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

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 is crucial for elucidating 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 short-term plate assays revealed only slight tolerance improvements, long-term hydroponic Cd treatments (0.5 µM and 1 µM) demonstrated significant enhancement in CV-expressing lines. Notably, the ubiquitously expressing p35S-CV line 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 than wild-type (Col-0) and pRBCS1A lines at 1 µM Cd. Subcellular analysis using Leadmium Green dye showed Cd preferentially localized in the peripheral cytoplasm, exhibiting strong co-localization with chloroplasts in Col-0 mesophyll cells. Conversely, CV expression effectively redirected Cd to the central vacuole, confirming efficient sequestration by tonoplast-targeted MerC. The superior tolerance of the p35S-CV line, coupled with its reduced shoot Cd accumulation, strongly suggests that vacuolar Cd sequestration in roots plays a primary role in conferring robust Cd tolerance in Arabidopsis, while sequestration in shoots provides supportive but insufficient protection alone under these conditions.