RESUMEN
Plant development is continually fine-tuned based on environmental factors. How environmental perturbations are integrated into the developmental programs and how poststress adaptation is regulated remains an important topic to dissect. Vegetative to reproductive phase change is a very important developmental transition that is complexly regulated based on endogenous and exogenous cues. Proper timing of flowering is vital for reproductive success. It has been shown previously that AGAMOUS LIKE 16 (AGL16), a MADS-box transcription factor negatively regulates flowering time transition through FLOWERING LOCUS T (FT), a central downstream floral integrator. AGL16 itself is negatively regulated by the microRNA miR824. Here we present a comprehensive molecular analysis of miR824/AGL16 module changes in response to mild and recurring heat stress. We show that miR824 accumulates gradually in response to heat due to the combination of transient transcriptional induction and posttranscriptional stability. miR824 induction requires heat shock cis-elements and activity of the HSFA1 family and HSFA2 transcription factors. Parallel to miR824 induction, its target AGL16 is decreased, implying direct causality. AGL16 posttranscriptional repression during heat stress, however, is more complex, comprising of a miRNA-independent, and a miR824-dependent pathway. We also show that AGL16 expression is leaf vein-specific and overlaps with miR824 (and FT) expression. AGL16 downregulation in response to heat leads to a mild derepression of FT. Finally, we present evidence showing that heat stress regulation of miR824/AGL16 is conserved within Brassicaceae. In conclusion, due to the enhanced post-transcriptional stability of miR824, stable repression of AGL16 is achieved following heat stress. This may serve to fine-tune FT levels and alter flowering time transition. Stress-induced miR824, therefore, can act as a "posttranscriptional memory factor" to extend the acute impact of environmental fluctuations in the poststress period.
RESUMEN
To maintain the balance between long-term stem cell self-renewal and differentiation, dynamic signals need to be translated into spatially precise and temporally stable gene expression states. In the apical plant stem cell system, local accumulation of the small, highly mobile phytohormone auxin triggers differentiation while at the same time, pluripotent stem cells are maintained throughout the entire life-cycle. We find that stem cells are resistant to auxin mediated differentiation, but require low levels of signaling for their maintenance. We demonstrate that the WUSCHEL transcription factor confers this behavior by rheostatically controlling the auxin signaling and response pathway. Finally, we show that WUSCHEL acts via regulation of histone acetylation at target loci, including those with functions in the auxin pathway. Our results reveal an important mechanism that allows cells to differentially translate a potent and highly dynamic developmental signal into stable cell behavior with high spatial precision and temporal robustness.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Diferenciación Celular , Autorrenovación de las Células , Proteínas de Homeodominio/metabolismo , Ácidos Indolacéticos/metabolismo , Meristema/metabolismo , Células Madre Pluripotentes/metabolismo , Proliferación Celular , Meristema/citología , Brotes de la Planta , Plantas Modificadas Genéticamente , Células Madre Pluripotentes/citología , Transducción de SeñalRESUMEN
In some plant-virus interactions plants show a sign of healing from virus infection, a phenomenon called symptom recovery. It is assumed that the meristem exclusion of the virus is essential to this process. The discovery of RNA silencing provided a possible mechanism to explain meristem exclusion and recovery. Here we show evidence that silencing is not the reason for meristem exclusion in Nicotiana benthamiana plants infected with Cymbidium ringspot virus (CymRSV). Transcriptome analysis followed by in situ hybridization shed light on the changes in gene expression in the shoot apical meristem (SAM) on virus infection. We observed the down-regulation of meristem-specific genes, including WUSCHEL (WUS). However, WUS was not down-regulated in the SAM of plants infected with meristem-invading viruses such as turnip vein-clearing virus (TVCV) and cucumber mosaic virus (CMV). Moreover, there is no connection between loss of meristem function and fast shoot necrosis since TVCV necrotized the shoot while CMV did not. Our findings suggest that the observed transcriptional changes on virus infection in the shoot are key factors in tip necrosis and symptom recovery. We observed a lack of GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE (GAPDH) expression in tissues around the meristem, which likely stops virus replication and spread into the meristem.
