Project description:Circadian clocks are gene networks producing 24-h oscillations at the level of clock gene expression that is synchronized to environmental cycles via light signals. The ELONGATED HYPOCOTYL 5 (HY5) transcription factor is a signalling hub acting downstream of several photoreceptors and is a key regulator of photomorphogenesis. Here we describe a mechanism by which light quality could modulate the pace of the circadian clock through controlling abundance of HY5. We show that hy5 mutants display remarkably shorter period rhythms in blue but not in red light or darkness and blue light is more efficient than red to induce accumulation of HY5 at transcriptional and post-transcriptional levels. We demonstrate that the pattern and level of HY5 accumulation modulates its binding to specific promoter elements of majority of clock genes, but only a few of these show altered transcription in the hy5 mutant. Mathematical modelling suggests that the direct effect of HY5 on the apparently non-responsive clock genes could be masked by feed-back from the clock gene network. We conclude that the information on the ratio of blue and red components of the white light spectrum is decoded and relayed to the circadian oscillator, at least partially, by HY5.
Project description:Cryptochromes were identified in plants and animals where they function as either photoreceptors or circadian clock components. In the filamentous fungus Neurospora, the biological function of cryptochrome has not yet been explored. Here, we demonstrate that Neurospora crassa cryptochrome (Nc cry) is a DASH-type of cryptochrome, capable of binding FAD and MTHF, whose transcript and protein levels are both strongly induced by blue light in a wc-1 dependent manner. Although the Nc cry transcript is circadian-regulated and antiphasic to frq, knockout strains of Nc cry appears to have a normal clock phenotype. Whole genome microarray and RT-QPCR analysis confirm that Nc cry is not involved in the signal transduction of either early or late light responses and seems to have no transcriptional regulatory activity under our laboratory conditions. Our study concludes that the only cryptochrome in Neurospora crassa is dispensable for the well-characterized blue light sensing cascade and is not part of the circadian clock system. Keywords: light response
Project description:Circadian pace is modulated by light intensity, known as the Aschoff’s rule, with largely unrevealed mechanisms. Here we report that photoreceptor CRY2 mediates blue light input to circadian clock by directly interacting with clock core component PRR9 in blue light dependent manner. This physical interaction dually blocks the accessibility of PRR9 protein to its co-repressor TPL/TPRs and the resulting kinase PPKs. Notably, phosphorylation of PRR9 by PPKs is critical for its DNA binding and repressive activity, hence to ensure proper circadian speed. Given the labile nature of CRY2 in strong blue light, our findings provide a mechanistic explanation for Aschoff’s rule in plants, i.e., blue light triggers CRY2 turnover in proportional to its intensity, which accordingly releasing PRR9 to fine tune circadian speed. Our findings not only reveal a novel network mediating light input into circadian clock, but also unmask a mechanism by which Arabidopsis circadian clock sensing light intensity.
Project description:We investigated light dependent gene expression changes in the marine ochrophyte Nannochloropsis oceanica CCMP1779. These algae have several putative blue light photoreceptors but appear to lack red light photoreceptors. To study early light signaling in N. oceanica and avoid as much as possible secondary downstream events, we quantified gene expression changes in dark-adapted cells after a short blue or red light pulse. More genes were differentially expressed under blue than under red light. In addition, fold change in expression was smaller for the red light-treated samples. For example, the median fold change of induced genes was 3 for blue light and 2.5 for red light. Moreover, hierarchical cluster analysis showed that gene expression after red light treatment was more similar to the dark control than after blue light treatment.
Project description:Cryptochromes were identified in plants and animals where they function as either photoreceptors or circadian clock components. In the filamentous fungus Neurospora, the biological function of cryptochrome has not yet been explored. Here, we demonstrate that Neurospora crassa cryptochrome (Nc cry) is a DASH-type of cryptochrome, capable of binding FAD and MTHF, whose transcript and protein levels are both strongly induced by blue light in a wc-1 dependent manner. Although the Nc cry transcript is circadian-regulated and antiphasic to frq, knockout strains of Nc cry appears to have a normal clock phenotype. Whole genome microarray and RT-QPCR analysis confirm that Nc cry is not involved in the signal transduction of either early or late light responses and seems to have no transcriptional regulatory activity under our laboratory conditions. Our study concludes that the only cryptochrome in Neurospora crassa is dispensable for the well-characterized blue light sensing cascade and is not part of the circadian clock system. Keywords: light response Two-color microarray. Alexa Fluor 555 was consistently used to label cDNA synthesized from reference RNA, which is a mixture containing equal amounts of RNA samples harvested from different circadian time points and light treatment durations. The same batch of pooled RNA was used as a reference for each array experiment. Alexa Fluor 647 was used exclusively to label cDNA representing sample RNA.
