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The initiation of nocturnal dormancy in Synechococcus as an active process.

Takano S, Tomita J, Sonoike K, Iwasaki H - BMC Biol. (2015)

Bottom Line: Because Synechococcus is an obligate photoautotroph, it has been generally assumed that repression of the transcription in the dark (dark repression) would be caused by a nocturnal decrease in photosynthetic activities through the reduced availability of energy (e.g. adenosine triphosphate (ATP)) needed for mRNA synthesis.By contrast, when ATP levels were decreased by the inhibition of both photosynthesis and respiration, the transcriptional repression was attenuated through inhibition of RNA degradation.Even though the level of total mRNA dramatically decreased in the dark, Synechococcus cells were still viable, and they do not need de novo transcription for their survival in the dark for at least 48 hours.

View Article: PubMed Central - PubMed

Affiliation: Department of Electrical Engineering and Biological Science, Waseda University, TWIns, Shinjuku, Tokyo, 162-8480, Japan. sou.tacano@gmail.com.

ABSTRACT

Background: Most organisms, especially photoautotrophs, alter their behaviours in response to day-night alternations adaptively because of their great reliance on light. Upon light-to-dark transition, dramatic and universal decreases in transcription level of the majority of the genes in the genome of the unicellular cyanobacterium, Synechococcus elongatus PCC 7942 are observed. Because Synechococcus is an obligate photoautotroph, it has been generally assumed that repression of the transcription in the dark (dark repression) would be caused by a nocturnal decrease in photosynthetic activities through the reduced availability of energy (e.g. adenosine triphosphate (ATP)) needed for mRNA synthesis.

Results: However, against this general assumption, we obtained evidence that the rapid and dynamic dark repression is an active process. Although the addition of photosynthesis inhibitors to cells exposed to light mimicked transcription profiles in the dark, it did not significantly affect the cellular level of ATP. By contrast, when ATP levels were decreased by the inhibition of both photosynthesis and respiration, the transcriptional repression was attenuated through inhibition of RNA degradation. This observation indicates that Synechococcus actively downregulates genome-wide transcription in the dark. Even though the level of total mRNA dramatically decreased in the dark, Synechococcus cells were still viable, and they do not need de novo transcription for their survival in the dark for at least 48 hours.

Conclusions: Dark repression appears to enable cells to enter into nocturnal dormancy as a feed-forward process, which would be advantageous for their survival under periodic nocturnal conditions.

No MeSH data available.


Related in: MedlinePlus

Inhibition of photosynthetic electron transport mimicked dark-repression-like genome-wide transcription profiles under illumination without dramatic loss of ATP content. a Temporal expression profiles of representative dark-repressed or -induced genes using three independent northern hybridisation analyses. We normalized the data for dark-repressed genes to the average value of illuminated samples (0, 30, and 60 minutes), while the data for dark-induced genes were normalized to the average value of dark-incubated samples (30 and 60 minutes). Bars indicate the standard deviation. b Organisation of Synechococcus expression profiles in the light, dark, and light with two inhibitors, DCMU and DBMIB. The data of all genes were normalized to the value at time 0 (minutes), corresponding to 12 hours in the light, and sorted by induction levels in the dark. c Total mRNA pools estimated from the sum of mRNA hybridisation signals normalized to genomic DNA signals under each condition. We normalized the signals at time 0 in the light to 1,000. Plots indicate the results from each independent experiment (n = 2). d The plot of PCA scores. Upper plot shows the PC1 score of each profile only. Filled circles and open circles indicate the samples at 30- and 60-minutes incubation, respectively. L 0 indicates scores of samples at time 0. For panels B to D, we used averaged data from two independent experiments. e Transition of the ATP level when photosynthetic activity was inhibited partially or completely. We transferred cells grown in the light for 12 hours to each condition at time 0. We normalized the ATP levels to the average value of control samples collected in the light at Time −10 to 0 (minutes). Bars indicate the standard deviation from triplicate cultures DBMIB 2,5-dibromo-3-methyl-6-isopropylbenzoquinone, DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea, PCA principal component analysis
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Fig1: Inhibition of photosynthetic electron transport mimicked dark-repression-like genome-wide transcription profiles under illumination without dramatic loss of ATP content. a Temporal expression profiles of representative dark-repressed or -induced genes using three independent northern hybridisation analyses. We normalized the data for dark-repressed genes to the average value of illuminated samples (0, 30, and 60 minutes), while the data for dark-induced genes were normalized to the average value of dark-incubated samples (30 and 60 minutes). Bars indicate the standard deviation. b Organisation of Synechococcus expression profiles in the light, dark, and light with two inhibitors, DCMU and DBMIB. The data of all genes were normalized to the value at time 0 (minutes), corresponding to 12 hours in the light, and sorted by induction levels in the dark. c Total mRNA pools estimated from the sum of mRNA hybridisation signals normalized to genomic DNA signals under each condition. We normalized the signals at time 0 in the light to 1,000. Plots indicate the results from each independent experiment (n = 2). d The plot of PCA scores. Upper plot shows the PC1 score of each profile only. Filled circles and open circles indicate the samples at 30- and 60-minutes incubation, respectively. L 0 indicates scores of samples at time 0. For panels B to D, we used averaged data from two independent experiments. e Transition of the ATP level when photosynthetic activity was inhibited partially or completely. We transferred cells grown in the light for 12 hours to each condition at time 0. We normalized the ATP levels to the average value of control samples collected in the light at Time −10 to 0 (minutes). Bars indicate the standard deviation from triplicate cultures DBMIB 2,5-dibromo-3-methyl-6-isopropylbenzoquinone, DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea, PCA principal component analysis

