Eyespot Apparatus Research Articles

Zeaxanthin is required for eyespot formation and phototaxis in Euglena gracilis.

The eyespot apparatus is an organelle that forms carotenoid-rich globules in diverse flagellated microalgae and functions in phototaxis. The euglenophytes have structurally and functionally distinct eyespot apparatuses from chlorophytes. β-Carotene is the most abundant pigment detected in chlorophytes' eyespots, while xanthophylls such as zeaxanthin and diadinoxanthin have been suggested to function in euglenophytes' eyespots. Here, we investigated the association between carotenoid composition and eyespot formation via pathway-scale mutagenesis using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome editing in the euglenophyte Euglena gracilis. Lycopene cyclase (lcy) mutants exhibited sole lycopene accumulation, defective red eyespots, and phototactic insensitivity. Conversely, β-carotene hydroxylase (cytochrome P450 97h1, cyp97h1) mutants accumulated β-carotene and its hydroxylated products β-cryptoxanthin and zeaxanthin and formed phototactic eyespot apparatuses, while cyp97h1 cyp97f2 double mutants were deficient in β-carotene hydroxylation and mostly lacked functional eyespots. Thus, zeaxanthin is required for the stable formation of functional eyespots in E. gracilis, highlighting evolutionary differences between euglenophytes and chlorophytes in the metabolic regulation of photoreactive organelle formation.

Carotenoid accumulation in the eyespot apparatus required for phototaxis is independent of chloroplast development in Euglena gracilis

Euglena gracilis exhibits photomovements in response to various light stimuli, such as phototactic and photophobic responses. Our recent study revealed that carotenoids in the eyespot apparatus are required for triggering phototaxis in this alga. However, the role of chloroplasts in eyespot formation is not understood. Here, we isolated carotenoid-less (cl) strains of E. gracilis from cells silenced gene expression of phytoene synthase (EgcrtB). Unlike WT, the culture colors of cl1, cl3, and the non-photosynthetic mutant SM-ZK were orange, while that of cl4 was white. Electron microscope observations showed that SM-ZK, cl1, and cl3 had no developed chloroplast and formed a normal eyespot apparatus, similar to that of WT, but this was not the case for cl4. Carotenoids detected in WT were diadinoxanthin, neoxanthin, and β-carotene. However, the most abundant species of SM-ZK, cl1, and cl3 was zeaxanthin, and there was no diadinoxanthin or neoxanthin. Photomovement analysis showed that SM-ZK, cl1, and cl3 exhibited negative phototactic and photophobic responses, similar to those of WT, whereas cl4 lacked negative phototaxis. Taken together, the formation of the eyespot apparatus required for phototaxis is independent of chloroplast development in E. gracilis, suggesting that this property is different from other photosynthetic flagellates.

Carotenoids in the eyespot apparatus are required for triggering phototaxis in Euglena gracilis.

SummaryCarotenoids are the most universal and most widespread pigments in nature. They have played pivotal roles in the evolution of photosensing mechanisms in microbes and of vision in animals. Several groups of phytoflagellates developed a photoreceptive organelle called the eyespot apparatus (EA) consisting of two separable components: the eyespot, a cluster of carotenoid‐rich globules that acts as a reflector device, and actual photoreceptors for photobehaviors. Unlike other algal eyespots, the eyespot of Euglenophyta lacks reflective properties and is generally considered to act as a shading device for the photoreceptor (paraflagellar body, PFB) for major photomovements. However, the function of the eyespot of Euglenophyta has not yet been fully proven. Here, we report that the blocking carotenoid biosynthesis in Euglena gracilis by suppressing the phytoene synthase gene (crtB) caused a defect in eyespot function resulting in a loss of phototaxis. Raman spectroscopy and transmission electron microscopy suggested that EgcrtB‐suppressed cells formed eyespot globules but had a defect in the accumulation of carotenoids in those packets. Motion analysis revealed the loss of phototaxis in EgcrtB‐suppressed cells: a defect in the initiation of turning movements immediately after a change in light direction, rather than a defect in the termination of cell turning at the appropriate position due to a loss of the shading effect on the PFB. This study revealed that carotenoids are essential for light perception by the EA for the initiation of phototactic movement by E. gracilis, suggesting one possible photosensory role of carotenoids in the EA for the phototaxis.

