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Involvement of RNA-binding Proteins in the plant responses to drought and salinity

Final Activity Report Summary - SALT-DROUGHT (Involvement of RNA-binding Proteins in the plant responses to drought and salinity)

The hormone Abscisic acid (ABA) plays a central role in the regulation of plant responses to drought and salt stress. The ABH1 (ABA Hypersensitive 1) gene, that encodes the Arabidopsis homolog of a nuclear CAP binding protein, was isolated and characterised in the host institution (Hugouvieux et al. 2001, Cell, 2001, 106:477-488). ABH1 controls ABA signalling but their specific action is still unknown. The fellow holder developed an investigation aimed at elucidating the function of ABH1. He identified proteins that interact with ABH1 using an in vivo approach. One of these proteins, identified by in vivo immunoprecipitation, was the glycolytic enzyme Glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The interaction between ABH1 and GAPDH was further confirmed both by in vitro pull-down experiments and by the yeast two-hybrid system. GAPDH has recently been described in animal systems as a multifunctional enzyme able to participate in transcriptional regulation and programmed cell death cascades. In plants, such functions for the GAPDH have not yet been described, thus demonstrating the originality and innovation of the finding. The results are very promising, but further work is needed in order to successfully complete the project (a 3 year project proposal was presented but only funding for the first 18 months was requested). The host and home institutions are presently collaborating on this subject and an application for funding of the project has been made to the Spanish Ministry of Education and Science (Proyectos I+D presented by Dr Ros and Dr Juan Segura which are the fellow and the scientist in charge of the European Union (EU) fellowship).

During the course of the research conducted by the fellow in the host institution, he acquired in depth experience in several techniques, including novel proteomic, genomic and time-resolved imaging tools. These new techniques will be of great help to him in the functional characterisation of not only ABH1, but also other RNA binding proteins, thus demonstrating a solid potential for making many important contributions to the proposed field of study.

During the last six months of the project, the research has been conducted in the home institution. The characterisation of the RNA binding protein SATO1 has been initiated. The objective is to identify putative proteins and RNAs that interact with SATO1. The knowledge of these interacting substances will help in the elucidation of the role of SATO1 in plants and its relation to salt tolerance.

Finally, the fellow has been collaborating with the host institution in another subject not initially included in the project. He has participated in the functional characterization of a leucine rich repeat receptor kinase involved in the development of pollen. This investigation was finished and resulted in a publication in The Plant Cell journal (17:3350-3361). In addition, the fellow acquired experience in the identification and characterisation of double mutants from Arabidopsis. The characterisation of single and double mutants from SATO1 is one of the specific objectives included in the research project). In constitutive overexpression lines of MC1 fused with a myc-tag, in vivo cleavage of MC1 was detected. 35S-MC1-HIS lines were produced to purify and sequence the fragments to detect the exact autocleavage site.