Free Cellular Riboflavin is Involved in Phase Shifting by Light of the Circadian Clock in Neurospora crassa

Phase shifting by light of the circadian conidiation rhythm of the Neurospora crassa strain band, of the riboflavin-deficient double mutant band rib2 and of the temperature-sensitive double mutant band ribl was measured. Fluence response curves of the band strain exhibited two distinct steps, whereas those of band ribl and band rib2 revealed only one step. Maximum phase advances observed were 5.5 h in band and 10.4 h in the band rib strains. Sensitivity of band rib2 to light was proportional to the riboflavin concentration in the growth medium over a 100 fold range. Extracellular flavin in the medium did not sensitize the strains. Riboflavin applied after exposure to light showed no effect. Light sensitivity correlated with the level of cellular riboflavin. Four analogs of riboflavin, none of which can be phosphorylated, increased the sensitivity of Neurospora to light. Even at high riboflavin concentrations in the medium, the sensitivity of the band rib2 strain to light was not saturated. In addition, four riboflavin derivatives with bulky substituents at positions 3, 8 or 10 of the isoalloxazine nucleus sensitized both strains. From our data, we conclude, that a) a cellular flavin controls the sensitivity of Neurospora crassa to light; b) that this flavin compound is riboflavin; and c) that the active riboflavin is not protein bound.