Ubiquitous distribution of helmchrome in phototactic swarmers of the stramenopiles.

Most swarmers (swimming cells) of the stramenopile group, ranging from unicellular protist to giant kelps (brown algae), have two heterogeneous flagella: a long anterior flagellum (AF) and a relatively shorter posterior flagellum (PF). These flagellated cells often exhibit phototaxis upon light stimulation, although the mechanism by which how the phototactic response is regulated remains largely unknown. A flavoprotein concentrating at the paraflagellar body (PFB) on the basal part of the PF, which can emit green autofluorescence under blue light irradiance, has been proposed as a possible blue light photoreceptor for brown algal phototaxis although the nature of the flavoprotein still remains elusive. Recently, we identified helmchrome as a PF-specific flavoprotein protein in a LC-MS/MS-based proteomics study of brown algal flagella (Fu et al. 2014). To verify the conservation of helmchrome, in the present study, the absence or presence and the localization of helmchrome in swarmers of various algal species were investigated. The results showed that helmchrome was only detected in phototactic swarmers but not the non-phototactic ones of the stramenopile group. Electron microscopy further revealed that the helmchrome detectable swarmers bear a conserved PFB-eyespot complex, which may serve as structural basis for light sensing. It is speculated that all three conserved properties: helmchrome, the PFB structure, and the eyespot apparatus, will be essential parts for phototaxis of stramenopile swarmers.

A Cyclic GMP-Dependent K+ Channel in the Blastocladiomycete Fungus Blastocladiella emersonii.

Phototaxis in flagellated zoospores of the aquatic fungus Blastocladiella emersonii depends on a novel photosensor, Blastocladiella emersonii GC1 (BeGC1), comprising a type I (microbial) rhodopsin fused to a guanylyl cyclase catalytic domain, that produces the conserved second messenger cyclic GMP (cGMP). The rapid and transient increase in cGMP levels during the exposure of zoospores to green light was shown to be necessary for phototaxis and dependent on both rhodopsin function and guanylyl cyclase activity. It is noteworthy that BeGC1 was localized to the zoospore eyespot apparatus, in agreement with its role in the phototactic response. A putative cyclic nucleotide-gated channel (BeCNG1) was also identified in the genome of the fungus and was implicated in flagellar beating via the action of a specific inhibitor (l-cis-diltiazem) that compromised zoospore motility. Here we show that B. emersonii expresses a K(+) channel that is activated by cGMP. The use of specific channel inhibitors confirmed the activation of the channel by cGMP and its K(+) selectivity. These characteristics are consistent with the function of an ion channel encoded by the BeCNG1 gene. Other blastocladiomycete fungi, such as Allomyces macrogynus and Catenaria anguillulae, possess genes encoding a similar K(+) channel and the rhodopsin-guanylyl cyclase fusion protein, while the genes encoding both these proteins are absent in nonflagellated fungi. The presence of these genes as a pair seems to be an exclusive feature of blastocladiomycete fungi. Taken together, these data demonstrate that the B. emersonii cGMP-activated K(+) channel is involved in the control of zoospore motility, most probably participating in the cGMP-signaling pathway for the phototactic response of the fungus.

How the green alga Chlamydomonas reinhardtii keeps time

The unicellular green alga Chlamydomonas reinhardtii has two flagella and a primitive visual system, the eyespot apparatus, which allows the cell to phototax. About 40 years ago, it was shown that the circadian clock controls its phototactic movement. Since then, several circadian rhythms such as chemotaxis, cell division, UV sensitivity, adherence to glass, or starch metabolism have been characterized. The availability of its entire genome sequence along with homology studies and the analysis of several sub-proteomes render C. reinhardtii as an excellent eukaryotic model organism to study its circadian clock at different levels of organization. Previous studies point to several potential photoreceptors that may be involved in forwarding light information to entrain its clock. However, experimental data are still missing toward this end. In the past years, several components have been functionally characterized that are likely to be part of the oscillatory machinery of C. reinhardtii since alterations in their expression levels or insertional mutagenesis of the genes resulted in defects in phase, period, or amplitude of at least two independent measured rhythms. These include several RHYTHM OF CHLOROPLAST (ROC) proteins, a CONSTANS protein (CrCO) that is involved in parallel in photoperiodic control, as well as the two subunits of the circadian RNA-binding protein CHLAMY1. The latter is also tightly connected to circadian output processes. Several candidates including a significant number of ROCs, CrCO, and CASEIN KINASE1 whose alterations of expression affect the circadian clock have in parallel severe effects on the release of daughter cells, flagellar formation, and/or movement, indicating that these processes are interconnected in C. reinhardtii. The challenging task for the future will be to get insights into the clock network and to find out how the clock-related factors are functionally connected. In this respect, system biology approaches will certainly contribute in the future to improve our understanding of the C. reinhardtii clock machinery.

