Phototaxis in Chlamydomonas Reinhardtii

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 determine 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 flagellum 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, which is affected by photosynthetic activity, respectively. Both strains exhibited positive phototaxis after treatment with changes the phototactic sign of Chlamydomonas. However, 25 μMH2O2, and both showed negative phototaxis after treatment with 50 mM TEMPOL. singlet oxygen did not affect the sign of phototaxis, although it is generated by photosynthesis (23). Singlet oxygen in plants has been shown to function not as a toxin but as a signal to up- quenchers did not cause a significant change in cellular ATP regulate genes that induce stress responses (24). We surmise concentration. An inducer (Neutral Red) and a reducer (L-histi- that only ROS that function as a toxin may affect the photo- dine) of singlet oxygen did not affect the sign of phototaxis (Fig. tactic sign. 2C) (18, 19). This result indicates that only specific types of ROS What redox-sensitive factor or cellular process switches the affect the sign of phototaxis (Discussion). For a quantification of phototactic sign? Gene expression does not appear to be in- the sign of phototaxis after different treatments, the average volved in the switching, because cells change the phototactic sign < values of cosθ (Fig. 2 B and C) were measured as phototactic quite quickly ( 1 min) after the treatment with ROS or ROS indexes for individual cells (Fig. 3). The phototactic indexes were quenchers or after the washing out of these reagents. On the reversed when the wild-type cells were treated with TEMPOL or other hand, the cytoplasmic redox state may have widespread DMTU and when the agg1 cells were treated with H O or t- effects on cellular metabolism, although the ROS and ROS 2 2 quenchers used in this study apparently did not change the cel- BOOH. These data suggest that the sign of phototaxis is regulated fi by certain kinds of ROS. lular ATP level signi cantly as estimated from the motility of As mentioned earlier, a previous study has shown that the sign oda1 (Table S1). Thus, we cannot yet tell whether a redox sig- of phototaxis varies depending on the cell’s photosynthetic ac- naling pathway changes the phototactic sign directly or changes it tivity. To determine whether the redox-dependent change in indirectly as a result of simultaneous redox-dependent reactions. Nevertheless, we can ask whether the redox poise affects the phototactic sign observed in this study depends on some change phototactic sign by acting on flagella or on some process in the in photosynthetic activity, we examined dark-grown cells of y1a signal transduction pathway. If redox poise acts on the flagellar [a mutant lacking y1 (yellow when grown in the dark) gene], cells activity control, two previously reported proteins are worth in which chlorophyll synthesis is blocked (20). As shown in Fig. 4, considering as possible targets. First, a flavodoxin family protein, the dark-grown y1a cells showed the same change in the pho- Agg3p, present in the flagellar membrane/matrix, has been sug- totactic sign after treatment with the redox reagents as wild-type fi gested to be involved in the phototactic sign reversal (25). This and agg1 cells. These ndings suggest that ROS affected the sign possibility has arisen from the observation that Agg3p RNAi of phototaxis without involving a change in the photosynthetic strains showed strong negative phototaxis, like the mutant agg1 activity. Under control conditions, the dark-grown y1a cells used in this study. Flavodoxin acts as a reducer; that is, its oxi- y1a showed strong negative phototaxis. Because cells also dation is coupled with the reduction of other protein(s). If we showed strong negative phototaxis when the cells were grown assume that the oxidized state of Agg3p triggers the negative-to- under light and displayed photosynthesis (Fig. S1), it is likely that positive switching of the phototactic sign, the behavior of the y1a carries a mutation that produces negative phototaxis. Agg3 RNAi strain is consistent with our finding that negative What is the target of redox regulation in the phototaxis sig- phototaxis occurs under reduced cytoplasm conditions. The naling processes? Several steps have been postulated in the second protein to consider is Agg2p, a membrane protein im- phototaxis pathway: (i) photoreception of light at the eyespot; plicated in the regulation of the phototactic sign and localized (ii) cation influx through channelrhodopsins and local depo- at the base of flagella (25). Although we have shown that the − larization of the plasma membrane; (iii)Ca2+ influx (>10 7 M) properties of the Ca2+-dependent dominance change between into the flagella via flagellar voltage-dependent Ca2+ chan- the cis- and trans-flagella apparently are not influenced by the nels; (iv) activation of some Ca2+-sensitive flagellar/axonemal redox poise, some detergent-soluble components could regulate components; and (v) change in the beating balance between the the flagellar dominance and phototaxis in a redox-sensitive

