mRNA N6-methyladenosine is critical for cold tolerance in Arabidopsis

Abstract

Plants respond to low temperatures by altering the mRNA abundance of thousands of genes contributing to numerous physiological and metabolic processes that allow them to adapt. At the post-transcriptional level, these cold stress-responsive transcripts undergo alternative splicing, microRNA-mediated regulation and alternative polyadenylation, amongst others. Recently, m6A, m5C and other mRNA modifications that can affect the regulation and stability of RNA were discovered, thus revealing another layer of post-transcriptional regulation that plays an important role in modulating gene expression. The importance of m6A in plant growth and development has been appreciated, although its significance under stress conditions is still underexplored. To assess the role of m6A modifications during cold stress responses, methylated RNA immunoprecipitation sequencing was performed in Arabidopsis seedlings esposed to low temperature stress (4°C) for 24 h. This transcriptome-wide m6A analysis revealed large-scale shifts in this modification in response to low temperature stress. Because m6A is known to affect transcript stability/degradation and translation, we investigated these possibilities. Interestingly, we found that cold-enriched m6A-containing transcripts demonstrated the largest increases in transcript abundance coupled with increased ribosome occupancy under cold stress. The significance of the m6A epitranscriptome on plant cold tolerance was further assessed using the mta mutant in which the major m6A methyltransferase gene was mutated. Compared to the wild-type, along with the differences in CBFs and COR gene expression levels, the mta mutant exhibited hypersensitivity to cold treatment as determined by primary root growth, biomass, and reactive oxygen species accumulation. Furthermore, and most importantly, both non-acclimated and cold-acclimated mta mutant demonstrated hypersensitivity to freezing tolerance. Taken together, these findings suggest a critical role for the epitranscriptome in cold tolerance of Arabidopsis.