(Signaling the Organelle Unfolded Protein response)
1. The problem – Food and feed availability must clearly increase significantly to meet the future demands of the increasing world population. This places great strain on agriculture, since the available arable land is limited and climate change negatively affects crop production.
2. The proposed solution – One solution is to breed for higher yielding crops that are more tolerant to stresses, thus novel genes and allelic variants conferring higher tolerance to abiotic and biotic stressful conditions need to be identify. We will contribute to this aspect by investigating the molecular details of the newly discovered chloroplast and mitochondrial Unfolded Protein Response (UPR) pathways in Arabidopsis, regarded as major stress responses activated by abiotic stresses or biotic agents, upon the accumulation of misfolded or unfolded proteins in the two organelles. In particular, we propose an integrated functional genomic approach, which involves the use of Arabidopsis mutants with active chloroplast- and mitochondrial-UPR signaling pathways, triggered by defects in organelle protein homeostasis. A high-throughput quantitative proteomic analysis from Arabidopsis mutant and WT seedlings in combination with RNAseq will be performed with the aim to provide a comprehensive picture of how UPR impact organelle and nuclear gene expression to efficiently overcome protein homeostasis unbalances. Advanced bioinformatics tools will be employed to reconstruct protein-protein interaction (PPI) networks and evaluate their function to define a list of candidate genes/proteins playing major roles in organelle UPR signaling pathways. In the last part of the project, we will exploit the gained knowledge to mine the exome-capture data from 510 geo-referenced barley cultivars and landraces, representing worldwide barley genetic diversity, as a first step in transferring Arabidopsis-based information to agronomical relevant crops.
3. The research network – Five research units (RU) from universities and research institutes, selected on the basis of complementary technologies and expertise in diverse research areas are brought together for the realization of the experimental plan. Competences and facilities for functional genomic studies in Arabidopsis are provided by RU1 (chloroplast biology), RU2 (mitochondrial biology) and RU3 (cellular redox homeostasis). RU4 will contribute to the proposal with the proteomic platform, whereas RU5 will make available the skills and instrumentations, in terms of software, algorithms and computational power, for the RNAseq and Proteomic data analysis. RU5 and RU1 will also provide their expertise for the identification of allelic variants and barley accessions through the barley exome-capture data analysis.
4. The research plan – The research plan is set up in 4 experimental work-packages (WP), plus a fifth WP dedicated to coordination and dissemination activities: WP1 is dedicated to the identification and characterization of mutants with active chloroplast and mitochondrial UPR, to be used as genetic material for transcriptomic and proteomic analysis. WP2 will be devoted to the transcriptomic and proteomic studies, together with the bioinformatics data analysis. Furthermore, the characterization of the attap1 mutant, lacking a chloroplast peptide transporter, will be also carried out in this WP, with the aim to verify the possible involvement of peptides, obtained through the activity of chloroplast proteases and exported to the cytosol, in chloroplast-to-nucleus UPR signaling. The functional characterization of few candidate genes, obtained in WP2, will be carried out in WP3, whereas WP4 will be dedicated to the knowledge transfer from Arabidopsis to barley.
5. The outputs – At the end of the three-year project we expect to deliver:
i) A list of candidate genes (CGs) with a role in chloroplast and/or mitochondrial UPR
ii) A list of peptides with a role in chloroplast UPR signal transduction
iii) The detailed characterization of 3 CGs
iv) Barley allelic variants and barley accessions that will represent the starting genetic material for future proposals, where their performance under different environmental conditions will be tested.