School of Biological and Molecular Sciences, Oxford Brookes University, Headington, Oxford OX3 0BP, UK1
Bass Brewers, Technical Centre, PO Box 12, Cross Street, Burton-on-Trent DE14 1XH, UK2
Author for correspondence: Katherine A. Smart. Tel: +44 1865 483248. Fax: +44 1865 483242. e-mail: kasmart{at}brookes.ac.uk
Keywords: ageing, lifespan, yeast, senescence, brewing
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Overview |
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Background |
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That brewing yeast should lose viability due to necrosis is expected; however, it has not been recognized that in the absence of lethal doses of stress or DNA damage brewing yeast will progress through a structured and defined lifespan, eventually reaching a senescent phase which culminates in cell death. Primarily this is due to the fact that the term ageing has been incorrectly used to describe any population of cells exhibiting a deteriorated physiological state. In the brewing industry, yeast age is measured chronologically in terms of the number of times a yeast population is serially repitched (recycled for reuse in successive fermentations). In addition, yeast which has been stored (in the form of a highly concentrated cell suspension, termed a slurry), or maintained in extended stationary phase is often referred to by brewers as aged. Such populations exhibit impaired physiological states, although their phenotypic characteristics more closely resemble those associated with necrosis than senescence.
The metric of the brewing yeast lifespan is not chronological but relates to the number of divisions an individual cell has undertaken (Hayflick & Moorhead, 1961 ; Muller et al., 1980
). The number of daughters produced by a mother cell, termed the divisional age, indicates the relative age of the cell, while the maximum lifespan potential of a cell is referred to as the Hayflick limit (Hayflick, 1965
). Yeast longevity is determined by genes and influenced by environmental factors; however cells of the same genotype exhibit intrinsic variation in lifespan. Each yeast cell is capable of dividing a number of times before reaching senescence, in which no cell division occurs and death metabolism is initiated. This form of ageing is known as replicative senescence and is a phenomenon shared by both yeast and mammalian cells (Jazwinski, 1990a
, 1993a
, b
; Austriaco, 1996
; Campisi, 1996
; Campisi et al., 1996
; Kennedy & Guarente 1996
; Smith & Pereira-Smith, 1996
; Sinclair et al., 1998
). Senescence is a consequence of termination of replication and is therefore intimately associated with cell division and hence the cell cycle.
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The yeast cell cycle |
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The cell cycle may be divided into five phases (Fig. 1): G0, G1, S, G2 and M (Pringle & Hartwell, 1981
; Wheals, 1987
). DNA synthesis occurs during S phase, while segregation of chromosomes during mitosis takes place at M phase. G1 and G2 represent two gap periods of variable length, during which organelle production and normal cell processes such as growth and development take place. Subsequent to M phase cell division occurs, although complete separation is not a prerequisite for continued reproduction (Wheals, 1987
). Indeed, ineffective motherdaughter cell separation can result in chain formation, which is a characteristic phenotype of some brewing yeast strains.
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G1 precedes the initiation of chromosomal DNA replication and represents the phase of growth in which cells must reach a minimum size before progressing through the remainder of the cell cycle (Futcher, 1996 ; Kuntzel et al., 1996
; Breeden, 1996
). Slow-growing cells have a relatively long generation time and this additional time is spent in the G1 phase. In addition, daughter cells that have never produced a bud are smaller than their corresponding mother cells and so have longer G1 phases. This differential is known as motherdaughter asymmetry, where mother and daughter cells may be distinguished by both cell size and division rate (Futcher, 1993
; Lew & Reed, 1995
).
Towards the end of G1 a ring of chitin appears on the cell surface, indicating the location of bud formation. Saccharomyces cerevisiae regulates its cycle at G1 and particularly at a stage termed START (Hartwell et al., 1974 ; Wheals, 1987
). After START has occurred, environmental factors such as external stress and poor nutrients are no longer able to prevent division. Thus even during the initial stages of fermentation when oxygen becomes limiting and cell division is no longer favoured, yeast cells that have passed through START are forced to complete the division to which they have been committed (Wheals, 1987
). Budding is the obvious visible sign that a cell has initiated a round of division and in general, the larger the bud, the closer the cell is to completion of division (Wheals, 1987
). During exponential growth the proportion of cells that have progressed through START and those that have not is roughly equal, and may be represented by the occurrence of 50% budded and 50% unbudded cells (Wheals, 1987
; Futcher, 1993
). Failure to progress through START results in cell-division arrest and entry into the cell cycle stationary phase, which is also known as G0 (Forsburg, 1994
).
