Norihito Nakamichi

PSEUDO-RESPONSE REGULATORS 9, 7, and 5 Are Transcriptional Repressors in the Arabidopsis Circadian Clock

PSEUDORESPONSE REGULATOR9 (PRR9), PRR7, and PRR5 regulate the Arabidopsis circadian clock. PRR9, PRR7, and PRR5 proteins associate with CIRCADIAN CLOCK ASSOCIATED1 and LATE ELONGATED HYPOCOTYL promoters in a sequential manner to repress their expression. A conserved region common to the three PRR proteins is sufficient for repressor activity. 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.

Impact of clock-associated Arabidopsis pseudo-response regulators in metabolic coordination

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.

Transcriptional repressor PRR5 directly regulates clock-output pathways

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.

Phytochrome-interacting factor 4 and 5 (PIF4 and PIF5) activate the homeobox ATHB2 and auxin-inducible IAA29 genes in the coincidence mechanism underlying photoperiodic control of plant growth of Arabidopsis thaliana.

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.

Molecular Mechanisms Underlying the Arabidopsis Circadian Clock

A wide range of biological processes exhibit circadian rhythm, enabling plants to adapt to the environmental day–night cycle. This rhythm is generated by the so-called ‘circadian clock’. Although a number of genetic approaches have identified >25 clock-associated genes involved in the Arabidopsis clock mechanism, the molecular functions of a large part of these genes are not known. Recent comprehensive studies have revealed the molecular functions of several key clock-associated proteins. This progress has provided mechanistic insights into how key clock-associated proteins are integrated, and may help in understanding the essence of the clocks molecular mechanisms.

The Common Function of a Novel Subfamily of B-Box Zinc Finger Proteins with Reference to Circadian-Associated Events in Arabidopsis thaliana

Over 1,600 genes encoding putative transcription factors have been identified in the Arabidopsis genome sequence, however, their physiological functions are not yet fully understood. In this study, a small subfamily of double B-box zinc finger (DBB, DOUBLE B-BOX) genes, encoding eight putative transcription factors, were characterized with reference to the circadian rhythm and the early photomorphogenic regulation of hypocotyl elongation in response to light signals. Among these, it was found that the transcriptions of five DBB genes were under the control of circadian rhythm. To gain insight into the physiological roles of these putative transcription factors, forward and reverse genetic studies were carried out. The results suggested that they are commonly implicated in light signal transduction during early photomorphogenesis, however, their functions are not totally redundant, as judged by the fact that their circadian-expression profiles (or phases) were distinctive from each other, and by the fact that some DBBs (named DBB1a, DBB1b, STO, and STH) were apparently implicated in light signal transduction in a negative manner, whereas another (named DBB3) was implicated in a positive manner with regard to light-induced inhibition of elongation of hypocotyls. We also found that homologous B-box zinc finger genes are widely conserved in higher plants (e.g., Oryza sativa). Taking this altogether, it is probable that in addition to previously characterized bZIP-type transcription factors (e.g., HY5 and HYH) and bHLH-type transcription factors (e.g., PIF4 and PIF5/PIL6), a set of B-box zinc finger transcription factors should also be taken into consideration for a better understanding of the complex molecular mechanisms underlying the early photomorphogenic development of Arabidopsis thaliana.

Circadian Clock- and PIF4-Controlled Plant Growth: A Coincidence Mechanism Directly Integrates a Hormone Signaling Network into the Photoperiodic Control of Plant Architectures in Arabidopsis thaliana

The plant circadian clock generates rhythms with a period close to 24 h, and it controls a wide variety of physiological and developmental events, enabling plants to adapt to ever-changing environmental light conditions. In Arabidopsis thaliana, the clock regulates the diurnal and photoperiodic plant growth including the elongation of hypocotyls and petioles in a time-of-day-specific and short-day (SD)-specific manner. In this mechanism, the clock-regulated PHYTOCHROME-INTERACTING FACTOR 4 gene encoding a basic helix-loop-helix transcription factor, together with phytochromes (mainly phyB), plays crucial roles. This diurnal and photoperiodic control of plant growth is best explained by the accumulation of the PIF4 protein at the end of the night-time specifically under SDs, due to coincidence between the internal (circadian rhythm) and external (photoperiod) cues. In this model, however, the PIF4-controlled downstream factors are not fully identified, although it has been generally proposed that the auxin-mediated signal transduction is crucially implicated. Here, we identified a set of hormone-associated genes as the specific PIF4 targets implicated in the photoperiodic control of plant growth. They include not only auxin-associated genes (GH3.5, IAA19 and IAA29), but also genes associated with other growth-regulating hormones such as brassinosteroids (BR6ox2), gibberellic acids (GAI), ethylene (ACS8) and cytokinin (CKX5). The dawn- and SD-specific expression profiles of these genes are modified in a set of phyB and clock mutants, both of which compromise the coincidence mechanism. The results of this study suggest that the circadian clock orchestrates a variety of hormone signaling pathways to regulate the photoperiod-dependent morphogenesis in A. thaliana.

