Desiccation tolerance has been described in at least nine Gesneriaceae genera, implying that this is the angiosperm family with the largest number of DT genera. Nowadays, the Gesneriaceae family contains three subfamilies: Sanangoideae, Gesnerioideae, and Didymocarpoideae. All documented DT gesneriads are members of the Trichosporeae tribe from Didymocarpoideae^[1].

So far, mechanisms have been characterized among a significant number of studies, and in fact two gesneriads (Dorcoceras hyrgrometricum and Haberlea rhodopensis) are among the top five most studied resurrection angiosperms^[2]. Both species, together with Ramonda serbica and, to a lesser extent, others such as Paraboea rufencens, Paraboea crassifolia, Oreocharis mileensis, Ramonda myconi, and Ramonda nathaliae, have allowed researchers to unravel the mechanisms of desiccation tolerance in gesneriads^{[3][4][5][6][7]}. Even thought more studies are needed, currently available information suggests that most resurrection gesneriads share similar protective mechanisms, with minor species-specific differences^[8].

In response to desiccation, all resurrection gesneriads utilize a strategy known as homoiochlorophylly; that is, they retain chlorophyll (Chl) in the desiccated state. The opposite strategy, shown by some monocots, is poikilochlorophylly, which involves a complete degradation of the photosynthetic apparatus. However, in gesneriads, Chl retention is not always complete; for example, it has been reported to decrease by 20 to 70% in the desiccated state in Ramonda species^[9].

The rate of CO₂ assimilation is remarkably low in R. myconi, and it decreases concomitantly with the loss of water content in H. rhodopensis. This process is first due to stomatal closure, and afterwards to reduced photochemical activity^[6][10]. In parallel, there is a shift between linear electron flow (LEF) and cyclic electron flow (CEF), which may contribute to the absence of a drastic decreases in the NADP/NADPH ratio, although a slight increase in NADPH could be observed at 38% of water content^[11]. Once rehydration starts, the activity of PSI is more rapidly recovered than that of PSII^[10]. These observations suggest that the partial loss of Chl does not indicate the occurrence of damage in the photosynthetic apparatus. Furthermore, given that Chl molecules are a major source of reactive oxygen species (ROS) when photosynthesis is impeded, partial Chl degradation represents the activation of a photoprotective mechanism^[12]. As a consequence of the potential ROS generation within the photosynthetic apparatus, oxidative stress is one of the main challenges associated with the process of desiccation and subsequent rehydration^[13].

To counteract ROS production, resurrection gesneriads constitutively express large pools of antioxidant molecules, including ascorbate and glutathione^[7][14]. Other low-molecular-weight antioxidants are phenolic compounds (phenolic acids, polyphenols), whose biosynthetic route (Shikimate pathway) is overexpressed during desiccation in H. rhodopensis^[15]. A similar polyphenol composition has been described in all gesneriad species studied so far: H. rhodopensis, R. myconi, R. serbica, and D. hygrometricum^[16][17][18]. Thus, Georgieva et al. proposed that the high polyphenol content is a characteristic feature of gesneriads^[19].

The activity of all of these antioxidants is complemented by the action of antioxidant enzymes. For example, Gechev et al. found more genes in H. rhodopensis encoding superoxide dismutases, monodehydroascorbate reductases (MDHARs), and glutathione reductases than in most plant species with sequenced genomes, which gives a hint about its constitutive tolerance against desiccation^[20]. Mladenov et al. also reported on the accumulation of ascorbate peroxidase and glutathione peroxidase in response to desiccation, as was shown in R. nathalie. The antioxidant machinery that accumulates during desiccation is also maintained in the desiccated state to be used during the first stages of subsequent rehydration^[7][21].

One of the main targets of ROS are polyunsaturated fatty acids, whose oxidation gives rise to the formation of lipid peroxides that propagate in membranes through peroxidation chains. Paradoxically, polyunsaturated fatty acids enhance membrane fluidity in desiccated tissues. This is probably why the response of the unsaturation ratio varies so widely among the gesneriads studied: in H. rhodopensis, it was not affected by desiccation, while it increased in Boea hygroscopica and decreased in R. serbica^[22][23][24]. The main antioxidant involved in avoiding the propagation of such peroxidation chains is tocopherol, and in fact the enhancement of this antioxidant in response to desiccation is a general trait observed in most (if not all) DT plants, including gesneriads such as D. hygrometricum, R. myconi, and H. rhodopensis^{[6][13][23][25]}.

