PSEUDO-RESPONSE REGULATOR Research Articles

Genetic Linkages Between Circadian Clock-Associated Components and Phytochrome-Dependent Red Light Signal Transduction in Arabidopsis thaliana

The current best candidates for Arabidopsis thaliana clock components are CCA1 (CIRCADIAN CLOCK-ASSOCIATED 1) and its homolog LHY (LATE ELONGATED HYPOCOTYL). In addition, five members of a small family, PSEUDO-RESPONSE REGULATORS (including PRR1, PRR3, PRR5, PRR7 and PRR9), are believed to be another type of clock component. The originally described member of PRRs is TOC1 (or PRR1) (TIMING OF CAB EXPRESSION 1). Interestingly, seedlings of A. thaliana carrying a certain lesion (i.e. loss-of-function or misexpression) of a given clock-associated gene commonly display a characteristic phenotype of light response during early photomorphogenesis. For instance, cca1 lhy double mutant seedlings show a shorter hypocotyl length than the wild type under a given fluence rate of red light (i.e. hypersensitivity to red light). In contrast, both toc1 single and prr7 prr5 double mutant seedlings with longer hypocotyls are hyposensitive under the same conditions. These phenotypes are indicative of linkage between the circadian clock and red light signal transduction mechanisms. Here this issue was addressed by conducting combinatorial genetic and epistasis analyses with a large number of mutants and transgenic lines carrying lesions in clock-associated genes, including a cca1 lhy toc1 triple mutant and a cca1 lhy prr7 prr5 quadruple mutant. Taking these results together, we propose a genetic model for clock-associated red light signaling, in which CCA1 and LHY function upstream of TOC1 (PRR1) in a negative manner, in turn, TOC1 (PRR1) serves as a positive regulator. PRR7 and PRR5 also act as positive regulators, but independently from TOC1 (PRR1). It is further suggested that these signaling pathways are coordinately integrated into the phytochrome-mediated red light signal transduction pathway, in which PIF3 (PHYTOCHROME-INTERACTING FACTOR 3) functions as a negative regulator immediately downstream of phyB.

Arabidopsis Clock-Associated Pseudo-Response Regulators PRR9, PRR7 and PRR5 Coordinately and Positively Regulate Flowering Time Through the Canonical CONSTANS-Dependent Photoperiodic Pathway

Photoperiodism allows organisms to measure daylength, or external photoperiod, and to anticipate coming seasons. Daylength measurement requires the integration of light signal and temporal information by the circadian clock. In the long-day plant Arabidopsis thaliana, CONSTANS (CO) plays a crucial role in integrating the circadian rhythm and environmental light signals into the photoperiodic flowering pathway. Nevertheless, the molecular mechanism by which the circadian clock modulates the cyclic expression profile of CO is poorly understood. Here, we first showed that the clock-associated genes PSEUDO-RESPONSE REGULATOR (PRR) PRR9, PRR7 and PRR5 are involved in activation of CO expression during the daytime. Then, extensive genetic studies using CIRCADIAN CLOCK-ASSOCIATED1 (CCA1)/LATE ELONGATED HYPOCOTYL (LHY) double mutants (cca1/lhy) and prr7/prr5 were conducted. The results suggested that PRR genes act coordinately in a manner parallel with and antagonistic to CCA/LHY, upstream of the canonical CO-FLOWERING LOCUS T (FT) photoperiodic flowering pathway. Finally, we provided evidence to propose a model, in which CCA1/LHY repress CO through GIGANTEA (GI), while PRR9, PRR7 and PRR5 activate CO predominantly by repressing CYCLING DOF FACTOR1 (CDF1) encoding a DNA-binding transcriptional repressor.

Characterization of a Set of Phytochrome-Interacting Factor-Like bHLH Proteins inOryza sativa

The model dicotyledon Arabidopsis thaliana has a characteristic small sub-family of phytochrome-interacting bHLH (basic Helix-Loop-Helix) factors, which are collectively designated the PIL (or PIF) (PHYTOCHROME INTERACTING FACTOR-LIKE) family proteins. In this study, we identified and characterized a set of highly homologous members (designated OsPIL11 to OsPIL16) in the model monocotyledon rice (Oryza sativa). Some of them (OsPIL11, OsPIL12, and OsPIL13) showed the ability to interact with the putative OsPRR1 (PSEUDO-RESPONSE REGULATOR 1) clock component, as far as the results of yeast two-hybrid assays were concerned. It was found that the expression of OsPIL13 is under the control of circadian rhythms (clock), while the expression of OsPIL15 is negatively regulated by light upon the onset to light exposure of etiolated seedlings. When the rice genes (OsPIL11 to OsPIL15) were over-expressed in A. thaliana, the resulting transgenic seedlings displayed anomalous morphologies with very long hypocotyls during early photomorphogenesis. These results suggest the view that the identified OsPILs are functional counterparts (or orthologs) of AtPILs, which are known to play important roles in red light-mediated (phyA and/or phyB-dependent) signal transduction pathways at immediate positions downstream of the photoreceptor in A. thaliana.

