Reduction-oxidation poise regulates the sign of phototaxis in Chlamydomonas reinhardtii Ken-ichi Wakabayashi1, Yuka Misawa, Shota Mochiji, and Ritsu Kamiya Department of Biological Sciences, Graduate School of Science, University of Tokyo. 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 Japan Edited by Susan S. Golden, University of California at San Diego, La Jolla, CA, and approved May 26, 2011 (received for review January 12, 2011) In many phototrophic microorganisms and plants, chloroplasts known to change the cytoplasmic redox poise greatly (11, 12), we change their positions relative to the incident light to achieve surmised that redox balance might play a role in switching the optimal photosynthesis. In the case of motile green algae, cells sign of phototaxis. To test this hypothesis, we examined photo- change their swimming direction by switching between positive taxis in Chlamydomonas after artificially changing the cellular and negative phototaxis, i.e., swimming toward or away from the redox poise. We report here that the sign of phototaxis is, in fact, light source, depending on environmental and internal conditions. determined by the cellular redox poise. However, little is known about the molecular signals that de- termine the phototactic direction. Using the green alga Chlamydo- Results monas reinhardtii, we found that cellular reduction-oxidation To change the Chlamydomonas cellular redox poise, we used two (redox) poise plays a key role: Cells always exhibited positive pho- groups of membrane-permeable redox reagents: reactive oxygen totaxis after treatment with reactive oxygen species (ROS) and species (ROS), which cause oxidative stress (group 1), and always displayed negative phototaxis after treatment with ROS reagents that quench ROS and reduce oxidative stress (group 2). quenchers. The redox-dependent switching of the sign of photo- We used H2O2, tertiary butyl hydroperoxide (t-BOOH), and taxis may contribute in turn to the maintenance of cellular redox Neutral Red as group 1 and 4-hydroxy-2, 2, 6, 6-tetramethyl homeostasis. piperidine 1-oxyl (TEMPOL), dimethylthiourea (DMTU), and L-histidine as group 2. Cells were treated with these reagents at hototaxis is a behavior in which organisms move toward or concentrations sufficient to cause oxidative stress (group 1) (13) Paway from directional light (“positive” or “negative” photo- or reductive stress (group 2) (14). Following reagent treatment, PLANT BIOLOGY taxis, respectively). For motile phototrophic organisms such as phototactic behavior was observed by positioning a green light- − − green algae, switching between positive and negative phototaxis is emitting diode (LED) (λ = 525 nm, ∼50 μmol photons m 2 s 1) crucial for maintaining the optimal light exposure for photosyn- adjacent to one side of the culture dish. thesis: They need to harvest light energy, but when light intensity We observed both a wild-type and a negatively phototactic is far above the saturation point of photosynthesis, they suffer strain, agg1 (15). First, to observe the net movement of cells, cell from high-light stress (1). Thus, the direction or “sign” of pho- suspensions were illuminated with directional green light. Wild- totaxis should be tightly controlled. The biflagellate unicellular type cells showed positive phototaxis, whereas agg1 cells exhibited green alga Chlamydomonas reinhardtii provides useful advantages negative phototaxis (Fig. 1). However, after treatment with an for the study of the phototactic sign. Cells quickly change their ROS (H2O2), cells of both strains came to display positive pho- swimming direction following light perception by channelrho- totaxis. Conversely, both strains came to show negative phototaxis dopsin (2). Many phototaxis-deficient mutants have been iso- after treatment with an ROS quencher (TEMPOL) (Fig. 1). lated, including a strain that exhibits only negative phototaxis We next examined whether individual cells orient themselves [agg1 (phototactic aggregation), as discussed below]. to the direction of light, to ensure that the photo-accumulation Several extracellular factors have been shown to affect the sign was brought about by the cells’ phototaxis rather than by a pho- of phototaxis in Chlamydomonas (3). Most importantly, light tophobic response. For this purpose, we measured the angle (θ) intensity controls the sign, which is positive under low light and between individual cell’s swimming direction and the light di- negative under high light (4). Extracellular calcium concentra- rection 15 s after the onset of stimulation with directional light tion also affects the sign, an effect that is consistent with the (Fig. 2A). After treatment with two kinds of ROS, both wild-type calcium-dependent regulation of