Little information is available about the precise mechanisms and determinants of freeze resistance in baker's yeast, Saccharomyces cerevisiae. Genomewide gene expression analysis and Northern analysis of different freeze-resistant and freeze-sensitive strains have now revealed a correlation between freeze resistance and the aquaporin genes AQY1 and AQY2. Deletion of these genes in a laboratory strain rendered yeast cells more sensitive to freezing, while overexpression of the respective genes, as well as heterologous expression of the human aquaporin gene hAQP1, improved freeze tolerance. These findings support a role for plasma membrane water transport activity in determination of freeze tolerance in yeast. This appears to be the first clear physiological function identified for microbial aquaporins. We suggest that a rapid, osmotically driven efflux of water during the freezing process reduces intracellular ice crystal formation and resulting cell damage. Aquaporin overexpression also improved maintenance of the viability of industrial yeast strains, both in cell suspensions and in small doughs stored frozen or submitted to freeze-thaw cycles. Furthermore, an aquaporin overexpression transformant could be selected based on its improved freeze-thaw resistance without the need for a selectable marker gene. Since aquaporin overexpression does not seem to affect the growth and fermentation characteristics of yeast, these results open new perspectives for the successful development of freezeresistant baker's yeast strains for use in frozen dough applications.Bread making is one of the oldest food-manufacturing processes and involves the fermentative capacity of the yeast Saccharomyces cerevisiae for the leavening of the dough. Special types of dough, such as sweet or sour dough, present specific challenges to the leavening activity of the yeast, and specific strains with better performance under such conditions have been selected. However, no appropriate strains of yeast are available yet for use in frozen doughs, an important recent development in the bakery industry (2, 33).The use of frozen doughs is steadily increasing in all industrialized countries because it offers great convenience, automation, and economy of scale. However, significant reduction of the leavening capacity during freeze storage is a serious drawback. Minimizing this loss requires specialized equipment for cold and rapid mixing of the dough which is not available to artisanal bakers. Moreover, these optimized production conditions still cannot completely overcome the drop in leavening activity during long-term storage.Conditions for production of baker's yeast have been optimized in the past decades and nowadays allow yeast with a very high stress resistance to be produced. Active dry yeast, for instance, is guaranteed to maintain its activity during shelf storage at room temperature for 2 years. However, the preparation of frozen doughs presents an unusual challenge. Although marketed baker's yeast is highly stress resistant, it rapidly lose...
Revealing QTLs with a minor effect in complex traits remains difficult. Initial strategies had limited success because of interference by major QTLs and epistasis. New strategies focused on eliminating major QTLs in subsequent mapping experiments. Since genetic analysis of superior segregants from natural diploid strains usually also reveals QTLs linked to the inferior parent, we have extended this strategy for minor QTL identification by eliminating QTLs in both parent strains and repeating the QTL mapping with pooled-segregant whole-genome sequence analysis. We first mapped multiple QTLs responsible for high thermotolerance in a natural yeast strain, MUCL28177, compared to the laboratory strain, BY4742. Using single and bulk reciprocal hemizygosity analysis we identified MKT1 and PRP42 as causative genes in QTLs linked to the superior and inferior parent, respectively. We subsequently downgraded both parents by replacing their superior allele with the inferior allele of the other parent. QTL mapping using pooled-segregant whole-genome sequence analysis with the segregants from the cross of the downgraded parents, revealed several new QTLs. We validated the two most-strongly linked new QTLs by identifying NCS2 and SMD2 as causative genes linked to the superior downgraded parent and we found an allele-specific epistatic interaction between PRP42 and SMD2. Interestingly, the related function of PRP42 and SMD2 suggests an important role for RNA processing in high thermotolerance and underscores the relevance of analyzing minor QTLs. Our results show that identification of minor QTLs involved in complex traits can be successfully accomplished by crossing parent strains that have both been downgraded for a single QTL. This novel approach has the advantage of maintaining all relevant genetic diversity as well as enough phenotypic difference between the parent strains for the trait-of-interest and thus maximizes the chances of successfully identifying additional minor QTLs that are relevant for the phenotypic difference between the original parents.
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