite in vivo to form a functional complex. CYCA2;3 protein stability was found to be controlled by CCS52A1, an activator of the anaphase-promoting complex. We conclude that CCS52A1 participates in endocycle onset by down-regulating CDKB1;1 activity through the destruction of CYCA2;3.
Assuntos
Proteínas de Arabidopsis/fisiologia , Ciclo Celular/fisiologia , Ciclina A/fisiologia , Quinases Ciclina-Dependentes/fisiologia , Proteínas de Arabidopsis/análise , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/fisiologia , Divisão Celular/genética , Divisão Celular/fisiologia , Núcleo Celular/metabolismo , Ciclina A/análise , Ciclina A/genética , Ciclina A2 , Quinases Ciclina-Dependentes/análise , Quinases Ciclina-Dependentes/genética , Regulação para Baixo , Proteínas de Fluorescência Verde/análise , Estabilidade Proteica , Proteínas Recombinantes de Fusão/análiseRESUMO
Prenylated Rab acceptor 1 (PRA1) domain proteins are small transmembrane proteins that regulate vesicle trafficking as receptors of Rab GTPases and the vacuolar soluble N-ethylmaleimide-sensitive factor attachment receptor protein VAMP2. However, little is known about PRA1 family members in plants. Sequence analysis revealed that higher plants, compared with animals and primitive plants, possess an expanded family of PRA1 domain-containing proteins. The Arabidopsis (Arabidopsis thaliana) PRA1 (AtPRA1) proteins were found to homodimerize and heterodimerize in a manner corresponding to their phylogenetic distribution. Different AtPRA1 family members displayed distinct expression patterns, with a preference for vascular cells and expanding or developing tissues. AtPRA1 genes were significantly coexpressed with Rab GTPases and genes encoding vesicle transport proteins, suggesting an involvement in the vesicle trafficking process similar to that of their animal counterparts. Correspondingly, AtPRA1 proteins were localized in the endoplasmic reticulum, Golgi apparatus, and endosomes/prevacuolar compartments, hinting at a function in both secretory and endocytic intracellular trafficking pathways. Taken together, our data reveal a high functional diversity of AtPRA1 proteins, probably dealing with the various demands of the complex trafficking system.