Light-induced phase shifting of the circadian clock in Neurospora crassa requires ammonium salts at high pH

Abstract

Effects of external ionic conditions on light induced phase shifting of the circadian rhythm of conidiation in Neurospora crassa were examined in simple buffer solutions for discerning effects of individual ions. Mycelia were cultured to liquid media of different pHs and then transferted to 10 mM piperazine-N,N'-bis(2-ethanesulmonic acid) (Pipes) buffer of various pHs and irradiated with while light. The phase of the rhythm of dark controls was not changed by transfer from medium to buffer. When mycelia were cultured in media of pH above 6.7, light did not advance the phase of the clock in Pipes buffer alone. However, light-induced phase advance was restored when an ammonium salt was added to buffer of pH higher than 7.6. An amination-defective mutant, bd am, showed the same response to ammonium nitrate as the wild-type strain, bd. Ammonium must be present before light irradiation for restoration of phase shifting. Free-amino-acid pools in the cells were changed by treatment with Pipes buffer: aspartle acid, glutamic acid, ammonia, glutamine and ornithine levels decreased, while lysine and histidine increased. Addition of ammonium nitrate to Pipes buffer resulted in further changes in amino-acid pools; lysine, histidine, arginine, alanine and ornithine decreased, and glutamine levels increased. Irradiation did not result in significant changes in amino acid pools.