RESUMEN
Plants, as sessile organisms, face the crucial challenge of adjusting growth and development with ever-changing environmental conditions. Protein synthesis is the fundamental process that enables growth of all organisms. Since elevated temperature presents a substantial threat to protein stability and function, immediate adjustments of protein synthesis rates are necessary to circumvent accumulation of proteotoxic stress and to ensure survival. This review provides an overview of the mechanisms that control translation under high-temperature stress by the modification of components of the translation machinery in plants, and compares them to yeast and metazoa. Recent research also suggests an important role for cytoplasmic biomolecular condensates, named stress granules, in these processes. Current understanding of the role of stress granules in translational regulation and of the molecular processes associated with translation that might occur within stress granules is also discussed.
Asunto(s)
Biosíntesis de Proteínas , Plantas/metabolismo , Condensados Biomoleculares/metabolismo , Condensados Biomoleculares/fisiología , Adaptación Fisiológica , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Fenómenos Fisiológicos de las PlantasRESUMEN
The important role of translational control for maintenance of proteostasis is well documented in plants, but the exact mechanisms that coordinate translation rates during plant development and stress response are not well understood. In Arabidopsis, the translation elongation complex eEF1B consists of three subunits: eEF1Bα, eEF1Bß, and eEF1Bγ. While eEF1Bα and eEF1Bß have a conserved GDP/GTP exchange function, the function of eEF1Bγ is still unknown. By generating Arabidopsis mutants with strongly reduced eEF1Bγ levels, we revealed its essential role during plant growth and development and analysed its impact on translation. To explore the function of the eEF1B subunits under high temperature stress, we analysed their dynamic localization as green fluorescent protein fusions under control and heat stress conditions. Each of these fusion proteins accumulated in heat-induced cytoplasmic foci and co-localized with the stress granule marker poly(A)-binding protein 8-mCherry. Protein-protein interaction studies and co-expression analyses indicated that eEF1Bß physically interacted with both of the other subunits and promoted their recruitment to cytoplasmic foci. These data provide new insights into the mechanisms allowing for rapid adaptation of translation rates during heat stress response.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Factor 1 de Elongación Peptídica/genética , Factor 1 de Elongación Peptídica/análisis , Factor 1 de Elongación Peptídica/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulación de la Expresión Génica de las PlantasRESUMEN
The two paralogous Arabidopsis genes MAINTENANCE OF MERISTEMS (MAIN) and MAINTENANCE OF MERISTEMS LIKE1 (MAIL1) encode a conserved retrotransposon-related plant mobile domain and are known to be required for silencing of transposable elements (TE) and for primary root development. Loss of function of either MAIN or MAIL1 leads to release of heterochromatic TEs, reduced condensation of pericentromeric heterochromatin, cell death of meristem cells and growth arrest of the primary root soon after germination. Here, we show that they act in one protein complex that also contains the inactive isoform of PROTEIN PHOSPHATASE 7 (PP7), which is named PROTEIN PHOSPHATASE 7-LIKE (PP7L). PP7L was previously shown to be important for chloroplast biogenesis and efficient chloroplast protein synthesis. We show that loss of PP7L function leads to the same root growth phenotype as loss of MAIL1 or MAIN. In addition, pp7l mutants show similar silencing defects. Double mutant analyses confirmed that the three proteins act in the same molecular pathway. The primary root growth arrest, which is associated with cell death of stem cells and their daughter cells, is a consequence of genome instability. Our data demonstrate so far unrecognized functions of an inactive phosphatase isoform in a protein complex that is essential for silencing of heterochromatic elements and for maintenance of genome stability in dividing cells.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Elementos Transponibles de ADN/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Arabidopsis/crecimiento & desarrollo , Arabidopsis/fisiología , Proteínas de Arabidopsis/genética , Cloroplastos/metabolismo , Silenciador del Gen , Germinación , Heterocromatina/genética , Isoenzimas , Meristema/genética , Meristema/crecimiento & desarrollo , Meristema/fisiología , Mutación , Proteínas Nucleares/genética , Fenotipo , Fosfoproteínas Fosfatasas/genética , Raíces de Plantas/genética , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/fisiología , Retroelementos/genéticaRESUMEN
