A dynamic pattern of local auxin sources is required for root regeneration

Following removal of its stem cell niche, the root meristem can regenerate by recruitment of remnant cells from the stump. Regeneration is initiated by rapid accumulation of auxin near the injury site but the source of this auxin is unknown. Here, we show that auxin accumulation arises from the activity of multiple auxin biosynthetic sources that are newly specified near the cut site and that their continuous activity is required for the regeneration process. Auxin synthesis is highly localized and PIN-mediate transport is dispensable for auxin accumulation and tip regeneration. Roots lacking the activity of the regeneration competence factor ERF115, or that are dissected at a zone of low-regeneration potential, fail to activate local auxin sources. Remarkably, restoring auxin supply is sufficient to confer regeneration capacity to these recalcitrant tissues. We suggest that regeneration competence relies on the ability to specify new local auxin sources in a precise spatio-temporal pattern

Local auxin biosynthesis is required for root regeneration after wounding

The root meristem can regenerate following removal of its stem-cell niche by recruitment of remnant cells from the stump. Regeneration is initiated by rapid accumulation of auxin near the injury site but the source of this auxin is unknown. Here, we show that auxin accumulation arises from the activity of multiple auxin biosynthetic sources that are newly specified near the cut site and that their continuous activity is required for the regeneration process. Auxin synthesis is highly localized while PIN-mediated transport is dispensable for auxin accumulation and tip regeneration. Roots lacking the activity of the regeneration competence factor ERF115, or that are dissected at a zone of low regeneration potential, fail to activate local auxin sources. Remarkably, restoring auxin supply is sufficient to confer regeneration capacity to these recalcitrant tissues. We suggest that regeneration competence relies on the ability to specify new local auxin sources in a precise temporal pattern

A dynamic pattern of local auxin sources is required for root regeneration

Following removal of its stem cell niche, the root meristem can regenerate by recruitment of remnant cells from the stump. Regeneration is initiated by rapid accumulation of auxin near the injury site but the source of this auxin is unknown. Here, we show that auxin accumulation arises from the activity of multiple auxin biosynthetic sources that are newly specified near the cut site and that their continuous activity is required for the regeneration process. Auxin synthesis is highly localized and PIN-mediate transport is dispensable for auxin accumulation and tip regeneration. Roots lacking the activity of the regeneration competence factor ERF115, or that are dissected at a zone of low-regeneration potential, fail to activate local auxin sources. Remarkably, restoring auxin supply is sufficient to confer regeneration capacity to these recalcitrant tissues. We suggest that regeneration competence relies on the ability to specify new local auxin sources in a precise spatio-temporal pattern

Repositorio documental de Fisiología Vegetal

Memoria ID-299. Ayudas de la Universidad de Salamanca para la innovación docente, curso 2013-2014.[ES] Este proyecto docente ha servido para la creación de un repositorio documental de fisiología y patología molecular de plantas orientado al aprendizaje e identificación de los fenotipos fisiológicos, genéticos y moleculares utilizados en vegetales, empleando mutantes y plantas transgénicas. Además se han seleccionado, digitalizado e integrado en una base de datos, imágenes de plantas modelo de experimentación como Arabidopsis en distintas etapas del desarrollo vegetal, imágenes de microscopía óptica y confocal descritas acorde con los objetivos de las distintas asignaturas a las que está orientado el repositorio documental. Parte de este material básico, junto con una selección de documentos externos (páginas web, revistas electrónicas, animaciones, videos técnicos y conferencias) se ha utilizado en el presente curso en las distintas asignaturas del Grado en Biología, Biotecnología y Máster en Agrobiotecnología. El conjunto de los recursos está siendo adaptado a formatos de aprendizaje secuencial tutelado y evaluable que se ha integrado en la plataforma Studium de la Universidad de Salamanca y ha estará plenamente operativo en el segundo cuatrimestre del curso académico 2013-2014. Del mismo modo, parte del material generado ha sido empleado en la construcción de las páginas web del Máster en Agrobiotecnología y del Centro Hispano-Luso de Investigaciones Agrarias (CIALE), así como en los folletos divulgativos del citado Máster

Análisis funcional de proteín-fosfatasas de tipo 2-C en la dormición/germinación y las respuestas a estrés en Arabidopsis Thaliana