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Fig. 2. The effect of redox reagents on the phototactic sign was dose dependent and reversible. (A) Cell swimming direction was measured for 1.5 s following 15 s illumination with a green LED to avoid the effects of photophobic response occurring immediately after the onset of the LED illumination. (B and C) Polar histograms depicting the percentage of cells moving in a particular direction relative to light illuminated from the right, with or without treatment with redox reagents (12 bins of 30°; n = 50 cells per condition). Experiments were done at least three times per condition, and representative results are shown. (B) Both strains showed random swimming when not illuminated with a green LED. Wild-type cells exhibited positive phototaxis, and agg1 cells exhibited negative phototaxis without treatment with any reagents. Note that wild-type cells exhibited negative phototaxis after treatment with ROS scavengers, whereas agg1 cells showed positive phototaxis after treatment with ROS (histograms in yellow boxes). After washout of the reagents, each strain reverted to the same phototaxis direction as control cells, suggesting that the effects of the reagents are reversible. (C) Neutral Red (NR) and L-histidine (His), an inducer and a scavenger of singlet oxygen (a type of ROS), respectively, did not affect the sign of phototaxis.

manner. It is interesting that NAD- and NADP-dependent ma- larization, Ca2+ entry, and a change in the activity of the two late dehydrogenases are present in the flagellar membrane/ma- flagella. A change in the photoreception step actually has been trix from the Chlamydomonas flagellar proteome database (26). shown to cause a reversal of the steering direction (31). Thus, Malate is a compound used indirectly to transport excess re- the photoreceptor may well be the target of the redox control. ducing equivalents from the chloroplast to the cytosol (27). Detailed comparison of the movement and photoreception fl Photosynthetic activity may change the redox poise of agellar properties between positively phototactic cells (28) and nega- cytosol through the functions of those enzymes. tively phototactic cells after treatment with redox reagents may It also is possible, however, that the redox poise affects pho- provide some clues to the mechanism of the reversal of toreception by modulating the eyespot or the subsequent signal phototactic sign. transduction process that leads to a change in flagellar activity. In conclusion, we have shown that the sign of phototaxis in As stated above, the phototactic behavior of a Chlamydomonas Chlamydomonas is switched by the cytoplasmic redox poise that cell most likely is based on the turning of its swimming path, caused by a transient imbalance between the propulsive forces is changed by certain kinds of ROS. This redox-controlled produced by the two flagella (28–30). Because the cell rotates switching of phototaxis direction may help maintain the redox around its long axis at a frequency of ∼2 Hz while swimming, the homeostasis in the cytoplasm (but not in the chloroplast) via the two flagella alternate their positions periodically. Thus, the regulation of photosynthesis. When the cytoplasm is oxidized, turning direction should reverse if the onset of the transient cells migrate toward light and increase photosynthesis activity, beating imbalance advances or delays by ∼0.25 s. Such a delay and thus the cytoplasm is reduced. When the cytoplasm becomes or advance could be produced at any of the stages of signal overly reduced, cells switch to negative phototaxis, the photo- transduction, which include photoreception, membrane depo- synthesis decreases, and the cytoplasm becomes oxidized. Such

Wakabayashi et al. PNAS Early Edition | 3of5 Downloaded by guest on September 26, 2021 Fig. 3. Phototactic indexes change signiﬁcantly after treatment with certain types of redox reagents. The phototactic Index (± SD) was calculated as an average value of cosθ in Fig. 2 (n = 50). When cells are not illuminated and swim in random directions, the phototactic index should be ∼0. When 100% of cells show clear positive or negative phototaxis, the phototactic index is 1 or −1, respectively. Asterisks indicate a signiﬁcant differences from control (illuminated, −16 no reagents) conditions (P < 1.0 × 10 , t test). His, L-histidine; NR. Neutral Red.