Tidal wetlands have been increasingly recognized as long-term carbon sinks in recent years. Work on carbon sequestration and decomposition processes in tidal wetlands focused so far mainly on effects of global-change factors such as sea-level rise and increasing temperatures. However, little is known about effects of land use, such as livestock grazing, on organic matter decomposition and ultimately carbon sequestration. The present work aims at understanding the mechanisms by which large herbivores can affect organic matter decomposition in tidal wetlands. This was achieved by studying both direct animal-microbe interactions and indirect animal-plant-microbe interactions in grazed and ungrazed areas of two long-term experimental field sites at the German North Sea coast. We assessed bacterial and fungal gene abundance using quantitative PCR, as well as the activity of microbial exo-enzymes by conducting fluorometric assays. We demonstrate that grazing can have a profound impact on the microbial community structure of tidal wetland soils, by consistently increasing the fungi-to-bacteria ratio by 38-42%, and therefore potentially exerts important control over carbon turnover and sequestration. The observed shift in the microbial community was primarily driven by organic matter source, with higher contributions of recalcitrant autochthonous (terrestrial) vs. easily degradable allochthonous (marine) sources in grazed areas favoring relative fungal abundance. We propose a novel and indirect form of animal-plant-microbe interaction: top-down control of aboveground vegetation structure determines the capacity of allochthonous organic matter trapping during flooding and thus the structure of the microbial community. Furthermore, our data provide the first evidence that grazing slows down microbial exo-enzyme activity and thus decomposition through changes in soil redox chemistry. Activities of enzymes involved in C cycling were reduced by 28-40%, while activities of enzymes involved in N cycling were not consistently affected by grazing. It remains unclear if this is a trampling-driven direct grazing effect, as hypothesized in earlier studies, or if the effect on redox chemistry is plant mediated and thus indirect. This study improves our process-level understanding of how grazing can affect the microbial ecology and biogeochemistry of semi-terrestrial ecosystems that may help explain and predict differences in C turnover and sequestration rates between grazed and ungrazed systems.
Asunto(s)
Fenómenos Fisiológicos Bacterianos , Secuestro de Carbono , Hongos/fisiología , Herbivoria , Microbiología del Suelo , Suelo/química , Animales , Bacterias/genética , Bacterias/aislamiento & purificación , Hongos/genética , Hongos/aislamiento & purificación , Genes Bacterianos , Genes Fúngicos , Alemania , Ganado , Ovinos , HumedalesRESUMEN
Plant development requires accurate coordination of gene expression, both in actively dividing meristematic cells and differentiated cells. Cell fate establishment and maintenance, among others, are mediated by chromatin organization complexes that determine the stable transcriptional states of specific cell types. Here, we focus on MAIN-LIKE1 (MAIL1), one of three homologs of MAINTENANCE OF MERISTEMS (MAIN), which form a plant-specific gene family in Arabidopsis thaliana. We show that MAIL1 encodes a ubiquitously expressed nuclear protein. A mail1 loss-of-function mutant developed short primary roots, in which the meristematic cells accumulated DNA double-strand breaks and underwent massive cell death. In addition, mail1 mutant showed also cell differentiation defects in root and shoot tissues, and developed disorganized callus-like structures. The genetic interaction between main and mail1 mutants suggests that they act in the same pathway, and that both are essential for maintaining correct cell division acitivity in meristematic cells, while MAIL1 has an additional function in differentiating cells.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Arabidopsis/crecimiento & desarrollo , Arabidopsis/fisiología , Proteínas de Arabidopsis/genética , Diferenciación Celular , División Celular , Expresión Génica , Genes Reporteros , Meristema/genética , Meristema/crecimiento & desarrollo , Meristema/fisiología , Mutación , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fenotipo , Raíces de Plantas/genética , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/fisiología , Brotes de la Planta/genética , Brotes de la Planta/crecimiento & desarrollo , Brotes de la Planta/fisiología , Plantas Modificadas Genéticamente , Proteínas Recombinantes de Fusión , Plantones/genética , Plantones/crecimiento & desarrollo , Plantones/fisiologíaRESUMEN
