Modification of nucleosides on transfer RNA (tRNA) is important either for correct mRNA decoding process or for tRNA structural stabilization. Nucleoside methylations catalyzed by MTase (methyltransferase) are the most common type among all tRNA nucleoside modifications. Although tRNA modified nucleosides and modification enzymes have been extensively studied in prokaryotic systems, similar research remains preliminary in higher plants, especially in crop species, such as rice (Oryza sativa). Rice is a monocot model plant as well as an important cereal crop, and stress tolerance and yield are of great importance for rice breeding. In this study, we investigated how the composition and abundance of tRNA modified nucleosides could change in response to drought, salt and cold stress, as well as in different tissues during the whole growth season in two model plants–O. sativa and Arabidopsis thaliana. Twenty two and 20 MTase candidate genes were identified in rice and Arabidopsis, respectively, by protein sequence homology and conserved domain analysis. Four methylated nucleosides, Am, Cm, m1A and m7G, were found to be very important in stress response both in rice and Arabidopsis. Additionally, three nucleosides,Gm, m5U and m5C, were involved in plant development. Hierarchical clustering analysis revealed consistency on Am, Cm, m1A and m7G MTase candidate genes, and the abundance of the corresponding nucleoside under stress conditions. The same is true for Gm, m5U and m5C modifications and corresponding methylation genes in different tissues during different developmental stages. We identified candidate genes for various tRNA modified nucleosides in rice and Arabidopsis, especially on MTases for methylated nucleosides. Based on bioinformatics analysis, nucleoside abundance assessments and gene expression profiling, we propose four methylated nucleosides (Am, Cm, m1A and m7G) that are critical for stress response in rice and Arabidopsis, and three methylated nucleosides (Gm, m5U and m5C) that might be important during development.

Despite its superiority for evaluating gene expression, real-time quantitative polymerase chain reaction (qPCR) results can be significantly biased by the use of inappropriate reference genes under different experimental conditions. Reaumuria soongorica is a dominant species of desert ecosystems in arid central Asia. Given the increasing interest in ecological engineering and potential genetic resources for arid agronomy, it is important to analyze gene function. However, systematic evaluation of stable reference genes should be performed prior to such analyses. In this study, the stabilities of 10 candidate reference genes were analyzed under 4 kinds of abiotic stresses (drought, salt, dark, and heat) within 4 accessions (HG010, HG020, XGG030, and XGG040) from 2 different habitats using 3 algorithms (geNorm, NormFinder, and BestKeeper). After validation of the ribulose-1,5-bisphosphate carboxylase/oxygenase large unite (rbcL) expression pattern, our data suggested that histone H2A (H2A) and eukaryotic initiation factor 4A-2 (EIF4A2) were the most stable reference genes, cyclophilin (CYCL) was moderate, and elongation factor 1α (EF1α) was the worst choice. This first systematic analysis for stably expressed genes will facilitate future functional analyses and deep mining of genetic resources in R. soongorica and other species of the Reaumuria genus.

Stress induces changes of modified nucleosides in tRNA, and these changes can influence codon–anticodon interaction and therefore the translation of target proteins. Certain nucleoside modification genes are associated with regulation of stress tolerance and immune response in plants. In this study, we found a dramatic increase of 2′-O-methyladenosine (Am) nucleoside in rice seedlings subjected to salt stress and abscisic acid (ABA) treatment. We identified LOC_Os03g61750 (OsTRM13) as a rice candidate methyltransferase for the Am modification. OsTRM13 transcript levels increased significantly upon salt stress and ABA treatment, and the OsTrm13 protein was found to be located primarily to the nucleus. More importantly, OsTRM13 overexpression plants displayed improved salt stress tolerance, and vice versa, OsTRM13 RNA interference (RNAi) plants showed reduced tolerance. Furthermore, OsTRM13 complemented a yeast trm13Δ mutant, deficient in Am synthesis, and the purified OsTrm13 protein catalysed Am nucleoside formation on tRNA-Gly-GCC in vitro. Our results show that OsTRM13, encoding a rice tRNA nucleoside methyltransferase, is an important regulator of salt stress tolerance in rice.