The green algal eyespot apparatus: a primordial visual system and more?

Most flagellate green algae exhibiting phototaxis posses a singular specialized light sensitive organelle, the eyespot apparatus (EA). Its design principles are similar in all green algae and produce, in conjunction with the movement pattern of the cell, a highly directional optical device. It enables an oriented movement response with respect to the direction and intensity of light. The functional EA involves local specializations of different compartments (plasma membrane, cytosol, and chloroplast) and utilizes specialized microbial-type rhodopsins, which act as directly light-gated ion channels. Due to their elaborate structures and the presence of retinal-based photoreceptors in some lineages, algal EAs are thought to play an important role in the evolution of photoreception and are thus not only of interest to plant biologists. In green algae considerable progress in the molecular dissection of components of this primordial visual system has been made by genetic and proteomic approaches in recent years. This review summarizes general aspects of the green algal EA as well as recent progress in the identification of proteins related to it. Further, novel data supporting a link between eyespot globules and plastoglobules will be presented and potential additional roles of the EA besides those in photoreception will be discussed.

The Phosphoproteome of aChlamydomonas reinhardtiiEyespot Fraction Includes Key Proteins of the Light Signaling Pathway

Flagellate green algae have developed a visual system, the eyespot apparatus, which allows the cell to phototax. In a recent proteomic approach, we identified 202 proteins from a fraction enriched in eyespot apparatuses of Chlamydomonas reinhardtii. Among these proteins, five protein kinases and two protein phosphatases were present, indicating that reversible protein phosphorylation occurs in the eyespot. About 20 major phosphoprotein bands were detected in immunoblots of eyespot proteins with an anti-phosphothreonine antibody. Toward the profiling of the targets of protein kinases in the eyespot fraction, we analyzed its phosphoproteome. The solubilized proteins of the eyespot fraction were treated with the endopeptidases LysC and trypsin prior to enrichment of phosphopeptides with immobilized metal-ion affinity chromatography. Phosphopeptides were analyzed by nano-liquid chromatography-electrospray ionization-mass spectrometry (MS) with MS/MS as well as neutral-loss-triggered MS/MS/MS spectra. We were able to identify 68 different phosphopeptides along with 52 precise in vivo phosphorylation sites corresponding to 32 known proteins of the eyespot fraction. Among the identified phosphoproteins are enzymes of carotenoid and fatty acid metabolism, putative signaling components, such as a SOUL heme-binding protein, a Ca(2+)-binding protein, and an unusual protein kinase, but also several proteins with unknown function. Notably, two unique photoreceptors, channelrhodopsin-1 and channelrhodopsin-2, contain three and one phosphorylation sites, respectively. Phosphorylation of both photoreceptors occurs in the cytoplasmatic loop next to their seven transmembrane regions in a similar distance to that observed in vertebrate rhodopsins, implying functional importance for regulation of these directly light-gated ion channels relevant for the photoresponses of C. reinhardtii.

EVIDENCE FOR A SPECIALIZED LOCALIZATION OF THE CHLOROPLAST ATP‐SYNTHASE SUBUNITS α, β, AND γ IN THE EYESPOT APPARATUS OF CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE)1

