Loss of ALN Function in Arabidopsis thaliana Induces Allantoin Accumulation and Enhances Antioxidant Enzyme Responses to Copper Toxicity

Abstract

Environmental contamination with heavy metals severely impairs crop productivity, primarily by inhibiting growth and triggering oxidative damage. To counteract these adverse effects, plants undergo complex metabolic reprogramming. Among these adaptations, the accumulation of purine-derived ureides—particularly allantoin—has been proposed as a protective mechanism, though its role under copper (Cu) stress remains unexplored. Here, we demonstrate that wild-type (Arabidopsis thaliana, WT) plants accumulate allantoate—the product of allantoin hydrolysis catalyzed by allantoinase (ALN, EC 3.5.2.5)—rather than allantoin when exposed to Cu toxicity. However, blocking this downstream catabolic step via loss of ALN function results in allantoin accumulation and is associated with enhanced Cu tolerance. Under Cu stress, aln-1 and aln-4 loss-of-function mutants maintained higher biomass and accumulated lower levels of malondialdehyde, superoxide anions, and hydrogen peroxide compared to WT. Furthermore, aln lines exhibited a stronger Cu-induced activation of key antioxidant enzymes (SOD, CAT, POD, APX) alongside higher expression of genes encoding specific isoforms targeted to multiple cellular compartments: cytosolic (CSD1), chloroplastic (CSD2, tAPX), peroxisomal (APX3, CAT3), and apoplastic (Prx51, Prx47). These results reveal that the loss of ALN function promotes a multi-compartmental antioxidant response under Cu toxicity, highlighting the pivotal role of purine catabolism in plant redox homeostasis and pointing to ureide metabolism as a potential avenue for enhancing crop stress tolerance.