Stem cells in the root and shoot apical meristem provide the descendant cells required for growth and development throughout the lifecycle of a plant. We found that mutations in the Arabidopsis MAINTENANCE OF MERISTEMS (MAIN) gene led to plants with distorted stem cell niches in which stem cells are not maintained and undergo premature differentiation or cell death. The malfunction of main meristems leads to short roots, mis-shaped leaves, reduced fertility and partial fasciation of stems. MAIN encodes a nuclear-localized protein and is a member of a so far uncharacterized plant-specific gene family. As main mutant plants are hypersensitive to DNA-damaging agents, expression of genes involved in DNA repair is induced and dead cells with damaged DNA accumulate in the mutant meristems, we propose that MAIN is required for meristem maintenance by sustaining genome integrity in stem cells and their descendants cells.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Inestabilidad Genómica , Meristema/genética , Mutación , Proteínas Nucleares/genética , Proteínas de Arabidopsis/metabolismo , Diferenciación Celular/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Daño del ADN/genética , Regulación de la Expresión Génica de las Plantas , Proteínas Nucleares/metabolismo , Fenotipo , Raíces de Plantas/genética , Brotes de la Planta/genética , Semillas/genética , Semillas/crecimiento & desarrolloRESUMEN
Plastid translation occurs on bacterial-type 70S ribosomes consisting of a large (50S) subunit and a small (30S) subunit. The vast majority of plastid ribosomal proteins have orthologs in bacteria. In addition, plastids also possess a small set of unique ribosomal proteins, so-called plastid-specific ribosomal proteins (PSRPs). The functions of these PSRPs are unknown, but, based on structural studies, it has been proposed that they may represent accessory proteins involved in translational regulation. Here we have investigated the functions of five PSRPs using reverse genetics in the model plant Arabidopsis thaliana. By analyzing T-DNA insertion mutants and RNAi lines, we show that three PSRPs display characteristics of genuine ribosomal proteins, in that down-regulation of their expression led to decreased accumulation of the 30S or 50S subunit of the plastid ribosomes, resulting in plastid translational deficiency. In contrast, two other PSRPs can be knocked out without visible or measurable phenotypic consequences. Our data suggest that PSRPs fall into two types: (i) PSRPs that have a structural role in the ribosome and are bona fide ribosomal proteins, and (ii) non-essential PSRPs that are not required for stable ribosome accumulation and translation under standard greenhouse conditions.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Proteínas de Cloroplastos/metabolismo , Plastidios/metabolismo , Proteínas Ribosómicas/metabolismo , Ribosomas/metabolismo , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/ultraestructura , Proteínas de Arabidopsis/genética , Proteínas de Cloroplastos/genética , Cloroplastos/metabolismo , Regulación hacia Abajo/genética , Regulación de la Expresión Génica de las Plantas/genética , Técnicas de Inactivación de Genes , Genoma de Planta/genética , Mutagénesis Insercional , Fenotipo , Fotosíntesis/fisiología , Complejo de Proteína del Fotosistema II/metabolismo , Hojas de la Planta/genética , Hojas de la Planta/crecimiento & desarrollo , Hojas de la Planta/fisiología , Hojas de la Planta/ultraestructura , Plastidios/genética , Polirribosomas/genética , Polirribosomas/metabolismo , Interferencia de ARN , Genética Inversa , Proteínas Ribosómicas/genética , Ribosomas/genéticaRESUMEN
The coordination of cell division with stress response is essential for maintaining genome stability in plant meristems. Proteins involved in pre-mRNA splicing are important for these processes in animal and human cells. Based on its homology to the splicing factor SART1, which is implicated in the control of cell division and genome stability in human cells, we analyzed if MDF has similar functions in plants. We found that MDF associates with U4/U6.U5 tri-snRNP proteins and is essential for correct splicing of 2,037 transcripts. Loss of MDF function leads to cell division defects and cell death in meristems and was associated with up-regulation of stress-induced genes and down-regulation of mitotic regulators. In addition, the mdf-1 mutant is hypersensitive to DNA damage treatment supporting its role in coordinating stress response with cell division. Our analysis of a dephosphomutant of MDF suggested how its protein activity might be controlled. Our work uncovers the conserved function of a plant splicing factor and provides novel insight into the interplay of pre-mRNA processing and genome stability in plants.
