Noriko Ueki

Eyespot-dependent determination of the phototactic sign in Chlamydomonas reinhardtii

Significance The phototactic behavior of the unicellular green alga Chlamydomonas reinhardtii is thought to rely on photoreception by the eyespot apparatus. Here, we isolated an eyespot-less mutant that clearly exhibits phototaxis. Intriguingly, the phototactic sign (the direction of cell migration) in this mutant is opposite to that of the wild type after treatment with reagents that enhance the sign, a property that we also detected in previously reported eyespot-less mutants. The reversed phototactic-sign phenotype was attributed to the fact that the photoreceptors were exposed to condensed light from their rear side. This report demonstrates the importance of the eyespot, in which carotenoid layers shield the photoreceptors from light condensed by the cell body, which functions as a convex lens. 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.

Morphology and reproduction of Volvox capensis (Volvocales, Chlorophyceae) from Montana, USA

Abstract: Volvox capensis was recorded previously only from South Africa. Here we established culture strains of this species from a sample collected in Montana, USA. Morphological details of asexual and sexual spheroids and molecular phylogeny of these strains were studied. The present alga was identified as V. capensis on the basis of morphological characteristics of asexual spheroids and zygotes. However, differences between the Montana and South African materials were recognized in number of sperm packets in a sexual, monoecious spheroid as well as in mode of gametic union between sperm and eggs. Possible polyspermy was observed in eggs of V. capensis by 4′-6-diamidino-2-phenylidole staining. Genetic difference between these two entities was small based on sequences of internal transcribed spacer 2 region of nuclear ribosomal DNA.

Identification of the agg1 mutation responsible for negative phototaxis in a “wild-type” strain of Chlamydomonas reinhardtii

The unicellular green alga Chlamydomonas reinhardtii is a model organism for various studies in biology. CC-124 is a laboratory strain widely used as a wild type. However, this strain is known to carry agg1 mutation, which causes cells to swim away from the light source (negative phototaxis), in contrast to the cells of other wild-type strains, which swim toward the light source (positive phototaxis). Here we identified the causative gene of agg1 (AGG1) using AFLP-based gene mapping and whole genome next-generation sequencing. This gene encodes a 36-kDa protein containing a Fibronectin type III domain and a CHORD-Sgt1 (CS) domain. The gene product is localized to the cell body and not to flagella or basal body.

A New Morphological Type of Volvox from Japanese Large Lakes and Recent Divergence of this Type and V. ferrisii in Two Different Freshwater Habitats

Volvox sect. Volvox is characterized by having unique morphological characteristics, such as thick cytoplasmic bridges between adult somatic cells in the spheroids and spiny zygote walls. Species of this section are found from various freshwater habitats. Recently, three species of Volvox sect. Volvox originating from rice paddies and a marsh were studied taxonomically based on molecular and morphological data of cultured materials. However, taxonomic studies have not been performed on cultured materials of this section originating from large lake water bodies. We studied a new morphological type of Volvox sect. Volvox (“Volvox sp. Sagami”), using cultured materials originating from two large lakes and a pond in Japan. Volvox sp. Sagami produced monoecious sexual spheroids and may represent a new morphological species; it could be clearly distinguished from all previously described monoecious species of Volvox sect. Volvox by its small number of eggs or zygotes (5–25) in sexual spheroids, with short acute spines (up to 3 μm long) on the zygote walls and elongated anterior somatic cells in asexual spheroids. Based on sequences of internal transcribed spacer (ITS) regions of nuclear ribosomal DNA (rDNA; ITS-1, 5.8S rDNA and ITS-2) and plastid genes, however, the Volvox sp. Sagami lineage and its sister lineage (the monoecious species V. ferrisii) showed very small genetic differences, which correspond to the variation within a single biological species in other volvocalean algae. Since V. ferrisii was different from Volvox sp. Sagami, by having approximately 100–200 zygotes in the sexual spheroids and long spines (6–8.5 μm long) on the zygote walls, as well as growing in Japanese rice paddies, these two morphologically distinct lineages might have diverged rapidly in the two different freshwater habitats. In addition, the swimming velocity during phototaxis of Volvox sp. Sagami spheroids originating from large lakes was significantly higher than that of V. ferrisii originating from rice paddies, suggesting adaptation of Volvox sp. Sagami to large water bodies.

Detergent-extracted Volvox model exhibits an anterior–posterior gradient in flagellar Ca2+ sensitivity

Significance The multicellular green alga Volvox rousseletii displays phototaxis by changing its flagellar beating pattern in response to photoreception. However, the molecular mechanism underlying flagellar regulation is unknown. This study describes a method to demembranate whole spheroids using a nonionic detergent, with the addition of ATP reactivating flagellar motility. These reactivated spheroids swam like live spheroids. Flagellar beating direction was altered in a Ca2+-dependent manner, with a greater change in the anterior hemisphere than in the posterior hemisphere. These findings indicate that V. rousseletii has an anterior–posterior gradient of flagellar sensitivity to Ca2+, which likely plays a key role in V. rousseletii phototaxis. Volvox rousseletii is a multicellular spheroidal green alga containing ∼5,000 cells, each equipped with two flagella (cilia). This organism shows striking photobehavior without any known intercellular communication. To help understand how the behavior of flagella is regulated, we developed a method to extract the whole organism with detergent and reactivate its flagellar motility. Upon addition of ATP, demembranated flagella (axonemes) in the spheroids actively beat and the spheroids swam as if they were alive. Under Ca2+-free conditions, the axonemes assumed planar and asymmetrical waveforms and beat toward the posterior pole, as do live spheroids in the absence of light stimulation. In the presence of 10−6 M Ca2+, however, most axonemes beat three-dimensionally toward the anterior pole, similar to flagella in photostimulated live spheroids. This Ca2+-dependent change in flagellar beating direction was more conspicuous near the anterior pole of the spheroid, but was not observed near the posterior pole. This anterior–posterior gradient of flagellar Ca2+ sensitivity may explain the mechanism of V. rousseletii photobehavior.

Dynein-mediated photobehavioral responses in Chlamydomonas

Abstract The unicellular green alga Chlamydomonas reinhardtii is a model organism for the study of photo-behavioral responses. Upon photoreception at the eyespot, cells show phototaxis (swimming toward or away from the light source) or photoshock (transient stop or backward swimming) depending on the light intensity. During these photoresponses, the manner of flagellar beating is elaborately regulated, and many Chlamydomonas mutants that show altered photoresponses have contributed to our understanding of the regulatory mechanisms of axonemal dyneins. Here we discuss the role of inner- and outer-arm dyneins in the photoresponses of Chlamydomonas.