Effects of H2O2 on M. aeruginosa Circadian Rhythms Figure 2. Photosynthesis-related gene transcripts in M. aerugi- nosa under light and dark conditions after H2O2 treatment. The shaded areas correspond to the dark period. Symbols represent mean 6 SEM of triplicate cultures. The mRNA amount of all examined genes is normalized to 16S rDNA. The open circle corresponds to the control treatment; the filled circle corresponds to the L0 treatment; the filled square corresponds to the D0 treatment. A: The effect of H2O2 on psaB circadian transcript; B: the effect of H2O2 on the psbD1 circadian transcript; C: the effect of H2O2 on the rbcL circadian transcript. doi:10.1371/journal.pone.0033347.g002 we first used real-time PCR to analyze the effect of H2O2 treatment on the circadian expression of the clock genes, circadian genes (three photosynthesis-related genes and three microcystin- related genes) within a light-dark cycle. Since Liu et al. [25] introduced bacterial luciferase structural genes (luxAB) as a reporter of circadian gene expression in cyanobacteria, many researchers have used this method to investigate the circadian rhythms of circadian genes. Ishiura et al. [16] demonstrated that the transcript levels of kaiA and kaiBC in Synechococcus exhibit circadian rhythms, and both bioluminescence peaks occur after 9 to 12 h under LL conditions. However, bioluminescence reporters may exhibit a time lag due to transcription, translation and post-translation processes [26]. Straub et al. [21] used DNA microarrays and demonstrated that the transcription patterns of the kaiB, kaiC and sasA genes are similar, with a peak within 3 h after light exposure. In this study, the expression peaks of these clock genes appeared after 4 h of light exposure in the LD cycle. These results are similar to those found in previous studies of circadian rhythms in cyanobacteria [27,28]. This study also observed that H2O2 treatment affected the rhythms of the clock genes. It is known that the circadian rhythm generated by the clock genes is output to downstream target genes via an unknown molecular mechanism [14]. Therefore, we Figure 3. Microcystin-related gene transcripts in M. aeruginosa under light and dark condition after H2O2 treatment. The shaded areas correspond to the dark period. Symbols represent mean 6 SEM of triplicate cultures. The mRNA amount of all examined genes is normalized to 16S rDNA. The open circle corresponds to the control treatment; the filled circle corresponds to the L0 treatment; the filled square corresponds to the D0 treatment. A: The effect of H2O2 on mcyA circadian transcript; B: the effect of H2O2 on the mcyD circadian transcript; C: the effect of H2O2 on mcyH circadian transcript. doi:10.1371/journal.pone.0033347.g003 analyzed the circadian rhythms of target genes (physiological- related genes, including three photosynthesis-related genes and microcystin-related genes) after H2O2 treatment. Photosynthesis-related genes are demonstrated to transcript mRNA rhythmically for the highest efficient photosynthesis. For example, the psbA2 gene, which encodes the D1 protein homolog of photosystem II, reaches its maximum transcript levels after 6 h of light exposure [15]. Sto¨ckel et al. [23] demonstrated that most of the genes encoding the subunits of photosystem I and II are maximally expressed during light exposure and minimally expressed in the dark. In this study, we selected three photosynthesis-related genes, psaB, psbD1 and rbcL, and found that the transcripts of these three photosynthesis-related genes displayed maximum transcript abundances in the light cycle and minimum transcript abundances in the dark cycle under normal conditions. These results also demonstrated the opinion that the circadian clock could precisely regulate photosynthetic genes to be active during day and promote organism growth in unicellular cyanobacteria [29]. This consideration was also confirmed by the growth curve of M. aeruginosa, which M. aeruginosa grows quickly in the light time, but retards in the dark time (Figure 8). The change of the rhythms of the photosynthesis-related genes after H2O2 treatment could decline solar energy utilization, and affect the synthesis of carbohydrate and high energy molecules (ATP and NADPH), which are the necessary substances for cyanobacterial growth. Therefore, this rhythmical disturbance of the photosyn- PLoS ONE | www.plosone.org 4 March 2012 | Volume 7 | Issue 3 | e33347