A Mediator of Singlet Oxygen Responses in Chlamydomonas reinhardtii and Arabidopsis Identified by a Luciferase-Based Genetic Screen in Algal Cells

Abstract

ORCID ID: 0000-0001-7502-6940 (R.B.).All cells produce reactive oxygen species (ROS) as by-products of their metabolism. In addition to being cytotoxic, ROS act as regulators of a wide range of developmental and physiological processes. Little is known about the molecular mechanisms underlying the perception of ROS and initiation of cellular responses in eukaryotes. Using the unicellular green alga Chlamydomonas reinhardtii, we developed a genetic screen for early components of singlet oxygen signaling. Here, we report the identification of a small zinc finger protein, METHYLENE BLUE SENSITIVITY (MBS), that is required for induction of singlet oxygendependent gene expression and, upon oxidative stress, accumulates in distinct granules in the cytosol. Loss-of-function mbs mutants produce singlet oxygen but are unable to fully respond to it at the level of gene expression. Knockout or knockdown of the homologous genes in the higher plant model Arabidopsis thaliana results in mutants that are hypersensitive to photooxidative stress, whereas overexpression produces plants with elevated stress tolerance. Together, our data indicate an important and evolutionarily conserved role of the MBS protein in ROS signaling and provide a strategy for engineering stress-tolerant plants.

Uncoupling High Light Responses from Singlet Oxygen Retrograde Signaling and Spatial-Temporal Systemic Acquired Acclimation

“…One explanation for this is that flu 1 O 2 signals are thought to be uncoupled from HL responses and have previously been shown to have an immediate cytotoxic effect on chloroplast membrane integrity, which is absent when 1 O 2 is produced as a result of HL at PSII in Col-0 (Lee et al, 2007;Mullineaux and Baker, 2010;Kim et al, 2012). A commonly used 1 O 2 stress marker, AAAATPase, was similarly absent in these conditions as well as in HL treated wild-type tissues under the conditions used herein, and in other studies using similar light regimes (Rossel et al, 2007;Shao et al, 2013). Similarly, FER1 was not induced in local tissue; however, there was a small but significant induction in distal tissues in contrast to wild-type HL-treated tissue where this gene was strongly induced (Fig.…”

“…AAA-ATPase is therefore a useful marker to compare the HL conditions of this study with others that have used much harsher conditions to study cell death signaling (Supplemental Fig. S1; Shao et al, 2013). Very high induction of the 1 O 2 and SAA-responsive marker, NodL, was seen in treated and distal tissues (NodL, P , 0.01).…”

Entire Photodamaged Chloroplasts Are Transported to the Central Vacuole by Autophagy

“…An aliquot of the synthesized cDNA derived from 4.0 ng total RNA was subjected to qRT-PCR analysis using the KAPA SYBR FAST qPCR Kit (KAPA Biosystems) using a real-time PCR detection system (CFX96, Bio-Rad). Analyses of dark-inducible genes and photooxidative stress-marker genes were described in previous studies (Baena-González et al, 2007;Ramel et al, 2012;Shao et al, 2013). The level of 18S rRNA was measured as an internal standard (Izumi et al, 2012).…”

Photo-Oxidative Stress during Leaf, Flower and Fruit Development

“…Indeed, carotenoid oxidation products generated during 1 O 2 scavenging, such as b-cyclocitral, also have been shown to be essential in transducing signals triggered by photooxidative stress and specific 1 O 2 signatures (Ramel et al, 2012). Furthermore, a small zinc finger protein, MBS, is required to induce 1 O 2 -dependent gene expression (Shao et al, 2013). Interestingly, however, mbs1 mutants supplemented with b-cyclocitral are unable to positively regulate antioxidant-related genes that are usually activated in the wild type, in which the zinc finger transcription factor MBS1 is fully active and enhances plant tolerance to high levels of light (Shumbe et al, 2017).…”