Symbiodiniaceae is an important dinoflagellate family which lives in endosymbiosis
with reef invertebrates, including coral polyps, making them central to the holobiont.
With coral reefs currently under extreme threat from climate change, there is a pressing
need to improve our understanding on the stress tolerance and stress avoidance mechanisms
of Symbiodinium spp. Reactive oxygen species (ROS) such as singlet oxygen are central
players in mediating various stress responses; however, the detection of ROS using
specific dyes is still far from definitive in intact Symbiodinium cells due to the
hindrance of uptake of certain fluorescent dyes because of the presence of the cell
wall. Protoplast technology provides a promising platform for studying oxidative stress
with the main advantage of removed cell wall, however the preparation of viable protoplasts
remains a significant challenge. Previous studies have successfully applied cellulose-based
protoplast preparation in Symbiodiniaceae; however, the protoplast formation and regeneration
process was found to be suboptimal. Here, we present a microfluidics-based platform
which allowed protoplast isolation from individually trapped Symbiodinium cells, by
using a precisely adjusted flow of cell wall digestion enzymes (cellulase and macerozyme).
Trapped single cells exhibited characteristic changes in their morphology, cessation
of cell division and a slight decrease in photosynthetic activity during protoplast
formation. Following digestion and transfer to regeneration medium, protoplasts remained
photosynthetically active, regrew cell walls, regained motility, and entered exponential
growth. Elevated flow rates in the microfluidic chambers resulted in somewhat faster
protoplast formation; however, cell wall digestion at higher flow rates partially
compromised photosynthetic activity. Physiologically competent protoplasts prepared
from trapped cells in microfluidic chambers allowed for the first time the visualization
of the intracellular localization of singlet oxygen (using Singlet Oxygen Sensor Green
dye) in Symbiodiniaceae, potentially opening new avenues for studying oxidative stress.
