The role of RGGA RNA binding protein in plant response to osmotic stress

Osmotic stress critically limits plant growth and crop productivity. The identification of genes underlying the mechanisms of stress response is the subject of intense research in plant biology. Through microarray analyses we previously identified and isolated RGGA gene, coding for an RNA binding protein, whose expression was specifically induced in Solanumtuberosum cell cultures gradually exposed to osmotic stress. The aim of this study was to confirm the role of RGGA as a functional RNA binding protein required for a proper response to stress condition. We identified the RGGA orthologue in Arabidopsis thaliana (AtRGGA, At4g16830) and evaluated the influence of drought and salt stress on AtRGGA gene expression in cells and seedlings exposed to high concentrations of NaCl, PolyEthyleneGlycole (PEG) and abscisic acid (ABA).Interestingly, AtRGGA gene expression was up-regulated in seedlings after long-term exposure to NaCl and PEG, while short-term treatments with NaCl resulted in AtRGGA down- regulation. To investigate the protein sub-cellular localization, a YFP-RGGA fusion protein was used. Fluorescence signal indicated that RGGA is localized in the cytoplasm and the peri- nuclear region. In addition, a ?-glucuronidase (GUS) assay showed promoter activity in several tissues, including guard cells of stomata, the organs controlling transpiration. Electrophoresis Mobility Shift Assays with total RNA using recombinant His-RGGA clearly showed that RGGA is capable of binding RNA in vitro. To carry out a functional analysis, a gain- and loss-of-function approach was performed using rggaknock-out mutants and AtRGGA over-expressing plants. In addition, rggaknock-out mutant was hyper-sensitive to ABA in root growth and survival tests and to salt stress during germination and at the vegetative stage. Accordingly, the over- expressing plants showed a higher tolerance both in vitro and in soil and accumulated lower levels of proline when exposed to salt and drought stress conditions. Finally, a global analysis of gene expression using microarrays, revealed extensive alterations in the transcriptome of AtRGGA over-expressing plants and rgga mutants under osmotic stress, indicating that RGGA participates in the modulation of transcript abundance of several key genes involved in abiotic stress response. The data taken together provide compelling evidence that the RGGA gene is involved in important mechanisms of plant response to osmotic stress.