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An integrated approach to identify stress-related regulatory genes in cork oak

Project: Research


Portuguese cork oak forest is not only a very important source of income but it also plays a crucial ecological role within the forest ecosystem. Due to the cork oak strategic significance for Portugal and to the threats increasingly affecting the culture, FCT recently sponsored a national effort aiming to deliver ESTs from several tissues at different developmental stages and exposed to abiotic stresses and biotic interactions, and to build a CorkOak EST Database (CorkOakDB). The information gathered in this initiative is now available for the project SuberStress, in which it will be processed and confirmed by 4 partners involved in the stress and bioinformatics studies in the ESTs-consortium (ITQB/IBET, IICT, Univ. Minho, Univ. Algarve), plus an additional partner with expertise in cork oak in vitro culture and in plant stress response (Univ. Aveiro). Following the EST sequences analyses already started by our team, we will determine which unigenes of the CorkOakDB are up and down regulated in each condition (stress vs. control) (TASK1). In order to understand which genes may be involved in the cross-talk between different imposed stresses, we will carefully study their expression variations in all the previously tested conditions (Phytophthora cinnamomi infections, cold, heat, salt, oxidative/light and drought stresses). Each partner will carry out experiments for a different stress. Additionally, in vitro cultures will be established by Univ. Aveiro to be used in specific cases in which gene function characterization may require the imposition of additional stress conditions (eg.: ABA, jasmonic acid). The expression studies will be semi-quantitative in a first approach and for the most interesting genes also quantitative (real-time RT-PCR available to all partners). Expression data will be combined with physiological data (photosynthetic efficiency as a measure of the stress-induced damage) and with literature data in order to select the most relevant target genes to search for upstream regulators. The transcription factors identified using a yeast-1 hybrid approach will be characterized for their role as activators or repressors. We will also follow an epigenetic approach to investigate how methylation of the promoters (at the level of both DNA and histones) of the target genes contributes to their regulation under stress. This integrated study will hopefully provide clues about key genes controlling the stress response network in cork oak. The bio-informatics comparison of cork oak data with data publicly available for other plants will further allow to start building a cork oak stress-response gene network. The project has as consultants, C. Pinto Ricardo (expert in cork oak physiology and iochemistry), Michel Delseny (expert in integrative biology, specifically in plant gene expression and stress response), and José Leal (coordinator of the Bioinformatics Unit of LAO-Laboratório Associado de Oeiras). The involvement of several Universities where Master courses are being run will facilitate the integration of Master students in parts of SuberStress. This is expected for TASK 2 (stress application and physiological analyses) and TASKs 3 and 7 (expression studies), for which it would be desirable to allocate a higher number of researchers than the available budget allows. In this way, 3 additional Master thesis are expected to be associated with this project besides the two diploma researchers to be hired (TASKs 2/3.1, and 3.2/4, 1 year each) who are expected to be integrated in a Master’s program. Furthermore, the student to be hired (2 years) to search and to validate upstream regulators (TASKs 4,5,7) is expected to continue the studies in the framework of a PhD thesis. Also the two students to be hired for the in silico studies (12 months/TASK1) and epigenetic analyses (12 months/TASK6) should have qualifications high enough to proceed for PhD with the study of cork oak stress responses with any of the partners. Three common publications are envisaged, one with the in silico studies (end of TASK1), a second including the stress experiments, physiology studies and gene expression analyses (end of TASK3), and a third one with the new gene regulators and their expression study in all stress conditions and also including in silico correlations (end of TASK7). Specific papers are also planned with the data gathered at ITQB from TASKs 4, 5 and 6 (cDNA expression libraries, yeastone hybrid screening for the TFs regulating the promoters of the three target genes and study of their differential methylation under stress). After consensus in the team, other papers may also be prepared on the work of Master students conducting, e.g. a more detailed physiological/biochemical study of the plants under stress.

Effective start/end date10/02/121/02/14
  • Amaral, Joana (Researcher)
  • Oliveira, Maria Margarida (Principal Investigator)