Wine yeast is rarely found on grapes with an unbroken skin, presumably due to lack of moisture and nutrients, but viable cells are found inside approximately one-third of damaged berries, where colonies establish fermentation chambers (46
). Desiccated colonies entrapped within fragments of dried berries are potentially an important source of inocula following dispersal (11
). In this context, it is important to emphasize that there are numerous permutations of environmental conditions under which S. cerevisiae
could be subjected to water stress. Thus, a number of empirical decisions were made in designing our experiments to dry, desiccate, and rehydrate yeast, including decisions as to the stage of growth of the cells, the time and temperature of drying, the length of time of desiccation, and the mode of rehydration. The fact that there was substantial overlap in the transcriptional analysis of S.cerevisiae
BY4743 and a sample of commercially dried yeast, purchased at random (Fig. ), suggests that the stress responses reported here are of environmental relevance.
The fatty acid catabolism transcriptome was activated markedly under the conditions employed, most prominently the genes of peroxisomal biogenesis and protein targeting, fatty acid transport and activation, beta-oxidation, and peroxisome-mitochondrion acetyl-CoA shuttling. It is highly probable that this shift was due solely to the glucose-limiting conditions needed to dry the cells. However, at some point in time during the desiccation process, the cells will no longer be able to take up glucose, even when supplies are adequate, and must begin to shut down all processes. Our working hypothesis is that this shutdown procedure is coordinated and controlled. What is not known is the sequence of events leading to metabolic arrest. If glucose uptake is repressed early in this process, it may be that the fatty acid catabolism transcriptome will be up-regulated even when adequate supplies of glucose are present.
The interplay between pyruvate and formate and the role of hypoxic and glucose-limiting conditions during desiccation and rehydration need further investigation. Based upon results presented here, as well as the recently reported data for the mitochondria of potato tubers (10
), we hypothesize that a formate-producing pathway is present in yeast as well as other eukaryotes and is an important contributor to cell energetics under quiescent conditions. In bacteria, pyruvate is converted to formate and acetyl-CoA by PFL, a free radical-generating enzyme (3
). However, as the induction of formate dehydrogenase has been reported to occur under normoxic, glucose-limiting conditions (7
) and the PFLs found in the past are oxygen sensitive, an enzyme with an alternative mechanism may be at work.
We propose that under the glucose-limiting conditions employed in this study, trehalose is not available in the amounts necessary to provide significant biophysical protection of the membranes. An increase in trehalose levels was observed, and appeared to be increasing at the latter stages of rehydration, but the levels found were significantly lower than that generally reported for membrane protection of yeast (53
). Similar responses at the transcriptional level for trehalose and glycerol-related genes have been reported under glucose-limiting conditions (20
), suggesting that the response may be related to the amount of carbohydrate available to the organism. Limiting supplies, as either free sugar or stored glycogen, may make the overall cost of trehalose biosynthesis through gluconeogenesis too expensive. Trehalose may be produced during desiccation only when yeast is exposed to osmotic stress with ample glucose supplies.
Glycerol is the major osmolyte synthesized under hyperosmotic stress and is responsible for most of the osmotic adjustment in yeast (1
). Glycerol-3-phosphate dehydrogenase, encoded by GPD1
, is responsible for glycerol synthesis, and lack of the enzyme renders the yeast osmosensitive. Transcription of GPD1
was unaffected through T6
and then down-regulated at T7
, providing evidence for distinct differences in the responses of yeast to osmotic and matric water stress. There are examples of anhydrophiles that do not rely upon trehalose for survival. The cyanobacterium Nostoc commune
, for example, produces an extracellular polysaccharide in combination with a water stress protein to survive desiccation (51
), rather than producing significant quantities of trehalose. Bdelloid rotifers have also been shown to undergo anhydrobiosis without producing trehalose (33
Because there is no significant biosynthesis of trehalose or glycerol in desiccated yeast, we propose that a peptide or small protein such as the hydrophilin Sip18p could also function in a similar role. SIP18
was the most up-regulated gene in our experiments, and its product is known to bind to phospholipid (54
). The broad class of proteins referred to as hydrophilins include l
bundant (LEA) proteins and dehydrins and are characterized by their high hydrophilicities (>1.0) and high glycine (>6%) contents (22
). These criteria appear to correlate strongly with the transcriptional response of a particular hydrophilin gene to hyperosmotic conditions, and LEA proteins are implicated in providing desiccation tolerance to plants and plant seeds by an as-yet-unknown mechanism. A recent search of the yeast genome revealed 12 potential hydrophilin-like genes, and analysis of their transcript levels upon osmotic stress (not desiccation) indicated that 8 of them were up-regulated (22
). The Yeast Genome Database (18
) currently lists 10 genes under the category “response to desiccation.” However, in our studies, transcription of only 3 of the 10 listed genes was markedly enhanced during desiccation and rehydration: YJL144W
(up to 3-fold), YPL223C
) (up to 7.6-fold) and YMR175W
) (up to 9.3-fold). Genes encoding phospholipases A2
, B, C, and D showed no to only a slight change in level of transcription during the course of the experiment, in contrast to the marked elevation in the transcription of genes involved with fatty acid beta-oxidation. These observations may suggest that there is minimal to no mobilization of fatty acids from membranes of viable cells to fuel beta-oxidation.
One of the findings from our analyses was the change in transcription of genes involved in cell wall structure and organization (down-regulation of GAS1
, and protein O
-mannosyltransferase genes PMT1
) and lipid synthesis and binding (down-regulation of SER3
, and YBR238C
), in addition to down-regulation of TGL1
, encoding the major lipid particle protein. Fourteen of the 19 proteins identified by a proteomic analysis of tryptically digested cell walls of log-phase S. cerevisiae
) were down-regulated in our study at least twofold. In contrast, there was up-regulation of cell wall spore-specific genes YNL196C
. These data suggest that cell wall organization and composition are critical for desiccation tolerance. A reduction in cell wall rigidity may allow the cell to distribute stabilizing factors in a more effective manner.
In summary, we have presented the first transcriptional analysis of S. cerevisiae
during desiccation and rehydration. The sets of tools, resources, and data that have been generated will allow other laboratories to extend our understanding beyond the initial discoveries and hypotheses presented here. Knowledge of the structural, physiological, and molecular bases for desiccation tolerance will contribute to our basic understanding of the living cell and its inherent ability to enter into, and return from, a state of complete metabolic arrest. In addition, we believe our studies lay a strong foundation for the development of new biomimetic strategies for long-term storage of labile cells and cell components. A desiccation-like strategy may be extremely useful for the long-term goal of placing sensitive cells, of relevance to the biomedical and biodefense communities (4
), in a state of full metabolic arrest. Applications include circumventing the requirement for refrigerated storage used with vaccines and blood products, development of robust biosensors, and the long-term preservation and archiving of valuable cell lines and clone libraries.