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Quiescent yeast cells

Compared with actively growing cells, those in the G0 phase have enhanced thermotolerance, partly a result ofa thickened cell envelope and elevated levels of carbohydrate reserves, notably glycogen and trehalose (Gray et al., 2004), as might be expected for cells adopting a strategy for survival. The shift into quiescence occurs in response to nutrient limitation via the concerted action of, amongst other factors, the activities of a number of protein kinases that participate in cellular cascade control pathways (Smets et al., 2010). It has been assumed that the shift to quiescence only takes place in G1 phase cells (Pardee, 1974); however, other work has shown that cells may enter this phase from any point in the cell cycle (Wei et al., 1993; Laporte et al., 2011). In the latter paper, the authors grew yeast on a carbon-limited medium and noted that within the stationary phase population approximately 90% of the cells were unbudded and the remaining 10% budded. Examination of the two subpopulations revealed that both groups had the same characteristics in terms of markers of quiescent cells and both sets were capable of resuming proliferation when suitable nutrients were supplied. The authors went on to separate budded and unbudded quiescent cells by micromanipulation and tested their relative abilities to form colonies on solid nutrient medium. Both types were capable of this; however, the budded fraction were much less able (65% of budded cells compared with 95% unbudded). This mirrors observations made in relation to brewing propagation, where it has also been seen that cells in the stationary phase culture after aerobic growth on malt wort had a much higher budding index compared with the same yeast cropped from fermentation (Miller et al., 2012). These authors noted that when the new culture was pitched into the first generation fermentation, there was a lack of synchrony in terms of the move into growth between the budded and unbudded fraction, and it was suggested that this was the basis of the frequent observation that these first fermentations give slower cycle times compared with yeast of an older generation. Conversely, cropped pitching yeast has a much lower budding index, suggesting that in this case the transition to quiescence leads to a more homogeneous population (Miller et al., 2012). Perhaps this is linked to the fact that in a brewery fermentation growth extent may be limited by the quantity of oxygen supplied initially, whereas in propagation it is likely to be another nutrient, in most cases probably amino nitrogen or possibly zinc.

The question has been posed whether the passage into quiescence is the result of following a genetically based programme that, once committed to, must progress to completion, rather like START in the cell cycle; or whether it is simply a number of passive adaptations that occur in response to adverse conditions (Daignan-Fornier and Sagot, 2011). These authors, in reviewing their own work and that of others, make several pertinent points. In yeast, the quiescent stage is entered in response to nutrient limitation; however, profiles of mRNA in quiescent cells are different depending on the limiting nutrient and the transcription of only a few genes are common to different nutrient limitations. This suggests that there is not a signature quiescent gene profile. At the metabolomics level there is little commonality between cells starved for different nutrients, supporting the view that quiescence is an adaptive response to growth at a very low rate. However, it can also be demonstrated that, independent of the nature of the limiting nutrient, cells undergo a common series of cellular adaptions associated with quiescence and these occur in response to sensing systems that are able to detect declining levels of nutrients and predict the onset of starvation. In this sense, there may be a number of different quiescent states depending on the prior history of the cell.

Quiescent yeast cells undergo structural reorganization during the transition to non-growth. The actin cytoskeleton is reorganized to give so- called ‘actin bodies', which may act as actin reserves that can be rapidly changed back into a functional form when nutrients become available (Sagot et al., 2006). In addition, the proteasome moves from the nucleus to form a cytoplasmic structure termed the proteasome storage granule (Laporte et al., 2008). This is accompanied by the concentration of many cytosolic metabolic enzymes into discrete structures (Narayanaswamy et al., 2009). Other cellular components are also organized into pools from where they may be rapidly mobilized to facilitate re-entry into proliferation. For example, quiescent cells contain large cytosolic processing bodies where mRNA molecules are held ready to participate in translation when growth-permitting conditions arise. Similarly, it has been demonstrated that, although many genes are repressed in the transition to stationary phase, transcription is held in a state in which it can be rapidly reactivated when the need arises (Radonjic et al., 2006).

Population heterogeneities in stationary phase cells have been reported (Allen et al., 2006). These authors studied differences in cells in the stationary phase as they underwent the diauxic shift. They were able to separate quiescent and non-quiescent yeast cells based on differences in density. A fraction of the population recovered from a glucose-exhausted medium comprised small, dense, unbudded daughter cells. On completion of diauxy, these were able synchronously to re-enter mitotic division. The suggestion was that these daughter cells derived from cell division in the diauxic phase, after which they underwent changes leading to density increase, partly attributable to enhanced trehalose accumulation and a thickened cell wall. A smaller subpopulation of cells apparently did not enter the quiescent phase and these, although viable, rapidly lost the ability to undergo mitosis and became necrotic. A smaller group of cells within this subset exhibited properties of quiescent cells.