The production of ROS within the photosynthetic apparatus can be prevented by simply reducing light absorption within the photosystem antennae. To reduce photon absorption by Chl, H. rhodopensis constitutively expresses high levels of red anthocyanins in the abaxial side of the leaves^[26]. When the leaves curl, the anthocyanic layer becomes exposed to light, causing a decrease in light reaching the mesophyll. Another strategy to reduce ROS formation is to enhance the dissipation of energy absorbed by Chl as heat, the so-called non-photochemical quenching (NPQ). This mechanism is linked to the accumulation of certain proteins, such as PsbS, and the presence of zeaxanthin^[27]. PsbS is specifically induced by desiccation in H. rhodopensis, while zeaxanthin has been shown to be synthesized in response to desiccation in R. myconi^[6][28].

A second challenge linked to desiccation is mechanical/structural damage caused by cell shrinkage, which is an obvious consequence of desiccation and leaf curling. First of all, cell walls have to be flexible enough to allow correct folding and to follow such volume alterations^[29]. Changes in cell wall permeability and plasticity have been reported in H. rhodopensis^[30]. The plasticity of the cell wall mainly depends on the relationship between the cellulose-xyloglucan network and pectin polysaccharides, whereby changes in their composition and connection lead to changes in cell flexibility^[21].Indeed, Mladenov et al. reported downregulated levels of genes involved in lignin and cellulose synthesis, and increased levels of enzymes involved in cell wall remodelling.

Maintaining membrane integrity is the main objective of desiccation tolerance strategies. This is accomplished by profound lipid remodelling, and membrane stabilization is further strengthened by the interaction with proteins, sugars, and remaining water molecules^[13]. Consequently, degradation of lipids is a limited phenomenon among resurrection plants, and membranes are highly preserved. In chloroplasts of R. myconi, membrane stability is enhanced by a partial conversion of monogalactosyldiacylglycerol (MGDG) to digalactosyldiacylglycerol (DGDG), a bilayer-forming lipid^[6]. Stability is reinforced in H. rhodopensis by the presence of a dense luminal substance (DLS), most likely a phenolic compound that prevents conformational changes of thylakoids^[31]. Additionally, phospholipids, such as phosphatidylethanolamine and phosphatidylcholine, are degraded during desiccation and there is an increase in phospholipase D^[15]. The accumulation of other lipids, such as sitosterol, is also a species-specific response to desiccation in H. rhodopensis^[23].

In H. rhodopensis, the primary central vacuole disappears when the cell desiccates, and smaller secondary vacuoles emerge in the vicinity of the cell wall. At the same time, organelles take the place of the central primary vacuole^[14]. The increase in the number of smaller vacuoles, which have a greater area/volume ratio, makes it possible to maintain the membrane surface area during the volume reduction caused by water loss^[32]. Desiccated chloroplasts (termed desiccoplasts) adopt a rounded shape and they undergo an enhancement in the number and size of plastoglobules^[14].

The accumulation of compatible solutes, acting as osmoprotectants, is a general response to water loss. In D. hygrometricum, R. myconi, and H. rhodopensis, sucrose is the main compound accumulated in response to desiccation^[33]. Sucrose can be produced after starch degradation or can come from gluconeogenesis, as there is consumption of glycolytic intermediates directly related to the accumulation of sucrose^[14][23]. In D. hygrometricum, raffinose-family compounds also increase during desiccation^[8]. At the later stages of desiccation, the massive accumulation of sucrose is more directly related to membrane protection by preventing non-bilayer phase separation and membrane fusion^[13]. Coincident with their protective functions, both sucrose and raffinose sharply decline after rehydration^[14][23].

Once the water potential surpasses a certain threshold (−100 MPa), there is a process of vitrification and the cytoplasm reaches the so-called glassy state, an amorphous metastable state^[13]. In this situation, most metabolic activities cease and chemical reactivity is inhibited. Transition to the glass state implies positive aspects for DT organisms, as the diffusion of oxygen is greatly reduced, decreasing ROS generation and preventing further water loss. Vitrification was studied in R. myconi by Fernández-Marín et al., who showed that this stage can occur in nature, as it can be reached in desiccated leaves at 20 °C^[34].

