2.2.1. Morphological Changes
In response to stress, anhydrobiotes make significant morphological and physiological changes to prepare themselves for harsher conditions. In yeast, starvation stress leads to a thickened cell wall that is more thermotolerant and more resistant to enzymatic digestion [
32]. Their metabolic level is also significantly reduced [
33]. Stationary cells have more mitochondria because they have to use ethanol as their carbon source since glucose is being depleted. Most of the stationary cells also accumulate more lipid droplets, which consists primarily of the neutral lipids, triacylglycerols and steryl esters, as a reservoir for energy [
34]. When they are re-introduced into fresh nutrients, the lipids are quickly consumed before cells use external nutrients for cellular growth [
35].
Desiccation stress to stationary yeast cells causes further structural changes [
36,
37,
38]. These changes include altered cell walls, altered cellular and nuclear membranes and a reduction in cell size. Desiccation also induces oxidative stress, which in turn causes DNA and membrane damages [
39]. Using transmission electron microscopy (TEM), we recently showed that desiccation in yeast triggers endoplasmic reticulum (ER) stress and unfolded protein response [
12]. The ER is misfolded and the nuclear membrane is ruptured (
Figure 2). Vacuoles are not readily observed in desiccated yeast cells. In
C. elegans, structural damage, including damage to the cell and mitochondrial membrane, is observed in desiccation-sensitive dauers, or desiccation-tolerant dauers without proper preconditioning [
19].
2.2.2. Physiological Changes
Like structural changes in anhydrobiotes, many physiological changes occur during the preconditioning, or the initial low stress stage. Starvation stress leads to a significant decrease of metabolism within the cells and an increased accumulation of proteins and non-reducing disaccharides that function as stress effectors, including hydrophilins, heat shock proteins and trehalose. These proteins and non-reducing disaccharides help to preserve both membrane and protein structure as well as prevent protein misfolding [
10,
11].
Stress proteins: Stress proteins are composed of three intertwined protein families: heat shock proteins (HSPs), intrinsically disordered proteins (IDPs) and hydrophilins. HSPs are molecular chaperones that contribute to the maintenance of cellular homeostasis by facilitating the refolding of misfolded proteins. They are broadly categorized based on their molecular weights, including: Hsp40 (~40 kDa), Hsp60 (57–65 kDa), Hsp70 (68–80 kDa), Hsp90 (83–99 kDa), and small HSPs (15–40 kDa) [
40]. HSPs also play crucial roles in stress response. Many HSPs are induced upon stresses including heat, osmotic, oxidative and desiccation stress [
41,
42,
43]. The yeast small heat shock protein Hsp12p, for example, protects against desiccation stress by interacting with the plasma membrane and protecting the membrane structure during desiccation [
44]. The disaggregase Hsp104p also enhances yeast desiccation tolerance. Interestingly, this enhancement is accomplished only in cooperation with the disaccharide trehalose [
45]. Hsp70p levels are increased upon stress, but no correlation was observed between Hsp70p increase and desiccation tolerance in yeast [
46]. It would be interesting to see if Hsp70p requires other stress effectors to function. Correlation of Hsp70p level and desiccation has also been reported in other organisms. In the green algae
Klebsormidium, Hsp70 gene was upregulated in desiccation-tolerant strains upon desiccation stress [
27]. In the brine shrimp
Artemia, knockdown of the Hsp70 gene reduces the viability of desiccated cysts [
47].
Intrinsically disordered proteins (IDPs) are a large and functionally important family of proteins characterized by their lack of stable three-dimensional structures yet existent in multiple interconverting conformational states. Many IDPs can change to a fixed tertiary structure after binding to other proteins or RNA [
48]. IDPs play important roles in many biological processes, including cell organization, development, and stress tolerance [
10,
49]. The tardigrade-specific IDPs are essential and sufficient for desiccation tolerance [
10]. The late embryogenesis abundant (LEA) proteins are plant specific IDPs and facilitate the stabilization of membranes during desiccation, which enhances the desiccation tolerance of plant seeds [
50]. In yeast, the IDP Hsp12p interacts with the plasma membrane and protects membrane structures against desiccation stress [
44]. Hsp12p also synergizes with trehalose to mitigate protein aggregation and desiccation stress [
11].
Hydrophilins are a group of proteins characterized with high glycine content (> 6%) and high hydrophilicity index (> 1.0) [
51,
52]. Hydrophilins often include intrinsically disordered proteins (IDPs) partially due to their high content of glycine, a disorder-promoting residue [
11]. Genes encoding hydrophilins are induced by osmotic stress, suggesting that these proteins play a role in response to water loss, or desiccation stress. Twelve hydrophilin proteins have been identified in
Saccharomyces cerevisiae. Their possible involvement in desiccation stress is listed in
Table 1. While the molecular mechanism in stress protection of many hydrophilins is not well studied, it is believed that they act to stabilize proteins and membranes during desiccation [
53]. Studies have shown that both Sip18p [
52] and Stf2p [
53] enhance desiccation tolerance by reducing the ROS level during desiccation stress.
