Enhanced HSP70 binding to m6A-methylated RNAs facilitates cold stress adaptation in mango seedlings.

Background: Cold stress poses a serious challenge to tropical fruit production, particularly in mango. N⁶-methyladenosine (m⁶A) modifications are key regulators of gene expression, enabling plants to respond to stress responses, enhance adaptation and improve resilience to environmental challenges.

Results: In our study, transcriptome-wide m⁶A methylation profiling under cold stress identified 6,499 differentially methylated m⁶A peaks and 2,164 differentially expressed genes (DEGs) in mango seedlings. Among these genes, six exhibited both significant increases in m⁶A modification levels and gene expression, 21 showed a significant increase in m⁶A levels but a concurrent downregulation of gene expression, and 26 showed reduced m⁶A levels but exhibited increased gene expression, highlighting distinct regulatory patterns in m⁶A-mediated gene expression control. Gene Ontology (GO) enrichment analysis revealed significant involvement in pathways such as potassium ion import, nitrate response, and transcription regulation. Notably, HSP70 was one of the upregulated genes in response to cold stress. RNA immunoprecipitation (RNA-IP) assays confirmed the association of HSP70 with m⁶A-modified RNAs in vivo, supporting its role in regulating stress-responsive transcripts. Additionally, immunofluorescence analysis demonstrated the formation of HSP70 condensates in plant cells under cold stress, indicating a potential mechanism for localized RNA stabilization. Fluorescence polarization assays demonstrated that HSP70 binds preferentially to m⁶A-modified RNAs, suggesting its role in forming protective condensates under cold conditions. This interaction between m⁶A modification and HSP70 points to a potential mechanism that helps stabilize stress-responsive transcripts, contributing to the plant's enhanced cold tolerance.

Conclusions: m⁶A modifications play a vital role in regulating gene expression under cold stress, offering new insights into mango's stress responses and potential breeding strategies for cold tolerance.