ACYR-Mediated N4-Acetylcytidine Modification Regulates Cadmium Responses in Arabidopsis

Covalent modifications of RNA modulate plant growth, development, and stress responses. N4-acetylcytidine (ac4C), a conserved RNA modification deposited by N-ACETYLTRANSFERASES FOR CYTIDINE IN RNA (ACYRs), regulates RNA stability and translation, but its possible function in plant responses to cadmium (Cd) has not been explored. We report here that ACYR-mediated RNA ac4C modification negatively affects Cd tolerance during the vegetative growth of Arabidopsis (Arabidopsis thaliana). Cd treatment repressed the expression of ACYR1 and ACYR2, as well as the abundance of their encoded proteins, leading to lower overall ac4C-modified mRNA levels. Multi-omics data combined with biochemical assays revealed that ACYR-dependent ac4C modification of the IRON MAN 2 (IMA2) and IMA3 transcripts enhanced their stability and translation. Lower levels of mRNA ac4C modification, induced by Cd treatment or by the loss of ACYR function, resulted in decreased abundance of IMA2 and IMA3 transcripts and their encoded peptides, leading to the ubiquitination and accelerated degradation of the basic helix-loop-helix (bHLH) transcription factors bHLH105 and bHLH115. This, in turn, suppressed the expression of bHLH Ib subfamily genes (bHLH38, bHLH39, bHLH100, and bHLH101) and of IRON-REGULATED TRANSPORTER 1 (IRT1), thereby preventing Cd uptake. Notably, IMA2 overexpression effectively restores the Cd-sensitive phenotype of the acyr mutant. Our findings reveal a mechanism by which ACYR-mediated ac4C modification regulates Cd uptake in Arabidopsis, providing potential targets for breeding crops with increased tolerance to Cd.