In vivo spectroscopic study of photoreceptor pigments of Blepharisma japonicum red and blue cells

Abstract

In the coloured ciliate Blepharisma japonicum, step-up photophobic responses are triggered by the endogenous pigment blepharismin. Blepharismin, red in dark-grown cells, is intracellularly photooxidized into a blue form (oxyblepharismin), still acting as photosensing pigment. With the aim of correlating the spectroscopic properties of blepharismin and oxyblepharismin in vivo with their photophysiological features, optical absorption, steady-state and time-resolved fluorescence spectra have been measured on cell suspensions. Both in blepharismin and oxyblepharismin in their physiological molecular environment, three fluorescent species have been observed, with virtually the same lifetimes ( ∼ 0.2 ns, ∼ 1.0 ns, ∼ 3.5 ns), but significantly different relative amplitudes. In red cells the long-living component has a very low relative amplitude (∼ 4%) and the short-living one is largely predominant (> 78%), whereas in blue cells the slowly decaying species has a slightly higher relative amplitude (∼ 40%) than the intermediately (∼ 31%) and the fast decaying species (∼ 29%). Together with the spectral width of time-gated spectra, these data are discussed in connection with current hypotheses on the structures of the chromophores. No meaningful difference in the above-mentioned spectroscopic parameters was observed after 30 min of UV-B irradiation, showing that no significant difference exists between red and blue blepharismin as far as UV-B lability is concerned.