G0 is an off cycle state during which no net increase in cell number occurs (Wei et al., 1993 ; Werner-Washburne et al., 1993
; Forsburg, 1994
; Nurse, 1997
). Entry into G0 is triggered by adverse environmental factors, one of which is nutrient depletion (Werner-Washburne et al., 1993
). During the latter stages of fermentation the yeast enters stationary phase and remains in this state during storage and repitching (McCaig & Bendiak, 1985
; DAmore, 1992
; Hammond 1993
; Stewart 1996
). A characteristic of organisms in G0 phase is an increased resistance to stresses for an extended period of time (Pringle & Hartwell, 1981
; Wheals, 1987
), causing cells to retain viability and vitality during cold storage (McCaig & Bendiak, 1985
).
Given the correct environmental conditions, yeast cells exit G0 and re-enter the cell cycle to initiate a new round of cell division; therefore stationary phase is a reversible phenotype. Cells in extended stationary phase are often referred to as aged cultures, although in fact they are composed of individual cells exhibiting both young and aged phenotypes (Smart, 1999 ).
Providing the cell completes START and the remainder of the G1 phase, it may then progress to the S phase, where DNA is replicated and the nucleus migrates towards the neck separating mother from bud (Futcher, 1993 ; Wei et al., 1993
; Weinert et al., 1994
; OConnell & Nurse, 1994
). After another resting phase, G2, the cell enters the M phase where mitosis and nuclear division occurs (Nurse et al., 1998
), and the bud emerges from within the chitin ring. Cytokinesis and cell separation are believed to occur in early G1 (reviewed by Pringle & Hartwell, 1981
; Forsburg & Nurse, 1991
; Nurse, 1997
).
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Senescence and the cell cycle |
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Life expectancy and yeast cells |
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In contrast, a colony of yeast may be considered to be immortal because although individuals will senesce and die, the continual generation of new daughter cells ensures that the population is maintained indefinitely. Yeasts, like many other eukaryotes, reproduce using a replicative reset technique (Austriaco, 1996 ). Following mitosis, a mother and daughter cell are produced through an asymmetric division in which each cell can be readily distinguished (Fig. 2
). The mother exhibits her personal Hayflick limit, whilst the daughter cell is reset to zero and is capable of displaying a full replicative life span in accordance with the Hayflick limit of its strain (Fig. 3
). Continuous growth of a population is therefore possible under favourable environmental conditions.
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Phenotypic characteristics associated with ageing and senescence |
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For yeast, ageing results in irreversible modifications to cellular appearance. The senescent phenotype gradually appears throughout the lifespan and the occurrence of certain modifications can often act as biomarkers in the determination of cell age. Several morphological and physiological changes associated with the ageing process in haploid, diploid and polyploid laboratory strains, and polyploid brewing production strains of yeast have been described. These include an increase in bud-scar number and therefore chitin deposition (Egilmez et al., 1990 ), an increase in cell size (Bartholomew & Mittwer, 1953
; Mortimer & Johnston, 1959
; Johnson & Lu, 1975
; Egilmez et al., 1990
; Barker & Smart, 1996
), a granular appearance to aged cells (Mortimer & Johnston, 1959
), accumulation of surface wrinkles (Mortimer & Johnston, 1959
; Muller, 1971
; Barker & Smart, 1996
), and eventual cell lysis (Mortimer & Johnston, 1959
). Many other morphological and physiological changes have since been identified (Table 1
).
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Mechanisms of ageing and senescence in S. cerevisiae |
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Another hypothesis for cellular ageing is that division capacity is limited by the length of telomeres. The telomere hypothesis suggests that the number of cell divisions is registered by the gradual loss of telomeric sequences (Holliday, 1996 ; Chiu & Harley, 1997
). Telomeres stabilize the chromosome and ensure that complete replication occurs (Zakian, 1989
), although it is known that they also aid in the attachment of chromosomes to the nuclear matrix (Haber & Thornburn, 1984
; Hastie & Allshire, 1989
; Zakian, 1989
).
At the start of replication, DNA polymerase acts in the 5'3' direction, adding bases with the aid of a primer. The section of the telomere covered by the DNA polymerase is not reproduced, thus causing a shortening of DNA (Olovnikov, 1973 ), by around 50 base pairs per division (depending on the organism) at the 5' end (Campisi et al., 1996
). This phenomenon has been identified in many organisms (Harley et al., 1990
; Hastie et al., 1990
; Lindsey et al., 1991
) and may act as a biological clock determining the number of divisions prior to senescence in mammalian systems.
