Project Full Name : Trafficking to the Vacuoles in Plants

Project No.: 301419

Project Acronym: MC Vacuole

Project Full Name: Trafficking to the Vacuoles in Plants

Marie Curie Actions International Incoming Fellowships (IIF) Final Report

Period covered: from 01/07/2012 till 31/12/2013 Project beneficiary and principal investigator: Dr Abel Rosado Project Scientist in Charge: Dr Miguel A. Botella. Host institution: University of Malaga, Spain. Date of submission: 31/01/2014 1. Final Publishable Summary Report The ability for net carbohydrate and protein accumulation in storage tissues is at the core of the inorganic carbon fixation and energy accumulation processes in plants. Heterotrophic plant organs such as fruits, seeds, tubers, and roots contain specialized cell types that accumulate photo-assimilates and storage proteins as energy reserves in their central vacuole. These energy accumulation processes directly impact the overall productivity and final size of the plant, and also has nutritionally related significance for humans and livestocks. Due to the agronomic importance of storage proteins and metabolites accumulated in plant vacuoles, many studies have focused on this process. Comparatively, a lot of data in plant vacuolar trafficking have been generated, yet has fueled intense debate. Controversial points range from the number and nature of vacuolar compartments in different tissues, the presence of different vesicle mediated sorting routes to the vacuole within the same cell, and the nature of the receptors that recognize the sorting determinants of soluble cargo to package them into vesicles destined for the vacuole. The EU funded research project Trafficking to the vacuoles in plants was aimed to answer some of these questions and its long term goal was to obtain a mechanistic understanding of the cellular processes involved in the accumulation and mobilization of vacuolar targeted cargoes for biotechnological purposes. Throughout this project, the analysis of ribosomal protein mutants in the model plant Arabidopsis thaliana highlighted the importance of basic cellular mechanisms, such as translational regulation, for the proper delivery of vacuolar targeted cargoes. Thus, by using cell biology, physiology, and bioinformatic analyses, we were able to demonstrate that independent ribosomal mutations exerted translational regulation of vacuolar targeting pathways and cargoes through the control of transcription factors containing specific DNA sequences in their promoter region named upstream open reading frames (uORFs).

As important as the description of regulatory aspects of the vacuolar trafficking pathways was the development of analytical tools with potential to generate useful information for biotechnological purposes in the field of vacuolar trafficking. Thus, to obtain a global perspective of the processes involved in the vacuolar delivery of cargoes we aimed to determine the protein and metabolite composition of specific vacuolar targeted vesicles. To achieve this objective we established a protocol to affinity purify intact SYP21 vacuolar targeted vesicles that will allow the future use proteomics and metabolomics approaches for machineries and vesicle cargoes identification. The basic knowledge derived from this Research Project establish the foundation for the future use of translational regulators to increase the net carbohydrate and protein accumulation in storage tissues of commercial crops of interest for the European Union farmers. 2. Wider societal implications of the project The global area under crops grew by about 12% over 1960 to 2000, but cereal production increased by over 100%. This increased production was made possible by scientific advances in the field of seeds, fertilizers, pesticides, etc. and by improved techniques of production, storage and distributions of the agricultural products. Still, the OECD-FAO Agricultural Outlook 2012-2021 points out that agricultural production needs to increase by 60% over the next 40 years to meet rising food demand. Globally, the scope for expanding agricultural land is limited, so additional tools need to be implemented in order to maintain the burgeoning population increase. In this context, the development of biotechnologically enhanced crops with higher yield and tolerance towards stresses could be essential for the sustainable use of our limited land and water resources. Dissemination activities 1. Research Manuscripts. Part of the research activities under the Marie Curie Fellowship has been published or will be incorporated on scientific articles that are currently under preparation:

Rosado et al., The Phospholipid Binding Synaptotagmins 1 and 3 act redundantly during Cold Acclimation in Arabidopsis. Under preparation. Estimated submission to PNAS March 2014.

Rosado et al., (2012). Arabidopsis ribosomal proteins control developmental programs through translational regulation of auxin response factors. PNAS. 109(48):19537-44.

2. Invited Seminars

Title: The Arabidopsis Synaptotagmins 1 and 3 maintain lipid homeostasis during cold stress episodes Place: Umea, Suecia Date: January 2013 Organized by: Umea Plant Science Centre

Title: Membrane Contact Sites in Plants Place: Madrid, Spain Date: September 2013 Organized by: Consejo Superior de Investigaciones Científicas (CSIC)

3. Presentations at National and International Meetings Title: Analisis de genes implicados en la ruta de secrecion en la tolerancia a estreses abioticos Congress: V Reunion de la Red Estres Abiotico de Plantas Place: Murcia, España Date : 19-21 September 2012 Organized by: REAP

Title: Ribosomal mutants involvement in vacuolar trafficking responses Congress: 14th ENPER Meeting Place: Madrid, Spain Year: 28-30th August 2012 Organized by: European Network of Plant Endomembrane Research

Title: Synaptotagmins involvement in intracellular trafficking Congress: 15th ENPER Meeting Participant role: Attendant Place: Gent, Belgium Year: 28-31th August 2013 Organized by: European Network of Plant Endomembrane Research

3. General Public June 2013. Publication in the general public magazine UCIENCIA of the article “Primeros pasos hacia el tomate autorreparable”. This publication is a tool for researchers and staff of the University of Malaga aimed to publicize the projects and research infrastructures where they work every day. Uciencia thus becomes a vehicle for the transfer of knowledge and information to the society. This popular science magazine is distributed quarterly and free of charge with the newspaper Sur http://www.diariosur.es