Project description:Sun-loving plants have the ability to detect and avoid shading through sensing of both blue and red light wavelengths. Higher plant cryptochromes (CRYs) control how plants modulate growth in response to changes in blue light. For growth under a canopy, where blue light is diminished, CRY1 and CRY2 perceive this change and respond by directly contacting two bHLH transcription factors, PIF4 and PIF5. These factors are also known to be controlled by phytochromes, the red/far-red photoreceptors; however, transcriptome analyses indicate that the gene regulatory programs induced by the different light wavelengths are distinct. Our results indicate that CRYs signal by modulating PIF activity genome-wide, and that these factors integrate binding of different plant photoreceptors to facilitate growth changes under different light conditions.
Project description:The red-light regulated transcription factors FHY3 and FAR1 form a key point of light input to the plant circadian clock in positively regulating expression of genes within the central clock. However, the fhy3 mutant shows an additional red light-specific disruption of rhythmicity which is inconsistent with this role. Here we demonstrate that only fhy3 and not far1 mutants show this red specific disruption of rhythmicity. We examined the differences in rhythmic transcriptome in red versus white light and reveal differences in patterns of rhythmicity among the central clock proteins suggestive of a change in emphasis withing the central mechanism of the clock, changes which underlie the red specificity of the fhy3 mutant. In particular, changes in enrichment of promoter elements were consistent with a key role for the HY5 transcription factor, a known integrator of the ratio of red to blue light in regulation of the clock. Examination of differences in the rhythmic transcriptome in the fhy3 mutant in red light which identified specific disruption of the CCA1-regulated ELF3 and LUX central clock genes, while the CCA1 target TBS element, TGGGCC, was enriched among genes that became arrhythmic. Coupled with the known interaction of FHY3 but not FAR1 with CCA1 we propose that the red-specific circadian phenotype of fhy3 may involve disruption of the previously-demonstrated moderation of CCA1 activity by FHY3 rather than a disruption of its own transcriptional regulatory activity. Together, this evidence suggests a conditional redundancy between FHY3 and HY5 in the integration of red and blue light input to the clock in order to enable a plasticity in response to light and optimise plant adaptation. Furthermore, our evidence also suggests changes in CCA1 activity between red and white light transcriptomes. This, together with the documented interaction of HY5 with CCA1, leads us to propose a model whereby this integration of red and blue signals may at least partly occur via direct FHY3 and HY5 interaction with CCA1 leading to moderation of CCA1 activity.
Project description:Sun-loving plants have the ability to detect and avoid shading through sensing of both blue and red light wavelengths. Higher plant cryptochromes (CRYs) control how plants modulate growth in response to changes in blue light. For growth under a canopy, where blue light is diminished, CRY1 and CRY2 perceive this change and respond by directly contacting two bHLH transcription factors, PIF4 and PIF5. These factors are also known to be controlled by phytochromes, the red/far-red photoreceptors; however, transcriptome analyses indicate that the gene regulatory programs induced by the different light wavelengths are distinct. Our results indicate that CRYs signal by modulating PIF activity genome-wide, and that these factors integrate binding of different plant photoreceptors to facilitate growth changes under different light conditions. We performed whole-genome chromatin immunoprecipitation with sequencing (ChIP-Seq) analysis on 5 day old Flash-CRY2, PIF4-Flash and PIF5-Flash treated in low blue-light for 16h.
Project description:Although many regulatory components of light signaling have been functionally characterized, only a few of them have been reported to cross talk with other signaling cascades. In this study, we have analyzed the expression profiles of Arabidopsis genes in wild-type, atmyc2 mutant, cop1-6 mutant, and atmyc2 cop1-6 double mutant seedlings grown under constant dark, constant blue-light, and constant blue-light along with abscisic acid (ABA) to illustrate the interplay of negative regulators, AtMYC2 and COP1, in light and ABA signaling Keywords: Light and abscisic acid treatment Expression data for wild-type, atmyc2, cop1-6, and atmyc2 cop1-6 seedlings under constant dark, blue-light and blue-light along with abscisic acid Ten-day-old Arabidopsis (wild-type, atmyc2 mutant, cop1-6 mutant, and atmyc2 cop1-6 double mutant) seedlings grown under constant dark, constant blue-light (35 µmol/sec/m2), and constant blue-light along with ABA (0.5 µm) were used for RNA extraction and hybridization on Affymetrix microarrays. Two biological replicates of each sample were used for microarray analysis.