The circadian clock is an endogenous time-keeping mechanism that enables organisms to adapt to external daily cycles. The clock coordinates biological activities with these cycles, mainly through ...genome-wide gene expression. However, the exact mechanism underlying regulation of circadian gene expression is poorly understood. Here we demonstrated that an Arabidopsis PSEUDO-RESPONSE REGULATOR 5 (PRR5), which acts in the clock genetic circuit, directly regulates expression timing of key transcription factors involved in clock-output pathways. A transient expression assay and ChIP-quantitative PCR assay using mutated PRR5 indicated that PRR5 associates with target DNA through binding at the CCT motif in vivo. ChIP followed by deep sequencing coupled with genome-wide expression profiling revealed the direct-target genes of PRR5. PRR5 direct-targets include genes encoding transcription factors involved in flowering-time regulation, hypocotyl elongation, and cold-stress responses. PRR5-target gene expression followed a circadian rhythm pattern with low, basal expression from noon until midnight, when PRR9, PRR7, and PRR5 were expressed. ChIP-quantitative PCR assays indicated that PRR7 and PRR9 bind to the direct-targets of PRR5. Genome-wide expression profiling using a prr9 prr7 prr5 triple mutant suggests that PRR5, PRR7, and PRR9 repress these targets. Taken together, our results illustrate a genetic network in which PRR5, PRR7, and PRR9 directly regulate expression timing of key transcription factors to coordinate physiological processes with daily cycles.
An interlocking transcriptional-translational feedback loop of clock-associated genes is thought to be the central oscillator of the circadian clock in plants. TIMING OF CAB EXPRESSION1 (also called ...PSEUDO-RESPONSE REGULATOR1 PRR1) and two MYB transcription factors, CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY), play pivotal roles in the loop. Genetic studies have suggested that PRR9, PRR7, and PRR5 also act within or close to the loop; however, their molecular functions remain unknown. Here, we demonstrate that PRR9, PRR7, and PRR5 act as transcriptional repressors of CCA1 and LHY. PRR9, PRR7, and PRR5 each suppress CCA1 and LHY promoter activities and confer transcriptional repressor activity to a heterologous DNA binding protein in a transient reporter assay. Using a glucocorticoid-induced PRR5-GR (glucorticoid receptor) construct, we found that PRR5 directly downregulates CCA1 and LHY expression. Furthermore, PRR9, PRR7, and PRR5 associate with the CCA1 and LHY promoters in vivo, coincident with the timing of decreased CCA1 and LHY expression. These results suggest that the repressor activities of PRR9, PRR7, and PRR5 on the CCA1 and LHY promoter regions constitute the molecular mechanism that accounts for the role of these proteins in the feedback loop of the circadian clock.
The plant circadian clock generates rhythms with a period close to 24 h, and it controls a wide range of physiological and developmental oscillations in habitats under natural light/dark cycles. ...Among clock-controlled developmental events, the best characterized is the photoperiodic control of flowering time in Arabidopsis thaliana. Recently, it was also reported that the clock regulates a daily and rhythmic elongation of hypocotyls. Here, we report that the promotion of hypocotyl elongation is in fact dependent on changes in photoperiods in such a way that an accelerated hypocotyl elongation occurs especially under short-day conditions. In this regard, we provide genetic evidence to show that the circadian clock regulates the photoperiodic (or seasonal) elongation of hypocotyls by modulating the expression profiles of the PIF4 and PIF5 genes encoding phytochrome-interacting bHLH (basic helix–loop–helix) factors, in such a manner that certain short-day conditions are necessary to enhance the expression of these genes during the night-time. In other words, long-day conditions are insufficient to open the clock-gate for triggering the expression of PIF4 and PIF5 during the night-time. Based on these and other results, the photoperiodic control of hypocotyl elongation is best explained by the accumulation of PIF4 and PIF5 during the night-time of short days, due to coincidence between the internal (circadian rhythm) and external (photoperiod) time cues. This mechanism is a mirror image of the photoperiod-dependent promotion of flowering in that plants should experience long-day conditions to initiate flowering promptly. Both of these clock-mediated coincidence mechanisms may coordinately confer ecological fitness to plants growing in natural habitats with varied photoperiods.
