RESUMEN
In plants, postembryonic formation of new organs helps shape the adult organism. This requires the tight regulation of when and where a new organ is formed and a coordination of the underlying cell divisions. To build a root system, new lateral roots are continuously developing, and this process requires the tight coordination of asymmetric cell division in adjacent pericycle cells. We identified EXPANSIN A1 (EXPA1) as a cell wall modifying enzyme controlling the divisions marking lateral root initiation. Loss of EXPA1 leads to defects in the first asymmetric pericycle cell divisions and the radial swelling of the pericycle during auxin-driven lateral root formation. We conclude that a localized radial expansion of adjacent pericycle cells is required to position the asymmetric cell divisions and generate a core of small daughter cells, which is a prerequisite for lateral root organogenesis.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , División Celular , Raíces de Plantas , Arabidopsis/citología , Arabidopsis/enzimología , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/fisiología , División Celular/genética , División Celular/fisiología , Pared Celular/genética , Pared Celular/fisiología , Raíces de Plantas/citología , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/fisiología , TranscriptomaRESUMEN
In plants, many signalling molecules, such as phytohormones, miRNAs, transcription factors, and small signalling peptides, drive growth and development. However, very few small signalling peptides have been shown to be necessary for lateral root development. Here, we describe the role of the peptide RALFL34 during early events in lateral root development, and demonstrate its specific importance in orchestrating formative cell divisions in the pericycle. Our results further suggest that this small signalling peptide acts on the transcriptional cascade leading to a new lateral root upstream of GATA23, an important player in lateral root formation. In addition, we describe a role for ETHYLENE RESPONSE FACTORs (ERFs) in regulating RALFL34 expression. Taken together, we put forward RALFL34 as a new, important player in lateral root initiation.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Hormonas Peptídicas/genética , Factores de Transcripción/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , División Celular , Hormonas Peptídicas/metabolismo , Raíces de Plantas/genética , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/metabolismo , Factores de Transcripción/metabolismoRESUMEN
In plants, the generation of new cell types and tissues depends on coordinated and oriented formative cell divisions. The plasma membrane-localized receptor kinase ARABIDOPSIS CRINKLY 4 (ACR4) is part of a mechanism controlling formative cell divisions in the Arabidopsis root. Despite its important role in plant development, very little is known about the molecular mechanism with which ACR4 is affiliated and its network of interactions. Here, we used various complementary proteomic approaches to identify ACR4-interacting protein candidates that are likely regulators of formative cell divisions and that could pave the way to unraveling the molecular basis behind ACR4-mediated signaling. We identified PROTEIN PHOSPHATASE 2A-3 (PP2A-3), a catalytic subunit of PP2A holoenzymes, as a previously unidentified regulator of formative cell divisions and as one of the first described substrates of ACR4. Our in vitro data argue for the existence of a tight posttranslational regulation in the associated biochemical network through reciprocal regulation between ACR4 and PP2A-3 at the phosphorylation level.
Asunto(s)
Proteínas de Arabidopsis/fisiología , Arabidopsis/citología , División Celular/fisiología , Fosfoproteínas Fosfatasas/fisiología , Raíces de Plantas/citología , Proteínas Serina-Treonina Quinasas/fisiología , Receptores de Superficie Celular/fisiología , Diferenciación Celular , FosforilaciónRESUMEN
Key steps in understanding ethylene signaling have come from studying Arabidopsis mutants. The mechanisms of receptor signal output are still poorly understood and the discovery of new components has increased the apparent complexity. Not all receptors are equivalent and some appear to have unique functions. There are multiple CTR-like proteins in tomato, which interact with more than one receptor. Focusing on mutants of the Arabidopsis triple response, which is primarily a growth response, may not uncover the complete range of components involved in developmental responses to ethylene and it is also possible there are real differences between species.
Asunto(s)
Arabidopsis/metabolismo , Etilenos/metabolismo , Transducción de Señal , Solanum lycopersicum/metabolismo , Secuencia de Aminoácidos , Modelos Biológicos , Datos de Secuencia Molecular , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Mapeo de Interacción de Proteínas , Estructura Terciaria de ProteínaRESUMEN
Ethylene regulates many aspects of the plant life cycle, including seed germination, root initiation, flower development, fruit ripening, senescence, and responses to biotic and abiotic stresses. It thus plays a key role in responses to the environment that have a direct bearing on a plant's fitness for adaptation and reproduction. In recent years, there have been major advances in our understanding of the molecular mechanisms regulating ethylene synthesis and action. Screening for mutants of the triple response phenotype of etiolated Arabidopsis seedlings, together with map-based cloning and candidate gene characterization of natural mutants from other plant species, has led to the identification of many new genes for ethylene biosynthesis, signal transduction, and response pathways. The simple chemical nature of ethylene contrasts with its regulatory complexity. This is illustrated by the multiplicity of genes encoding the key ethylene biosynthesis enzymes 1-aminocyclopropane-1-carboxylic acid (ACC) synthase and ACC oxidase, multiple ethylene receptors and signal transduction components, and the complexity of regulatory steps involving signalling relays and control of mRNA and protein synthesis and turnover. In addition, there are extensive interactions with other hormones. This review integrates knowledge from the model plant Arabidopsis and other plant species and focuses on key aspects of recent research on regulatory networks controlling ethylene synthesis and its role in flower development and fruit ripening.