Asunto(s)
Cucumovirus/fisiología , Regulación de la Expresión Génica de las Plantas , Nicotiana/virología , Enfermedades de las Plantas/virología , Transcriptoma , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/metabolismo , Meristema/virología , Enfermedades de las Plantas/genética , Brotes de la Planta , Interferencia de ARN , Nicotiana/genética , Nicotiana/metabolismoRESUMEN
To produce seeds, flowering plants need to specify somatic cells to undergo meiosis. Here, we reveal a regulatory cascade that controls the entry into meiosis starting with a group of redundantly acting cyclin-dependent kinase (CDK) inhibitors of the KIP-RELATED PROTEIN (KRP) class. KRPs function by restricting CDKA;1-dependent inactivation of the Arabidopsis Retinoblastoma homolog RBR1. In rbr1 and krp triple mutants, designated meiocytes undergo several mitotic divisions, resulting in the formation of supernumerary meiocytes that give rise to multiple reproductive units per future seed. One function of RBR1 is the direct repression of the stem cell factor WUSCHEL (WUS), which ectopically accumulates in meiocytes of triple krp and rbr1 mutants. Depleting WUS in rbr1 mutants restored the formation of only a single meiocyte.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/embriología , Proteínas de Homeodominio/metabolismo , Óvulo Vegetal/embriología , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular , Proteínas Inhibidoras de las Quinasas Dependientes de la Ciclina/genética , Proteínas Inhibidoras de las Quinasas Dependientes de la Ciclina/metabolismo , Proteínas de Homeodominio/genética , Meiosis/genética , Meiosis/fisiología , Mutación , Óvulo Vegetal/genética , Óvulo Vegetal/metabolismoRESUMEN
RNA silencing is one of the main defense mechanisms employed by plants to fight viruses. In change, viruses have evolved silencing suppressor proteins to neutralize antiviral silencing. Since the endogenous and antiviral functions of RNA silencing pathway rely on common components, it was suggested that viral suppressors interfere with endogenous silencing pathway contributing to viral symptom development. In this work, we aimed to understand the effects of the tombusviral p19 suppressor on endogenous and antiviral silencing during genuine virus infection. We showed that ectopically expressed p19 sequesters endogenous small RNAs (sRNAs) in the absence, but not in the presence of virus infection. Our presented data question the generalized model in which the sequestration of endogenous sRNAs by the viral suppressor contributes to the viral symptom development. We further showed that p19 preferentially binds the perfectly paired ds-viral small interfering RNAs (vsiRNAs) but does not select based on their sequence or the type of the 5' nucleotide. Finally, co-immunoprecipitation of sRNAs with AGO1 or AGO2 from virus-infected plants revealed that p19 specifically impairs vsiRNA loading into AGO1 but not AGO2. Our findings, coupled with the fact that p19-expressing wild type Cymbidium ringspot virus (CymRSV) overcomes the Nicotiana benthamiana silencing based defense killing the host, suggest that AGO1 is the main effector of antiviral silencing in this host-virus combination.
Asunto(s)
Nicotiana/genética , Nicotiana/virología , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/virología , Proteínas de Plantas/metabolismo , Tombusvirus/genética , Proteínas Virales/genética , Northern Blotting , Western Blotting , Ensayo de Cambio de Movilidad Electroforética , Secuenciación de Nucleótidos de Alto Rendimiento , Inmunoprecipitación , Plantas Modificadas Genéticamente , ARN de Planta/genética , ARN Interferente Pequeño/genéticaRESUMEN
A major feature of embryogenesis is the specification of stem cell systems, but in contrast to the situation in most animals, plant stem cells remain quiescent until the postembryonic phase of development. Here, we dissect how light and metabolic signals are integrated to overcome stem cell dormancy at the shoot apical meristem. We show on the one hand that light is able to activate expression of the stem cell inducer WUSCHEL independently of photosynthesis and that this likely involves inter-regional cytokinin signaling. Metabolic signals, on the other hand, are transduced to the meristem through activation of the TARGET OF RAPAMYCIN (TOR) kinase. Surprisingly, TOR is also required for light signal dependent stem cell activation. Thus, the TOR kinase acts as a central integrator of light and metabolic signals and a key regulator of stem cell activation at the shoot apex.
Asunto(s)
Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Luz , Meristema/crecimiento & desarrollo , Brotes de la Planta/crecimiento & desarrollo , Células Madre/metabolismo , Células Madre/efectos de la radiación , Meristema/genética , Meristema/metabolismo , Meristema/efectos de la radiación , Brotes de la Planta/genética , Brotes de la Planta/metabolismo , Brotes de la Planta/efectos de la radiaciónRESUMEN
The presence of a large central vacuole is one of the hallmarks of a prototypical plant cell, and the multiple functions of this compartment require massive fluxes of molecules across its limiting membrane, the tonoplast. Transport is assumed to be energized by the membrane potential and the proton gradient established by the combined activity of two proton pumps, the vacuolar H(+)-pyrophosphatase (V-PPase) and the vacuolar H(+)-ATPase (V-ATPase). Exactly how labor is divided between these two enzymes has remained elusive. Here, we provide evidence using gain- and loss-of-function approaches that lack of the V-ATPase cannot be compensated for by increased V-PPase activity. Moreover, we show that increased V-ATPase activity during cold acclimation requires the presence of the V-PPase. Most importantly, we demonstrate that a mutant lacking both of these proton pumps is conditionally viable and retains significant vacuolar acidification, pointing to a so far undetected contribution of the trans-Golgi network/early endosome-localized V-ATPase to vacuolar pH.