HmWRKY22-HmABCG1 regulatory module mediates aluminum tolerance through enhanced translocation and antioxidant defense

Aluminum (Al) toxicity in acidic soils limits crop productivity about 30-40% of the world's arable land. Certain plant species, such as Hydrangea macrophylla, have developed advanced mechanisms for Al tolerance. The HmABCG1, an ATP-binding cassette (ABC) transporter, has been functionally characterized from hydrangea that is crucial for Al tolerance. Phylogenetic analysis classifies HmABCG1 alongside the established clade of plant ABCG transporters, preserving all conserved functional features, including the Walker A and Walker B, ABC signature motif, Q, D, and H-loop structures. An increased HmABCG1 expression was observed in hydrangea roots and flowers under Al stress. Heterologous expression in yeast showed that HmABCG1 enhances cellular resistance to Al. Functional validation through overexpression (OE) and RNA interference (RNAi) techniques in hydrangea leaf discs and Arabidopsis revealed that HmABCG1 improves Al tolerance by facilitating the Al translocation from Arabidopsis roots to shoots. The Arabidopsis-OE plants exhibited 2-3 folds increase in Al content within the shoots, reduction in root retention, elevated chlorophyll and anthocyanin contents, and increased antioxidant enzymes (SOD, POD, and CAT) activities, alongside a decreased production of reactive oxygen species (ROS) under Al stress. Conversely, RNAi Arabidopsis plants exhibited greater Al retention in their roots, increased oxidative damage, and were more sensitive to Al. Direct promoter binding was confirmed through Y1H assay, and in vivo transcriptional activation was validated by dual-luciferase reporter assay in intact plant cell, demonstrating that HmWRKY22 directly binds and activates HmABCG1 transcription. These results elucidate a novel mechanism of Al tolerance characterized by transporter-mediated metal redistribution and enhanced antioxidant defense, thereby providing substantial insights into the biotechnological improvement for Al-tolerant crops.