N⁶-methyladenosine mRNA methylation positively regulated the response of poplar to salt stress

As the most abundant form of methylation modification in messenger RNA (mRNA), the distribution of N⁶-methyladenosine (m⁶A) has been preliminarily revealed in herbaceous plants under salt stress, but its function and mechanism in woody plants were still unknown. Here, we showed that global m⁶A levels increased during poplar response to salt stress. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) revealed that m⁶A significantly enriched in the coding sequence region and 3'-untranslated regions in poplar, by recognising the conserved motifs, AGACU, GGACA and UGUAG. A large number of differential m⁶A transcripts have been identified, and some have been proved involving in salt response and plant growth and development. Further combined analysis of MeRIP-seq and RNA-seq revealed that the m⁶A hypermethylated and enrich in the CDS region preferred to positively regulate expression abundance. Writer inhibitor, 3-deazaneplanocin A treatment increased the sensitivity of poplar to salt stress by reducing mRNA stability to regulate the expression of salt-responsive transcripts PagMYB48, PagGT2, PagNAC2, PagGPX8 and PagARF2. Furthermore, we verified that the methyltransferase PagFIP37 plays a positively role in the response of poplar to salt stress, overexpressed lines have stronger salt tolerance, while RNAi lines were more sensitive to salt, which relied on regulating mRNA stability in an m⁶A manner of salt-responsive transcripts PagMYB48, PagGT2, PagNAC2, PagGPX8 and PagARF2. Collectively, these results revealed the regulatory role of m⁶A methylation in poplar response to salt stress, and revealed the importance and mechanism of m⁶A methylation in the response of woody plants to salt stress for the first time.