Asunto(s)
Arabidopsis/metabolismo , Etilenos/biosíntesis , Regulación de la Expresión Génica de las Plantas , Arabidopsis/enzimología , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismoRESUMEN
Arabidopsis AtTRP1 is an orthologue of SlTPR1, a tomato tetratricopeptide repeat protein that interacts with the tomato ethylene receptors LeETR1 and NR in yeast 2-hybrid assays and in vitro, and modulates plant development. AtTRP1 is encoded by a single copy gene in the Arabidopsis genome, and is related to TCC1, a human protein that competes with Raf-1 for Ras binding, and distantly related to the immunophilin-like FK-binding proteins TWD1 and PAS1. The former is involved in auxin transport and the latter is translocated to the nucleus in response to auxin. AtTRP1 interacted preferentially with the Arabidopsis ethylene receptor ERS1 in yeast two-hybrid assays. This association was confirmed by in vivo co-immunoprecipitation. AtTRP1 promoter-GUS was highly expressed in vascular tissue, mature anthers, the abscission zone, and was induced by ACC. Overexpression of AtTRP1 in wild-type Arabidopsis resulted in dwarf plants with reduced fertility, altered leaf/silique morphology, and enhanced expression of the ethylene responsive gene AtChitB. Exogenous GA did not reverse the dwarf habit. Etiolated transgenic seedlings overexpressing AtTRP1 displayed enhanced sensitivity to low ACC and this was correlated with the transgene expression. Seedlings overexpressing AtTRP1 at high levels exhibited shortened and swollen hypocotyls, inhibited root growth, and an altered apical hook. Plants overexpressing AtTRP1 also showed a reduced response to exogenous IAA and altered expression of a subset of auxin early responsive genes. These results indicated that overexpression of AtTRP1 affects cross-talk between ethylene and auxin signalling and enhances some ethylene responses and alters some auxin responses. A model for AtTRP1 action is proposed.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Regulación del Desarrollo de la Expresión Génica , Receptores de Superficie Celular/metabolismo , Proteínas de Unión a Telómeros/metabolismo , Secuencia de Aminoácidos , Arabidopsis/química , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Secuencia de Bases , Proteínas Portadoras , Etilenos/metabolismo , Regulación de la Expresión Génica de las Plantas , Ácidos Indolacéticos/metabolismo , Datos de Secuencia Molecular , Receptores de Superficie Celular/química , Receptores de Superficie Celular/genética , Alineación de Secuencia , Transducción de Señal , Proteínas de Unión a Telómeros/química , Proteínas de Unión a Telómeros/genéticaRESUMEN
The gaseous hormone ethylene is perceived by a family of ethylene receptors which interact with the Raf-like kinase CTR1. SlTPR1 encodes a novel TPR (tetratricopeptide repeat) protein from tomato that interacts with the ethylene receptors NR and LeETR1 in yeast two-hybrid and in vitro protein interaction assays. SlTPR1 protein with a GFP fluorescent tag was localized in the plasmalemma and nuclear membrane in Arabidopsis, and SlTPR1-CFP and NR-YFP fusion proteins were co-localized in the plasmalemma and nuclear membrane following co-bombardment of onion cells. Overexpression of SlTPR1 in tomato resulted in ethylene-related pleiotropic effects including reduced stature, delayed and reduced production of inflorescences, abnormal and infertile flowers with degenerate styles and pollen, epinasty, reduced apical dominance, inhibition of abscission, altered leaf morphology, and parthenocarpic fruit. Similar phenotypes were seen in Arabidopsis overexpressing SlTPR1. SlTPR1 overexpression did not increase ethylene production but caused enhanced accumulation of mRNA from the ethylene responsive gene ChitB and the auxin-responsive gene SlSAUR1-like, and reduced expression of the auxin early responsive gene LeIAA9, which is known to be inhibited by ethylene and to be associated with parthenocarpy. Cuttings from the SlTPR1-overexpressors produced fewer adventitious roots and were less responsive to indole butyric acid. It is suggested that SlTPR1 overexpression enhances a subset of ethylene and auxin responses by interacting with specific ethylene receptors. SlTPR1 shares features with human TTC1, which interacts with heterotrimeric G-proteins and Ras, and competes with Raf-1 for Ras binding. Models for SlTPR1 action are proposed involving modulation of ethylene signalling or receptor levels.