regulation may function as a negative-feedback mechanism that For dish assays, cell suspensions (∼107 cells/mL) were put in petri dishes maintains the moderately reduced state of the cytoplasm. (30 mm in diameter, 10 mm thick), illuminated with a green LED from one side for 15 min, and photographed (DMC-LX2; Panasonic). Materials and Methods For single-cell analysis, petri dishes were placed on the stage of an inverted − microscope (IX70; Olympus) and illuminated with a green LED. The behavior Cell Culture and Strains. Chlamydomonas reinhardtii strains CC124 (nit1 − − − − of the cells was observed with dim red light (λ >600 nm, ∼5 μmol photons (nitrate reductase), nit2 , agg1 ,mt(mating type)) (15), CC125 (nit1 , − − m 2 s 1) and was video recorded using a CCD camera (XC-75; Sony). The nit2−,mt+) CC1168 (y1−,mt+) (20), and CC2228 (oda1−,mt+) (16) were used angle (θ) between the light direction and the swimming direction was and are referred to as “agg1,”“wild type,”“y1a,” and “oda1,” respec- measured during 1.5 s following illumination with a green LED for 15 s. tively. Cells were grown in tris-acetate phosphate medium (32) with aera- Images of swimming cells were auto-tracked using Image Hyper software tion at 20 °C on a 12-h/12-h light/dark cycle. The y1a cells were grown in (Science Eye). The angles were calculated from the cell trajectories. 24-h darkness. Beat Frequency Analysis. Flagellar beat frequency was determined from the ′ Reagents. DMTU, Neutral Red, Igepal CA-630, and 5,5 -dithiobis(2-nitrobenzoic power spectra of cell body vibration signals in microscope images of a pop- acid) (DTNB) were purchased from Sigma-Aldrich. ATP, creatine kinase, and ulation of cells (36). Median frequency was obtained from the power spectra creatine phosphate were purchased from Roche Diagnostics. All other averaged for 20 s. chemicals were purchased from Wako Pure Chemical Industries. Reactivation of Demembranated Cell Models. Cell models were prepared and Phototaxis Assay. The phototactic behaviors of Chlamydomonas cells were analyzed by themethod of Kamiyaand Witman (6), with modifications. Calcium fi quanti ed by recording and analyzing their behavior under continuous buffers for reactivation were prepared according to Wakabayashi et al. (37), unidirectional illumination using a green LED (λ =25nm,∼50 μmol pho- with modifications. In brief, after cells were washed with a buffer containing 10 −2 −1 tons m s ). The photon flux density was measured with an LI-1000 Data mM Hepes (pH 7.4) and 4% sucrose, cells were demembranated with HMKP

Logger (LI-COR Inc.). The wavelength of the light source was selected to be buffer [30 mM Hepes, 5 mM MgSO4, 50 mM K-acetate, 1% polyethylene glycol close to the maximum absorption wavelength (500 nm) of Chlamydomo- (Mr 20,000)] containing 0.1% Igepal CA-630. Cell models were reactivated in nas channelrhodopsin 1, the photoreceptor functioning in phototaxis (33– four different reactivation buffers with either Ca2+-free or Ca2+ buffer and with 35). Cells were washed with an experimental solution [5 mM Hepes (pH either reducing or oxidizing buffer. All buffers were made in the HMKP buffer 7.4), 0.2 mM EGTA, 1 mM KCl, 0.3 mM CaCl2] (22), shaken gently under containing 1 mM ATP, 70 U/mL creatine kinase, and 5 mM creatine phosphate. white light for 1 h, and kept under dim red light for 15 min before the To change the concentration of free Ca2+, either 1 mM EGTA or 5.2 mM EGTA 2+ −8 phototaxis assays. plus 3.2 mM CaCl2 [which results in a final free-Ca concentration of ∼3.9 × 10 M(∼pCa7.4)] was added. These buffers are referred to as “Ca2+-free buffer” and “+Ca2+ buffer,” respectively. To change the redox state, either 1 mM DTT or 10 μM DTNB (17) was added. These buffers are referred to as “reducing buffer” and “oxidizing buffer,” respectively. Cell models reactivated in either buffer solution were observed under a dark-field microscope. Cells that were rotating in small areas by predominantly beating one of the two flagella were chosen, and the dominance of the cis-ortrans-flagellum (the flagellum closer to the eyespot or farther from the eyespot) over the other was determined by visual inspection (6).

Fig. 4. The redox reagents changed the phototactic sign of a chlorophyll- ACKNOWLEDGMENTS. We thank Benjamin D. Engel for his critical reading deﬁcient mutant. The y1a strain has a defect in chlorophyll synthesis when of the manuscript, Takahiro Ide and Yuta Mitani for assistance with the phototaxis assays, and Drs. Ichiro Terashima and Ko Noguchi for the grown in the dark. Dark-grown y1a cells showed strong negative phototaxis measurement of photosynthetic activity of Chlamydomonas cells and for without treatment with redox reagents. Treatment of y1a cells with redox helpful discussions. This work was supported by grants from the Japan So- reagents changed the phototactic sign in the same manner as in agg1 cells. ciety for Promotion of Sciences and the Asahi Glass Foundation (to K.W.) and These data suggest that the phototactic sign was changed directly by the a by grant from the Ministry of Education, Culture, Sports, Science and cytoplasmic redox poise, not by a change in photosynthetic activity. Technology (to K.W. and R.K.).

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