In yeast, however, strains displaying telomeres of various lengths have exhibited similar longevities (Dmello & Jazwinski, 1991 ; Austriaco & Guarente, 1997
) and it has been suggested that telomere shortening may not even occur in yeast cells (Dmello & Jazwinski, 1991
; Smeal et al., 1996
).
However, cytoplasmic senescent factors, specific senescence and youth genes, DNA damage and repair, oxidative damage, and mitochondrial integrity have all been implicated in the mechanism of progression through ageing and senescence.
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Cytoplasmic senescence factors |
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One candidate for this cytoplasmic senescence factor is extrachromosomal rDNA circles (ERCs). Yeast rDNA located on chromosome XII gives rise to circular forms of single rDNA repeats known as pop outs or ERCs (Sinclair et al., 1998 ). Recently it has been observed that ERCs accumulate in ageing mother cells (Sinclair & Guarente, 1997
) and prematurely induce the senescent phenotype if inserted into young cells (Sinclair & Guarente, 1997
; Sinclair et al., 1997
).
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Genetics of ageing |
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It has been postulated that ageing occurs either by a genetic inhibition of metabolically essential proteins and enzymes, such as DNA repair enzymes or antioxidants (Johnson, 1997 ) or alternatively by gene activation causing the production of proteins that directly inhibit DNA synthesis (Goletz et al., 1994
).
Genetic control over lifespan can be investigated by identifying changes within the cell during the lifespan that are not the product of senescence, but rather the cause of it (Kenyon, 1996 ). A number of genes that influence longevity in yeast have already been identified (Table 2
).
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The RAS genes, which are part of the signal-transduction pathways involved in sensing the nutritional status of the cell and in responding to stresses, have also been reported to affect longevity. Overexpression of RAS1 has been found to have no effect on lifespan, while deletion of RAS1 and overexpression of RAS2 have been found to increase lifespan by around 30% (Sun et al., 1994 ). These long-lived yeasts show an increased efficiency in terms of metabolic capacity and productivity (Jazwinski, 1996
).
Genetic analysis of individuals exhibiting increased lifespan during starvation stress led to the identification of four youth genes (Kennedy et al., 1995 ; Kennedy & Guarente, 1996
). One of these, SIR4, had several functions including chromatic silencing (Osiewacz, 1997
) and repression of genes placed near telomeres (telomere position effect) (Aparicio et al., 1991
; Shore, 1995
). Kennedy et al. (1995)
demonstrated that SIR4 was important in lifespan regulation and may trigger senescence by silencing a stress-response gene known as AGE. The SIR4 gene belongs to a family of SIR genes. SIR proteins are involved in DNA repair and nucleolus protection. Towards the end of the lifespan the nucleolus becomes enlarged and fragments (Sinclair et al., 1998
); thus it appears that silencing, DNA repair and genome stability are important in determining lifespan (Sinclair et al., 1998
).
Other genes involved in DNA repair and integrity have been implicated in the mechanism of yeast ageing and senescence. DNA helicases have been demonstrated to contribute to premature ageing syndromes in humans. The SGS1 (Slow-Growth Suppresser) gene, which encodes a yeast DNA helicase is also a longevity-assurance gene (Sinclair & Guarente, 1997 ) and its deletion reduces the lifespan of S. cerevisiae. RAD9, a gene involved in cell-cycle checkpoint regulation is also thought to be a youth gene, as deletion shortens lifespan (Kennedy et al., 1994
).
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Oxidative stress and the free-radical theory of ageing |
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Organisms undergoing aerobic respiration are frequently subjected to slow but continuous damage to their cellular components caused by free-radical stress (Harman, 1956 , 1981
, 1992
). Free radicals can occur naturally due to radiation or from the use of oxygen by aerobic cells. Oxygen is a highly reactive molecule that can form ROS, including the superoxide anion (O2·-), hydrogen peroxide (H2O2) and the hydroxyl group (OH) (Jamieson, 1995
). These free radicals cause damage to almost all cellular constituents. The most susceptible are proteins and lipid membranes (Wolff et al., 1986
), although more substantial damage can be caused due to the mutagenesis of DNA (Storz et al., 1987
).
