An RT-qPCR approach to study the expression of genes responsible for sugar assimilation during rehydration of active dry yeast.
A short reactivation period in aqueous media is required for active dry yeast (ADY) to be utilised in winemaking. Rehydration restores the active metabolic conditions necessary for good fermentative and competitive abilities. We used a reverse transcription-quantitative PCR (RT-qPCR) method with relative quantification to investigate the expression of seven hexose transporter genes (HXT1-7) and one invertase-encoding gene (SUC2) during ADY rehydration in water with or without sucrose. For this, seven candidate reference genes were evaluated, and the three most stably expressed genes were selected and used for mRNA normalisation. The results show that, during the rehydration in the presence of sucrose, yeast cells are able to immediately hydrolyse this sugar into glucose and fructose as soon as they are introduced in the medium. Subsequently, differential glucose/fructose uptake occurs, which is mediated by hexose transporters. At the transcriptomic level, there is a strong induction of the high-affinity transporters, HXT2 and HXT4, and the low-affinity transporters, HXT3 and HXT1, when ADY is rehydrated with sucrose, while HXT5 and HXT6/7 are expressed at high levels with a moderate tendency to decrease. In water, the HXT2 gene was the only one of the transporter genes studied that showed significant variations. These results suggest that during rehydration, expression is not simply regulated by the affinity to hexose but is also controlled by other mechanisms that allow the cell to bypass glucose control. Moreover, the expression of SUC2 showed little variation in media with sucrose, suggesting that other invertases and/or posttranscriptional controls exist.
Vaudano
Enrico
E
CRA-Centro di Ricerca per l'Enologia, Via Pietro Micca, 35, 14100 Asti, Italy. biologia.molecolare@isenologia.it
Costantini
Antonella
A
Noti
Olta
O
Garcia-Moruno
Emilia
E
D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism; D005285:Fermentation; D005516:Food Microbiology; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D005947:Glucose / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins / Q000235:genetics* / Q000378:metabolism; D012331:RNA, Fungal / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism; D014867:Water / Q000378:metabolism; D014920:Wine / Q000382:microbiology*; D043324:beta-Fructofuranosidase / Q000235:genetics / Q000378:metabolism
D004271:DNA, Fungal; D005656:Fungal Proteins; D009004:Monosaccharide Transport Proteins; D012331:RNA, Fungal; D029701:Saccharomyces cerevisiae Proteins; D014867:Water; D043324:beta-Fructofuranosidase; D005947:Glucose