Among abiotic stresses, salt stress adversely affects growth and development in rice. Contrasting salt tolerant (CSR27), and salt sensitive (MI48) rice varieties provided information on an array of genes that may contribute for salt tolerance of rice. Earlier studies on transcriptome and proteome profiling led to the identification of salt stress-induced serine hydroxymethyltransferase-3 (SHMT3) gene. In the present study, the SHMT3 gene was isolated from salt-tolerant (CSR27) rice. OsSHMT3 exhibited salinity-stress induced accentuated and differential expression levels in different tissues of rice. OsSHMT3 was overexpressed in Escherichia coli and assayed for enzymatic activity and modeling protein structure. Further, Arabidopsis transgenic plants overexpressing OsSHMT3 exhibited tolerance toward salt stress. Comparative analyses of OsSHMT3 vis a vis wild type by ionomic, transcriptomic, and metabolic profiling, protein expression and analysis of various traits revealed a pivotal role of OsSHMT3 in conferring tolerance toward salt stress. The gene can further be used in developing gene-based markers for salt stress to be employed in marker assisted breeding programs. HIGHLIGHTS simple - The study provides information on mechanistic details of serine hydroxymethyl transferase gene for its salt tolerance in rice. Keywords: rice, Arabidopsis, salinity, serine, glycine, aquaporins Introduction Rice (Oryza sativa) is a staple food for ∼90% of the Asian population (Mohanty et al., 2013). Production of rice in 2011–2014 was 495.63 million tons (MT) (FAO, 2014 1). The global population is projected to increase by 25% by 2050 (United Nations World Population Prospects, 2017 2). Therefore, there is a need for a commensurate increase in rice production to meet the ever-increasing demands of the growing population (FAOSTAT, 2009 3). Although India has the largest acreage under rice [44.5 million ha (Mha)], the average productivity (2.5 tons ha-1) is abysmally low compared with China (Singh et al., 2016). This low productivity could be attributed to harsh conditions in different agroclimatic regions of India under which rice is cultivated and often subjected to various abiotic stresses (salinity, drought, flood, and nutrient deficiencies). In India, a large proportion of agriculture land (6.73 Mha) is affected by high saline content (Singh et al., 2016). Salinity adversely affects growth and development, particularly during seedling and flowering stages, and consequently the yield potential of rice (Krishnamurthy et al., 2016; Zeng et al., 2018; Morton et al., 2019). The salinity-tolerant plants have evolved an array of adaptive strategies (exclusion, compartmentation, and secretion of Na+) to mitigate the toxic effects of salinity stress (Arzani and Ashraf, 2016; Zeng et al., 2018). Potassium (K) and calcium (Ca) signaling also exert significant influences toward conferring tolerance toward salinity stress in rice (Shabala and Cuin, 2008; Frouin et al., 2018). Reactive oxygen species (ROS) and osmolytes (choline, glycine betaine, sugars, etc.) have also been implicated in conferring variable levels of tolerance toward salt stress (Hanson et al., 2000). Complete sequencing of rice genome (IRGSP, 2005) has expedited the process of deciphering the molecular entities, which play pivotal roles in conferring tolerance to salt stress. Availability of salt-tolerant and salt-sensitive rice varieties and state-of-the-art omics technologies further provided the fillip in this endeavor. Serine hydroxymethyltransferase (SHMT), a pyridoxal phosphate-dependent enzyme, plays a pivotal role in cellular one-carbon pathways by catalyzing the reversible conversions of L-serine to glycine and tetrahydrofolate to 5,10-methylenetetrahydrofolate in lower and higher organisms (Schirch and Szebenyi, 2005). Structurally conserved SHMT has been implicated in various roles across lower and higher organisms (Moreno et al., 2005; Voll et al., 2006). In plants, SHMT activity has been detected in different organelles (mitochondria, cytosol, plastids, and nuclei) suggesting their diverse roles in metabolic pathways (Besson et al., 1995; Neuburger et al., 1996; Zhang et al., 2010). In rice, the SHMT family comprises five members (OsSHMT1-5). OsSHMT1, encoding the largest subunit of SHMT and an ortholog of Arabidopsis thaliana SHM1, was identified by employing enhancer trapping and the characterization of chlorophyll-deficient mutant (oscdm1) (Wu et al., 2015), photorespiratory mutant osshm1 and map-based cloning (Wang et al., 2015). In Arabidopsis also, the mutation in AtSHM1 (At4g37930) caused an aberration in mitochondrial SHMT activity and exhibited a lethal photorespiratory phenotype during growth at ambient CO2 (Somerville and Ogren, 1981; Voll et al., 2006). These studies suggested conserved function of SHMT1 in photorespiration in taxonomically diverse rice and Arabidopsis. Interestingly, in soybean, SHMT plays a pivotal role in cyst nematode (SCN) resistance (Liu et al., 2012; Kandoth et al., 2017). In another study, overexpression of ApSHMT from halotolerant cyanobacteria Aphanothece halophytica in Escherichia coli (E. coli) triggered a higher accumulation of glycine betaine due to elevated levels of precursors, choline and serine, and consequently augmented tolerance toward salinity-stress (Waditee-Sirisattha et al., 2012). These studies thus suggested roles of SHMT not only in photorespiration but also in other metabolic pathways in different plant species. The recombinant inbred line (RIL) population of salt-tolerant (CSR27) and salt-sensitive (MI48) rice genotypes were screened for their extreme tolerance and sensitivity toward salt stress, and bulk segregant analysis together with a gene expression study led to the identification of an array of differentially regulated salt stress responsive genes including OsSHMT3 (Pandit et al., 2010) using bulk tolerant (BT) and bulk sensitive (BS) RILs. In a subsequent study, these BT and BS populations were also analyzed for their proteomic profiling, which revealed high expression of several proteins including serine hydroxymethyltransferase-3 (OsSHMT3) in the former (Mishra et al., 2016). Overexpression of salt-tolerance-related genes as well as stress-inducible transcription factors has led to the transgenic plants with enhanced salt tolerance (Arzani and Ashraf, 2016). Overexpression of OsCYP94C2, (gene from Cyt450 family) and C-terminal centrin-like domain (OsCCD1) conferred tolerance toward salt stress (Kurotani et al., 2015; Jing et al., 2016). In this context, it is intriguing whether overexpression of OsSHMT3 would elicit any tolerance toward salt stress. Here, we examined the role of SHMT in conferring salt stress tolerance by mediating biosynthetic pathway of glycine to serine interconversion and synthesis of amino acids. In the present study the OsSHMT3 was amplified from salt-tolerant rice. OsSHMT3 was overexpressed in E. coli and assayed for enzymatic activity and modeling protein structure. Further, transgenic Arabidopsis overexpressing OsSHMT3 (OEs) lines were tested for their tolerance toward salt stress. Comprehensive and comparative analyses of the wild-types and OEs for their ionomic, transcriptomic and metabolic profiles, protein expression and different traits were employed to study the role of OsSHMT3 in conferring tolerance toward salt stress in Arabidopsis.