Mechanistic Insights into d‑Alanine-Induced Plant Growth Inhibition: Synergistic Roles of Transport, Transcriptional Regulation, and Metabolic Perturbations

D-amino acids (D-AAs) are detrimental to plant growth yet also have physiological roles in plants. To unravel the molecular mechanism underlying D-AA inhibition, we examined responses to d-alanine (d-Ala), a central metabolite in D-AA metabolism, in Marchantia polymorpha. We show that d-Ala is transported by MpLHT1 and partially metabolized by transaminase MpDAT. Transcriptomics identified MpBZIP4 as a d-Ala-responsive transcription factor that negatively regulates d-Ala tolerance. Additionally, an enriched flavonoid in M. polymorpha, luteolin, exacerbates d-Ala-induced growth inhibition, possibly by acting as a potent inhibitor of carbonic anhydrase (CA). Strikingly, ectopic expression of Arabidopsis thaliana 1-aminocyclopropane-1-carboxylic acid oxidase 4 (AtACO4) intensified d-Ala’s inhibitory effects through dysregulation of luteolin metabolites and altering the CA subfamily (MpαCALs) expression. These findings provide a multilayered mechanism for d-Ala-induced growth suppression, integrating transporter-mediated uptake, transcriptional responses, and metabolic perturbations. This work advances our understanding of D-AA phytotoxicity and uncovers conserved and novel mechanisms in land plants.