Asunto(s)
Etilenos/metabolismo , Ácidos Indolacéticos/metabolismo , Proteínas de Plantas/metabolismo , Receptores de Superficie Celular/metabolismo , Solanum lycopersicum/crecimiento & desarrollo , Solanum lycopersicum/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Secuencia de Bases , Regulación del Desarrollo de la Expresión Génica , Solanum lycopersicum/química , Solanum lycopersicum/genética , Datos de Secuencia Molecular , Proteínas de Plantas/química , Proteínas de Plantas/genética , Unión Proteica , Receptores de Superficie Celular/química , Receptores de Superficie Celular/genética , Alineación de Secuencia , Técnicas del Sistema de Dos HíbridosRESUMEN
Fluorescent protein labelling technologies enable dynamic protein actions to be imaged in living cells and can also be used in conjunction with other methods such as Forster resonance energy transfer and biomolecular fluorescence complementation. In this report, we describe the generation of a series of 23 novel GATEWAY-compatible vectors based on pGreenII and pDH51 backbones with the latest fluorescent protein tags (Cerulean, EGFP and Venus) and the choice of three in planta selection markers. These vectors can be obtained from the Nottingham Arabidopsis Stock Centre (N9819-N9846) and should be a powerful tool box for transgenic research in plants.
Asunto(s)
Vectores Genéticos , Proteínas Luminiscentes/genética , Plantas/genética , Transformación Genética , Transferencia de Energía , Datos de Secuencia Molecular , Reacción en Cadena de la PolimerasaRESUMEN
Ethylene is required for climacteric fruit ripening. Inhibition of ethylene biosynthesis genes, 1-aminocyclopropane-1-carboxylate (ACC) synthase and ACC oxidase, prevents or delays ripening, but it is not known how these genes are modulated during normal development. LeHB-1, a previously uncharacterized tomato homeobox protein, was shown by gel retardation assay to interact with the promoter of LeACO1, an ACC oxidase gene expressed during ripening. Inhibition of LeHB-1 mRNA accumulation in tomato fruit, using virus-induced gene silencing, greatly reduced LeACO1 mRNA levels, and inhibited ripening. Conversely, ectopic overexpression of LeHB-1 by viral delivery to developing flowers elsewhere on injected plants triggered altered floral organ morphology, including production of multiple flowers within one sepal whorl, fusion of sepals and petals, and conversion of sepals into carpel-like structures that grew into fruits and ripened. Our findings suggest that LeHB-1 is not only involved in the control of ripening but also plays a critical role in floral organogenesis.
Asunto(s)
Flores/crecimiento & desarrollo , Flores/metabolismo , Proteínas de Homeodominio/metabolismo , Solanum lycopersicum/metabolismo , Aminoácido Oxidorreductasas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Flores/genética , Frutas/metabolismo , Regulación de la Expresión Génica de las Plantas/fisiología , Silenciador del Gen , Proteínas de Homeodominio/química , Proteínas de Homeodominio/genética , Datos de Secuencia Molecular , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regiones Promotoras Genéticas , Unión ProteicaRESUMEN
In the model plant Arabidopsis, members of a family of two-component system His kinase-like ethylene receptors have direct protein-protein interactions with a single downstream Ser/Thr kinase CTR1. These components of the ethylene signalling network found in Arabidopsis are conserved in the climacteric fruit tomato, but both the ethylene receptors and CTR1-like proteins (LeCTRs) in tomato are encoded by multigene families. Here, using a yeast two-hybrid interaction assay, it is shown that the tomato receptors LeETR1, LeETR2, and NEVER-RIPE (NR) can interact with multiple LeCTRs. In vivo protein localization studies with fluorescent tagged proteins revealed that the ethylene receptor NR was targeted to the endoplasmic reticulum (ER) when transiently expressed in onion epidermal cells, whereas the four LeCTR proteins were found in the cytoplasm and nucleus. When co-expressed with NR, three LeCTRs (1, 3, and 4), but not LeCTR2, also adopted the same ER localization pattern in an NR receptor-dependent manner but not in the absence of NR. The receptor-CTR interactions were confirmed by biomolecular fluorescence complementation (BiFC) showing that NR could form a protein complex with LeCTR1, 3, and 4. This suggested that ethylene receptors recruit these LeCTR proteins to the ER membrane through direct protein-protein interaction. The receptor-CTR interactions and localization observed in the study reinforce the idea that ethylene receptors transmit the signal to the downstream CTRs and show that a single receptor can interact with multiple CTR proteins. It remains unclear whether the different LeCTRs are functionally redundant or have unique roles in ethylene signalling.
