CRISPR/Cas9-mediated knockout of ZmHMA3 reveals its essential role in zinc homeostasis and high-zinc stress tolerance in maize

Excessive Zn is toxic to maize (Zea mays L.). The heavy metal ATPase gene ZmHMA3 is associated with heavy metal transport, but its function in maize tolerance to high Zn stress has not been fully characterized. In this study, CRISPR/Cas9 technology was used to generate zmhma3 knockout mutants to investigate its function under high Zn stress. High Zn stress significantly induced the expression of ZmHMA3 in maize leaves and roots. Phenotypic analysis showed that, compared to the WT plants, the zmhma3 mutants exhibited significantly reduced tolerance to excessive Zn, manifested as severe growth inhibition, impaired root structure, decreased activity of key antioxidant enzymes (CAT, POD, SOD), and aggravated membrane damage. Furthermore, the mutants accumulated significantly higher levels of Zn in both roots and leaves, accompanied by disordered subcellular Zn distribution, indicating disrupted intracellular Zn homeostasis. Our results demonstrate that ZmHMA3 is a key positive regulator in maize's response to high Zn stress, likely by coordinating Zn compartmentalization and alleviating oxidative damage. This study provides new genetic and physiological insights into the molecular mechanisms of Zn stress tolerance in maize and offers a potential target for breeding new maize varieties tolerant with improved high-Zn-efficiency.