Sodium citrate induces apoptosis in biocontrol yeast Cryptococcus laurentii

Aim: To provide the observation that sodium citrate induced apoptosis in biocontrol yeast Cryptococcus laurentii. Methods and Results: The viability of the yeast cells was evaluated using the percentage of colony-forming units (CFU) of treated cells. The induction of cell death was dependent on the concentration of sodium citrate and exhibited typical apoptotic markers such as phosphatidylserine (PS) translocation as shown by annexin V coupled with fluorescein isothiocyanate (FITC) labelling and DNA fragmentation as detected by TdT-mediated dUTP-biotin nick end labelling (TUNEL) assay. The annexin V-positive cells reached the maximum (14.8%) on the third day, whereas TUNEL-positive cells increased gradually from 5.92 to 27.9% within 5 days of incubation in sodium citrate. In addition, confocal laser microscopy and flow cytometric analysis revealed that the induction of apoptosis was associated with the production of reactive oxygen species (ROS) that reached the highest intracellular level in the first day, before the peak of the early event (PS exposure) in apoptosis. The apoptosis was delayed by the addition of antioxidant glutathione (GSH), suggesting that ROS generated in this process plays a key role in the regulation of the apoptosis in C.laurentii cells. Conclusions: This study indicated that the apoptotic signals in C.laurentii are dependent on citrate ions and/or sodium ions, the concentration and initial acidity of sodium citrate. Induction of ROS in response to sodium citrate plays a significant role in apoptosis. Significance and Impact of the Study: Yeast Cryptococcus laurentii has been selected as an effective biocontrol indicator for the postharvest diseases because of its competition for nutrients and space with the pathogen in the wound of fruits. This study presents a convenient method for commercial production of yeast as biocontrol agent.