Integrated physiological, transcriptome-wide m⁶A methylation and proteome analysis reveals molecular mechanisms of tomato root response to cadmium stress

Soil cadmium pollution has increasingly become a serious problem for crop production, which drastically attenuates plant growth and food safety. Although N⁶-methyladenosine (m⁶A) methylation is crucial for plant response to various stresses, the regulatory mechanism underlying m⁶A modification during cadmium (Cd) stress remains unclear. This study investigated the physiological responses, transcriptome-wide m⁶A methylome, and proteome changes in tomato roots exposed to 50 μmol · L⁻¹ CdCl₂. Excess Cd restricted plant growth, altered the antioxidant system and disrupted mineral nutrient absorption. We identified a negative correlation between m⁶A levels and gene transcription for that 150 out of 198 differentially expressed genes (DEGs) were hypomethylated but mRNA up-regulated. Cd stress also enhanced translational efficiency, particularly for differentially abundant proteins (DAPs). Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that differentially m⁶A modified genes (DMGs), DEGs, and DAPs were commonly enriched in phenylpropanoid biosynthesis, glutathione metabolism, and ABC transporters, reflecting cell wall barriers, chelation, and transport of Cd, respectively. Finally, we confirmed the Cd-transport activity of eight putative metal transporters identified in DMGs, DEGs, or DAPs by yeast complementaion experiments, and pharmacologically investigated the effect of m⁶A modification on their expression. Treatment with the m⁶A methylation inhibitor 3-deazaneplanocin A (3-DA) reduced SlIRT1/2 expression and increased SlNRAMP3/SlZIP4 expression, while the m⁶A demethylase inhibitor meclofenamic acid (MA) treatment decreased SlNRAMP3 expression but elevated SlIRT2 expression under Cd stress. Our findings provide novel insights into the interplay between m⁶A modification, transcription, and translation under Cd stress and the associated plant stress response.