Pak. J. Bot., 52(5): 1639-1646, 2020. DOI: http://dx.doi.org/10.30848/PJB2020-5(38) THE OVER-EXPRESSION OF A GLYCINE-RICH RNA-BINDING PROTEIN IN NICOTIANA TABACUM AND ITS ROLES IN PLANT STRESS TOLERANCE AMNA MUHAMMAD 1 , FARIHA KHAN 2* , BUSHRA JAVAID 1 , TEHSEEN RUBBAB 1 , AZKA NOUREEN 1 AND S.M. SAQLAN NAQVI 1,3* 1 Department of Biochemistry, PMAS Arid Agriculture University Rawalpindi, 46300 Rawalpindi. Pakistan. 2 Department of Biosciences, COMSATS University Islamabad, 45550 Islamabad, Pakistan 3 Bacha Khan University, Charsadda, 24420, Khyber Pakhtunkhwa, Pakistan *Corresponding author's email: saqlan@uaar.edu.pk, fariha.khan@comsats.edu.pk Abstract Glycine-rich RNA-binding proteins (GR-RBPs) have been explored in different plant species for their role in development and environmental stress responses. NtGR-RBP1, a tobacco glycine-rich RNA-binding protein has recently been suggested as RNA chaperone with reference to protective role in bacteria upon exposure to various stresses. In the present study, we aimed to test the roles of NtGR-RBP1 in plants. The cDNA sequence of NtGR-RBP1 was amplified and cloned into entry vector using pENTR/D-TOPO cloning kit followed by sub-cloning into a plant GATEWAY® expression vector resulting in construction of pH7WG2-NtGR-RBP1. NtGR-RBP1 recombinants were confirmed through colony PCR and sequencing. Further, pH7WG2- NtGR-RBP1 was cloned into Agrobacterium tumefaciens AGL1 competent cells and confirmed by PCR after screening on streptomycin and hygromycin. The culture was subsequently used for the transformation of Nicotiana tabacum cv samsun by Agrobacterium-mediated plant transformation using the leaf disc method. Explants were screened using hygromycin as selection marker and later confirmed through PCR. Seed germination assay showed that NtGR-RBP1 had significant role in response to cold, heat and drought stress, while salt stress has negative effect on germination. Transformed plants will be used further for in depth functional characterization of NtGR-RBP1. Key words: Glycine-rich RNA-binding Protein, Nicotiana tabacum, RNA chaperone. Introduction Plant Glycine Rich RNA-Binding proteins (GR-RBPs) were isolated owing to their differential expression and modulations towards stimuli such as biotic and abiotic stresses about two decades ago. Belonging to the Class IVA of RBPs, these bipartite proteins contain an RNA recognition motif (RRM) at N-terminus and a glycine rich region at the C-terminus (Mangeon et al., 2010). GR-RBPs play crucial roles towards growth and development of different plant species (Staiger et al., 2003). Their multifunctional roles include transport and localization of RNA, mRNA splicing, post-transcriptional metabolism of RNA, polyadenylation, translation and degradation (Glisovic et al., 2008). GR-RBPs ubiquitously exist in plants and the expression of GR-RBP encoding genes is regulated in response to a variety of abiotic stresses, i.e. cold, heat, drought, salinity, wounding and flooding, biotic stress, such as pathogen attack (Gomez et al., 1998; Maruyama et al., 1999; Glisovic et al., 2008; Califice et al., 2012; Khan et al., 2013). Other stress conditions that also regulate the expression of these proteins include phytohormones, viral infection, UV irradiation, and heavy metals (Naqvi et al., 1998; Sachetto-Martins et al., 2000; Aneeta et al., 2002; Mangeon et al., 2010). During cold stress canonical cold shock domain proteins (CSPs) along with GR-RBPs play protective role in plant adaptation (Nakaminami et al., 2006). In various studies, heterologous expression of GR-RBPs rescued reduced growth rates of plants during cold adaptation that had their GR-RBP genes deleted. (Kim et al., 2010). Arabidopsis thaliana and Oryza sativa have been investigated deeply for illuminating the response of GR-RBPs in encounter to different stress conditions, for example, a CCHC-type zinc finger motif containing protein at its C-terminus conferred freezing tolerance in Arabidopsis, while when expressed, it has a negative impact on seed germination and seedling growth upon dehydration and salt stress (Kim et al., 2007a). For instance, over-expression of GR-RBP2 during salt stress had no effect on seedling growth while it improved seed germination (Kim et al., 2007b). The RNA chaperone activity of AtGR-RBP2 was confirmed in E. coli in response to cold acclimation (Kim et al., 2007c). Over- expression of another GR-RBP i.e. AtGR-RBP7, during cold stress caused transport of mRNAs to the cytoplasm from the nucleus and promoted freezing tolerance by regulation of stomatal apertures (Kim et al., 2008). A rice OsGR-RBP4 was reported to be involved in heat stress (Sahi et al., 2007) while, over-expression of SaGR-RBP1 from Suaeda asparagoides conferred salt tolerance in Arabidopsis (Ayarpadikannan et al., 2014). Furthermore, seven BnGR-GRPs were found in Brassica napus with the almost same sequence of N-terminal RRM domain while there were significant differences in the sequence of C- terminal glycine rich region. Transcript expression of all BnGRPs was remarkably elevated in response to cold, but was seen down-regulated in response to dehydration and salt stress. The functional roles of BnGRP1 as RNA chaperone were determined by over-expression of BnGRP1 in cold sensitive mutant BX04 Escherichia coli, which resulted in improved cold tolerance, while the heterologous expression of the same gene in Arabidopsis showed the elevated seed germination rate and plants showed freezing tolerance (Kim et al., 2012). The stress related role of NtGR-RBP1 was previously explored by checking the transcript expression of NtGR- RBP1in response to cold, salt and wounding stress, the level of mRNA was upregulated in response to cold stress and it was modulated in salt stress, while wounding caused no effect, so it was concluded that NtGR-RBP1 has a