As the most abundant internal modification of mRNA, N 6 -methyladenosine (m 6 A) methylation of RNA is emerging as a new layer of epitranscriptomic gene regulation in cellular processes, including embryo development, flowering-time control, microspore generation and fruit ripening, in plants. However, the cellular role of m 6 A in plant responses to environmental stimuli remains largely unexplored. In this study, we show that m 6 A methylation plays an important role in salt stress tolerance in Arabidopsis. All mutants of m 6 A writer components, including MTA, MTB, VIRILIZER (VIR) and HAKAI, displayed salt-sensitive phenotypes in an m 6 A-dependent manner. The vir mutant, in which the level of m 6 A was most highly reduced, exhibited salt-hypersensitive phenotypes. Analysis of the m 6 A methylome in the vir mutant revealed a transcriptomewide loss of m 6 A modification in the 3ʹ untranslated region (3ʹ-UTR). We demonstrated further that VIR-mediated m 6 A methylation modulates reactive oxygen species homeostasis by negatively regulating the mRNA stability of several salt stress negative regulators, including ATAF1, GI and GSTU17, through affecting 3ʹ-UTR lengthening linked to alternative polyadenylation. Our results highlight the important role played by epitranscriptomic mRNA methylation in the salt stress response of Arabidopsis and indicate a strong link between m 6 A methylation and 3ʹ-UTR length and mRNA stability during stress adaptation.

Despite the importance of pentatricopeptide repeat (PPR) proteins in organellar RNA metabolism and plant development, the functions of many PPR proteins remain unknown. Here, we determined the role of a mitochondrial PPR protein (At1g52620) comprising 19 PPR motifs, thus named PPR19, in Arabidopsis thaliana. The ppr19 mutant displayed abnormal seed development, reduced seed yield, delayed seed germination, and retarded growth, indicating that PPR19 is indispensable for normal growth and development of Arabidopsis thaliana. Splicing pattern analysis of mitochondrial genes revealed that PPR19 specifically binds to the specific sequence in the 3'-terminus of the NADH dehydrogenase 1 (nad1) transcript and stabilizes transcripts containing the second and third exons of nad1. Loss of these transcripts in ppr19 leads to multiple secondary effects on accumulation and splicing of other nad1 transcripts, from which we can infer the order in which cis- and trans-spliced nad1 transcripts are normally processed. Improper splicing of nad1 transcripts leads to the absence of mitochondrial complex I and alteration of the nuclear transcriptome, notably influencing the alternative splicing of a variety of nuclear genes. Our results indicate that the mitochondrial PPR19 is an essential component in the splicing of nad1 transcripts, which is crucial for mitochondrial function and plant development.

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Ribosomal RNA (rRNA) methylation is a pivotal process in the assembly and activity of ribosomes, which in turn play vital roles in the growth, development, and stress responses of plants. Although few methyltransferases responsible for rRNA methylation have been identified in plant chloroplasts, the nature and function of these enzymes in chloroplasts remain largely unknown. In this study, we characterized Arabidopsis RsmD (At3g28460), an ortholog of the methyltransferase responsible for N2-methylguanosine (m2G) modification of 16S rRNA in Escherichia coli. Confocal microscopic analysis of an RsmD-GFP fusion protein revealed that RsmD is localized to chloroplasts. Primer extension analysis indicated that RsmD is responsible for m2G methylation at position 915 in the 16S rRNA of Arabidopsis chloroplasts. Under cold stress, rsmd mutant plants exhibited retarded growth, i.e., had shorter roots, lower fresh weight, and pale-green leaves, compared with wild-type plants. However, these phenotypes were not detected in response to drought or salt stress. Notably, the rsmd mutant was hypersensitive to erythromycin or lincomycin and accumulated fewer chloroplast proteins compared with the wild type, suggesting that RsmD influences translation in chloroplasts. Complementation lines expressing RsmD in the rsmd mutant background recovered wild-type phenotypes. Importantly, RsmD harbored RNA methyltransferase activity. Collectively, the findings of this study indicate that RsmD is a chloroplast 16S rRNA methyltransferase responsible for m2G915 modification that plays a role in the adaptation of Arabidopsis to cold stress.