Cross-Talk between the Cellular Redox State and the Circadian System in Neurospora

The circadian system is composed of a number of feedback loops, and multiple feedback loops in the form of oscillators help to maintain stable rhythms. The filamentous fungus Neurospora crassa exhibits a circadian rhythm during asexual spore formation (conidiation banding) and has a major feedback loop that includes the FREQUENCY (FRQ)/WHITE COLLAR (WC) -1 and -2 oscillator (FWO). A mutation in superoxide dismutase (sod)-1, an antioxidant gene, causes a robust and stable circadian rhythm compared with that of wild-type (Wt). However, the mechanisms underlying the functions of reactive oxygen species (ROS) remain unknown. Here, we show that cellular ROS concentrations change in a circadian manner (ROS oscillation), and the amplitudes of ROS oscillation increase with each cycle and then become steady (ROS homeostasis). The ROS oscillation and homeostasis are produced by the ROS-destroying catalases (CATs) and ROS-generating NADPH oxidase (NOX). cat-1 is also induced by illumination, and it reduces ROS levels. Although ROS oscillation persists in the absence of frq, wc-1 or wc-2, its homeostasis is altered. Furthermore, genetic and biochemical evidence reveals that ROS concentration regulates the transcriptional function of WCC and a higher ROS concentration enhances conidiation banding. These findings suggest that the circadian system engages in cross-talk with the cellular redox state via ROS-regulatory factors

Light signals are transduced to the phosphorylation of 15 kDa proteins in Neurospora crassa

AbstractA microsomal fraction prepared from the mycelia of the band (bd) strain of Neurospora crassa showed enhanced phosphorylation of two small proteins with molecular masses of around 15 kDa (ps15) by the irradiation of the reaction mixture containing [γ-32P]ATP at 0°C for 1 s with blue light (450 nm, 6 μmol/m2/s or 420 nm, 80 μmol/m2/s). The reaction was stopped at 5 s of incubation at 0°C after blue light irradiation. The light effect could not be detected in ps15, when a microsomal fraction from a blind mutant, wc-1 or wc-2 was used. The mixing followed by homogenization of the microsomal fractions from wc-1 and wc-2 restored the activity to stimulate the phosphorylation of ps15 by blue light. The phosphorylated amino acid residue of ps15 was unstable when the proteins on a nylon membrane were exposed to an acid or alkaline solution, suggesting that the phosphorylated residue was aspartic acid. The other phosphorylated protein with a molecular mass of 70 kDa (p70) showed no light effect in the phosphorylation and the phosphorylated residue was estimated to be histidine, since it was stable in alkaline solution

Circadian oscillation and light-induced changes in the concentration of cyclic nucleotides in Neurospora

Rhythmic oscillations in the concentration of cAMP in myeclia of wild type 74A, bd, bd frq-1 and bd frq-2 strains of Neurospora crassa grown in liquid media in darkness were detected. The period lengths of the rhythm of cAMP concentrations were about 21 to 22 h in 74A and bd, and 14≈20 and 19 h in bd frq-1 and bd frq-2. The concentration of cGMP oscillated slightly. In parallel experiments using solid medium, conidiation occurred about 22 h after the peak of cAMP concentration. Exposure of bd mycelia to white light (3.9 J/m2 ·s) at 7∼12 h after the onset of continuous darkness, when the concentration of cAMP was high, reduced the concentrations of CAMP and cGMP, whereas after 18 h of darkness when the concentration of CAMP was low, only the concentration of cGMP was reduced. The reduction in the concentrations of cAMP and cGMP by light occurred within 60 s. Exposure of mycelia to constant light resulted in an oscillation of cAMP concentration with a period length from 60 to 90 min. After 9 h of continuous darkness, exposure of bd mycelia to 0.5 mM cAMP or 0.5 mM cGMP for 1 h and subsequent transfer to solid media resulted in phase advances of the conidiation rhythm of 2.1 h and 1.2 h, respectively. Exposure to light for 1 h, however, caused a 4.3 h phase delay. These results strongly suggest that cAMP and possibly cGMP are factors controlling the circadian rhythm

Partial characterization and light-induced regulation of GTP-binding proteins in Lemna paucicostata.

Binding of GTP-binding proteins with [35S]GTP7S in the extract containing membrane components of Lemna paucicostata 441 was inhibited by red or far red light by 20 to 25%, but blue light showed no or little effect. The plant used for the preparation of the extract was subjected to single darkness for 8 h, as both red and far red light inhibit flowering. The extract treated with 1% Lubrol was fractionated by gel filtration. Four species of GTP-binding proteins, GL1, GL2, GL3 and GL4 were detected with Km values 3, 7, 80 and 4 nM, respectively. GL1, GL2 and GL3 were ADP-ribosylated by pertussis toxin. The extract activated by [35S]GTP-γS in darkness, under red light or under far red light was treated with 1% Lubrol and subsequent gel filtration of the extracts made it possible to detect GTP-binding protein with a small molecular weight only in an extract labeled in darkness. The reduction in the molecular weight of GTP-binding protein from the larger molecule associated with the binding of [35S]GTPγS was confirmed by rechromatography of the larger molecule activated by [35S]GTPγS in darkness. The binding of GL2 and/or GL3 with [35S]GTPγS was suggested to be inhibited by red or far red light