The eyespot apparatus (EA) of Chlamydomonas reinhardtii P. A. Dang. consists of two layers of carotenoid‐rich lipid globules subtended by thylakoids. The outermost globule layer is additionally associated with the chloroplast envelope membranes and the plasma membrane. In a recent proteomic approach, we identified 202 proteins from isolated EAs of C. reinhardtii via at least two peptides, including, for example, structural components, signalling‐related proteins, and photosynthetic‐related membrane proteins. Here, we have analyzed the proteins of the EA with regard to their topological distribution using thermolysin to find out whether the arrangement of globules and membranes provides protection mechanisms for some of them. From about 230 protein spots separated on two‐dimensional gels, the majority were degraded by thermolysin. Five major protein spots were protected against the action of this protease. These proteins and some that were degradable were identified by mass spectrometry. Surprisingly, the thermolysin‐resistant proteins represented the α and β subunits of the soluble CF1 complex of the chloroplast ATP synthase. Degradable proteins included typical membrane proteins like LHCs, demonstrating that thermolysin is not in general sterically prevented by the EA structure from reaching membrane‐associated proteins. A control experiment showed that the CF1 complex of thylakoids is efficiently degraded by thermolysin. Blue native PAGE of thermolysin‐treated EAs followed by SDS‐PAGE revealed that the α and β subunits are present in conjunction with the γ subunit in a thermolysin‐resistant complex. These results provide strong evidence that a significant proportion of these ATP‐synthase subunits have a specialized localization and function within the EA of C. reinhardtii.

Proteomic Analysis of the Eyespot ofChlamydomonas reinhardtiiProvides Novel Insights into Its Components and Tactic Movements

Flagellate green algae have developed a visual system, the eyespot apparatus, which allows the cell to phototax. To further understand the molecular organization of the eyespot apparatus and the phototactic movement that is controlled by light and the circadian clock, a detailed understanding of all components of the eyespot apparatus is needed. We developed a procedure to purify the eyespot apparatus from the green model alga Chlamydomonas reinhardtii. Its proteomic analysis resulted in the identification of 202 different proteins with at least two different peptides (984 in total). These data provide new insights into structural components of the eyespot apparatus, photoreceptors, retina(l)-related proteins, members of putative signaling pathways for phototaxis and chemotaxis, and metabolic pathways within an algal visual system. In addition, we have performed a functional analysis of one of the identified putative components of the phototactic signaling pathway, casein kinase 1 (CK1). CK1 is also present in the flagella and thus is a promising candidate for controlling behavioral responses to light. We demonstrate that silencing CK1 by RNA interference reduces its level in both flagella and eyespot. In addition, we show that silencing of CK1 results in severe disturbances in hatching, flagellum formation, and circadian control of phototaxis.

Subfractionation of eyespot apparatuses from the green alga Spermatozopsis similis: isolation and characterization of eyespot globules.

Despite the well-characterized function of the green-algal eyespot apparatus as a combined absorption/reflection screen for the photoreceptor for phototaxis, little is known about the proteins involved in the formation of this complex organelle. We therefore purified the carotenoid-rich lipid globules, which are the most conspicuous component of the eyespot sensu strictu from Spermatozopsis similis Preisig et Melkonian. Electron microscopy and an average carotenoid:chlorophyll ratio of 51, confirmed the high purity of the fraction. The diameter of isolated globules (approx. 112 nm) fell within their in vivo range (90-120 nm). Absorption spectra in aqueous media peaked at 535 nm. The predominant carotenoids were beta/psi-, beta, beta- and delta-carotene. Freeze-fracture studies with cells and whole-mount electron microscopy of isolated globules demonstrated regularly arranged particles at the globule surface. Sodium dodecyl sulfate polyacrylamide gel electrophresis revealed specific enrichment of 10 tightly bound major proteins and several minor proteins with the globules. Proteases were used to analyze their topology and function. Upon treatment with thermolysin, globules were released from a fraction enriched in isolated eyespot apparatuses. Major proteins of these globules, and those treated with thermolysin after isolation, were identical. However, the purified proteins were sensitive to thermolysin, indicating that domains of them are normally hidden in the globule matrix. In contrast, pronase degraded all globule-associated proteins in situ. These globules were not stable and easily fused, whereas thermolysin-treated globules were relatively stable. Lipase did not affect globule stability. These results indicate that the five thermolysin-resistant proteins (apparent Mr values: 56, 52, 32, 29, 27 kDa) are close to the surface and might be crucial for globule stabilization, whereas the thermolysin-accessible proteins are probably involved in globule/globule interactions and/or globule/eyespot-membrane interactions.

