Freezing-Induced Biomineralization of Calcium Associated with Amino-Acid-Like Groups in a Cold-Tolerant Cactus

Background and Aims Under abiotic stress, biominerals may transcend structural functions to confer environmental resilience. Crucially, cold stress induces substantial metabolic adjustments in plants, particularly in cold-tolerant species. These metabolites such as amino or organic acids may directly influence crystal polymorph selection, yet their real-time impact on in vivo crystallization kinetics remains uncharacterized. To test this, we employ an integrative analytical framework across a controlled thermal gradient (-2°C to 25°C) in Mammillaria schumannii (cold-tolerant) and Hylocereus undulatus (cold-sensitive). Our objectives are to decipher the mechanism of cold-induced crystal formation and elucidate its potential roles, thereby providing new insight into the possible functional linkage between mineral deposition and cold adaptation in Cactaceae. Methods Using a combined analytical framework, we compared biomineralization in Mammillaria schumannii and Hylocereus undulatus: Morphological analysis: Crystal architecture via scanning electron microscopy (SEM). Elemental composition: Stoichiometry by energy-dispersive X-ray spectroscopy (EDS). Molecular structure: Functional group identification via Raman spectroscopy. Dynamic monitoring: Crystal formation induced by freezing, or dissolution during rewarming. Key Results Our work provides several key discoveries: A novel mineral phenotype: Freezing induces intracellular crystals with unusual elemental stoichiometry (C:O:Ca ≈ 57:42:1) and distinctive morphologies that differ fundamentally from classical calcium oxalate biominerals.Evidence for amino acid–mediated biomineralization: Raman spectroscopy indicates strong enrichment of amino acid functional groups, suggesting that these structures represent calcium interacting with amino-acid–like groups rather than conventional mineral crystals.Dynamic calcium redistribution during recovery: Upon warming, intracellular crystals dissolve while extracellular crystals appear in intertubercular tissues, indicating reversible calcium shuttling associated with freezing recovery.Evidence for evolutionary adaptation: This response is absent in the tropical cactus Hylocereus undulatus, suggesting that freezing-induced biomineralization may represent a specialized mechanism of freezing tolerance. Conclusions We propose a hypothesis of “cryo-mineral switching”, describing reversible mineral formation and dissolution during freezing–thawing cycles that may buffer ionic imbalance and contribute to cellular protection during freeze–thaw stress.