Eyespot-dependent determination of the phototactic sign in Chlamydomonas reinhardtii

Abstract

The biflagellate green alga Chlamydomonas reinhardtii exhibits both positive and negative phototaxis to inhabit areas with proper light conditions. It has been shown that treatment of cells with reactive oxygen species (ROS) reagents biases the phototactic sign to positive, whereas that with ROS scavengers biases it to negative. Taking advantage of this property, we isolated a mutant, lts1-211, which displays a reduction-oxidation (redox) dependent phototactic sign opposite to that of the wild type. This mutant has a single amino acid substitution in phytoene synthase, an enzyme that functions in the carotenoid-biosynthesis pathway. The eyespot contains large amounts of carotenoids and is crucial for phototaxis. Most lts1-211 cells have no detectable eyespot and reduced carotenoid levels. Interestingly, the reversed phototactic-sign phenotype of lts1-211 is shared by other eyespot-less mutants. In addition, we directly showed that the cell body acts as a convex lens. The lens effect of the cell body condenses the light coming from the rear onto the photoreceptor in the absence of carotenoid layers, which can account for the reversed-phototactic-sign phenotype of the mutants. These results suggest that light-shielding property of the eyespot is essential for determination of phototactic sign.

Keywords: Chlamydomonas; carotenoids; eyespot; lens; phototaxis.

Fig. 1.

Schematic diagrams of a Chlamydomonas cell and its phototactic behavior. (Top) The eyespot is located near the cell equator and contains the carotenoid granule layers (red) and photoreceptor proteins, channelrhodopsins (ChR1 and ChR2; blue). The carotenoid layers reflect a light beam (orange arrows) and amplify the light signal from the outside of the cell on ChR (the “front side”) and block the light from the inside of the cell (the “rear side”). The flagellum closest to the eyespot is called the cis-flagellum, whereas the other one is called the trans-flagellum. Modified from refs. and . (Bottom) As the cell swims with self-rotation, the eyespot apparatus scans the incident light around the cell’s swimming path. After photoreception by the channelrhodopsins, the cell changes the beating balance of the two flagella and exhibits either positive or negative phototaxis (swimming toward or away from the light source, respectively).

Fig. 2.

The lts1-211 mutant lacks eyespots and exhibits the opposite sign of phototaxis relative to the wild type. (A) Dish phototaxis assays of the wild type, lts1-211, and lts1-211R (rescued strain) with or without treatment with redox reagents. Cell suspensions in Petri dishes were photographed after illumination with a green light-emitting diode (LED) from the side for 5 min (green arrows). The areas without cells on the horizontal axis (e.g., ROS scavenger-treated lts1-211R) are likely caused by the photophobic responses of some cells. (B) Polar histograms representing the percentage of cells moving in a particular direction relative to light illumination from the right (12 bins of 30°; n = 20–30 cells per condition) for 1.5 s following 15-s illumination. (C) The sign of phototactic index in lts1-211 (gray) is opposite to that of WT or lts-211R (black) with or without treatment with redox reagents. The phototactic index was calculated as an average value of cosθ in B. 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. (D) lts1-211 produces less carotenoids than the wild type. β-Carotene and lutein levels in each strain (PSY null mutants lts1-202 and lts1-30 cells were grown in the dark) are shown [average values ± SEM for six (WT, lts1-211 and lts1-211R) or three (PSY null mutants) independently prepared samples]. Asterisks represent significant differences (P < 0.05, paired t test). (E) Bright-field images of the wild-type, lts1-211, and lts1-211R cells. Note that lts1-211 is eyespot-less.

Fig. 3.

Phyotene synthtase gene in lts1-211 and genetic/phenotypic differences from the other lts1 alleles. (A) Structure of the Chlamydomonas PSY gene and the mutation in lts1-211 (mid). DNA and amino acid sequences in the vicinity of the mutation in exon 2 in the wild-type and lts1-211 genomes (Top) are shown. For the rescue experiment, lts1-211 was transformed with a 6,000-kb DNA fragment containing the PSY gene, which was cloned into pSI103 plasmid (Bottom) (42). (B) Domain structure of PSY. The P159I mutation in lts1-211 occurs in the catalytic domain of PSY. Mutations in the previously reported PSY null mutants are also shown as follows: In lts1-30, W123 is substituted for a stop codon, whereas in lts1-202 (previously called FN68), a frameshift occurs (16). (C) Part of the carotenoid-biosynthesis pathway in Chlamydomonas modified from ref. . PSY (boxed) synthesizes phytoene from geranylgeranyl-diphosphate. β-Carotene and lutein, the two major carotenoids in Chlamydomonas analyzed in Fig. 2D, are underlined. (D) Growth phenotypes of the wild type, lts1-211, and two PSY null mutants. Cell suspensions from each mutant containing ∼10⁵ cells were spotted onto TAP-agar plates and incubated in the light (∼50 µmol photons⋅m⁻²⋅sec⁻¹; Top) or dark (Bottom) for 3 d.