Asunto(s)
Arabidopsis , Ribonucleoproteína Nuclear Pequeña U5 , Animales , Humanos , Arabidopsis/genética , Arabidopsis/metabolismo , División Celular/genética , Inestabilidad Genómica , Ribonucleoproteína Nuclear Pequeña U4-U6/genética , Ribonucleoproteína Nuclear Pequeña U4-U6/metabolismo , Ribonucleoproteína Nuclear Pequeña U5/genética , Ribonucleoproteína Nuclear Pequeña U5/metabolismo , Precursores del ARN/genética , Precursores del ARN/metabolismo , Factores de Empalme de ARN/genéticaRESUMEN
Ribosome biogenesis is a key process in all eukaryotic cells that requires hundreds of ribosome biogenesis factors (RBFs), which are essential to build the mature ribosomes consisting of proteins and rRNAs. The processing of the required rRNAs has been studied extensively in yeast and mammals, but in plants much is still unknown. In this study, we focused on a RBF from A. thaliana that we named NUCLEOLAR RNA CHAPERONE-LIKE 1 (NURC1). NURC1 was localized in the nucleolus of plant cell nuclei, and other plant RBF candidates shared the same localization. SEC-SAXS experiments revealed that NURC1 has an elongated and flexible structure. In addition, SEC-MALLS experiments confirmed that NURC1 was present in its monomeric form with a molecular weight of around 28 kDa. RNA binding was assessed by performing microscale thermophoresis with the Arabidopsis internal transcribed spacer 2 (ITS2) of the polycistronic pre-rRNA precursor, which contains the 5.8S, 18S, and 25S rRNA. NURC1 showed binding activity to the ITS2 with a dissociation constant of 228 nM and exhibited RNA chaperone-like activity. Our data suggested that NURC1 may have a function in pre-rRNA processing and thus ribosome biogenesis.
Asunto(s)
Arabidopsis , Proteínas de Plantas , Animales , Proteínas Nucleares , Dispersión del Ángulo Pequeño , Difracción de Rayos X , Arabidopsis/genética , ARN , Precursores del ARN , MamíferosRESUMEN
The transition from vegetative to generative development is a major developmental switch in flowering plants and is critical for reproductive success. This transition requires reprogramming of lateral primordia at the shoot apical meristem, which leads to the formation of determinate floral meristems instead of leaves. In Arabidopsis, flowering is induced by a network of interacting pathways. In the photoperiod-dependent pathway, the two key elements mediating the effect of day length on flowering time are the transcription factors CONSTANS (CO) and the phloem mobile flowering signal FLOWERING LOCUS T (FT). Here, we identify a factor that is critically involved in this flowering response. The gene, which we named LATE FLOWERING (LATE), encodes a C(2)H(2) -type zinc-finger transcriptional regulator, and is expressed in the leaf vasculature and the vegetative shoot apical meristem. Ectopic expression of LATE in all tissues results in a dose-dependent phenotype characterized by late flowering, altered floral organ identity and sterile flowers. Using tissue-specific promoters, we further show that LATE controls the transition to flowering at two levels: first, it regulates the expression of flowering time genes in the leaf vasculature, and second, it interferes with floral meristem identity genes at the apex.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Flores/crecimiento & desarrollo , Proteínas Represoras/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Flores/genética , Regulación de la Expresión Génica de las Plantas , Meristema/genética , Meristema/crecimiento & desarrollo , Fotoperiodo , Proteínas Represoras/genética , Dedos de ZincRESUMEN
Ribonuclease E (RNase E) represents a key enzyme in bacterial RNA metabolism. It plays multifarious roles in RNA processing and also initiates degradation of mRNA by endonucleolytic cleavage. Plastids (chloroplasts) are derived from formerly free-living bacteria and have largely retained eubacterial gene expression mechanisms. Here we report the functional characterization of a chloroplast RNase E that is encoded by a single-copy nuclear gene in the model plant Arabidopsis thaliana. Analysis of knockout plants revealed that, unlike in bacteria, RNase E is not essential for survival. Absence of RNase E results in multiple defects in chloroplast RNA metabolism. Most importantly, polycistronic precursor transcripts overaccumulate in the knockout plants, while several mature monocistronic mRNAs are strongly reduced, suggesting an important function of RNase E in intercistronic processing of primary transcripts from chloroplast operons. We further show that disturbed maturation of a transcript encoding essential ribosomal proteins results in plastid ribosome deficiency and, therefore, provides a molecular explanation for the observed mutant phenotype.
