Universität zu Köln

Bashir, Faiza, Kovacs, Sandor, Abraham, Agnes, Nagy, Krisztina, Ayaydin, Ferhan, Valkony-Kelemen, Ildiko, Ferenc, Gyorgyi, Galajda, Peter ORCID: 0000-0003-3308-088X, Toth, Szilvia Z. ORCID: 0000-0003-3419-829X, Sass, Laszlo, Kos, Peter B., Vass, Imre and Szabo, Milan (2022). Viable protoplast formation of the coral endosymbiont alga Symbiodinium spp. in a microfluidics platform. Lab Chip, 22 (16). S. 2986 - 3000. CAMBRIDGE: ROYAL SOC CHEMISTRY. ISSN 1473-0189

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Abstract

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.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Bashir, Faiza UNSPECIFIED UNSPECIFIED UNSPECIFIED Kovacs, Sandor UNSPECIFIED UNSPECIFIED UNSPECIFIED Abraham, Agnes UNSPECIFIED UNSPECIFIED UNSPECIFIED Nagy, Krisztina UNSPECIFIED UNSPECIFIED UNSPECIFIED Ayaydin, Ferhan UNSPECIFIED UNSPECIFIED UNSPECIFIED Valkony-Kelemen, Ildiko UNSPECIFIED UNSPECIFIED UNSPECIFIED Ferenc, Gyorgyi UNSPECIFIED UNSPECIFIED UNSPECIFIED Galajda, Peter UNSPECIFIED orcid.org/0000-0003-3308-088X UNSPECIFIED Toth, Szilvia Z. UNSPECIFIED orcid.org/0000-0003-3419-829X UNSPECIFIED Sass, Laszlo UNSPECIFIED UNSPECIFIED UNSPECIFIED Kos, Peter B. UNSPECIFIED UNSPECIFIED UNSPECIFIED Vass, Imre UNSPECIFIED UNSPECIFIED UNSPECIFIED Szabo, Milan UNSPECIFIED UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-681618
DOI:	10.1039/d2lc00130f
Journal or Publication Title:	Lab Chip
Volume:	22
Number:	16
Page Range:	S. 2986 - 3000
Date:	2022
Publisher:	ROYAL SOC CHEMISTRY
Place of Publication:	CAMBRIDGE
ISSN:	1473-0189
Language:	English
Faculty:	Unspecified
Divisions:	Unspecified
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language SINGLET OXYGEN; OXIDATIVE STRESS; MICROADRIATICUM FREUDENTHAL; ULTRAVIOLET-RADIATION; THERMAL TOLERANCE; THECAL PLATES; SP NOV.; DINOFLAGELLATE; PHOTOSYNTHESIS; BIOTECHNOLOGY Multiple languages Biochemical Research Methods; Chemistry, Multidisciplinary; Chemistry, Analytical; Nanoscience & Nanotechnology; Instruments & Instrumentation Multiple languages
URI:	http://kups.ub.uni-koeln.de/id/eprint/68161