Cadmium Dose-Responsive Transcriptomics Prioritizes CvHMA15 and CvHMA16 in Cardamine violifolia

Cadmium (Cd) responses in metal-accumulating plants involve coordinated stress programs and specialized metal transporters, but the relationship between these levels remains poorly resolved in Cardamine violifolia. Here, seeds were germinated for seven days under 0, 10, 25, or 100 mg L−1 CdCl2, followed by root phenotyping and dose-resolved transcriptome analysis. Root growth showed a biphasic pattern: root length increased at 10 mg L−1, whereas growth at 100 mg L−1 was strongly reduced relative to the low-dose treatment. RNA-seq of 12 libraries identified 5665 genes that responded in at least one comparison and resolved six expression trajectories. Pathway analysis indicated low-dose metabolic adjustment, strong activation of phenylpropanoid, signaling, and xenobiotic-response pathways at 25 mg L−1, and broader transcriptional repression at 100 mg L−1. Cross-referencing the transcriptome with 16 reference-identified CvHMA loci prioritized CvHMA15 and CvHMA16 as a 99.87 identical protein pair with divergent regulation: CvHMA15 was moderately down-regulated across the Cd gradient, whereas CvHMA16 was induced and met the combined differential-expression threshold at 100 mg L−1. In a pYES2.0-based yeast spot assay, expression of either CvHMA15 or CvHMA16 maintained colony growth across five cell densities on medium containing 50 μM CdCl2, whereas the empty-vector control was strongly inhibited; growth was comparable among transformants in the absence of Cd. These results establish a transcriptome-guided framework for candidate prioritization while leaving transport direction and in planta mechanism to direct functional tests.