Asunto(s)
Arabidopsis/genética , Cloroplastos/enzimología , Endorribonucleasas/metabolismo , Poliadenilación , ARN del Cloroplasto/metabolismo , Ribosomas/metabolismo , Arabidopsis/enzimología , Arabidopsis/crecimiento & desarrollo , ADN de Plantas/genética , Regulación de la Expresión Génica de las Plantas , Técnicas de Inactivación de Genes , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Plantas Modificadas Genéticamente/enzimología , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/crecimiento & desarrolloRESUMEN
Tidal wetlands are effective carbon sinks, mitigating climate change through the long-term removal of atmospheric CO2. Studies along surface-elevation and thus flooding-frequency gradients in tidal wetlands are often used to understand the effects of accelerated sea-level rise on carbon sequestration, a process that is primarily determined by the balance of primary production and microbial decomposition. It has often been hypothesized that rates of microbial decomposition would increase with elevation and associated increases in soil oxygen availability; however, previous studies yield a wide range of outcomes and equivocal results. Our mechanistic understanding of the elevation-decomposition relationship is limited because most effort has been devoted to understanding the terminal steps of the decomposition process. A few studies assessed microbial exo-enzyme activities (EEAs) as initial and rate-limiting steps that often reveal important insight into microbial energy and nutrient constraints. The present study assessed EEAs and microbial abundance along a coastal ecotone stretching a flooding gradient from tidal flat to high marsh in the European Wadden Sea. We found that stabilization of exo-enzymes to mineral sediments leads to high specific EEAs at low substrate concentrations in frequently flooded, sediment-rich zones of the studied ecotone. We argue that the high background activity of a mineral-associated enzyme pool provides a stable decomposition matrix in highly dynamic, frequently flooded zones. Furthermore, we demonstrate that microbial communities are less nutrient limited in frequently flooded zones, where inputs of nutrient-rich marine organic matter are higher. This was reflected in both increasing exo-enzymatic carbon versus nutrient acquisition and decreasing fungal versus bacterial abundance with increasing flooding frequency. Our findings thereby suggest two previously unrecognized mechanisms that may contribute to stimulated microbial activity despite decreasing oxygen availability in response to accelerated sea-level rise.