the beating balance between and agg1 cells exhibited positive phototaxis; in contrast, after the two flagella (5, 6). However, even under light and medium treatment with the two kinds of ROS quenchers, both strains conditions that are known to change the phototactic sign, there exhibited negative phototaxis (Fig. 2B and Movies S1, S2, S3, and almost always is a population of cells that shows phototaxis of the S4). The threshold concentrations of the ROS and ROS opposite sign. In addition, after prolonged light stimulation, cells quenchers needed for the sign switching varied slightly from one exhibit adaptation and become less sensitive to the light signal. experiment to another, presumably depending on the culture Thus, the effect of the extracellular factors must vary depending conditions. However, the difference in the threshold concen- on the internal condition of each cell. Factors that have been trations was always within ∼50% of the concentrations indicated thought to affect the internal conditions include circadian in Fig. 2B. Under all conditions, cells of oda1 (outer dynein arm), rhythm and photosynthetic activity (7–9). Circadian rhythm dy- a mutant that lacks the redox-sensitive outer arm dynein (14, namically affects the magnitude of phototaxis, but whether it also 16, 17), displayed almost the same flagellar beat frequency as affects the sign of phototaxis is not known (7). In contrast, the control cells (Table S1), indicating that the ROS or ROS photosynthetic activity clearly affects the sign: Cells of a certain wild-type Chlamydomonas that show negative phototaxis under normal light conditions show positive phototaxis when treated Author contributions: K.W. and R.K. designed research; K.W., Y.M., and S.M. performed with an inhibitor of photosynthesis, 3-(3′,4′-dichlorophenyl)-l,l- research; K.W., Y.M., and S.M. analyzed data; and K.W. and R.K. wrote the paper. dimethylurea (DCMU) (8). The molecular mechanism under- The authors declare no conflict of interest. lying this phenomenon is unknown. This article is a PNAS Direct Submission. Eukaryotic cells possess multiple systems for maintaining the 1To whom corresponding should be addressed. E-mail: [email protected]. “ cytoplasm in a moderately reduced state called reduction- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. oxidation (redox) homeostasis” (10). Because photosynthesis is 1073/pnas.1100592108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1100592108 PNAS Early Edition | 1of5 Downloaded by guest on September 26, 2021 two flagella, which causes the phototactic turning (21). The fifth step may occur because the sensitivity to Ca2+ differs in the flagellum closer to the eyespot (the cis-flagellum) and the fla- gellum farther from the eyespot (the trans-flagellum). Previous studies showed that the cis-flagellar axoneme beats more strongly than the trans-axoneme when demembranated cells are reac- − tivated with ATP at <10 8 MCa2+, whereas the trans-axoneme − − beats more strongly than the cis-axoneme at 10 7 Mto10 6 M Ca2+ (6, 22). Because this dominance switching may well reflect the mechanism of phototactic turning, we examined whether it is affected by redox. However, we found that the redox reagents did not change the flagellar dominance in either wild-type or agg1 cells (Fig. S2). This result suggests that redox might affect some process or processes upstream of the flagellar dominance control (Discussion). Discussion We have demonstrated clear switching of the phototactic sign in Fig. 1. The redox reagents changed the Chlamydomonas swimming direc- Chlamydomonas tions. (A) Typical ROS generated from oxygen and ROS-quenching reagents. with ROS and their quenchers. These data Various ROS are produced by partial reduction of oxygen: a superoxide show that cytoplasmic redox poise that is affected by certain − − anion (O2 ), H2O2, and a hydroxyl radical (·OH). t-BOOH has an effect similar kinds of ROS (H2O2 and O2 ) regulates the sign of phototaxis at 1 to that of H2O2. Singlet oxygen, O2, is generated by photosensitizers such as both cell-population and single-cell levels. Previously, photo- chlorophylls in vivo. Membrane-permeable ROS and ROS quenchers used in synthetic activity has been shown to affect the sign of phototaxis this study are shown in red and blue, respectively. (B) Dish phototaxis assay. (8); however, how photosynthesis affects the sign was unclear. Cell suspensions, with or without redox reagents, were photographed after The results of experiments using various redox modulators and illumination from the right side for 15 min (green arrows). Without redox experiments using a photosynthesis-deficient strain suggest that reagents, wild-type and agg1 cells showed positive and negative phototaxis, the redox poise,