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Genoma de Planta/genética , Pirofosfatasa Inorgánica/metabolismo , ATPasas de Translocación de Protón Vacuolares/metabolismo , Vacuolas/enzimología , Aclimatación , Arabidopsis/citología , Arabidopsis/genética , Arabidopsis/fisiología , Proteínas de Arabidopsis/antagonistas & inhibidores , Proteínas de Arabidopsis/genética , Frío , Endosomas/enzimología , Flores/citología , Flores/enzimología , Flores/genética , Flores/fisiología , Concentración de Iones de Hidrógeno , Pirofosfatasa Inorgánica/antagonistas & inhibidores , Pirofosfatasa Inorgánica/genética , Meristema/citología , Meristema/enzimología , Meristema/genética , Meristema/fisiología , Mutagénesis Insercional , Fenotipo , Raíces de Plantas/citología , Raíces de Plantas/enzimología , Raíces de Plantas/genética , Raíces de Plantas/fisiología , Plantones/citología , Plantones/enzimología , Plantones/genética , Plantones/fisiología , Análisis de Secuencia de ADN , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , ATPasas de Translocación de Protón Vacuolares/genética , Red trans-Golgi/enzimologíaRESUMEN
Cell-cell communication is essential for multicellular development and, consequently, evolution has brought about an array of distinct mechanisms serving this purpose. Consistently, induction and maintenance of stem cell fate by noncell autonomous signals is a feature shared by many organisms and may depend on secreted factors, direct cell-cell contact, matrix interactions, or a combination of these mechanisms. Although many basic cellular processes are well conserved between animals and plants, cell-to-cell signaling is one function where substantial diversity has arisen between the two kingdoms of life. One of the most striking differences is the presence of cytoplasmic bridges, called plasmodesmata, which facilitate the exchange of molecules between neighboring plant cells and provide a unique route for cell-cell communication in the plant lineage. Here, we provide evidence that the stem cell inducing transcription factor WUSCHEL (WUS), expressed in the niche, moves to the stem cells via plasmodesmata in a highly regulated fashion and that this movement is required for WUS function and, thus, stem cell activity in Arabidopsis thaliana. We show that cell context-independent mobility is encoded in the WUS protein sequence and mediated by multiple domains. Finally, we demonstrate that parts of the protein that restrict movement are required for WUS homodimerization, suggesting that formation of WUS dimers might contribute to the regulation of apical stem cell activity.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas de Homeodominio/genética , Meristema/metabolismo , Células Madre/metabolismo , Arabidopsis/citología , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Comunicación Celular/genética , Transferencia Resonante de Energía de Fluorescencia , Regulación de la Expresión Génica de las Plantas , Proteínas de Homeodominio/química , Proteínas de Homeodominio/metabolismo , Hibridación in Situ , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Meristema/citología , Microscopía Confocal , Tallos de la Planta/citología , Tallos de la Planta/genética , Tallos de la Planta/metabolismo , Plantas Modificadas Genéticamente , Plasmodesmos/metabolismo , Multimerización de Proteína , Transporte de Proteínas/genética , Transducción de Señal/genéticaRESUMEN
Plants continuously maintain pluripotent stem cells embedded in specialized tissues called meristems, which drive long-term growth and organogenesis. Stem cell fate in the shoot apical meristem (SAM) is controlled by the homeodomain transcription factor WUSCHEL (WUS) expressed in the niche adjacent to the stem cells. Here, we demonstrate that the bHLH transcription factor HECATE1 (HEC1) is a target of WUS and that it contributes to SAM function by promoting stem cell proliferation, while antagonizing niche cell activity. HEC1 represses the stem cell regulators WUS and CLAVATA3 (CLV3) and, like WUS, controls genes with functions in metabolism and hormone signaling. Among the targets shared by HEC1 and WUS are phytohormone response regulators, which we show to act as mobile signals in a universal feedback system. Thus, our work sheds light on the mechanisms guiding meristem function and suggests that the underlying regulatory system is far more complex than previously anticipated.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Homeodominio/metabolismo , Reguladores del Crecimiento de las Plantas/metabolismo , Tallos de la Planta/genética , Transducción de Señal/genética , Factores de Transcripción/metabolismo , Transcripción Genética , Proteínas de Arabidopsis/genética , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Citocininas/genética , Citocininas/metabolismo , Regulación de la Expresión Génica de las Plantas/genética , Proteínas de Homeodominio/genética , Meristema/genética , Meristema/metabolismo , Reguladores del Crecimiento de las Plantas/genética , Brotes de la Planta/genética , Brotes de la Planta/metabolismo , Tallos de la Planta/metabolismo , Células Madre/citología , Células Madre/metabolismo , Factores de Transcripción/genéticaRESUMEN
Analysis of gene activity with high spatial resolution is a prerequisite for deciphering regulatory networks which underlie developmental programs. Over many years, in situ hybridization has become the gold standard for the identification of in vivo expression patterns of endogenous mRNAs. Nonetheless, the method has several limitations, and the detection of lowly expressed transcripts is still a challenge. Here, we present a robust protocol for sensitive analysis of expression patterns in inflorescence tissue of Arabidopsis thaliana. We describe how the samples are fixed, embedded, and sectioned in preparation for in situ hybridization, how RNA probes are prepared, and how hybridization and detection is carried out. While the described protocol is optimized for inflorescence meristems, it can possibly be used for other tissues as well.