V0 and V1 domains of vacuolar H+-ATPase play distinct roles in cadmium tolerance of Broussonetia papyrifera.

Cadmium (Cd) contamination poses a major threat to forest ecosystems, and Broussonetia papyrifera is a promising woody species with strong Cd tolerance and accumulation capacity. Vacuolar H+-ATPase (V-ATPase) is central to ion homeostasis and heavy metal detoxification, yet the organ- and domain-specific roles of its subunits in Cd responses remain unclear. We integrated physiological measurements, transcript profiling, enzyme activity assays and genome-wide analyses to characterize V-ATPase subunits (BpVHAs) and their functions under a 30-day Cd treatment in roots, stems and leaves of B. papyrifera. Cd stress caused only mild electrolyte leakage but triggered substantial V-ATPase accumulation and enhanced activity. V-ATPase activity displayed a tissue-graded temporal pattern, with roots responding earliest and most strongly. We identified 22 BpVHA genes belonging to conserved V0 and V1 domains, whose promoters were enriched in stress- and hormone-related cis-elements. Most genes exhibited a rise-then-fall expression trend during Cd exposure, with V0 subunits induced earlier, more strongly and for longer than V1 subunits. Mantel tests showed that V1 expression correlated more strongly with V-ATPase activity in roots, whereas V0 expression dominated in stems and leaves. Our findings highlight BpVHA-a2, BpVHA-a3 and BpVHA-c1 as key contributors to Cd detoxification. We propose a model in which roots rely on rapid V1-driven ATP hydrolysis, while aerial tissues depend on sustained V0-mediated proton pumping to support vacuolar Cd sequestration. This study provides new insights into heavy metal tolerance in woody plants and identifies promising molecular targets for breeding and phytoremediation in Cd-contaminated environments.