Riassunti dei progetti del corso di dottorato di ricerca in biologia molecolare e cellulare
Project leader: LUCIA COLOMBO (lucia.colombo@unimi.it)
Download 336.57 Kb.
|
Project leader: LUCIA COLOMBO (lucia.colombo@unimi.it)Location: Department of Biosciences, University of Milan, ItalyRESEARCH PROJECT SUMMARYGenetic and epigenetic control of seed number in Arabidopsis The understanding of the genetic control of organ primordia formation and differentiation are one of the most challenging aspect of the developmental biology. In this project we combine the study of a very fascinating and still unknown process with its possible application for improving plant yield. The seed are formed from the ovule upon fertilization. Several important events are required for successful seed settings: the ovule primordia have to be formed, followed by pattern formation and morphogenesis and finally after fertilization seed has to develop. The seeds number is one of the most important trait in plant breeding. Yield enhancement is required to meet increasing food demand. In addition, there is an increasing demand for plant-derived products for non-food purposes, such as energy production. The genetic networks controlling ovule number and fertility are strictly connected and a restricted number of master genes control these developmental processes such as SEEDSTICK (STK) and CUP SHAPED COTYLEDONS (CUCs) encode for transcription factors. This proposal aims to study the molecular network underpinning the ovule number and fertility using Arabidopsis as model. The objectives will be reached using an integrative approach base on advance technology such as laser microscopy, genome wide target identification and genome wide expression profile. Project leader: ALEX COSTA (alex.costa@unimi.it)Location: Department of Biosciences, University of Milan, ItalyRESEARCH PROJECT SUMMARYFunctional characterization of Arabidopsis thaliana iGluRs (glutamate receptors) channels Ionotropic glutamate receptors (iGluRs) are ligand-gated cation channels that mediate neurotransmission in animal nervous systems. Homologous proteins in plants (20 members in Arabidopsis, Lacombe et al., 2001 Science) have been implicated in root development, ion transport, and several metabolic and signaling pathways. A recent work demonstrate the involvement of two members of the plant GLR family, GLR3.3 and GLR3.6, in long-distance wound signaling (Mousavi et al., 2013 Nature). Moreover, another member of this family, the GLR1.2 is specifically expressed in pollen and its activity is crucial for proper Ca2+ fluxes (formation of the Ca2+ tip gradient), and ultimately for the proper pollen tube growth and fertility (Michard et al., 2011, Science). Analyses of animal iGluRs, analyzed in heterologous expression system such as Xenopus oocytes have shown that these channels have varying conductances for sodium Na+, potassium K+ and Ca2+. They were therefore classified as non-selective cation channels (NSCC). Studies conducted thus far suggest that also plant iGluRs are NSCCs. Importantly, in a recent study Vincill et al. established HEK cells as a system for studying iGluR function (Vincill et al., 2012 Plant Physiol). In their work they found that AtGLR3.4 is an amino acid gated channel that is suggested to be selective to Ca2+ and is capable of inducing cytosolic Ca2+ peaks in response to asparagine, glycine, or serine. A second work using a different heterologous expression system, Xenopus oocytes, found that another Arabidopsis iGluR homolog, AtGLR1.4, functioned as a ligand-gated, nonselective, Ca2+-permeable cation channel that responded to an even broader range of amino acids, none of which are agonists of animal iGluRs (Tapken et al., 2013 Sci Signal). At the electrophysiological level only few members of this large plant iGluR family have been functionally characterized. In this regard future research is needed to analyze in detail conductance properties of different plant iGluRs. The aim of this project will be the identification, molecular cloning, functional characterization and study of the physiological role of different Ca2+ permeable iGluR channels from Arabidopsis. Public microarray data show that at least 13 members of the iGluR family are expressed in the different tissues of the root tip cells with, in some cases distinct expression patterns. Arabidopsis plants expressing the Ca2+ probe Cameleon will be treated with the 20 standard amino acids, and the Ca2+ responses will be monitored in root tip cells by using both an wide field fluorescence microscopy (for an initial screening) and the SPIM-FRET set up (recently developed in our lab; Costa et al., 2013 PLOS ONE) for single cell analyses. This analyses will allow to correlate the amino acids-induced cytosolic Ca2+ rises with the expression patterns of the 13 members the root expressed iGluRs. This series of experiments will provide useful information about which are the effective iGluRs ligands and narrow down the number of interesting candidates. The predicted plasma membrane localized iGluRs will be cloned in the pcDNA3 expression vector (for mammalian cells) and in order to study their functionality in the heterologous mammalian system (HEK293T cells) will be co-expressed individually with the Ca2+ probe Cameleon for imaging analyses. The cells co-expressing the different channels together with the Cameleon will be treated with the amino acids reported to induce cytosolic Ca2+ rises in root cells. This approach will enable a first fast screening of the iGluRs candidates. Only those, in which the treatment with the different amino acids, will show a cytosolic Ca2+ increase will be further electrophysiologically characterized. In this latter case, a combined imaging and electrophysiological set up will also permit to perform at the same time and in the same cell a detail electrical characterization of the channel and the study of its Ca2+ permeability. Finally the identified and selected channels will be also studied in Xenopus oocytes in which a more detailed analysis of the selectivity and other properties will be carried out. A second part of the project will be devoted to the study of the physiological role of the functional iGluR channels. For this aim the in planta expression pattern of the channel/s will be performed by qRT-PCR and through the cloning of promoter/s region/s (in order to generate GUS transgenic reporters lines). The plasma membrane localization will be confirmed by the generation of GFP fusion proteins. Isolation and phenotypic characterization of knock out mutant/s for any given channel/s, will then shed light on the physiological role played by the channel/s. In the isolated KO mutant/s the cytosolic localized Cameleon probe will be introduced and the response to the aminoacid evaluated. Download 336.57 Kb. Share with your friends:
|