Dejana Milić

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.

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.

Plants are inevitably exposed to extreme climatic conditions that lead to a disturbed balance between the amount of absorbed energy and their ability to process it. Variegated leaves with photosynthetically active green leaf tissue (GL) and photosynthetically inactive white leaf tissue (WL) are an excellent model system to study source–sink interactions within the same leaf under the same microenvironmental conditions. We demonstrated that under excess excitation energy (EEE) conditions (high irradiance and lower temperature), regulated metabolic reprogramming in both leaf tissues allowed an increased consumption of reducing equivalents, as evidenced by preserved maximum efficiency of photosystem II (Ф_PSII) at the end of the experiment. GL of the EEE-treated plants employed two strategies: (i) the accumulation of flavonoid glycosides, especially cyanidin glycosides, as an alternative electron sink, and (ii) cell wall stiffening by cellulose, pectin, and lignin accumulation. On the other hand, WL increased the amount of free amino acids, mainly arginine, asparagine, branched-chain and aromatic amino acids, as well as kaempferol and quercetin glycosides. Thus, WL acts as an important energy escape valve that is required in order to maintain the successful performance of the GL sectors under EEE conditions. Finally, this role could be an adaptive value of variegation, as no consistent conclusions about its ecological benefits have been proposed so far.