A light microscopical and ultrastructural investigation and validation of Khawkinea pertyi comb. nova (Euglenophyta)

This study deals with the validation of the colourless euglenoid flagellate Khawkinea pertyi (PRINGSHEIM et HOVASSE) PRINGSHEIM ex ANGELER comb, nov. previously assigned to the genus Astasia and then invalidly transferred to Khawkinea. The assignment to Khawkinea is justified because of the presence of an eyespot apparatus. The re-examination of this species, with an accompanied documentation of its ultrastructure, reveals overwhelming similarities in comparison with Khawkinea quartana (MOROFF) JAHN et Mc KIBBEN, especially at the fine structure level. Hence, current hypotheses of the evolutionary origin of Khawkinea species, due to apochlorosis events, are discussed and taxonomic remarks are given.

Light- and Ca 2+ -Modulated Heterotrimeric GTPases in the Eyespot Apparatus of a Flagellate Green Alga

Light- and Ca 2+ -Modulated Heterotrimeric GTPases in the Eyespot Apparatus of a Flagellate Green Alga

A microspectrophotometric study of the shielding properties of eyespot and cell body in Chlamydomonas

The eyespot apparatus of the unicellular alga Chlamydomonas exhibits a clear directivity, i.e., it perceives light from different directions with different sensitivity. Using a newly constructed confocal microscope we have studied how absorption and reflection of eyespot and cell body shape this directivity. In agreement with previous results the eyespot was found to be highly reflectant, owing to its interference reflector design, but only for yellow light. Light of 490 nm, the maximum of absorption of the photoreceptor, was hardly reflected at all, even when the reflector was "tuned" to lower wavelengths by tilting it relative to the incoming light. The absorption of the carotenoids in the interference reflector also contributed little to the shielding properties of the cell, leaving the major contribution to the cell body. Thus most of the attenuation of light reaching the eyespot from the rear is due to chlorophyll and other pigments within the cell. In its peak around 490 nm the "contrast-ratio" reached a value of 8-10.

Carotenoids in the eyespot apparatus of the flagellate green alga Spermatozopsis similis: Adaptation to the retinal-based photoreceptor

Isolated intact eyespot apparatuses, the photoreceptive organelles involved in blue-light-mediated photoresponses of flagellate green algae, were analyzed regarding their carotenoid composition. Carotenoids from the eyespot apparatuses of Spermatozopsis similis were identified by high-performance liquid chromatography, visible-light absorption spectra, mass spectroscopy and by 1H-nuclear magnetic resonance spectroscopy (carotenes), and compared with those of whole-cell extracts. Both extracts contained β,β-carotene, β,ψ-carotene (formerly γ-carotene), lycopene, lutein, zeaxanthin, violaxanthin and all-E-and 9′-Z-neoxanthin. The relative carotenoid compositions, however, differed significantly. A twofold relative increase in the total carotene level was evident in the fraction enriched in eyespot apparatuses. This was mainly due to an increase in the monocyclic β,ψ-carotene and the aliphatic lycopene, whereas the relative content of β,β-carotene remained unchanged. On the other hand a relative decrease in the total xanthophyll content, especially of lutein and the epoxidic carotenoid neoxanthin, was observed in the eyespot apparatuses compared with the whole-cell extracts. The decrease of the latter resulted almost solely from a reduction of the 9′-Z-rather than the all-E-isomer. The bulk of the carotenes is thought to be localized in the highly organized eyespot lipid globules, which act as a combined quarter-wave interference reflector and absorption screen for the photoreceptor in green algae. The enrichment of β,ψ-carotene and lycopene in the eyespot apparatuses, extending the range of visible light absorption to longer wavelengths, represents an adaptation of the screen to the retinal-based photoreceptor of flagellate green algae and is one of the prerequisites for maximal directional sensitivity of the eyespot apparatus.

Functional analysis of the eyespot in Chlamydomonas reinhardtii mutant ey 627, mt ?

The function of the eyespot in phototaxis of the flagellate green alga Chlamydomonas reinhardtii Dangeard was studied using quantitative reflection confocal laser scanning microscopy and photoelectric measurements. The reflective properties of the eyespot and the photoreceptor current of the C. reinhardtii eyespot mutant ey 627, mt (-) were compared with those of Chlamydomonas strains possessing a well-developed eyespot. Under growth conditions in which strongly disorganized eyespots were observed in the mutant by electron microscopy, there was a significant reduction in the reflection intensity of the eyespot and in the amplitude ratio (500∶440 nm) of photoreceptor currents induced by flashes of 500- and 440-nm light in non-oriented cells. Photoelectrical responses of pre-oriented cells revealed that the latter effect is caused by an altered directional sensitivity of the antenna complex, whereas the functional state of the photoreceptor pigment is not strongly affected in mutant cells. Both the reflection intensity and the amplitude ratio of photoreceptor currents increased to the level of reference strains under conditions supporting the development of a well-organized eyespot in the mutant. Furthermore, incubation of the mutant with high concentrations of all-trans-retinal (10 μM), independent of whether carotenoid biosynthesis was inhibited or not, was found to increase the reflection intensity of the eyespot. An increase in the rate of photoorientation of the mutant occurred concomitant with the increase in the reflective properties of the mutant eyespot. These observations demonstrate the importance of an intact eyespot for interference reflection and absorption of phototactically active light, and thus for the directional sensitivity of the eyespot apparatus.