The plant circadian clock generates rhythms with a period close to 24 h, and it controls a wide variety of physiological and developmental events. Among clock-controlled developmental events, the ...best characterized is the photoperiodic control of flowering time, which is mediated through the CONSTANS (CO)-FLOWERING LOCUS T (FT) pathway in Arabidopsis thaliana. The clock also regulates the diurnal plant growth including the elongation of hypocotyls in a short day (SDs)-specific manner. In this mechanism, phytochromes (mainly phyB) and the PHYTOCHROME-INTERACTING FACTOR4 (PIF4) and PIF5, encoding phytochrome-interacting basic helix-loop-helix (bHLH) transcription factors, play crucial roles. The time of day-specific and photoperiodic control of hypocotyl elongation is best explained by the accumulation of the PIF4 and PIF5 proteins during night-time before dawn, especially under SDs, due to coincidence between the internal (circadian rhythm) and external (photoperiod) time cues. However, the PIF4- and/or PIF5-controlled downstream factors have not yet been identified. Here, we provide evidence that ARABIDOPSIS THALIANA HOMEOBOX PROTEIN2 (ATHB2), together with auxin-inducible IAA29, is diurnally expressed with a peak at dawn under the control of PIF4 and PIF5 specifically in SDs. This coincidentally expressed transcription factor serves as a positive regulator for the elongation of hypocotyls. The expression profiles of ATHB2 were markedly altered in certain clock and phytochrome mutants, all of which show anomalous phenotypes with regard to the photoperiodic control of hypocotyl elongation. Taken together, we propose that an external coincidence model involving the clock-controlled PIF4/PIF5-ATHB2 pathway is crucial for the diurnal and photoperiodic control of plant growth in A. thaliana.
We analyzed global gene expression in Arabidopsis in response to various hormones and in related experiments as part of the AtGenExpress project. The experimental agents included seven basic ...phytohormones (auxin, cytokinin, gibberellin, brassinosteroid, abscisic acid, jasmonate and ethylene) and their inhibitors. In addition, gene expression was investigated in hormone-related mutants and during seed germination and sulfate starvation. Hormone-inducible genes were identified from the hormone response data. The effects of each hormone and the relevance of the gene lists were verified by comparing expression profiles for the hormone treatments and related experiments using Pearson's correlation coefficient. This approach was also used to analyze the relationships among expression profiles for hormone responses and those included in the AtGenExpress stress-response data set. The expected correlations were observed, indicating that this approach is useful to monitor the hormonal status in the stress-related samples. Global interactions among hormones-inducible genes were analyzed in a pairwise fashion, and several known and novel hormone interactions were detected. Genome-wide transcriptional gene-to-gene correlations, analyzed by hierarchical cluster analysis (HCA), indicated that our data set is useful for identification of clusters of co-expressed genes, and to predict the functions of unknown genes, even if a gene's function is not directly related to the experiments included in AtGenExpress. Our data are available online from AtGenExpressJapan; the results of genome-wide HCA are available from PRIMe. The data set presented here will be a versatile resource for future hormone studies, and constitutes a reference for genome-wide gene expression in Arabidopsis.
In higher plants, the circadian clock controls a wide range of cellular processes such as photosynthesis and stress responses. Understanding metabolic changes in arrhythmic plants and determining ...output-related function of clock genes would help in elucidating circadian-clock mechanisms underlying plant growth and development. In this work, we investigated physiological relevance of PSEUDO-RESPONSE REGULATORS (PRR 9, 7, and 5) in Arabidopsis thaliana by transcriptomic and metabolomic analyses. Metabolite profiling using gas chromatography-time-of-flight mass spectrometry demonstrated well-differentiated metabolite phenotypes of seven mutants, including two arrhythmic plants with similar morphology, a PRR 9, 7, and 5 triple mutant and a CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1)-overexpressor line. Despite different light and time conditions, the triple mutant exhibited a dramatic increase in intermediates in the tricarboxylic acid cycle. This suggests that proteins PRR 9, 7, and 5 are involved in maintaining mitochondrial homeostasis. Integrated analysis of transcriptomics and metabolomics revealed that PRR 9, 7, and 5 negatively regulate the biosynthetic pathways of chlorophyll, carotenoid and abscisic acid, and α-tocopherol, highlighting them as additional outputs of pseudo-response regulators. These findings indicated that mitochondrial functions are coupled with the circadian system in plants.
