N6-methyladenosine RNA modification regulates cotton drought response in a Ca²⁺ and ABA-dependent manner

N⁶ -methyladenosine (m⁶ A) is the most prevalent internal modification present in mRNAs, and is considered to participate in a range of developmental and biological processes. Drought response is highly regulated at the genomic, transcriptional and post-transcriptional levels. However, the biological function and regulatory mechanism of m⁶ A modification in the drought stress response is still poorly understood. We generated a transcriptome-wide m⁶ A map using drought-resistant and drought-sensitive varieties of cotton under different water deficient conditions to uncover patterns of m⁶ A methylation in cotton response to drought stress. The results reveal that m⁶ A represents a common modification and exhibit dramatic changes in distribution during drought stress. More 5'UTR m⁶ A was deposited in the drought-resistant variety and was associated with a positive effect on drought resistance by regulating mRNA abundance. Interestingly, we observed that increased m⁶ A abundance was associated with increased mRNA abundance under drought, contributing to drought resistance, and vice versa. The demethylase GhALKBH10B was found to decrease m⁶ A levels, facilitating the mRNA decay of ABA signal-related genes (GhZEP, GhNCED4 and GhPP2CA) and Ca²⁺ signal-related genes (GhECA1, GhCNGC4, GhANN1 and GhCML13), and mutation of GhALKBH10B enhanced drought resistance at seedling stage in cotton. Virus-induced gene silencing (VIGS) of two Ca²⁺ -related genes, GhECA1 and GhCNGC4, reduced drought resistance with the decreased m⁶ A enrichment on silenced genes in cotton. Collectively, we reveal a novel mechanism of post-transcriptional modification involved in affecting drought response in cotton, by mediating m⁶ A methylation on targeted transcripts in the ABA and Ca²⁺ signalling transduction pathways.