Tonoplast-targeted bacterial transporter MerC enhances cadmium tolerance in Arabidopsis via vacuolar sequestration and cytoplasmic protection.

Key message: Vacuolar sequestration via tonoplast-targeted MerC enhances Cd tolerance; expression in roots plays a primary role, while expression in mesophyll is also crucial for protecting the chloroplasts. Targeting metal transporters to the vacuole via genetic engineering offers a strategy to enhance plant tolerance to toxic metals like cadmium (Cd) by promoting vacuolar sequestration. Understanding the influence of different expression patterns of metal transporters is crucial for elucidating Cd-tolerance mechanisms. This study investigated how ubiquitous (p35S promoter) versus mesophyll-specific (pRBCS1A promoter) expression of a tonoplast-targeted bacterial metal transporter, MerC-AtVAM3 (CV), impacts Cd tolerance, nutrient homeostasis, and subcellular Cd distribution in Arabidopsis. While the short-term plate assays revealed only slight tolerance improvements, the long-term hydroponic Cd treatments (0.5 µM and 1 µM) resulted in significant enhancement in the CV-expressing lines. Notably, the p35S-CV line, which ubiquitously expresses the transgene, exhibited stronger tolerance (improved growth, mitigated chlorosis confirmed by higher SPAD values) compared to the mesophyll-specific pRBCS1A-TCV lines, particularly under 1 µM Cd. Nutritional profiling indicated that CV expression alleviated some Cd-induced nutrient imbalances. Although root Cd accumulation was similar across lines, p35S-CV shoots displayed approximately 30% lower Cd concentration compared to the wild-type (Col-0) and pRBCS1A lines under 1 µM Cd conditions. In Col-0 mesophyll cells, subcellular analysis using the Leadmium Green dye showed Cd was preferentially localized in the peripheral cytoplasm. The Leadmium Green signal also exhibited strong co-localization with chloroplasts. Conversely, CV expression effectively redirected Cd to the central vacuole, confirming efficient sequestration by the tonoplast-targeted MerC. The superior tolerance of the p35S-CV line strongly suggests that vacuolar Cd sequestration in roots plays a primary role in conferring robust Cd tolerance in Arabidopsis, while vacuolar sequestration in shoots provides supportive protection.