Ramonda serbica Panc. is an ancient resurrection plant able to survive a long desiccation period and recover metabolic functions upon watering. The accumulation of protective late embryogenesis abundant proteins (LEAPs) is a desiccation tolerance hallmark. To propose their role in R. serbica desiccation tolerance, we structurally characterised LEAPs and evaluated LEA gene expression levels in hydrated and desiccated leaves. By integrating de novo transcriptomics and homologues LEAP domains, 318 R. serbica LEAPs were identified and classified according to their conserved motifs and phylogeny. The in silico analysis revealed that hydrophilic LEA4 proteins exhibited an exceptionally high tendency to form amphipathic α-helices. The most abundant, atypical LEA2 group contained more hydrophobic proteins predicted to fold into the defined globular domains. Within the desiccation-upregulated LEA genes, the majority encoded highly disordered DEH1, LEA1, LEA4.2, and LEA4.3 proteins, while the greatest portion of downregulated genes encoded LEA2.3 and LEA2.5 proteins. While dehydrins might chelate metals and bind DNA under water deficit, other intrinsically disordered LEAPs might participate in forming intracellular proteinaceous condensates or adopt amphipathic α-helical conformation, enabling them to stabilise desiccation-sensitive proteins and membranes. This comprehensive LEAPs structural characterisation is essential to understanding their function and regulation during desiccation aiming at crop drought tolerance improvement.

The resurrection plant Ramonda serbica Panc. survives long desiccation periods and fully recovers metabolic functions within one day upon watering. This study aimed to identify key candidates and pathways involved in desiccation tolerance in R. serbica. We combined differential transcriptomics and proteomics, phenolic and sugar analysis, FTIR analysis of the cell wall polymers, and detailed analysis of the photosynthetic electron transport (PET) chain. The proteomic analysis allowed the relative quantification of 1192 different protein groups, of which 408 were differentially abundant between hydrated (HL) and desiccated leaves (DL). Almost all differentially abundant proteins related to photosynthetic processes were less abundant, while chlorophyll fluorescence measurements implied shifting from linear PET to cyclic electron transport (CET). The levels of H₂O₂ scavenging enzymes, ascorbate-glutathione cycle components, catalases, peroxiredoxins, Fe-, and Mn superoxide dismutase (SOD) were reduced in DL. However, six germin-like proteins (GLPs), four Cu/ZnSOD isoforms, three polyphenol oxidases, and 22 late embryogenesis abundant proteins (LEAPs; mainly LEA4 and dehydrins), were desiccation-inducible. Desiccation provoked cell wall remodeling related to GLP-derived H₂O₂/HO^● activity and pectin demethylesterification. This comprehensive study contributes to understanding the role and regulation of the main metabolic pathways during desiccation aiming at crop drought tolerance improvement.

The photosynthetically active green leaf (GL) and non-active white leaf (WL) tissues of variegated Pelargonium zonale provide an excellent model system for studying processes associated with photosynthesis and sink-source interactions, enabling the same microenvironmental conditions. By combining differential transcriptomics and metabolomics, we identified the main differences between these two metabolically contrasting tissues. Genes related to photosynthesis and associated pigments, the Calvin–Benson cycle, fermentation, and glycolysis were strongly repressed in WL. On the other hand, genes related to nitrogen and protein metabolism, defence, cytoskeletal components (motor proteins), cell division, DNA replication, repair and recombination, chromatin remodelling, and histone modifications were upregulated in WL. A content of soluble sugars, TCA intermediates, ascorbate, and hydroxybenzoic acids was lower, while the concentration of free amino acids (AAs), hydroxycinnamic acids, and several quercetin and kaempferol glycosides was higher in WL than in GL. Therefore, WL presents a carbon sink and depends on photosynthetic and energy-generating processes in GL. Furthermore, the upregulated nitrogen metabolism in WL compensates for the insufficient energy from carbon metabolism by providing alternative respiratory substrates. At the same time, WL serves as nitrogen storage. Overall, our study provides a new genetic data resource for the use of this excellent model system and for ornamental pelargonium breeding and contributes to uncovering molecular mechanisms underlying variegation and its adaptive ecological value.

Vegetative desiccation tolerance requires specialised cell wall (CW) adaptations to withstand severe mechanical stress during dehydration and rehydration. While intracellular protective strategies in resurrection plants, including Ramonda serbica , are well documented, the CW response remains poorly understood. Here, we integrated immunocytochemical profiling, FTIR spectroscopy, quantification of CW-bound phenolics, transcriptomics, and ionically bound CW proteomics across hydrated (HL), desiccated (DL), and rehydrated states (R1–1h, R2–24h, R3–48h). Reversible CW folding was facilitated by condensed arabinogalactan-proteins (AGPs) and extensins, alongside a site-specific balance between pectin methylesterification and demethylesterification. Structural compaction was further reinforced by the accumulation of CW-bound hydroxycinnamates, which persisted through R1 phase. Moreover, basic 7S globulin, miraculin, α-galactosidase, and two LEA4 protein family members were strongly accumulated during DL and R1, providing the first evidence of ionically CW-bound LEA proteins. Initial rewatering (R1) triggered a rapid transcriptomic reactivation of pectin-degrading/modifying enzymes, carbohydrate-active enzymes, and subtilases, accompanied by unesterified pectin enrichment. By 24–48 h (R2–R3), CW-bound hydroxycinnamic acids declined, and CW architecture, gene expression, and proteome profiles returned to baseline levels. Overall, our findings reveal a coordinated spatiotemporal apoplastic network—driven by glycoproteins, pectin modulation, CW-bound hydroxycinnamates, and LEA4 proteins—essential for rapid desiccation recovery.

Desiccation, an extreme form of dehydration, reduces the cellular water content to below 5 % and poses a major challenge for most plants. Ramonda serbica , a tertiary relict and homoiochlorophyllous resurrection plant, is an exceptional model for investigating vegetative desiccation tolerance. Late Embryogenesis Abundant (LEA) proteins are strongly involved in this adaptive trait, but their exact molecular function is still unclear. In this study, we report the first successful recombinant production of the desiccation-induced LEA protein, RsLEAP30, from a dicotyledonous resurrection plant species using an Escherichia coli expression system. By employing immobilised metal affinity and size-exclusion chromatography, we achieved to purify RsLEAP30 to purity over 95 %, providing a robust and scalable method for producing other LEA proteins. Structural characterisation by circular dichroism spectroscopy, combined with in silico modelling, revealed that RsLEAP30 is predominantly disordered but contains α-helical regions. We suggest that this structural duality underpins the protective role of RsLEAP30 in chloroplasts, likely via interactions with thylakoids and desiccation-sensitive proteins within photosynthesis-associated proteins. This function may be crucial for the rapid recovery of photosynthetic components upon rehydration. Our study provides new insights into the structure–function relationship of LEA proteins in resurrection plants and establishes a foundation for future investigations. Understanding the protective mechanisms of RsLEAP30 will pave the way for bioengineering strategies aimed at improving the drought tolerance of crops.