RESUMEN
Transposable elements (TEs) are prevalent in most eukaryotes, and host genomes have devised silencing strategies to rein in TE activity. One of these, transcriptional silencing, is generally associated with DNA methylation and short interfering RNAs. Here we show that the Arabidopsis genes MAIL1 and MAIN define an alternative silencing pathway independent of DNA methylation and short interfering RNAs. Mutants for MAIL1 or MAIN exhibit release of silencing and appear to show impaired condensation of pericentromeric heterochromatin. Phylogenetic analysis suggests not only that MAIL1 and MAIN encode a retrotransposon-related plant mobile domain, but also that host plant mobile domains were captured by DNA transposons during plant evolution. Our results reveal a role for Arabidopsis proteins with a transposon-related domain in gene silencing.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Elementos Transponibles de ADN/genética , Silenciador del Gen , Proteínas Nucleares/genética , Dominios Proteicos , Retroelementos/genética , Centrómero/metabolismo , Metilación de ADN , Evolución Molecular , Heterocromatina/metabolismo , ARN Interferente PequeñoRESUMEN
DNA topoisomerase II is required for mitotic chromosome condensation and segregation. Here we characterize the effects of inhibiting DNA topoisomerase II activity in plant cells using the non-DNA damaging topoisomerase II inhibitor ICRF-193. We report that ICRF-193 abrogated chromosome condensation in cultured alfalfa (Medicago sativa L.) and tobacco (Nicotiana tabaccum L.) mitoses and led to bridged chromosomes at anaphase. Moreover, ICRF-193 treatment delayed entry into mitosis, increasing the frequency of cells having a pre-prophase band of microtubules, a marker of late G2 and prophase, and delaying the activation of cyclin-dependent kinase. These data suggest the existence of a late G2 checkpoint in plant cells that is activated in the absence of topoisomerase II activity. To determine whether the checkpoint-induced delay was a result of reduced cyclindependent kinase activity, mitotic cyclin B2 was ectopically expressed. Cyclin B2 bypassed the ICRF-193-induced delay before mitosis, and correspondingly, reduced the frequency of interphase cells with a pre-prophase band. These data provide evidence that plant cells possess a topoisomerase II-dependent G2 cell cycle checkpoint that transiently inhibits mitotic CDK activation and entry into mitosis, and that is overridden by raising the level of CDK activity through the ectopic expression of a plant mitotic cyclin.
Asunto(s)
Ciclo Celular , Ciclina B/biosíntesis , ADN-Topoisomerasas de Tipo II/metabolismo , Fase G2 , Medicago sativa/metabolismo , Nicotiana/metabolismo , Piperazinas/farmacología , Antineoplásicos/farmacología , Cromosomas/metabolismo , Dicetopiperazinas , Inhibidores Enzimáticos/farmacología , Citometría de Flujo , Microscopía Fluorescente , Microtúbulos/efectos de los fármacos , Mitosis/efectos de los fármacos , Plásmidos/metabolismo , Factores de TiempoRESUMEN
Treatment of Allium cepa meristematic cells in metaphase with the topoisomerase II inhibitor ICRF-193, results in bridging of the sister chromatids at anaphase. Separation of the sisters in experimentally generated acentric chromosomal fragments was also inhibited by ICRF-193, indicating that some non-centromeric catenations also persist in metaphase chromosomes. Thus, catenations must be resolved by DNA topoisomerase II at the metaphase-to-anaphase transition to allow segregation of sisters. A passive mechanism could maintain catenations holding sisters until the onset of anaphase. At this point the opposite tension exerted on sister chromatids could render the decatenation reaction physically more favorable than catenation. But this possibility was dismissed as acentric chromosome fragments were able to separate their sister chromatids at anaphase. A timing mechanism (a common trigger for two processes taking different times to be completed) could passively couple the resolution of the last remaining catenations to the moment of anaphase onset. This possibility was also discarded as cells arrested in metaphase with microtubule-destabilising drugs still displayed anaphase bridges when released in the presence of ICRF-193. It is possible that a checkpoint mechanism prevents the release of the last catenations linking sisters until the onset of anaphase. To test whether cells are competent to fully resolve catenations before anaphase onset, we generated multinucleate plant cells. In this system, the nuclei within a single multinucleate cell displayed differences in chromosome condensation at metaphase, but initiated anaphase synchronously. When multinucleates were treated with ICRF-193 at the metaphase-toanaphase transition, tangled and untangled anaphases were observed within the same cell. This can only occur if cells are competent to disentangle sister chromatids before the onset of anaphase, but are prevented from doing so by a checkpoint mechanism.