Cells exhibit defined anti-oxidant defences that are depleted throughout the life cycle and are comprised of enzymic and non-enzymic antioxidants (Jamieson, 1998 ). These defence mechanisms act to scavenge free radicals or repair enzymes by removing and replacing damaged molecules. Under normal conditions, antioxidant defence mechanisms are capable of maintaining ROS at harmless levels, but prolonged exposure to free radicals can eventually result in an inability to prevent cellular damage (Jamieson, 1995
). The extent of oxidative damage incurred and the subsequent susceptibility to further damage increases with age (Kale & Jazwinski, 1996
). It is likely that the cause of these phenomena are due to an increase in oxidant production, a decline in antioxidant defence mechanisms or a decline in the efficiency of repair mechanisms (Sohal & Weindruch, 1996
). Recent advances in this field have shown that deletion of genes responsible for antioxidant defence mechanisms cause a reduction in lifespan (Barker et al., 1999
; Wawryn et al., 1999
).
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Mitochondrial DNA damage and ageing |
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The role of mitochondria in ageing is becoming increasingly apparent. It has been demonstrated that the mitochondrial transmembrane proteins encoded by BAP37 and PHB1 (Coates et al., 1997 ) are required to ensure longevity, and mitochondrial DNA is known to be necessary for resistance to oxidative stress in yeast (Grant et al., 1997
).
Lifespan analysis of respiratory-deficient yeast suggests that the impact of petite mutations on longevity may be strain specific (Kirchman et al., 1999 ). As the mitochondria are the source of ROS, it has been suggested that mitochondrial dysfunction could lead to reduced levels of stress (Longo et al., 1996
; Guidot et al., 1993
) and therefore an enhanced longevity. Alternatively, the energetic requirement of free-radical scavengers implies that lifespan would be extended by the presence of fully functional mitochondria (Grant et al., 1997
). Berger & Yaffe (1998)
observed a 40% reduction in lifespan for petite mutants, supporting the latter hypothesis. Kirchman et al. (1999)
also reported a reduced lifespan for several petite mutants; however analysis of a long-lived subpopulation of a yeast strain, YPK9, identified these individuals as being respiratory deficient.
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Measuring yeast cell age |
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Ageing in brewing yeast |
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For brewing yeast, very little has been reported regarding the physiological modifications that occur as a function of individual cell ageing. Our laboratory reported an extensive increase in cell size throughout the lifespan (Barker & Smart, 1996 ; Fig. 5
). Interestingly, the increase in size of the mother cell did not result in a corresponding increase in size for each successive bud produced (Barker & Smart, 1996
), except in very rare instances when the newly formed bud could equal the size of its ageing mother. At the end of brewery fermentations, the vessel contents are cooled to 24 °C, which facilitates the sedimentation of yeast into the fermenter cone. This settling process is known to be rate zonal in nature, with larger cells settling faster than their smaller companions. Deans et al. (1997)
demonstrated that older cells were indeed more abundant in lower than in higher regions of the cone (Fig. 6
), supporting the hypothesis put forward by Barker & Smart (1996)
that age stratification could occur as a result of the preferential sedimentation of the older cell population. The presence of an age gradient within the cone becomes of significance when considering the mechanism by which yeast is removed from the vessel prior to storage and subsequent serial repitching. Typically, the cropped yeast is run down from the fermenter in two fractions, termed cuts. The first cut, consisting of larger, older and dead cells together with protein debris (trub) is discarded as waste. The second cut, consisting of middle-aged and virgin cells, is transferred to a yeast collection vessel prior to repitching into a fresh fermentation. An alternative mechanism is to remove yeast from the cone prior to vessel cooling so that some yeast remains in suspension (Loveridge et al., 1999
). This procedure, known as warm or early cropping, would be anticipated to result in a greater proportion of aged individuals to be selected for serial repitching (Loveridge et al., 1999
). Successively cropping and repitching a specific layer of yeast in this way could inadvertently select for cells of a particular size and therefore age. During laboratory batch culturing, a normal cell population consists of approximately 50% virgin cells, 25% first generation cells, 12·5% second generation cells and so on; therefore it is possible that cropping could have implications for subsequent fermentations due to selective pressures and the disruption of population dynamics (Barker & Smart, 1996
). It is likely that as genes are differentially expressed throughout the lifespan, a good age balance would result in optimum fermentation performance. Seeding fermentations with a population skewed towards an imbalance of either young or aged cells could result in an inappropriate expression of genes. This could influence a number of metabolic parameters, ultimately affecting the fermentation profile, leading to an inconsistent process and a product of poorer quality.
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In conclusion, individual cell ageing in brewing strains may influence fermentation performance where yeast handling in the brewery allows for the inadvertent selection of yeast populations enriched for very young or aged cells.
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ACKNOWLEDGEMENTS |
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