AUTHOR=Tamaki Shun , Sato Ryosuke , Koshitsuka Yuki , Asahina Masashi , Kodama Yutaka , Ishikawa Takahiro , Shinomura Tomoko 

TITLE=Suppression of the Lycopene Cyclase Gene Causes Downregulation of Ascorbate Peroxidase Activity and Decreased Glutathione Pool Size, Leading to H2O2 Accumulation in Euglena gracilis

JOURNAL=Frontiers in Plant Science

VOLUME=12

YEAR=2021

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2021.786208

DOI=10.3389/fpls.2021.786208

ISSN=1664-462X

ABSTRACT=<p>Carotenoids are photosynthetic pigments and hydrophobic antioxidants that are necessary for the survival of photosynthetic organisms, including the microalga <italic>Euglena gracilis</italic>. In the present study, we identified an uncharacterized gene encoding the <italic>E. gracilis</italic> β-carotene synthetic enzyme lycopene cyclase (EgLCY) and discovered a relationship between EgLCY-mediated carotenoid synthesis and the reactive oxygen species (ROS) scavenging system ascorbate-glutathione cycle. The <italic>EgLCY</italic> cDNA sequence was obtained <italic>via</italic> homology searching <italic>E. gracilis</italic> transcriptome data. An enzyme assay using <italic>Escherichia coli</italic> demonstrated that EgLCY converts lycopene to β-carotene. <italic>E. gracilis</italic> treated with <italic>EgLCY</italic> double-stranded RNA (dsRNA) produced colorless cells with hypertrophic appearance, inhibited growth, and marked decrease in carotenoid and chlorophyll content, suggesting that EgLCY is essential for the synthesis of β-carotene and downstream carotenoids, which are abundant and physiologically functional. In <italic>EgLCY</italic> dsRNA-treated cells, the ascorbate-glutathione cycle, composed of ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), monodehydroascorbate reductase (MDAR), and glutathione reductase (GR), was unusually modulated; APX and GR activities significantly decreased, whereas DHAR and MDAR activities increased. Ascorbate content was significantly increased and glutathione content significantly decreased in <italic>EgLCY</italic> dsRNA-treated cells and was correlated with their recycling enzyme activities. Fluorescent imaging demonstrated that <italic>EgLCY</italic> dsRNA-treated cells accumulated higher levels of H<sub>2</sub>O<sub>2</sub> compared to wild-type cells. Taken together, this study revealed that EgLCY-mediated synthesis of β-carotene and downstream carotenoid species upregulates APX activity and increases glutathione pool size for H<sub>2</sub>O<sub>2</sub> scavenging. Our study suggests a possible relationship between carotenoid synthesis and the ascorbate-glutathione cycle for ROS scavenging in <italic>E. gracilis</italic>.</p>