Exploring Nucleotide Dynamics in Resurrection Plant Haberlea rhodopensis During Desiccation and Rehydration Using HILIC-LC-hrMS/MS

Resurrection plants are incredibly tolerant to drought. When dried out, they temporarily halt photosynthesis and respiration while protecting cellular structures. Upon rehydration, they rapidly resume normal function, allowing their cells and tissues to become re-energized. Nucle-otides play a crucial role as energy and information carriers in all living organisms. However, the dynamics and functioning of these vital compounds in resurrection plants have not been exten-sively studied. Nucleotide, nucleoside, and nucleobase extraction and analysis are technically challenging, so improved methods are of continued interest, especially techniques that can dis-tinguish the nucleotides’ degree of phosphorylation. Here, we describe an accurate mass, high-resolution hydrophilic interaction chromatography (HILIC-LC-hrMS/MS)- based method for quantitative profiling of nucleotides and their phosphate forms in the resurrection plant Haberlea rhodopensis Friv. The proposed ion-pair free method, exploiting a gradient of both organic com-ponent and buffer salt concentrations, was applied on tissue samples from leaves and roots of H. rhodopensis exposed to dehydration and subsequent rehydration. In the proposed method, 20 analyses (nucleosides and nucleotides) were separated by HILIC. This preliminary study identified remarkable features in nucleotide phosphate content and dynamics during desiccation and sub-sequent rehydration suggesting alternative energy metabolism pathways in drought tolerance of resurrection plants.