Increasing intracellular trehalose is sufficient to confer desiccation tolerance to Saccharomyces cerevisiae

Diverse organisms capable of surviving desiccation, termed anhydrobiotes, include species from bacteria, yeast, plants, and invertebrates. However, most organisms are sensitive to desiccation, likely due to an assortment of different stresses such as protein misfolding and aggregation, hyperosmotic stress, membrane fracturing, and changes in cell volume and shape leading to an overcrowded cytoplasm and metabolic arrest. The exact stress(es) that cause lethality in desiccation-sensitive organisms and how the lethal stresses are mitigated in desiccation-tolerant organisms remain poorly understood. The presence of trehalose in anhydrobiotes has been strongly correlated with desiccation tolerance. In the yeast Saccharomyces cerevisiae, trehalose is essential for survival after long-term desiccation. Here, we establish that the elevation of intracellular trehalose in dividing yeast by its import from the media converts yeast from extreme desiccation sensitivity to a high level of desiccation tolerance. This trehalose-induced tolerance is independent of utilization of trehalose as an energy source, de novo synthesis of other stress effectors, or the metabolic effects of trehalose biosynthetic intermediates, indicating that a chemical property of trehalose is directly responsible for desiccation tolerance. Finally, we demonstrate that elevated intracellular maltose can also make dividing yeast tolerant to short-term desiccation, indicating that other disaccharides have stress effector activity. However, trehalose is much more effective than maltose at conferring tolerance to long-term desiccation. The effectiveness and sufficiency of trehalose as an antagonizer of desiccation-induced damage in yeast emphasizes its potential to confer desiccation tolerance to otherwise sensitive organisms.Water is an essential molecule whose absence can lead to a variety of detrimental and often lethal effects on cells and organisms (1–3). Severe water removal, termed desiccation, has been proposed to lead a variety of detrimental stresses (3). Which of these stresses leads to lethality in desiccation-sensitive organisms is unclear. Organisms capable of surviving desiccation, commonly termed anhydrobiotes, are found among bacteria, fungi, plants, and invertebrates (1, 3). Anhydrobiotes harbor stress effectors that are known or postulated to mitigate the different stresses associated with desiccation (2, 4). These stress effectors include osmolytes, heat shock proteins, redox balancing enzymes, nonreducing disaccharides (trehalose, sucrose), and hydrophilins (short unstructured hydrophilic proteins—also known as LEAs) (1). A reasonable assumption might be that many if not all of these stress effectors are necessary for desiccation tolerance given the multitude of stresses imposed by desiccation. However, this assumption is challenged by the uncertainty in the number and degree of lethal stresses generated by desiccation and the versatility and coordination/cooperation of multiple stress effectors in ameliorating such lethal stresses. Thus, a critical question in the anhydrobiosis field is whether a single stress effector is sufficient to promote desiccation tolerance.One of the most studied desiccation-associated stress effectors is the simple nonreducing disaccharide, trehalose (α,α-1,1-glucoside) (5). It is found in extremely high concentrations in most anhydrobiotes, including in the model organism Saccharomyces cerevisiae (6, 7). In this yeast, exponentially dividing cells have very low levels of trehalose and are extremely desiccation sensitive (8). However, in saturated cultures, yeast cells accumulate high levels of a number of stress effectors, including extremely high levels of trehalose (up to 15% of dry cell mass) (6, 7). We recently showed that high levels of trehalose are necessary for yeast cells in saturated cultures to survive weeks to months of desiccation (long term), but not a few days (short term) (9). Trehalose dispensability during short-term desiccation is due in part to overlapping functions with the heat shock factor Hsp104. This overlap led us to discover that trehalose functions as a chemical chaperone capable of preventing the aggregation of both membrane and cytoplasmic proteins (9). Work in the nematode Caenorhabditis elegans demonstrated that worms unable to synthesize trehalose display hallmarks of membrane damage, consistent with trehalose playing a role in preserving membrane structure (10). Indeed, trehalose has been found to be lipidated in nematodes and these “maradolipids” are required for efficient desiccation tolerance (11). Due to the different and versatile mechanisms by which trehalose confers desiccation tolerance in anhydrobiotes, we hypothesize that trehalose, in the absence of other stress effectors, will be sufficient in conferring desiccation tolerance.A simple way to address this hypothesis is to increase the intracellular levels of trehalose in desiccation/dehydration-sensitive cells or organisms then assess whether they acquire desiccation tolerance. Two strategies for increasing intracellular trehalose have been previously used. These were engineering high level expression of trehalose biosynthetic enzymes or importing extracellular trehalose via fusion with lipid vesicles (12–16). Both methods only generated small increases in trehalose levels and minor increases in dehydration but not desiccation tolerance. This weak effect could reflect the need for additional stress effectors. Alternatively, trehalose alone could indeed be sufficient for desiccation tolerance but was missed for two reasons. First, high physiological levels of trehalose observed in desiccation-tolerant organisms were not reached so a potential critical threshold level of trehalose was not met. Second, the biosynthetic strategy not only increased trehalose but also trehalose-6-phosphate, a potent regulator of glucose metabolism that has deleterious effects on cell and organism fitness. Thus, it remains untested whether trehalose alone is sufficient for generating desiccation tolerance.The correlative evidence for trehalose being sufficient for desiccation tolerance was provided from our previous study comparing desiccation sensitivity of saturated and exponentially dividing cultures of yeast (8). Cells in a saturated culture rapidly lose desiccation tolerance when they divide upon dilution into fresh media. Shortly, after dilution, the levels of many stress factors, including trehalose, diminish. Trehalose levels drop as a consequence of activation of two intracellular trehalases, NTH1 and NTH2, and the inhibition of the trehalose biosynthetic enzyme Tps1 (6, 7). The diluted cells retained their desiccation tolerance significantly longer when trehalose depletion was slowed by inactivation of the trehalases (9). This result is consistent with the notion that sustaining high trehalose levels, while reducing the levels of other stress effectors, is sufficient to promote desiccation tolerance. Encouraged by this result, we decided to investigate further the potential sufficiency of trehalose for desiccation tolerance, exploiting the ability of the AGT1 sugar transporter to import extracellular trehalose (17). Here, we show that when AGT1 overexpressing cells are grown in the presence of trehalose, they acquire high levels of intracellular trehalose and desiccation tolerance similar to that of saturated cultures. We characterize this novel acquisition of desiccation tolerance and provide important insights into the roles of trehalose concentration and trehalose structure in both short- and long-term desiccation tolerance.