Mentions: To examine whether inhibition of photosynthesis triggers dark repression/induction even under light, we applied two photosynthesis electron transport inhibitors, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropylbenzoquinone (DBMIB) (for target sites, see Additional file 1: Figure S1A), and analysed the effects on dark-repressed/induced gene expression profiles. Note that we have confirmed that either 2 μM DCMU or 10 μM DBMIB was sufficient to block electron transport completely by monitoring the effective quantum yield of Photosystem II (ΦII), (Fm′–Fo)/Fm′ becoming approximately equal to zero in the light (see Additional file 1: Figure S1B). Cells were grown in the light, acclimated to two 12 hour/12 hour light–dark (LD) cycles, and then returned to the light. At 12 hours in the light after the LD cycles, we kept cells in the light, acclimated them to the dark, or treated them with each of the inhibitors under the light for 30 or 60 minutes, and then subjected the cells to transcription analyses. We observed the changes after 30 minutes and 60 minutes from the addition of the stimuli because the rate of transcript variability is highest in 30 minute dark incubation, and total mRNA decreased up to about 50 % within 60 minutes [2]. Initially, we performed northern blot analyses on three representative dark-repressed genes (for information on genes, see Additional file 2: Table S1), which were remarkably repressed and induced upon light-to-dark transition within 30 minutes, respectively (Fig. 1a). In the cells treated with DCMU or DBMIB under illuminated conditions, the levels of expression of dark-repressed genes, petJ, kaiBC, and rbp3, decreased, while those of dark-induced genes, gifA, syc1260_c, and hspA, increased (Fig. 1a). As exemplified by hspA, which was less up-regulated by DCMU than by DBMIB, the extent of dark repression/induction seemed generally greater with DBMIB than with DCMU. These data suggest changes in the expression of all six genes through the cessation of photosynthesis in the dark. Note that longer treatments with DCMU or DBMIB up to eight hours also down-regulated and up-regulated the expression of kaiBC and gifA under illumination (Additional file 3: Figure S2). Although the level of expression of kaiBC was not reduced to zero, even without DBMIB it is reduced within eight hours because of its circadian clock function [6]. Moreover, treatment with DBMIB [7, 8] and DCMU [9] affects circadian clock function, possibly through antagonising the function of KaiA by enhancing the phosphorylation of KaiC. Therefore, longer treatment with these inhibitors may cause complex effects on transcriptional profiles of clock-controlled gene expression, as exemplified by kaiBC and many other representative dark-repressed genes. To avoid this confusion, we further focused on the transcriptional and metabolic changes within one hour after dark-acclimation or inhibitor treatment, which should be sufficient to dissect the mechanism triggering dark-induced global expression changes.Fig. 1


The initiation of nocturnal dormancy in Synechococcus as an active process.

