Abstract

The Arabidopsis enolase2 (AtENO2) gene produces both the glycolytic enzyme enolase and an N-terminal truncated isoform, termed the transcriptional repressor Arabidopsis cMyc-Binding Protein 1 (AtMBP-1), through alternative translation. Maintaining AtENO2 homeostasis via feedback repression by AtMBP-1 is essential for its biological functions; however, the underlying regulatory mechanism remains unclear. Cadmium (Cd), a toxic soil pollutant, strongly inhibits plant growth and development. Interestingly, Cd induces enolase accumulation in plants, and AtMBP-1 binds to metal-responsive elements (MREs) that confer a Cd response in Arabidopsis. Here, we demonstrate that AtMBP-1-mediated repression via an MRE in the AtENO2 promoter maintains the AtENO2 expression balance, which is required for Arabidopsis Cd resistance. Knockdown of AtENO2 via antisense RNA increased Cd sensitivity, which was accompanied by altered intermediates of the tryptophan pathway (notably those involved in auxin biosynthesis), elevated Cd accumulation in the shoots and roots, and increased lipid peroxidation. We further showed that AtENO2 utilizes its 5'-UTR as a Cd-responsive alternative promoter, driving a predominant short transcript that is efficiently translated into the AtENO2 protein. Accordingly, Cd stress increased AtENO2 protein accumulation. An MRE located within the 5'-UTR promoter was functional: CRISPR/Cas9-mediated deletion of a 50 bp MRE-containing fragment in vivo increased AtENO2 abundance and significantly enhanced Arabidopsis growth under Cd stress. AtMBP-1 binds to this MRE to repress AtENO2 promoter activity, thereby forming an autoregulatory loop that fine-tunes AtENO2 levels and prevents its overaccumulation during prolonged Cd stress. Our findings reveal a previously unknown mechanism underlying AtENO2 function in plant adaptation to heavy metal stress.