Proteins that are not HSPs, IDPs or hydrophilins may also be involved in membrane stability. The cortical ER protein Ist2p, for example, connects ER to plasma membrane and stabilizes the membrane during desiccation stress. Ist2p also plays a key role in sustaining or restoring the plasma membrane structure during rehydration [
54].
Table 1.
Yeast hydrophilins and their possible roles in desiccation stress.
Table 1.
Yeast hydrophilins and their possible roles in desiccation stress.
GENE ID |
GENE NAME |
POSSIBLE ROLE IN (DESICCICATION) RESISTANCE |
REFERENCES |
YJL184W |
GON7 |
Involved in cell wall mannoprotein biosynthesis and osmotic stress response. |
[55] |
YMR175W |
SIP18 |
Involved in osmotic and desiccation stress response. |
[56,57] |
YMR260C |
TIF11 |
Localizes to cytoplasmic stress granule; desiccation resistance increases in mutant. |
[8,58] |
YFL014W |
HSP12 |
Plant LEA-like protein, involved in plasma membrane organization and response to multiple stresses, including desiccation stress. |
[44] |
YDL213C |
NOP6 |
Required for desiccation-rehydration process. |
[53] |
YGR008C |
STF2 |
Involved in cellular response to desiccation, oxidation, and DNA replication stress. |
[53] |
YBR016W |
CPP1 |
Involved in the adaptive response to hyperosmotic stress. Detailed biological function unknown. |
[59] |
YPL223C |
GRE1 |
Paralog to SIP18. Involved in response to multiple stresses, including osmotic, oxidative, heat shock and desiccation. |
[60] |
YFL010C |
WWM1 |
Biological function unknown. Interacts with the caspase-related protease Mca1p. |
[61] |
YJL144W |
ROQ1 |
Regulator of the Ubr1p E3 ubiquitin ligase; Involved in osmotic, DNA replication and desiccation stress. |
[61,62] |
YNL162W |
RPL42A |
Subunit of the large ribosomal 60S subunit. Function in desiccation stress unknown. |
[53,63] |
YNL190W |
|
Cell wall protein; essential for desiccation stress response. |
[53] |
Trehalose: Trehalose is one of the most well-studied stress effectors in desiccation biology. A study showed that both the structure and the dynamics of the dehydrated matrices differ significantly between trehalose (a non-reducing disaccharide) and sucrose (a reducing disaccharide). The dehydrated trehalose matrix is homogeneous whereas the dehydrated sucrose forms a heterogeneous matrix. This unique structure makes trehalose a more effective stabilizer for proteins under stress [
64,
65].
High concentration of trehalose is found in many anhydrobiotes [
66]. In budding yeast, elevated levels of trehalose are found only in stationary cells, which are desiccation tolerant. The trehalose concentration is very low in exponentially growing cells, and these cells are very sensitive to desiccation [
22,
24]. Stationary yeast cells have a metabolic mode distinct from the exponentially growing cells. Cells growing exponentially use fermentative glycolysis to meet their energetic and biosynthetic needs. When glucose is depleted, cells are transitioned to respiratory metabolism, primarily using ethanol as their energy source [
67].
In yeast, both glycolytic and respiratory activities are very low during the stationary phase, when trehalose has accumulated [
68]. The glyoxylate shunt, or glyoxylate cycle, plays an essential role in trehalose synthesis in both stationary yeast cells and dauer larva of the
C. elegans [
69]. The glyoxylate shunt works by bypassing the steps in the citric acid cycle to produce trehalose using ethanol or acetate as its carbon source. Fatty acids, which are abundant in stationary yeast, could also be used as a carbon source of the glyoxylate cycle [
69,
70].
Trehalose often cooperates with other stress proteins to establish desiccation tolerance. In yeast, for example, trehalose works together with Hsp12p to promote both short-term and long-term desiccation tolerance [
11]. It has also been shown that trehalose cooperates with Hsp104p to promote short-term desiccation tolerance while trehalose is responsible for long-term survival [
45]. In tardigrade, while trehalose levels are relatively low, the effector still plays a crucial role in promoting desiccation tolerance by working synergistically with the tardigrade-specific disordered protein CAHS D [
21].
Glycerol: Loss of water alters the osmolarity of cells and therefore induces osmotic stress. The high osmolarity glycerol (HOG) pathway is activated upon osmotic stress and increases the production of glycerol, which is the main osmolyte in yeast [
71]. However, studies have shown that glycerol does not affect desiccation tolerance [
22,
72]. Our preliminary data showed that the addition of glycerol to culture medium prior to desiccation does enhance the desiccation tolerance (Zhang, unpublished data). More studies are needed in elucidating possible roles of glycerol in the desiccation stress response.
Proline: Proline is an amino acid that is unique among the standard amino acids in which it does not have a free α-amino group. In bacteria or plants, proline accumulates in response to osmotic stress and functions as an osmoprotectant [
73]. In yeast, increasing proline level by either deleting the proline-oxidase (PUT1) or externally adding proline enhances both freezing and desiccation resistance [
74]. However, in yeast, no proline increase was observed upon stress, unlike in bacteria or plants [
73]. Like glycerol, the involvement of proline in desiccation stress warrants further investigations.