Читать книгу Handbook of Enology: Volume 1 - Pascal Ribéreau-Gayon - Страница 29

When the diploid cells of spore‐forming yeast are in a hostile nutrient medium (for example, depleted of fermentable sugar, poor in nitrogen, and very aerated), they stop multiplying. Some transform into a kind of sac with a thick cell wall. These sacs are called asci. Each one contains four haploid ascospores arising from meiotic division of the nucleus. Grape must and wine are not propitious to yeast sporulation and, in principle, it never occurs in this medium. Yet Mortimer et al. (1994) observed the sporulation of certain wine yeast strains, even in sugar‐rich media. Our researchers have often observed asci in “old” agar culture media stored for several weeks in the refrigerator or at ambient temperatures (Figure 1.12). The natural conditions under which wild wine yeasts sporulate and the frequency of this phenomenon are not known. In the laboratory, the agar or liquid media conventionally used to provoke sporulation have a sodium acetate base (1%). In S. cerevisiae, aptitude for sporulation varies greatly from strain to strain. Wine yeasts, both wild and selected, do not sporulate easily, and when they do, they often produce nonviable spores.

FIGURE 1.12 Scanning electron microscope photograph of S. cerevisiae cells kept on a sugar‐agar medium for several weeks. Asci containing ascospores can be observed.

(Source: Photograph from M. Mercier, Department of Electron Microscopy, Université de Bordeaux 1.)

Meiosis in yeasts and in higher eukaryotes (Figure 1.13) has some similarities. Several hours after the transfer of diploid vegetative cells to a sporulation medium, the SPB splits during the S phase of DNA replication. A dense body (DB) appears simultaneously in the nucleus near the nucleolus. The DB evolves into synaptonemal complexes—structures permitting the coupling and recombination of homologous chromosomes. After eight to nine hours in the sporulation medium, the two SPBs separate and the spindle begins to form. This stage is called metaphase I of meiosis. At this stage, the chromosomes are not yet visible. Then, while the nuclear membrane remains intact, the SPB divides. At metaphase II, a second mitotic spindle stretches itself while the ascospore cell wall begins to form. Spindle stretching and cell wall development of ascospores go hand in hand. Nuclear buds, cytoplasm, and organelles migrate into the ascospores. At this point, edification of the cell wall is completed. The spindle then disappears when the division is completed.

FIGURE 1.13 Meiosis in S. cerevisiae (Tuite and Oliver, 1991). (a) Cell before meiosis; (b) dividing of SPB; (c) synaptonemal complexes appear; (d) separation of SPBs; (e) constitution of spindle (metaphase I of meiosis); (f) dividing of the SPBs; (g) metaphase II of meiosis; (h) end of meiosis; formation of ascospores.

Placed under favorable conditions, i.e. sugar‐enriched nutrient media, the ascospores germinate, breaking the cell wall of the ascus, and begin to multiply. In S. cerevisiae, the haploid cells have two mating types: a and α. The ascus contains two a ascospores and two α ascospores (Figure 1.14). Type a (MATa) cells produce a mating pheromone a. This peptide made up of 12 amino acids is called mating factor α. In the same manner, type α cells produce mating factor α, a peptide made up of 13 amino acids. The a factor, emitted by the MATa cells, stops the reproduction of MATα cells in G1, and reciprocally, the α factor produced by α cells stops the biological cycle of type a cells. Mating occurs between two cells of the opposite mating type. Their agglutination enables cellular and nuclear fusion and makes use of cell wall glycoproteins, called a and α agglutinins. The vegetative diploid cell that is formed (a/α) can no longer produce mating pheromones and is insensitive to their action; it reproduces by budding.

Some strains, from a monosporic culture, can be maintained in a stable haploid state. Their mating type remains constant for many generations. They are heterothallic. Others change mating type during cell division, causing diploid cells to appear in the descendants of an ascospore. They are homothallic and have an HO gene that inverses mating type at a high frequency during vegetative division. This interconversion (Figure 1.15) occurs in the mother cell at the G1 stage of the biological cycle, after the first budding but before the DNA replication phase. In this manner, a type α M ascospore divides to produce two α cells (M and the first daughter cell, D1). During the following cell division, M produces two cells (M and D2) that have become a cells. In the same manner, the D1 cell produces two α cells after the first division and two a cells during its second budding. Laboratory strains that are deficient or missing the HO gene have a stable mating type. Heterothallism can therefore be considered the result of a mutation of the HO gene or of genes that control its functioning (Herskowitz et al., 1992).

FIGURE 1.14 Reproduction cycle of a heterothallic yeast strain. a, α: spore mating types.

Most wild and selected winemaking strains that belong to the S. cerevisiae species are diploid and homothallic. This is also true of almost all of the strains that have been isolated in vineyards of the Bordeaux region. Moreover, recent studies carried out by Mortimer et al. (1994) in Californian and Italian vineyards have shown that the majority of strains (80%) are homozygous for the HO character (HO/HO); heterozygosis (HO/ho) is in the minority. Heterothallic strains (ho/ho) are rare (less than 10%). We have made the same observations for yeast strains isolated in the Bordeaux region; for example, the F10 strain, which is fairly prevalent in spontaneous fermentations in certain Bordeaux wines, is HO/HO. In other words, the four spores arising from an ascus give monoparental diploids, capable of forming asci when placed in a pure culture. This generalized homozygosis for the HO character of wild winemaking strains is probably an important factor in their evolution, according to the genome renewal phenomenon proposed by Mortimer et al. (1994) (Figure 1.16). According to this author, the continuous reproduction of a yeast strain in its natural environment is accompanied by the accumulation of heterozygotic damage to the DNA. Certain slow‐growth or functional loss mutations of certain genes decrease strain vigor in the heterozygous state. Sporulation, however, produces haploid cells containing various combinations of these heterozygotic characters. All of these spores will become homozygous diploid cells with a series of genotypes because of the homozygosity of the HO character. Certain diploids that prove to be more vigorous than others will in time supplant the parents and less vigorous collaterals. This very persuasive model is supported by the characteristics of the wild winemaking strains analyzed. In these, the spore viability rate is an inverse function of the heterozygosis rate for a certain number of mutations. The completely homozygous strains present the highest spore viability and vigor.

FIGURE 1.15 Mating type interconversion model of haploid yeast cells in a homothallic strain (Herskowitz et al., 1992). * designates cells capable of changing mating type at the next cell division or cells already having undergone budding. M, initial cell carrying the HO gene; D1, D2, daughter cells of M; D1.1, daughter cell of D1.

In conclusion, we can question whether sporulation of strains under natural conditions is indispensable to ensure their growth and fermentation performance. We can also raise the question of conserving selected strains of active dry yeasts (ADYs) for use as yeast starters. It may be necessary to regenerate them periodically to eliminate possible mutations from their genome, which could diminish their vigor.