Li et al. (2013), again working with glucose- grown cells undergoing diauxic shift, used flow cytometry to separate the three distinct cell types described by Allen et al. (2006). They confirmed that within these groups was a subpopulation of quiescent cells that were small, had thickened cell walls, and were extremely heat-tolerant. These cells comprised a population of daughter cells formed as a consequence of an asymmetric budding event triggered by the diminishing glucose concentration. Time was required for cells to respond to changes in glucose concentration. Indeed, if the supply was abruptly removed the budding event resulting in the generation of resistant daughter cells was disrupted (Li et al., 2013). The quiescent daughter cells were homogeneous and arrested in the G: phase and highly resistant to applied stresses. In the quiescent state they have very long lifespans and are able to begin synchronous proliferation, should conditions change to growth-permitting (Li et al., 2009). The falling glucose concentration triggers a survival response in which there is a lengthening of the G1 phase, and the quiescent daughter cells have an altered metabolism in which accumulation of both glycogen and trehalose are favoured (Miles et al., 2013). These storage carbohydrates provide a source of carbon and energy to allow survival in the starvation phase and during the subsequent transition from quiescence to growth. In addition, trehalose has a protective role as a stabilizer of membranes and proteins against stresses such as heat and desiccation (Sillje et al., 1999; Elbein et al., 2003). Although these carbohydrates clearly have important roles in survival of potential starvation conditions, they are not determinative of the quiescent state since not all cells with elevated intracellular levels can give rise to quiescent daughters (Li et al., 2013). The second and third subpopulations comprised a much smaller fraction of mother cells that had undergone the shift to quiescence and non-quiescent mother cells.

The transition to quiescence has been shown in the work described above to be related to the ability of cells to sense impending glucose exhaustion (Ozcan et al., 1996; Grose et al., 2007; Conrad et al., 2014). In the case ofbrewery fermentations, the growth-determining nutrient is usually unknown and probably varies with different types of wort; however, it is never sugar. Where high levels of sugar adjuncts are used, nitrogen-containing nutrients, notably free amino nitrogen, can be low and may be limiting, although this has rarely been subject to proper investigation. In many brewery fermentations, it is often assumed that oxygen is the limiting substrate via its role in anaerobic pitching yeast as substrate for synthesis of sterols and unsaturated fatty acids (see ‘Response of yeast to oxygen', below). How does this relate to the transition to quiescence? Using chemostat cultures, Hazelwood et al. (2009) investigated the effects of growth under different nutrient limitations on accumulation of trehalose and glycogen. They observed that, in addition to glucose, limitation of ammonia, phosphate, sulfur, and zinc were all influential and by inference storage carbohydrate accumulation was not simply a result of glucose excess. Glucose and ammonia limitation gave 10- to 14-fold more glycogen than growth under conditions of glucose excess. In addition to glycogen, trehalose accumulation was favoured by ammonia limitation. The responses were attributed to post-transcriptional regulation rather than transcriptional regulation alone. Li et al. (2013) studied the events occurring in diauxy and concluded that glucose limitation resulted in differentiation into three cell types. One fraction comprised very small quiescent daughter cells that arose from highly asymmetric budding. These cells acquired enhanced thermotolerance and accumulated high levels of reserve carbohydrates. Differentiation between quiescent and non-quiescent cells occurred shortly after diauxy and it was concluded that post-transcriptional regulation of mRNA played a crucial role.

The passage into quiescence, as judged by formation of proteasome storage bodies and actin re-ordering, is seemingly dependent on lack of carbon. Laporte et al. (2011) reported that nitrogen-starved yeast cells acquired thermotolerance but did not exhibit the actin skeleton and protea- some modifications. Both starvation of carbon and transfer to distilled water did, presumably indicating the primacy of the glucose signal.

The yeast cell wall is highly plastic in nature and changes in its structure occur in response to environmental changes (Klis et al., 2002, 2006). Remodelling of cell wall structure is regulated by the cell wall integrity (CWI) pathway (Levin, 2011). This system responds to signals received at the cell surface, either as a consequence of normal growth or as environmental challenges, via the appropriate sensors and transmits these to the intracellular targets that mediate the appropriate response. The key element is a G protein, termed Rho1. This switching protein integrates signals from the cell surface and the cell budding cycle, and controls cell wall biogenesis and actin organization.

With regard to entry into quiescence, the increased density of small quiescent daughter cells is in part due to thickening of the cell wall and this change is related to enhanced longevity (Li et al., 2009). Cells become stronger mechanically and less porous. These changes are in part a consequence of the presence of proteins that, based on the observation that they can be removed by treatment with dithiothreitol, are anchored by disulfide bonds (Shimoi et al., 1998; Jansen, 2009). There is a 6- to 7-fold increase in the level of disulfide linkages in stationary phase cells compared with those growing exponentially (de Nobel et al., 1990). The most abundant cell wall protein in quiescent cells is a GPI cell wall protein termed Sed1 (Shimoi et al., 1998). GPI (glycophosphate phosphatidylino- sitol) cell wall proteins are those in which the lipid moiety is synthesized in the endoplasmic reticulum and from there used to transport proteins to the cell membrane where they may then be further modified and inserted into the cell wall. Here they are covalently bonded to cell wall glucans (Pittet and Conzelmann, 2007). The function of some of these appears to be to deliver mRNA to P bodies, where they are stored and therefore help maintain the quiescent cell wall phenotype. Release of these at the appropriate time facilitates re-entry into growth.

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