[EN] Plant seed is the result of ovule maturation and it is formed by an embryo surrounded by protective structures and storage tissues such as endosperm or perisperm, in those species which cotyledons do not accumulate reserves. Seed plays a key role in plant life cycle regarding its survival as a species. It is the dispersal unit of the plant, which is able to survive the period between seed maturation and the establishment of the next generation as a seedling after it has germinated. For this survival, the seed, mainly in a dry state, is well equipped to sustain extended periods of unfavourable conditions. Germination is a process that begins with water uptake by the seed (imbibition) and ends with the emergence of the embryonic axis, usually the radicle, through the structures surrounding it. On the other hand, dormancy is the temporary failure of a seed to complete germination under favourable conditions and it allows for the dispersal of seeds in space and time. To optimise germination over time, the seed enters in a dormant state. Dormancy prevents pre-harvest germination as well. Numerous studies have been performed to better understand how germination is controlled by various environmental factors and plant hormone. Nevertheless, many aspects about the process of germination are still unknown. The transition from dormancy to germination depends on several factors that affect seeds simultaneously, in Arabidopsis it requires the removal of several “blocks” to germination, such as after-ripening, chilling and exposure to nitrate and light, but the termination of dormancy and the triggering of germination is not due to only one factor but several, that generate a cross talk between them and hormonal regulation and provide an integrated network that leads to the the decision of germinate or stay dormant depending on ecological opportunities. Hormonal regulation is due to abscisic acid (ABA) and gibberellins (GA), the first one has an inhibitory and the second one a promotive effect in the induction of germination. Genetic evidence of this antagonistic effect was provided by molecular genetics through the isolation of ABA-deficient mutations as suppressors of non-germination due to GA deficiency and the GA response mutant sleepy as suppressor of abi11-1. Dormancy maintenance is due to de novo synthesis of ABA during imbibition. ABA is produced via carotenoid pathway and NCED is the enzyme that catalyzes the limiting step of the synthesis: the conversion of 9´-cis-neoxanthin and 9-cis-violaxanthin to xanthoxin by 9-cis-epoxycarotenoid dioxygenase. It regulates the rate of ABA production, associated with the induction and maintenance of dormancy. Release from dormancy, on the other hand, is correlated with ABA catabolism by an other key hormone, CYP707A2 and also to increased GA synthesis of GA3ox1, GA3ox2 and decreased expression of inactivating enzyme GA2ox. But the relative activities of ABA and GA not only depend on its quantity but also on the perception of hormones by its receptors and subsequent signalling transduction pathways, that eventually influence gene expression. Therefore, the identification of hormone transduction pathways is critical to unravelling these processes where posttranslational modifications of signalling proteins by phosphorylation or dephosphorylation, and ubiquitination, which affects their stability, play a critical role. Phosphorylation is one of the major reversible signal transduction control mechanisms, mediated by protein kinases and protein phosphatases. PP2Cs are universally distributed group of phosphatases, forming the largest phosphatase family in plants. It is divided in ten groups from A to J. Members of PP2Cs from cluster A and D are the main characters of this dissertation. Cluster A PP2Cs (A-PP2Cs), known for years as major negative regulators of ABA signalling, have been revealed as components of the its receptor complex. In conditions of non-ABA, PP2Cs continuously dephosphorylate SnRKs, blocking the transduction response to ABA. When ABA is present, PYR/PYL/RCAR proteins, ABA receptors, are able to bound to ABA and thereby stably interact with PP2Cs and inhibit its dephosphorylation by disabling its catalytic function. On the other hand, cluster D PP2Cs (D-PP2Cs), largely unknown have been recently related with elongation processes. In the first chapter of this dissertation, A-PP2Cs and ABA receptors, PYR/PYL/RCAR proteins, are characterized in oxidative stress responses in germination. To accomplish it, germination assays in the presence of methyl-viologen, a reactive oxygen species generator, are performed in the presence of several mutant lines (with overexpression, knock-out or hipermorphic phenotypes) of A-PP2Cs and PYR/PYL/RCAR. Also, the activity of the most important enzymes of the plant antioxidant system, superoxide dismutase, catalase and glutathione reductase, are measured in seeds of all these lines to further unravel the function of both types of proteins. In the second part, a Yeast Two Hybrid (Y2H) is performed to find D-PP2Cs interacting proteins. The interactions are fully characterized by in planta verification with Bimolecular complementation assays (BiFC). A triple D-PP2C phosphatase mutant is generated and characterized in germination assays. Multiple hormone assays are performed as well to elucidate the relationship between D-PP2Cs and hormones signalling pathway. One of the D-PP2Cs interacting proteins is also characterized in the same conditions, showing the clear effect of the interaction in biological function

The Nuclear Interactor PYL8/RCAR3 ofFagus sylvaticaFsPP2C1 Is a Positive Regulator of Abscisic Acid Signaling in Seeds and Stress