LIGHT-REGULATED WD1 and PSEUDO-RESPONSE REGULATOR9 Form a Positive Feedback Regulatory Loop in the Arabidopsis Circadian Clock

Plant growth and development rely on proper operation of the circadian clock. Two clock genes, LIGHT-REGULATED WD1 (LWD1) and LWD2, maintain the function of the Arabidopsis circadian clock by adjusting the light input signal and regulating the expression of central oscillator genes. Our study also supports the presence of a positive feedback loop within the Arabidopsis circadian clock. In Arabidopsis thaliana, central circadian clock genes constitute several feedback loops. These interlocking loops generate an ~24-h oscillation that enables plants to anticipate the daily diurnal environment. The identification of additional clock proteins can help dissect the complex nature of the circadian clock. Previously, LIGHT-REGULATED WD1 (LWD1) and LWD2 were identified as two clock proteins regulating circadian period length and photoperiodic flowering. Here, we systematically studied the function of LWD1/2 in the Arabidopsis circadian clock. Analysis of the lwd1 lwd2 double mutant revealed that LWD1/2 plays dual functions in the light input pathway and the regulation of the central oscillator. Promoter:luciferase fusion studies showed that activities of LWD1/2 promoters are rhythmic and depend on functional PSEUDO-RESPONSE REGULATOR9 (PRR9) and PRR7. LWD1/2 is also needed for the expression of PRR9, PRR7, and PRR5. LWD1 is preferentially localized within the nucleus and associates with promoters of PRR9, PRR5, and TOC1 in vivo. Our results support the existence of a positive feedback loop within the Arabidopsis circadian clock. Further mechanistic studies of this positive feedback loop and its regulatory effects on the other clock components will further elucidate the complex nature of the Arabidopsis circadian clock.

A Circadian Clock- and PIF4-Mediated Double Coincidence Mechanism is Implicated in the Thermosensitive Photoperiodic Control of Plant Architectures in Arabidopsis thaliana

In Arabidopsis thaliana, the circadian clock regulates diurnal and photoperiodic plant growth including the elongation of hypocotyls in a time-of-day-specific and short-day (SD)-specific manner. The clock-controlled PHYTOCHROME-INTERACTING FACTOR 4 (PIF4) encoding a basic helix-loop-helix (bHLH) transcription factor plays crucial roles in this regulation. PIF4 is transcribed precociously at the end of the night in SDs, under which conditions the protein product is stably accumulated, while PIF4 is expressed exclusively during the daytime in long days (LDs), under which conditions the protein product is degraded by light-activated phytochrome B. The dawn- and SD-specific elongation of hypocotyls is best explained by the coincident accumulation of the active PIF4 protein during the night-time before dawn specifically in SDs. However, this coincidence model was challenged with the recent finding that the elongation of hypocotyls is markedly promoted at high growth temperature (i.e. 28°C) even under LDs in a PIF4-dependent manner. Here, we reconciled these apparently conflicting facts by showing that the transcription of PIF4 occurs precociously at the end of the night-time at 28°C in LDs, similarly to in SDs. Both the events resulted in the same consequence, i.e. that a set of PIF4 target genes (ATHB2, GH3.5, IAA19, IAA29, BRox2, GAI, ACS8 and CKX5) was induced accordingly in a time-of-day-specific manner. Taken together, we propose an extended double coincidence mechanism, by which the two environmental cues (i.e. photoperiods and temperatures), both of which vary on a season to season basis, are integrated into the same clock- and PIF4-mediated output pathway and regulate a hormone signaling network to fit plant architectures properly to domestic habitats.

Insight into Missing Genetic Links Between Two Evening-Expressed Pseudo-Response Regulator Genes TOC1 and PRR5 in the Circadian Clock-Controlled Circuitry in Arabidopsis thaliana

In Arabidopsis thaliana, many circadian clock-associated genes have been identified. Among them, the evening-expressed TOC1 (TIMING OF CAB EXPRESSION 1) gene plays a role by forming a transcriptional feedback core loop together with the morning-expressed CCA1 (CIRCADIAN CLOCK-ASSOCIATED 1) gene and its homologous LHY (LATE ELONGATED HYPOCOTYL) gene. TOC1 encodes a member of the PSEUDO-RESPONSE REGULATOR (PRR) family, including PRR9, PRR7, PRR5, PRR3,and PRR1/TOC1. The PRR genes other than TOC1 (or PRR1) also appear to be crucial for certain circadian-associated events. To clarify missing genetic linkages amongst these PRR genes, here we constructed a toc1 prr5 double knockdown mutant. In free-running circadian rhythms, the resulting toc1-2 prr5-11 mutant plants showed an extremely short period and reduced amplitude phenotype, which was more severe than that of the toc1-2 single mutant plant, suggesting a non-linear genetic interaction between TOC1 and PRR5. Surprisingly, the hallmark early flowering phenotype of toc1-2 in the short-day conditions had been converted to a markedly late flowering phenotype in the long-day conditions, when combined with the prr5-11 allele, which itself showed a subtle flowering phenotype. This unexpected genetic result (i.e. phenotypic sign conversion) suggested that the TOC1 and PRR5 genes are coordinately implicated in a non-linear and closed genetic circuitry. In the toc1-2 prr5-11 double mutant, the diurnal expression profile of CDF1 (CYCLING DOF FACTOR 1) was markedly de-repressed in the evening in the long-day conditions. These and other results of this study led us to propose the novel view that TOC1 might play bipartite roles in the control of flowering time within a closed circuitry; the one is a GI (GIGANTEA)-dependent negative role through CCA1/LHY, and the other is a CDF1-dependent positive role through cooperating closely with PRR5.