Takano S, Tomita J, Sonoike K, Iwasaki H - BMC Biol. (2015)

Inhibition of photosynthetic electron transport mimicked dark-repression-like genome-wide transcription profiles under illumination without dramatic loss of ATP content. a Temporal expression profiles of representative dark-repressed or -induced genes using three independent northern hybridisation analyses. We normalized the data for dark-repressed genes to the average value of illuminated samples (0, 30, and 60 minutes), while the data for dark-induced genes were normalized to the average value of dark-incubated samples (30 and 60 minutes). Bars indicate the standard deviation. b Organisation of Synechococcus expression profiles in the light, dark, and light with two inhibitors, DCMU and DBMIB. The data of all genes were normalized to the value at time 0 (minutes), corresponding to 12 hours in the light, and sorted by induction levels in the dark. c Total mRNA pools estimated from the sum of mRNA hybridisation signals normalized to genomic DNA signals under each condition. We normalized the signals at time 0 in the light to 1,000. Plots indicate the results from each independent experiment (n = 2). d The plot of PCA scores. Upper plot shows the PC1 score of each profile only. Filled circles and open circles indicate the samples at 30- and 60-minutes incubation, respectively. L 0 indicates scores of samples at time 0. For panels B to D, we used averaged data from two independent experiments. e Transition of the ATP level when photosynthetic activity was inhibited partially or completely. We transferred cells grown in the light for 12 hours to each condition at time 0. We normalized the ATP levels to the average value of control samples collected in the light at Time −10 to 0 (minutes). Bars indicate the standard deviation from triplicate cultures DBMIB 2,5-dibromo-3-methyl-6-isopropylbenzoquinone, DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea, PCA principal component analysis
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4494158&req=5

Fig1: Inhibition of photosynthetic electron transport mimicked dark-repression-like genome-wide transcription profiles under illumination without dramatic loss of ATP content. a Temporal expression profiles of representative dark-repressed or -induced genes using three independent northern hybridisation analyses. We normalized the data for dark-repressed genes to the average value of illuminated samples (0, 30, and 60 minutes), while the data for dark-induced genes were normalized to the average value of dark-incubated samples (30 and 60 minutes). Bars indicate the standard deviation. b Organisation of Synechococcus expression profiles in the light, dark, and light with two inhibitors, DCMU and DBMIB. The data of all genes were normalized to the value at time 0 (minutes), corresponding to 12 hours in the light, and sorted by induction levels in the dark. c Total mRNA pools estimated from the sum of mRNA hybridisation signals normalized to genomic DNA signals under each condition. We normalized the signals at time 0 in the light to 1,000. Plots indicate the results from each independent experiment (n = 2). d The plot of PCA scores. Upper plot shows the PC1 score of each profile only. Filled circles and open circles indicate the samples at 30- and 60-minutes incubation, respectively. L 0 indicates scores of samples at time 0. For panels B to D, we used averaged data from two independent experiments. e Transition of the ATP level when photosynthetic activity was inhibited partially or completely. We transferred cells grown in the light for 12 hours to each condition at time 0. We normalized the ATP levels to the average value of control samples collected in the light at Time −10 to 0 (minutes). Bars indicate the standard deviation from triplicate cultures DBMIB 2,5-dibromo-3-methyl-6-isopropylbenzoquinone, DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea, PCA principal component analysis
Mentions: To examine whether inhibition of photosynthesis triggers dark repression/induction even under light, we applied two photosynthesis electron transport inhibitors, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropylbenzoquinone (DBMIB) (for target sites, see Additional file 1: Figure S1A), and analysed the effects on dark-repressed/induced gene expression profiles. Note that we have confirmed that either 2 μM DCMU or 10 μM DBMIB was sufficient to block electron transport completely by monitoring the effective quantum yield of Photosystem II (ΦII), (Fm′–Fo)/Fm′ becoming approximately equal to zero in the light (see Additional file 1: Figure S1B). Cells were grown in the light, acclimated to two 12 hour/12 hour light–dark (LD) cycles, and then returned to the light. At 12 hours in the light after the LD cycles, we kept cells in the light, acclimated them to the dark, or treated them with each of the inhibitors under the light for 30 or 60 minutes, and then subjected the cells to transcription analyses. We observed the changes after 30 minutes and 60 minutes from the addition of the stimuli because the rate of transcript variability is highest in 30 minute dark incubation, and total mRNA decreased up to about 50 % within 60 minutes [2]. Initially, we performed northern blot analyses on three representative dark-repressed genes (for information on genes, see Additional file 2: Table S1), which were remarkably repressed and induced upon light-to-dark transition within 30 minutes, respectively (Fig. 1a). In the cells treated with DCMU or DBMIB under illuminated conditions, the levels of expression of dark-repressed genes, petJ, kaiBC, and rbp3, decreased, while those of dark-induced genes, gifA, syc1260_c, and hspA, increased (Fig. 1a). As exemplified by hspA, which was less up-regulated by DCMU than by DBMIB, the extent of dark repression/induction seemed generally greater with DBMIB than with DCMU. These data suggest changes in the expression of all six genes through the cessation of photosynthesis in the dark. Note that longer treatments with DCMU or DBMIB up to eight hours also down-regulated and up-regulated the expression of kaiBC and gifA under illumination (Additional file 3: Figure S2). Although the level of expression of kaiBC was not reduced to zero, even without DBMIB it is reduced within eight hours because of its circadian clock function [6]. Moreover, treatment with DBMIB [7, 8] and DCMU [9] affects circadian clock function, possibly through antagonising the function of KaiA by enhancing the phosphorylation of KaiC. Therefore, longer treatment with these inhibitors may cause complex effects on transcriptional profiles of clock-controlled gene expression, as exemplified by kaiBC and many other representative dark-repressed genes. To avoid this confusion, we further focused on the transcriptional and metabolic changes within one hour after dark-acclimation or inhibitor treatment, which should be sufficient to dissect the mechanism triggering dark-induced global expression changes.Fig. 1