The functional protein phosphatase type 2C from beechnut (Fagus sylvatica; FsPP2C1) was a negative regulator of abscisic acid (ABA) signaling in seeds. In this report, to get deeper insight on FsPP2C1 function, we aim to identify PP2C-interacting partners. Two closely related members (PYL8/RCAR3 and PYL7/RCAR2) of the Arabidopsis (Arabidopsis thaliana) BetV I family were shown to bind FsPP2C1 in a yeast two-hybrid screening and in an ABA-independent manner. By transient expression of FsPP2C1 and PYL8/RCAR3 in epidermal onion (Allium cepa) cells and agroinfiltration in tobacco (Nicotiana benthamiana) as green fluorescent protein fusion proteins, we obtained evidence supporting the subcellular localization of both proteins mainly in the nucleus and in both the cytosol and the nucleus, respectively. The in planta interaction of both proteins in tobacco cells by bimolecular fluorescence complementation assays resulted in a specific nuclear colocalization of this interaction. Constitutive overexpression of PYL8/RCAR3 confers ABA hypersensitivity in Arabidopsis seeds and, consequently, an enhanced degree of seed dormancy. Additionally, transgenic 35S:PYL8/RCAR3 plants are unable to germinate under low concentrations of mannitol, NaCl, or paclobutrazol, which are not inhibiting conditions to the wild type. In vegetative tissues, Arabidopsis PYL8/RCAR3 transgenic plants show ABA-resistant drought response and a strong inhibition of early root growth. These phenotypes are strengthened at the molecular level with the enhanced induction of several ABA response genes. Both seed and vegetative phenotypes of Arabidopsis 35S:PYL8/RCAR3 plants are opposite those of 35S:FsPP2C1 plants. Finally, double transgenic plants confirm the role of PYL8/RCAR3 by antagonizing FsPP2C1 function and demonstrating that PYL8/RCAR3 positively regulates ABA signaling during germination and abiotic stress responses

Local auxin biosynthesis is required for root regeneration after wounding

The root meristem can regenerate following removal of its stem-cell niche by recruitment of remnant cells from the stump. Regeneration is initiated by rapid accumulation of auxin near the injury site but the source of this auxin is unknown. Here, we show that auxin accumulation arises from the activity of multiple auxin biosynthetic sources that are newly specified near the cut site and that their continuous activity is required for the regeneration process. Auxin synthesis is highly localized while PIN-mediated transport is dispensable for auxin accumulation and tip regeneration. Roots lacking the activity of the regeneration competence factor ERF115, or that are dissected at a zone of low regeneration potential, fail to activate local auxin sources. Remarkably, restoring auxin supply is sufficient to confer regeneration capacity to these recalcitrant tissues. We suggest that regeneration competence relies on the ability to specify new local auxin sources in a precise temporal pattern

The Nuclear Interactor PYL8/RCAR3 of Fagus sylvatica FsPP2C1 Is a Positive Regulator of Abscisic Acid Signaling in Seeds and Stress1[C][W][OA]

The functional protein phosphatase type 2C from beechnut (Fagus sylvatica; FsPP2C1) was a negative regulator of abscisic acid (ABA) signaling in seeds. In this report, to get deeper insight on FsPP2C1 function, we aim to identify PP2C-interacting partners. Two closely related members (PYL8/RCAR3 and PYL7/RCAR2) of the Arabidopsis (Arabidopsis thaliana) BetV I family were shown to bind FsPP2C1 in a yeast two-hybrid screening and in an ABA-independent manner. By transient expression of FsPP2C1 and PYL8/RCAR3 in epidermal onion (Allium cepa) cells and agroinfiltration in tobacco (Nicotiana benthamiana) as green fluorescent protein fusion proteins, we obtained evidence supporting the subcellular localization of both proteins mainly in the nucleus and in both the cytosol and the nucleus, respectively. The in planta interaction of both proteins in tobacco cells by bimolecular fluorescence complementation assays resulted in a specific nuclear colocalization of this interaction. Constitutive overexpression of PYL8/RCAR3 confers ABA hypersensitivity in Arabidopsis seeds and, consequently, an enhanced degree of seed dormancy. Additionally, transgenic 35S:PYL8/RCAR3 plants are unable to germinate under low concentrations of mannitol, NaCl, or paclobutrazol, which are not inhibiting conditions to the wild type. In vegetative tissues, Arabidopsis PYL8/RCAR3 transgenic plants show ABA-resistant drought response and a strong inhibition of early root growth. These phenotypes are strengthened at the molecular level with the enhanced induction of several ABA response genes. Both seed and vegetative phenotypes of Arabidopsis 35S:PYL8/RCAR3 plants are opposite those of 35S:FsPP2C1 plants. Finally, double transgenic plants confirm the role of PYL8/RCAR3 by antagonizing FsPP2C1 function and demonstrating that PYL8/RCAR3 positively regulates ABA signaling during germination and abiotic stress responses

iPLANT II: Consolidación del Laboratorio virtual de Fisiología y Fitopatología Molecular de Plantas

Memoria ID-081. Ayudas de la Universidad de Salamanca para la innovación docente, curso 2010-2011.Este proyecto docente ha servido para la documentación en video y edición de varios protocolos básicos de utilidad en un laboratorio de Fisiología Vegetal y Fitopatología Molecular, que comprenden: técnicas de laboratorio con plantas modelo, protocolos de transformación estable y transitoria de plantas, obtención de mutantes y plantas transgénicas, análisis de genes reportadores, interacción proteína-proteína por doble híbrido y complementación bimolecular fluorescente (BiFC) in vivo y finalmente distintos protocolos transcriptómicos. Además se han seleccionado, digitalizado e integrado en una base de datos, imágenes de plantas modelo de experimentación como Arabidopsis y tomate en distintas etapas del desarrollo vegetal, imágenes de microscopía óptica y confocal descritas acorde con los objetivos de las distintas asignaturas a las que está orientado el laboratorio virtual