Autofluorescence-based high-throughput isolation of nonbleaching Cyanidioschyzon merolae strains under nitrogen-depletion

Nozomi Takeue; Ayaka Kuroyama; Yoshiharu Hayashi; Kan Tanaka; Sousuke Imamura

doi:10.3389/fpls.2022.1036839

Autofluorescence-based high-throughput isolation of nonbleaching Cyanidioschyzon merolae strains under nitrogen-depletion

Front Plant Sci. 2022 Dec 14:13:1036839. doi: 10.3389/fpls.2022.1036839. eCollection 2022.

Authors

Nozomi Takeue^{1

2}, Ayaka Kuroyama³, Yoshiharu Hayashi³, Kan Tanaka², Sousuke Imamura^{2

4}

Affiliations

¹ School of Life Science and Technology, Tokyo Institute of Technology, Midori-ku, Yokohama, Japan.
² Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology, Midori-ku, Yokohama, Japan.
³ Product and Business Planning Section, Planning and Marketing Department, Life Science Business Division, Medical Business Group, Sony Corporation, Nishi-ku, Yokohama, Japan.
⁴ Space Environment and Energy Laboratories, Nippon Telegraph and Telephone Corporation, Musashino-shi, Tokyo, Japan.

Abstract

Photosynthetic organisms maintain optimum levels of photosynthetic pigments in response to environmental changes to adapt to the conditions. The identification of cyanobacteria strains that alleviate bleaching has revealed genes that regulate levels of phycobilisome, the main light-harvesting complex. In contrast, the mechanisms of pigment degradation in algae remain unclear, as no nonbleaching strains have previously been isolated. To address this issue, this study attempted to isolate nonbleaching strains of the unicellular red alga Cyanidioschyzon merolae after exposure to nitrogen (N)-depletion based on autofluorescence information. After four weeks under N-depletion, 13 cells from 500,000 cells with almost identical pre- and post-depletion chlorophyll a (Chl a) and/or phycocyanin autofluorescence intensities were identified. These nonbleaching candidate strains were sorted via a cell sorter, isolated on solid medium, and their post-N-depletion Chl a and phycocyanin levels were analyzed. Chl a levels of these nonbleaching candidate strains were lower at 1-4 weeks of N-depletion similar to the control strains, however, their phycocyanin levels were unchanged. Thus, we successfully isolated nonbleaching C. merolae strains in which phycocyanin was not degraded under N-depletion, via autofluorescence spectroscopy and cell sorting. This versatile method will help to elucidate the mechanisms regulating pigments in microalgae.

Keywords: flow cytometer; nitrogen-depletion; nonbleaching strain; phycocyanin; red alga.