Asunto(s)
Anafase/genética , Núcleo Celular/genética , Cromátides/genética , ADN de Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Genes cdc/fisiología , Cebollas/genética , Inhibidores de Topoisomerasa II , Anafase/efectos de los fármacos , Benzamidas/farmacología , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Núcleo Celular/efectos de los fármacos , Cromátides/efectos de los fármacos , Cromosomas/efectos de los fármacos , Cromosomas/genética , Fragmentación del ADN/efectos de los fármacos , Fragmentación del ADN/genética , ADN-Topoisomerasas de Tipo II/metabolismo , ADN de Plantas/efectos de los fármacos , Dicetopiperazinas , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Genes cdc/efectos de los fármacos , Células Gigantes/citología , Células Gigantes/efectos de los fármacos , Células Gigantes/metabolismo , Hidroxiurea/farmacología , Metafase/efectos de los fármacos , Metafase/genética , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Cebollas/efectos de los fármacos , Piperazinas/farmacologíaRESUMEN
MAIN-LIKE1 (MAIL1) is a ubiquitously expressed nuclear protein, which has a crucial function during root development. We have recently described loss of function mutants for MAIL1, in which the organization and function of the primary root meristem is lost soon after germination. Moreover cell differentiation is impaired resulting in primary root growth arrest soon after emergence. Here we show that mail1 mutants form several anchor roots from the hypocotyl to root junction. These anchor roots show similar defects in the organization of the stem cell niche as the primary root. In contrast, differentiation processes are not impaired and thus anchor roots seem to be able to compensate for the loss of primary root function. Our data show that MAIL1 is essential for specification of cell fate in the primary root but not in anchor roots.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Proteínas Nucleares/metabolismo , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/metabolismo , Mutación/genética , Plantones/crecimiento & desarrollo , Plantones/metabolismoRESUMEN
Cell division depends on the fine control of both microtubule dynamics and microtubule organisation. The microtubule bundling protein MAP65 is a ;midzone MAP' essential for the integrity of the anaphase spindle and cell division. Arabidopsis thaliana MAP65-1 (AtMAP65-1) binds and bundles microtubules by forming 25 nm cross-bridges. Moreover, as AtMAP65-1 bundles microtubules in interphase, anaphase and telophase but does not bind microtubules in prophase or metaphase, its activity through the cell cycle must be under tight control. Here we show that AtMAP65-1 is hyperphosphorylated during prometaphase and metaphase and that CDK and MAPK are involved in this phosphorylation. This phosphorylation inhibits AtMAP65-1 activity. Expression of non-phosphorylatable AtMAP65-1 has a negative effect on mitotic progression resulting in excessive accumulation of microtubules in the metaphase spindle midzone causing a delay in mitosis. We conclude that normal metaphase spindle organisation and the transition to anaphase is dependent on inactivation of AtMAP65-1.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Ciclo Celular/fisiología , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Arabidopsis/citología , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Células Cultivadas , Quinasas Ciclina-Dependientes/metabolismo , Recuperación de Fluorescencia tras Fotoblanqueo , Proteínas Asociadas a Microtúbulos/genética , Microtúbulos/ultraestructura , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fosforilación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal/fisiología , Huso Acromático/metabolismo , Nicotiana/citologíaRESUMEN
Mitotic progression is timely regulated by the accumulation and degradation of A- and B-type cyclins. In plants, there are three classes of A-, and two classes of B-type cyclins, but their specific roles are not known. We have generated transgenic tobacco plants in which the ectopic expression of a plant cyclin B2 gene is under the control of a tetracycline-inducible promoter. We show that the induction of cyclin B2 expression in cultured cells during G2 phase accelerates the entry into mitosis and allows cells to override the replication checkpoint induced by hydroxyurea in the simultaneous presence of caffeine or okadaic acid, drugs that are known to alleviate checkpoint control. These results indicate that in plants, a B2-type cyclin is a rate-limiting regulator for the entry into mitosis and a cyclin B2-CDK complex might be a target for checkpoint control pathways. The cyclin B2 localization and the timing of its degradation during mitosis corroborate these conclusions: cyclin B2 protein is confined to the nucleus and during mitosis it is only present during a short time window until mid prophase, but it is effectively degraded from this timepoint onwards. Although cyclin B2 is not present in cells arrested by the spindle checkpoint in metaphase, cyclin B1 is accumulating in these cells. Ectopic expression of cyclin B2 in developing plants interferes with differentiation events and specifically blocks root regeneration, indicating the importance of control mechanisms at the G2- to M-phase transition during plant developmental processes.