Desiccation induces the expression of genes encoding several sets of protective proteins. This is the case of early light-induced proteins (ELIPs) and PsbS, which are involved in the maintenance of chloroplasts and the regulation of photosynthesis. Their expression is maintained during the rehydration process, and some of them are still present 7 days after the onset of rehydration. Desiccation also induces massive expression of late embryogenesis abundant (LEA) proteins in H. rhodopensis, R. serbica, and D. hygrometricum^[20][21]. These proteins act as water replacement molecules to maintain the structure of the membranes and organelles, as they have little possibility of interacting with other molecules due to their low capacity for forming hydrogen bonds^[35].

Damaged DNA is another key aspect of maintaining genome integrity. It can be repaired by several processes, including nucleotide excision repair, in which genes have been reported to be specifically activated during dehydration in H. rhodopensis. In case cellular damage occurs, autophagy is also an option^[15]. By inducing autophagy, cell death can be inhibited in order to recycle damaged structures to create new structures needed for the protective response of the organism against water deficiency stress, as has been observed in D. hygrometricum^[25]. The promotion of autophagy is in accordance with the decreasing transcription and protein accumulation of AMC4 in H. rhodopensis during desiccation, and works as a promoter of programmed cell death under stress^[21][36]. This is how DT plants suppress senescence when dehydrated^[13].

Cross-tolerance to freezing and desiccation is somewhat logical, since dehydration prevents the formation of ice crystals inside cells. In fact, it has been documented that H. rhodopensis has the ability to dehydrate rapidly under freezing temperatures^[37]. Consequently, most responses are shared between drought-induced desiccation (DID) and freezing-induced desiccation (FID)^[38]. However, there are slight differences between these two processes; for example, FID is characterized by faster recovery of PSII compared to DID^[39]. In addition, during FID, secondary vacuoles are reported to appear at 60% RWC. This process is similar to the one that happens after DID, but it occurs more rapidly after FID, most likely because of the RWC recovery rate enabled by the environment and the influence of freezing^[38].

In addition to FID, it has been documented in manipulative experiments that hydrated leaves of R. myconi freeze at a relatively high temperature (−2 °C). Tissue freezing involves an abrupt reduction of photochemical efficiency (Fv/Fm), which correlates with the enzymatic formation of zeaxanthin. Interestingly, the glass transition in hydrated leaves occurs at −15 °C, meaning that enzyme activity is possible in frozen leaves of R. myconi^[34]. This justifies the induction of antioxidant enzymes in frozen leaves of H. rhodopensis^[37]. Recovery of Fv/Fm is initially fast upon transfer to warm conditions and is completely restored in 6 h. This means that even in winter, R. myconi is photosynthetically active whenever the temperature is above the freezing point^[6].

Apart from the obvious fact that desiccation protects the plant from ice formation, in order to fully withstand winter stress, resurrection gesneriads have to develop a profound metabolic reconfiguration through a process of seasonal acclimation. Only a few studies have addressed the specific mechanisms that H. rhodopensis and R. myconi employ to cope with low-temperature stress. These include substantial induction of thermal energy dissipation, which is in agreement with a high accumulation of zeaxanthin, and the PsbS protein^[6][10]. Other stress proteins that accumulate in H. rhodopensis in response to low temperature are Lhcb5, Lhcb6, dehydrins, and ELIPs^[38]. Genes encoding lipocalins are also upregulated^[26]. Lipocalins are small ligand-binding proteins that can be found in both the cell and the chloroplast membrane. In winter, there is also a substantial accumulation of low-molecular-weight metabolites, such as polyphenols^[37]; sugars, such as trehalose, maltose, raffinose, sucrose, and glucose; amino acids, such as proline, glycine, serine, alanine, asparagine, and aspartate; polyamines, such as putrescine and ornithine; and antioxidants of the glutathione-ascorbate system^{[6][26][28][37]}. The joint action of all of these mechanisms decreases photosynthesis, lowers osmotic potential, and keep plants in a state primed for freezing protection^[26].

3. Concluding Remarks

Overall, most physiological mechanisms are shared in terms of the responses to desiccation and low-temperature stresses (Figure 1). This is perhaps the reason why tertiary paleotropical relict gesneriads were able to survive in sheltered habitats of southern Europe during the quaternary glaciations. The question of whether freezing tolerance is a constitutive trait in resurrection gesneriads or evolved in temperate species as a result of climate cooling deserves further studies.