Characterization of the eyespot regions of "blind" Chlamydomonas mutants after restoration of photophobic responses

ABSTRACT Chlamydomonas reinhardtii exhibits photophobic and positive and negative phototactic responses that can be defined for cell populations using computerized cell tracking and motion analysis. Mutants CC‐2359 and FN68 are pigment deficient mutants that are blocked in carotenoid synthesis and lack these photo responses. In particular, neither mutant exhibits flash‐induced photophobic responses to visible light stimuli to which wild‐type gametic cells exhibit a strong response, with several behavioral stages. Upon addition of all‐trans retinal to these mutants, the photophobic responses are restored with minor quantitative differences from wild‐type populations. Using both light and electron microscopy, we have compared the ultrastructural characteristics of wild‐type C. reinhardtii to those of both mutants. As previously described, wild‐type cells contain an eyespot consisting of 2–4 layers of pigmented granules encased within thylakoid membranes, located between the distal extremities of the flagellar root. This structure is also visible as an orange‐red spot in light microscopy. The photoreceptor is thought to be concentrated in the plasma membrane above the eyespot. The mutant, CC‐2359, lacks this eyespot as seen by both light and electron microscopy, even when the photophobic response has been restored. FN68‐like mutants studied earlier by Morel‐Laurens and Feinlieb and others contain an eyespot which can be seen only by electron microscopy. In FN‐68, the eyespot generally has the same dimensions as in wt cells, differing mainly in pigment granule appearance. Consistent with these findings, several laboratories have shown that the full range of phototactic responses can be reconstituted in FN68 and CC‐2359, but that negative phototaxis requires a significantly stronger light stimulus in the latter strain. We confirm the suggestion that the eyespot is not necessary for the photophobic response, and is not critical for the appropriate assembly and function of the photophobic response receptor in the membrane. Furthermore, the locus of reconstitution of the functional receptor is not the eyespot. Because of the definitive demonstration of the absence of the eyespot in CC‐2359, however, the eyespot may play a role in negative phototaxis.