Expresión y caracterización de la proteína mutante OPEN

Los canales iónicos destacan por su importancia biológica al participar en numerosos y vitales procesos entre los que se encuentran la transmisión nerviosa o la secreción de determinados compuestos. La alteración de su función desemboca en importantes enfermedades que en general se conocen como canalopatías. Por todo ello, son objeto de estudio para multitud de grupos de investigación. Dentro de los canales iónicos, los canales de potasio se han estudiado profundamente gracias a la proteína KcsA, un canal de potasio modelo aislado a partir del microorganismo Streptomyces lividans y cuya estructura cristalográfica está resuelta facilitando así su estudio. El KcsA presenta dos compuertas conectadas alostéricamente, una primera cuya apertura es dependiente del pH y una segunda compuerta, el filtro de selectividad, que determina qué iones pueden atravesar el canal. El filtro de selectividad puede encontrarse en dos estados, conductor e inactivado, siendo ésta última conformación el objeto de estudio en este trabajo. En estado de reposo, el canal tiene la primera compuerta cerrada, mientras que el filtro está en su conformación conductora. La apertura de la primera compuerta a pH ácido permite el paso de iones a través del canal en un primer momento (canal en estado abierto). Sin embargo, esta apertura desencadena de manera alostérica la inactivación del filtro de selectividad, entrando en un estado en el que se impide el paso de iones (canal en estado inactivado). En este trabajo se ha realizado un estudio de la conformación del filtro de selectividad en estado inactivado haciendo uso de un mutante en el que la primera compuerta está permanentemente abierta incluso a pH neutro, permitiendo así una comparación directa con el estado en reposo nativo del canal a ese mismo pH, ya previamente caracterizado. Con este fin, se ha expresado y purificado el mutante OPEN de KcsA, para luego hacer una caracterización de la estructura de su filtro de selectividad mediante experimentos de unión de diferentes iones conductores y no conductores.Ion channels stand out because their biological importance since they take part of a large number of life processes as nervous transmission or secretion of certain compounds. Alteration of these channels results in serious diseases known as channelopathies. Therefore, ion channels are under consideration for a multitude of research groups. As part of ion channels, potassium channels have been studied in detail thanks to the KcsA protein, which is a model potassium channel isolated from the microorganism Streptomyces lividans, whose structure has been determined crystallographically, making easier its study. KcsA has two gates allosterically connected. The opening of the first one is regulated by pH. The second one is the selectivity filter which determines the kind of ions which can pass through the channel. The selectivity filter has two states, conductive and inactivated, being this last conformation our subject of study in this project. At the resting state, the channel has its first gate closed while the selectivity filter remains in its conductive conformation. Opening of the first gate at low pH allows the passage of ions through the channel in first instance (channel in an open state). However, this opening triggers, in an allosteric way, the inactivation of the selectivity filter which enters a state which blocks the ionic conduction (channel in an inactivated state). In this project, a conformational study of the selectivity filter in its inactivated state has been performed. For this purpose, a mutant protein whose first gate is constantly open, even in a neutral pH, has been used since its study allows the direct comparison with the native resting state of the channel at the same pH, previously characterized. To this end, the OPEN mutant of KcsA has been expressed and purified in order to characterize its selectivity filter structure by performing binding experiments with different conductive and non-conductive ions

Genes involved in auxin biosynthesis, transport and signalling underlie the extreme adventitious root phenotype of the tomato aer mutant

The use of tomato rootstocks has helped to alleviate the soaring abiotic stresses provoked by the adverse effects of climate change. Lateral and adventitious roots can improve topsoil exploration and nutrient uptake, shoot biomass and resulting overall yield. It is essential to understand the genetic basis of root structure development and how lateral and adventitious roots are produced. Existing mutant lines with specific root phenotypes are an excellent resource to analyse and comprehend the molecular basis of root developmental traits. The tomato aerial roots (aer) mutant exhibits an extreme adventitious rooting phenotype on the primary stem. It is known that this phenotype is associated with restricted polar auxin transport from the juvenile to the more mature stem, but prior to this study, the genetic loci responsible for the aer phenotype were unknown. We used genomic approaches to define the polygenic nature of the aer phenotype and provide evidence that increased expression of specific auxin biosynthesis, transport and signalling genes in different loci causes the initiation of adventitious root primordia in tomato stems. Our results allow the selection of different levels of adventitious rooting using molecular markers, potentially contributing to rootstock breeding strategies in grafted vegetable crops, especially in tomato. In crops vegetatively propagated as cuttings, such as fruit trees and cane fruits, orthologous genes may be useful for the selection of cultivars more amenable to propagation.The research was supported by BBSRC—UKRI funding; the RootLINK (BB/L01954X/1) and AdRoot (BB/S007970/1) projects