Asunto(s)
Ciclina B/genética , Daño del ADN/genética , Regulación del Desarrollo de la Expresión Génica/genética , Regulación de la Expresión Génica de las Plantas/genética , Genes cdc/fisiología , Mitosis/genética , Nicotiana/crecimiento & desarrollo , Proteínas de Plantas/genética , Cafeína/farmacología , Diferenciación Celular/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cisteína Endopeptidasas/metabolismo , Fase G2 , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Genes cdc/efectos de los fármacos , Hidroxiurea/farmacología , Mitosis/efectos de los fármacos , Complejos Multienzimáticos/antagonistas & inhibidores , Complejos Multienzimáticos/metabolismo , Ácido Ocadaico/farmacología , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/metabolismo , Regiones Promotoras Genéticas/efectos de los fármacos , Regiones Promotoras Genéticas/genética , Complejo de la Endopetidasa Proteasomal , Regeneración/efectos de los fármacos , Regeneración/genética , Huso Acromático/genética , Nicotiana/genética , Nicotiana/metabolismoRESUMEN
In plants after the disassembly of mitotic spindle, a specific cytokinetic structure called the phragmoplast is built, and after cytokinesis, microtubules populate the cell cortex in an organized orientation that determines cell elongation and shape. Here, we show that impaired cyclin B1 degradation, resulting from a mutation within its destruction box, leads to an isodiametric shape of epidermal cells in leaves, stems, and roots and retarded growth of seedlings. Microtubules in these misshaped cells are grossly disorganized, focused around the nucleus, whereas they were entirely missing or abnormally organized along the cell cortex. A high percentage of cells expressing nondestructible cyclin B1 had doubled DNA content as a result of undergoing endomitosis. During anaphase the cytokinesis-specific syntaxin KNOLLE could still localize to the midplane of cell division, whereas NPK1-activating kinesin-like protein 1, a cytokinetic kinesin-related protein, was unable to do so, and instead of the formation of a phragmoplast, the midzone microtubules persisted between the separated nuclei, which eventually fused. In summary, our results show that the timely degradation of mitotic cyclins in plants is required for the reorganization of mitotic microtubules to the phragmoplast and for proper cytokinesis. Subsequently, the presence of nondegradable cyclin B1 leads to a failure in organizing properly the cortical microtubules that determine cell elongation and shape.
Asunto(s)
Ciclina B/metabolismo , Nicotiana/crecimiento & desarrollo , Nicotiana/metabolismo , Secuencia de Bases , División Celular , Ciclina B/genética , Ciclina B1 , Quinasas Ciclina-Dependientes/metabolismo , ADN de Plantas/genética , ADN de Plantas/metabolismo , Expresión Génica , Genes de Plantas , Microtúbulos/metabolismo , Mitosis , Mutación , Fenotipo , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Poliploidía , Plantones/metabolismo , Huso Acromático/metabolismo , Nicotiana/genéticaRESUMEN
Microarray transcript profiling and RNA interference are two new technologies crucial for large-scale gene function studies in multicellular eukaryotes. Both rely on sequence-specific hybridization between complementary nucleic acid strands, inciting us to create a collection of gene-specific sequence tags (GSTs) representing at least 21,500 Arabidopsis genes and which are compatible with both approaches. The GSTs were carefully selected to ensure that each of them shared no significant similarity with any other region in the Arabidopsis genome. They were synthesized by PCR amplification from genomic DNA. Spotted microarrays fabricated from the GSTs show good dynamic range, specificity, and sensitivity in transcript profiling experiments. The GSTs have also been transferred to bacterial plasmid vectors via recombinational cloning protocols. These cloned GSTs constitute the ideal starting point for a variety of functional approaches, including reverse genetics. We have subcloned GSTs on a large scale into vectors designed for gene silencing in plant cells. We show that in planta expression of GST hairpin RNA results in the expected phenotypes in silenced Arabidopsis lines. These versatile GST resources provide novel and powerful tools for functional genomics.