Fig. S1.

Identification of the mutant gene in lts1-211. (A) lts1-211 was mapped to a 131-kb region on chromosome 11. (B) A series of magnified screen shots from genome viewer IGV showing the genomes of lts1-211, WT, and CC124, which were compared with the genome sequence database based on CC503/cw92 (Top; blue bars indicate genes, and thick bars indicate exons). Gray bars in each genome indicate the paired-end reads of Illumina sequencing, in which colored regions indicate mutations, as detected via comparisons to the database. Visual comparison of the data revealed only one missense mutation in PSY (CCC to ATC, leading to a substitution of isoleucine for proline) in the mapped region.

Fig. S2.

Dish phototaxis assay of the wild type, lts1-211 and lts1-211R without redox reagents at various light intensities. Cell suspensions in Petri dishes were photographed after illumination with a green LED from the side for 5 min (green arrows). Against weak light (∼0.3 μmol photons⋅m⁻²⋅s⁻¹), WT and lts1-211R showed positive phototaxis, whereas lts1-211 did not show significant phototaxis (Left). As the light intensity gets stronger (Middle and Right), more cells of WT and lts1-211R showed negative, and those of lts1-211 showed positive phototaxis.

Fig. S3.

Motility analyses of lts1-211. Swimming velocity (average ± SD: n = 47 for WT and n = 60 for lts1-211) (A) and flagellar beat frequency (average ± SEM of three independent experiments) (B). Analysis showing that lts1-211 swims at almost the same speed as, or slightly faster than, the wild type. (C) Dominance between the two flagella in demembranated cell models, reactivated with 1 mM ATP with and without submicromolar Ca²⁺ (pCa 7.4). No significant differences were detected between the wild-type and lts1-211 strains. Average data from three independent experiments are shown (n = 32–121 per experiment).

Fig. 4.

All eyespot-deficient mutants show a redox-dependent sign of phototaxis opposite to that of the wild type. (A) Cell images, dish phototaxis assays, and polar histograms of eye1-1, eye2-1, and eye3, with or without treatment with redox reagents (12 bins of 30°; n = 24–56 cells per condition). (B) Phototactic index calculated as an average value of cosθ measured in A. After treatment with redox reagents, all eyeless mutants showed signs of phototaxis opposite to those of strains with eyespots (wild type and lts1-211R) and same as lts1-211 (Fig. 2C).

Fig. 5.

The Chlamydomonas cell body has a lens effect. (A) Wild-type and lts1-211 cells were observed with bright-field illumination (Left) or with sideways illumination (Middle and Right; yellow arrows indicate the direction of illumination). A small bright area is observed in each cell on the side opposite the light source. (B) The letter “P” (for “photo”) set on a field stop ring of the microscope was imaged through the cells of both strains by the lens effect. The letter “P” appeared on each cell as the plane of focus was moved from the cells (Left) to above the cells (Right). (C) The setting of the microscope and a hypothetical optical path are shown. I, image; L, cell as a lens; O, object.

Fig. 6.

Model illustrating the effect of light illumination on the photoreceptors and the phototactic sign of the wild type (Top) and eyeless mutants (Bottom). Carotenoid layers (red) reflect and amplify the light signal (orange arrows) onto the photoreceptors (blue) when the eyespot faces the light source. These layers shield the photoreceptors from the light condensed by the lens effect of the cell when the eyespot faces the side opposite the light source. The photoreceptors in an eyeless mutant cell localize to several patches around the “correct” position but function normally (24). The photoreceptors receive stronger light stimulation when facing away from the light source, i.e., in an opposite manner to that of wild-type photoreception. When the wild type cells are illuminated by strong light, they show negative phototaxis by beating the cis-flagellum (C) stronger than the trans-flagellum (T) when the eyespot faces the light source (Top Left). In contrast, the eyeless mutant cells show positive phototaxis by beating the cis-flagellum stronger than the trans-flagellum when the eyespot faces the side opposite the light source (Bottom Right).

Fig. S4.

Estimation of the refractive index of Chlamydomonas cell. Approximate focal length, f = 21.13 μm, can be evaluated with the magnification (M = y′/y) and the distance from the object to the lens a. (The value for y′ was measured when “P” seemed smallest and best focused.) When the cell acts as a ball lens, radius r, relative refractive index n, and the focal length f are related by f = nr/2(n−1) from well-known lens-maker's formula. Using the value r = 3.98 μm (the average of the longest and shortest radii of WT cells) and the refractive index of water n₁ = 1.333, the refractive index of the cell n₂ is estimated to be 1.47.