STRUCTURAL AND EVOLUTIONARY ASPECTS OF THE FLAGELLAR APPARATUS IN GREEN ALGAE AND LAND PLANTS

SummaryUltrastructural features of the flagellar apparatus in green algae and land plants seem to be of considerable phylogenetic significance. Based on functional considerations details of the flagellar apparatus can be used to construct tentative lines of evolution within the green algae. An “ancestral” asymmetric zooflagellate possibly gave rise to a green unicell after a primary endosymbiotic event involving a green prokaryote. During the transition from phagotrophic to phototrophic nutrition in most green algae (the Chlorophyceae and Ulvaphyceae sensu Stewart & Mattox and most Prasinophyceae sensu Christensen) the original asymmetric flagellar apparatus was transformed into a 180° rotational symmetric flagellar apparatus with 4 flagella and a flagellar root system of the X‐2‐X‐2‐type. Most significant in this transition was presumably the evolution of the phototactic apparatus and of an apical flagellar groove. In the Charophyceae sensu Stewart & Mattox (including those green algae related to the ancestry of the archegoniate land plants) the early transition towards a terrestrial life, lack of an eyespot apparatus and development of a special type of oogamy possibly led to the evolution of the unilateral flagellar root in these organisms. It is suggested that the evolution of the unilateral MLS‐root in the Charophyceae sensu Stewart & Mattox can be traced back to an “ancestral green flagellate” before the evolution of the typical X‐2‐X‐2 flagellar root system. A tentative evolutionary scheme of the green algae mainly based on motile cell structure and mitotic and cytokinetic mechanisms is presented.

On the presence of plastids and the eyespot apparatus in a porfiromycin-bleached strain ofEuglena gracilis

Ultrastructural studies were performed on a strain ofEuglena gracilis bleached with the antibiotic porfiromycin. A limited number of organelles could be identified as plastids by their possession of a double envelope and a stroma containing bunches of minute thylakoids, DNA-fibrils and ribosomes. A stigma was situated close to the reservoir. Opposite the stigma a paraflagellar body was observed in the main flagellum.

Structure and function in the euglenoid eyespot apparatus: The fine structure, and response to environmental changes

The microanatomy of the eyespot apparatus of Euglena gracilis Z was examined with the electron microscope. The stigma was found to be a membrane-bounded organelle showing no close homology with the chloroplast or any other organelle. The structure and pigment content of the stigma both diminish with extended hetrotrophic growth, and quickly regain normal dimensions upon exposure to light. Synthesis of the red pigment is particularly sensitive to inhibition by chloramphenicol, whereas construction of the structure itself is specifically inhibited by cycloheximide.The paraflagellar body appears to consist of two sets of parallel 80 Å striations intersecting at 60°. It is within the flagellar membrane, but separated from the axoneme by another structure, the paraflagellar rod. This elongated structure has an ordered substructure which appears as intersecting sets of parallel striations; part of its basal portion projects as a circular flange which makes contact with the paraflagellar body.

Structure and Function of the Euglenoid Eyespot

With Euglena gracilis neither the action spectrum for photokinesis nor that for phototaxis corresponds to the absorption spectrum of the stigma, as has been previously claimed. The phototaxis action spectrum may be interpreted instead as a combination of the photokinesis action spectrum, stigma absorption spectrum, and chloroplast absorption spectrum. This supports the contention that the paraflagellar body rather than the stigma is the probable photoreceptor of the eyespot apparatus, and indicates that both the stigma and chloroplasts provide the directional Tight screen'. It also suggests that the absorption spectrum of this photoreceptor is the photokinesis action spectrum.