Bottom Line: Because Synechococcus is an obligate photoautotroph, it has been generally assumed that repression of the transcription in the dark (dark repression) would be caused by a nocturnal decrease in photosynthetic activities through the reduced availability of energy (e.g. adenosine triphosphate (ATP)) needed for mRNA synthesis.By contrast, when ATP levels were decreased by the inhibition of both photosynthesis and respiration, the transcriptional repression was attenuated through inhibition of RNA degradation.Even though the level of total mRNA dramatically decreased in the dark, Synechococcus cells were still viable, and they do not need de novo transcription for their survival in the dark for at least 48 hours.

View Article: PubMed Central - PubMed

Affiliation: Department of Electrical Engineering and Biological Science, Waseda University, TWIns, Shinjuku, Tokyo, 162-8480, Japan. sou.tacano@gmail.com.

ABSTRACT

Background: Most organisms, especially photoautotrophs, alter their behaviours in response to day-night alternations adaptively because of their great reliance on light. Upon light-to-dark transition, dramatic and universal decreases in transcription level of the majority of the genes in the genome of the unicellular cyanobacterium, Synechococcus elongatus PCC 7942 are observed. Because Synechococcus is an obligate photoautotroph, it has been generally assumed that repression of the transcription in the dark (dark repression) would be caused by a nocturnal decrease in photosynthetic activities through the reduced availability of energy (e.g. adenosine triphosphate (ATP)) needed for mRNA synthesis.

Results: However, against this general assumption, we obtained evidence that the rapid and dynamic dark repression is an active process. Although the addition of photosynthesis inhibitors to cells exposed to light mimicked transcription profiles in the dark, it did not significantly affect the cellular level of ATP. By contrast, when ATP levels were decreased by the inhibition of both photosynthesis and respiration, the transcriptional repression was attenuated through inhibition of RNA degradation. This observation indicates that Synechococcus actively downregulates genome-wide transcription in the dark. Even though the level of total mRNA dramatically decreased in the dark, Synechococcus cells were still viable, and they do not need de novo transcription for their survival in the dark for at least 48 hours.

Conclusions: Dark repression appears to enable cells to enter into nocturnal dormancy as a feed-forward process, which would be advantageous for their survival under periodic nocturnal conditions.

No MeSH data available.


Related in: MedlinePlus