Integrated transcriptomic and metabolomic analyses reveal differential responses of the calcium-secreting plant Ceratostigma willmottianum to CaCl2 and Ca(NO3)2

Calcium salts are widely present in soil, and excessive concentrations can reduce crop yields. Under calcium salt stress, plants are simultaneously affected by both anions and cations, making it difficult to accurately assess which ion causes the damage and how plants respond to each. In this study, the calcium-secreting plant Ceratostigma willmottianum was treated with iso-osmotic 100 mM CaCl₂ and Ca(NO₃)₂, and the molecular mechanisms in response to different calcium salts and specific ions were explored based on transcriptomic and metabolomic approaches. Results showed that CaCl₂ induced the accumulation of reactive oxygen species, chloroplast destruction, and growth inhibition. Accordingly, CaCl₂ primarily upregulated genes involved in saccharide metabolism and flavonoid biosynthesis, leading to the accumulation of soluble sugars and flavonoid compounds. In contrast, Ca(NO₃)₂ did not cause stress damage, which may be related to the mitigating effect of NO₃⁻ as a nitrogen source. Genes related to nitrogen assimilation and chlorophyll synthesis were upregulated by NO 3 ⁻, leading to the accumulation of proline, glutathione, and chlorophyll. Additionally, calcium salt treatment increased Ca²⁺ content in leaves and enhanced both Ca²⁺ compartmentalization and secretion capacity in the salt glands. Ultrastructural analysis revealed a corresponding increase in the number of vesicles. Concurrently, genes associated with Ca²⁺ transporters, Ca²⁺-binding proteins, and vesicle transport components were upregulated, suggesting their important role in facilitating ion secretion. Overall, this study provides insights into the adaptive strategies of calcium-secreting plants to high-calcium environments and elucidates the molecular mechanisms underlying their responses to Cl⁻, NO₃⁻, and Ca²⁺. • C. willmottianum exhibits sensitivity to CaCl₂ but not to Ca(NO₃)₂. • NO₃⁻ likely mitigates calcium stress by acting as a nitrogen source. • Calcium salts increase the Ca²⁺ sequestration and secretion of salt glands. • The expression of genes related to Ca²⁺ transport may contribute to Ca²⁺ adaptation.