Formation and Properties of the Glassy Matrix: Vitrification Mechanism in Haberlea rhodopensis and the Resurrection Plants

Cytoplasmic vitrification is the standard explanation for how resurrection plants endure drying, yet the glass is rarely examined in tissue—its composition, glass transition and residual water remain unresolved. We characterize the glassy matrix of Haberlea rhodopensis by integrating transcriptomic and metabolomic data, biophysical modeling and targeted experiments. Sugar-metabolism genes, such as SUCROSE-PHOSPHATE SYNTHASES and SUCROSE SYNTHASES, respond to desiccation with strong concordance across three studies (Kendall’s W = 0.872; 81 genes measured in all three) and, with the metabolites, define a four-component glass-former set—sucrose plus the raffinose family oligosaccharides raffinose, stachyose and verbascose, with sucrose dominant (~92%). Apparent contradictions are plausibly explained: INVERTASE induction is consistent with compartmental separation from accumulating sucrose and declining trehalose with trehalose-6-phosphate (T6P) signaling, not vitrification. A parameterized state diagram traces the desiccation–rehydration path through vitrification and predicts that unfreezable water is locked throughout the glass. Desiccated tissue survived liquid nitrogen (−196 °C), suggesting that freezing tolerance reflects vitrification. Rewetting dried tissue was exothermic, consistent with an amorphous glass. We argue the residual water is functionally bound, that late embryogenesis abundant (LEA) proteins help shape the glass, and hypothesize that natural deep eutectic solvent (NaDES)-forming metabolites provide a transient reaction medium.