Arabidopsis PSEUDO RESPONSE REGULATOR (PRR) genes are components of the circadian clock mechanism. In order to understand the scope of genome-wide transcriptional regulation by PRR genes, a ...comparison survey of gene expression in wild-type Arabidopsis and a prr9-11 prr7-10 prr5-10 triple mutant (d975) using mRNA collected during late daytime was conducted using an Affymetrix ATH-1 GeneChipsup(R). The expression of 'night genes' increased and the expression of 'day genes' decreased toward the end of the diurnal tight phase, but expression of these genes was essentially constant in d975. The expression levels of 'night genes' were lower, whereas the expression of 'day genes' was higher in d975 than in the wild type. Bioinformatics approaches have indicated that the set of up-regulated genes in d975 and the set of cold-responsive genes have significant overlap. We found that d975 is more tolerant to cold, high salinity and drought stresses than the wild type. In addition, dehydration-responsive element B1/C-repeat-binding factor (DREB1/CBF), which is expressed around mid-day, is more highly expressed in d975. Raffinose and L-proline accumulated at higher levels in d975 even when plants were grown under normal conditions. These results suggest that PRR9, PRR7 and PRR5 are involved in a mechanism that anticipates diurnal cold stress and which initiates a stress response by mediating cyclic expression of stress response genes, including DREB1/CBF.
Mass measurement using relay feedback of velocity and restoring force compensation is investigated for determining the mass of an object under weightless conditions. In the measurement system, the ...velocity of the object is fed back through a relay with hysteresis and the force acting on the object is switched from a positive value to a negative value when the velocity reaches a positive threshold and vice versa. As a result, a limit cycle is induced in the measurement system and the mass is estimated based on the period of the limit cycle. In addition, restoring force compensation with a spring is introduced to avoid the drift of the trajectory. This compensation makes the static equilibrium state unique. However, the trajectory still drifts slightly. It causes some error in measurement when a simple formula of estimating mass is applied. To eliminate such an error, a new formula is derived to estimate the mass independently of the position of the trajectory that is determined by the switching positions in the relay actions. When the switching positions deflect from the origin at which the spring is in the natural length, the trajectory is not at the center and becomes asymmetric. It is analytically shown that the period of the limit cycle is minimum when the switching positions are at the origin. It indicates that mass is overestimated with the simple estimation formula when the trajectory is not at the center. The validity of the modified formula and the analytical results are confirmed experimentally.
Arabidopsis thaliana has 11 members belonging to the typical type-B ARR (authentic response regulator) family. Among them, seven highly homologous members appear also to be conserved in rice (Oryza ...sativa), but others are not. It was suggested that these seven ARRs are commonly implicated as DNA-binding transcription factors in the phosphorelay-mediated cytokinin signal transduction network in higher plants. To gain an insight into the functions of the cytokinin-associated type-B ARRs, we previously investigated an arr1 arr10 arr12 triple mutant and reported that it exhibited stunted growth and abnormality in vascular development. Based on this fact, here we attempted to characterize the mutant intensively with reference to cytokinin-associated phenotypes through the life cycle. We showed that the observed cytokinin-associated phenotypes of arr1 arr10 arr12 were very severe and highly analogous to those observed for certain ahk2 ahk3 ahk4/cre1 triple mutants, which have virtually no cytokinin receptor to propagate the phosphorelay signal transduction network. Among the seven ARR members belonging to the cytokinin-associated type-B ARR subfamily, it was thus suggested that ARR1, ARR10 and ARR12 together play essential (or general) roles in cytokinin signal transduction. It is therefore conceivable that the other type-B ARRs (ARR2, ARR11, ARR14 and ARR18) might play more specific roles spatially and temporally in plants.
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