Asunto(s)
Retículo Endoplásmico/metabolismo , Proteínas de Plantas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Superficie Celular/metabolismo , Solanum lycopersicum/metabolismo , Fluorescencia , Regulación de la Expresión Génica de las Plantas/fisiología , Solanum lycopersicum/genética , Proteínas de Plantas/genética , Unión Proteica , Proteínas Serina-Treonina Quinasas/genética , Transporte de Proteínas , Receptores de Superficie Celular/genética , Transducción de Señal/genética , Técnicas del Sistema de Dos HíbridosRESUMEN
Arabidopsis AtCTR1 is a Raf-like protein kinase that interacts with ETR1 and ERS and negatively regulates ethylene responses. In tomato, several CTR1-like proteins could perform this role. We have characterized LeCTR2, which has similarity to AtCTR1 and also to EDR1, a CTR1-like Arabidopsis protein involved in defence and stress responses. Protein-protein interactions between LeCTR2 and six tomato ethylene receptors indicated that LeCTR2 interacts preferentially with the subfamily I ETR1-type ethylene receptors LeETR1 and LeETR2, but not the NR receptor or the subfamily II receptors LeETR4, LeETR5 and LeETR6. The C-terminus of LeCTR2 possesses serine/threonine kinase activity and is capable of auto-phosphorylation and phosphorylation of myelin basic protein in vitro. Overexpression of the LeCTR2 N-terminus in tomato resulted in altered growth habit, including reduced stature, loss of apical dominance, highly branched inflorescences and fruit trusses, indeterminate shoots in place of determinate flowers, and prolific adventitious shoot development from the rachis or rachillae of the leaves. Expression of the ethylene-responsive genes E4 and chitinase B was upregulated in transgenic plants, but ethylene production and the level of mRNA for the ethylene biosynthetic gene ACO1 was unaffected. The leaves and fruit of transgenic plants also displayed enhanced susceptibility to infection by the fungal pathogen Botrytis cinerea, which was associated with much stronger induction of pathogenesis-related genes such as PR1b1 and chitinase B compared with the wild-type. The results suggest that LeCTR2 plays a role in ethylene signalling, development and defence, probably through its interactions with the ETR1-type ethylene receptors of subfamily I.
Asunto(s)
Etilenos/metabolismo , Proteínas Quinasas/metabolismo , Transducción de Señal , Solanum lycopersicum/enzimología , Solanum lycopersicum/crecimiento & desarrollo , Botrytis/patogenicidad , Regulación de la Expresión Génica de las Plantas , Solanum lycopersicum/genética , Reguladores del Crecimiento de las Plantas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/enzimología , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Mapeo de Interacción de Proteínas , Proteínas Quinasas/genética , ARN de Planta/genéticaRESUMEN
There are at least five lipoxygenases (TomloxA, TomloxB, TomloxC, TomloxD, and TomloxE) present in tomato (Lycopersicon esculentum Mill.) fruit, but their role in generation of fruit flavor volatiles has been unclear. To assess the physiological role of TomloxC in the generation of volatile C6 aldehyde and alcohol flavor compounds, we produced transgenic tomato plants with greatly reduced TomloxC using sense and antisense constructs under control of the cauliflower mosaic virus 35S promoter. The expression level of the TomloxC mRNA in some transgenic plants was selectively reduced by gene silencing or antisense inhibition to between 1% and 5% of the wild-type controls, but the expression levels of mRNAs for the four other isoforms were unaffected. The specific depletion of TomloxC in transgenic tomatoes led to a marked reduction in the levels of known flavor volatiles, including hexanal, hexenal, and hexenol, to as little as 1.5% of those of wild-type controls following maceration of ripening fruit. Addition of linoleic or linolenic acid to fruit homogenates significantly increased the levels of flavor volatiles, but the increase with the TomloxC-depleted transgenic fruit extracts was much lower than with the wild-type control. Confocal imaging of tobacco (Nicotiana tabacum) leaf cells expressing a TomloxC-GFP fusion confirmed a chloroplast localization of the protein. Together, these results suggest that TomloxC is a chloroplast-targeted lipoxygenase isoform that can use both linoleic and linolenic acids as substrates to generate volatile C6 flavor compounds. The roles of the other lipoxygenase isoforms are discussed.