The cellular reactive oxygen species (ROS) mediate stability and amplification of circadian rhythm

生物钟由许多反馈回路组成。丝状真菌神经孢子菌（Neurospora crassa）有一个主要的反馈回路，由频率(FRQ)/白领(WC) -1和-2振荡器（FWO）和假定的FRQ-less振荡器（FLOs），如依赖于频率的FLO （WC-FLO）组成。细胞内活性氧被认为与生物钟有关。然而，这种参与的机制仍然未知。在这里，我们表明细胞ROS浓度和ROS破坏过氧化氢酶（CATs）的活动以昼夜节律的方式振荡。ROS的振幅随周期的增加而增加，此后保持稳定，而细胞CAT活性的振幅则下降。细胞ROS由NADPH氧化酶（NOX）产生，并通过cat通过FLOs振荡，但其振幅由two调节。在没有FRQ的情况下，Cat-1的表达会振荡，但在wc-1或wc-2中则不会。此外，遗传和生化证据表明，较高的ROS浓度可促进WCC的转录功能并增强分生带。由WCC和CAT-1通过ROS组成的反馈环可能是WC-FLO的候选环。这些发现表明，细胞ROS连接了振荡子，如two和FLOs，使它们能够交流并稳定昼夜节律。

Circadian clocks are composed of a number of feedback loops. The filamentous fungus Neurospora crassa has a major feedback loop that consists of the FREQUENCY (FRQ)/WHITE COLLAR (WC) -1 and -2 oscillator (FWO) and presumptive FRQ-less oscillators (FLOs) such as WC-dependent FLO (WC-FLO). Intracellular ROS are thought to be involved in the circadian clocks. However, the mechanisms underlying this involvement remain unknown. Here, we show that cellular ROS concentrations and the ROS destroying catalases (CATs) activities oscillate in a circadian manner. The amplitudes of ROS increase with each cycle and remain steady thereafter, while those of cellular CAT activity decrease. The cellular ROS are generated by NADPH oxidase (NOX) and oscillate via FLOs using CATs, but their amplitudes are regulated by FWO. cat-1 expression oscillates in the absence of frq but does not in wc-1 or wc-2. Furthermore, genetic and biochemical evidence reveals that a higher ROS concentration promotes the WCC's transcriptional function and enhances conidiation banding. The feedback loop composed of the WCC and CAT-1 via ROS may be a candidate for the WC-FLO. These findings suggest that cellular ROS connect oscillators such as the FWO and FLOs, allowing them to communicate and stabilize the circadian rhythm.