mRNA N6-methyladenosine is critical for cold tolerance in Arabidopsis

Authors: GOVINDAN, GANESAN - Oklahoma State University; SHARMA, BISHWAS - University Of Pennsylvania; LI, YONGFANG - Oklahoma State University; ARMSTRONG, CHRISTOPHER - University Of Pennsylvania; MERUM, PANDRANGAIAH - Oklahoma State University; Rohila, Jai; GREGORY, BRIAN - University Of Pennsylvania; SUNKAR, RAMANJULU - Oklahoma State University

Submitted to: Plant Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/13/2022
Publication Date: 6/16/2022
Citation: Govindan, G., Sharma, B., Li, Y., Armstrong, C.D., Merum, P., Rohila, J.S., Gregory, B.D., Sunkar, R. 2022. mRNA N6-methyladenosine is critical for cold tolerance in Arabidopsis. Plant Journal. https://doi.org/10.1111/tpj.15872.
DOI: https://doi.org/10.1111/tpj.15872

Interpretive Summary: The central dogma of biology is that for gene expressions DNA blueprint provides instructions to RNA, but recently in breakthrough research it was discovered that it is not as simple as just reading the DNA blueprint. It could involve one more layer of regulation in the form of biochemical markers, such as methylation, on the mRNA and provides finer control on the gene expressions. This phenomenon is known as epitranscriptional regulation. Through decades of research, it has been established that gaining abiotic stress tolerance in plants are highly complex in nature and DNA alone was not able to explain many of the abiotic stress tolerance mechanisms fully. Thus, we hypothesized that epitranscriptional regulation may have implications on cold stress tolerance in plants. To test our hypothesis, we used Arabidopsis plant and exposed it to cold stress and specifically detected and characterized the methylation of mRNA in cold treated plants compared with the control (non-treated) plants. This experiment revealed a large-scale shift in mRNA methylation in response to cold treatment of the plants. Earlier research has shown that m6A methylation of mRNA (i.e., methylation of the adenosine base at the nitrogen-6 positions of the mRNA) affects transcript stability/degradation and its translation process. Thus, we decided to investigate these possibilities for cold stress tolerance in Arabidopsis and found that in cold stress treated plants the abundance of m6A-containing transcripts had increased significantly and also these methylated mRNA had increased ribosome occupancy for the translation event. To validate the results, we used a mutant line of Arabidopsis, called mta mutant, which had a known defect for the m6A methylation of mRNA. When the wild type and the mta mutant of Arabidopsis were exposed to cold stress it was found that the mta mutant plant was highly susceptible to cold stress, had poor growth and development, and exhibited significant differences in gene expression levels of cold-tolerance genes (e.g., CBF and COR genes). Based on all the experimental evidence we conclude that the m6A methylation of mRNA is critical for cold tolerance in plants. Thus, mRNA modifications provide new avenues to scientists for increasing abiotic stress tolerance and climate resiliency of crop plants.

Technical Abstract: Plants respond to low temperatures by altering the mRNA abundance for 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 among others. Recently, m6A, m5C and other mRNA modifications that can affect the regulation and stability of RNA was discovered, thus revealing another layer of post-transcriptional regulation that plays an important role in modulating the gene expression. The importance of m6A in plant growth and development has been appreciated but its significance under stress conditions is still underexplored. To assess the role of m6A modifications during cold stress responses, MeRIP-seq was performed in Arabidopsis seedlings exposed to low temperature stress (4oC) 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 displayed 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 ROS accumulation. Furthermore, and most importantly, both non-acclimated and cold-acclimated mta mutant displayed hypersensitivity to freezing tolerance. Taken together, these findings suggest a critical role for the epitranscriptome in cold tolerance of Arabidopsis.