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Plant cuticle lipids form outer protective layers to resist environmental stresses; however, the relationship between cuticle properties and cold tolerance is unclear. Here, the extremophyte Thellungiella salsuginea was stressed under cold conditions (4 °C) and the cuticle of rosette leaves was examined in terms of epicuticular wax crystal morphology, chemical composition, and cuticle-associated gene expression. The results show that cold induced formation of distinct lamellas within the cuticle ultrastructure. Cold stress caused 14.58% and 12.04% increases in the amount of total waxes and cutin monomer per unit of leaf area, respectively, probably associated with the increase in total fatty acids. The transcriptomic analysis was performed on rosette leaves of Thellungiella exposed to cold for 24 h. We analyzed the expression of 72 genes putatively involved in cuticle lipid metabolism, some of which were validated by qRT-PCR (quantitative reverse transcription PCR) after both 24 h and one week of cold exposure. Most cuticle-associated genes exhibited higher expression levels under cold conditions, and some key genes increased more dramatically over the one week than after just 24 h, which could be associated with increased amounts of some cuticle components. These results demonstrate that the cuticle provides some aspects of cold adaptation in T. salsuginea. [ABSTRACT FROM AUTHOR]