Characterization of the Rice Circadian Clock-Associated Pseudo-Response Regulators inArabidopsis thaliana

Members of the small family of Arabidopsis PSEUDO-RESPONSE REGULATORS (PRR1/TOC1, PRR3, PRR5, PRR7, and PRR9) play roles close to the circadian clock in Arabidopsis thaliana. We have reported that the rice (Oryza sativa) genome also encodes a set of PRR counterparts (designated OsPRR1, OsPRR37, OsPRR59, OsPRR73, and OsPRR95 respectively). To gain new insight into the molecular functions of OsPRRs, we carried out genetic complementation analyses by introducing two representative rice genes, OsPRR1 and OsPRR37, into the corresponding Arabidopsis loss-of-function mutants (toc1 and prr7 respectively). The results showed that these OsPRR and AtPRR genes are genetically interchangeable at least in part, suggesting the conserved clock-associated function of these OsPRRs.

The two-component signal system in rice ( Oryza sativa L.): A genome-wide study of cytokinin signal perception and transduction

In this report we define the genes of two-component regulatory systems in rice through a comprehensive computational analysis of rice ( Oryza sativa L.) genome sequence databases. Thirty-seven genes were identified, including 5 HKs (cytokinin-response histidine protein kinase) ( OsHK1–4, OsHKL1), 5 HPs (histidine phosphotransfer proteins) ( OsHP1–5), 15 type-A RRs (response regulators) ( OsRR1–15), 7 type B RR genes ( OsRR16–22), and 5 predicted pseudo-response regulators ( OsPRR1–5). Protein motif organization, gene structure, phylogenetic analysis, chromosomal location, and comparative analysis between rice, maize, and Arabidopsis are described. Full-length cDNA clones of each gene were isolated from rice. Heterologous expression of each of the OsHKs in yeast mutants conferred histidine kinase function in a cytokinin-dependent manner. Nonconserved regions of individual cDNAs were used as probes in expression profiling experiments. This work provides a foundation for future functional dissection of the rice cytokinin two-component signaling pathway.

Mutants of Circadian-AssociatedPRRGenes Display a Novel and Visible Phenotype as to Light Responses during De-Etiolation ofArabidopsis thalianaSeedlings

In Arabidopsis thaliana, it is currently accepted that certain mutants with lesions in clock-associated genes commonly display hallmarked phenotypes with regard to three characteristic biological events: (i) altered rhythmic expression of circadian-controlled genes, (ii) changes in flowering time, and (iii) altered sensitivity to red light in elongation of hypocotyls. During the course of examination of the clock-associated mutants of PSEUDO-RESPONSE REGULATORS, PRRs, including TOC1 (PRR1), we found that they commonly show another visible phenotype of anomalous greening responses upon the onset to light exposure of etiolated seedlings. These findings are indicative of a novel link between circadian rhythms and chloroplast development.

Characterization of Circadian-Associated Pseudo-Response Regulators: I. Comparative Studies on a Series of Transgenic Lines Misexpressing Five Distinctive PRR Genes inArabidopsis thaliana

Every member of a small family of Pseudo-Response Regulator (PRR) genes, including Timing of Cab Expression 1 (TOC1 [or PRR1]), are believed to play roles close to the circadian clock in the model higher plant Arabidopsis thaliana. In this study we established a transgenic line that misexpresses (or overexpresses) the PRR7 gene. As compared with wild-type plants, the resulting PRR7-misexpressing plants (designated PRR7-ox) showed characteristic phenotypes as to hallmarked circadian-associated biological events: (i) early flowering in a manner independent of photoperiodicity, (ii) hypersensitive response to red light during early photomorphogenesis, and (iii) altered free-running rhythms with long period of clock-associated genes. Finally, a series of all transgenic lines (PRR1-ox, PRR3-ox, PRR5-ox, PRR7-ox, and PRR9-ox) were characterized comparatively with regard to their clock-associated roles. The results suggested that the five homologous PRR factors play coordinate roles, distinctively from one another, and closely to the circadian clock in higher plants.

Characterization of Circadian-Associated Pseudo-Response Regulators: II. The Function of PRR5 and Its Molecular Dissection inArabidopsis thaliana

Together with PRR1/TOC1, PRR5 belongs to the small family of Pseudo-Response Regulators (PRRs), which function as clock components of Arabidopsis thaliana. We employed a set of transgenic lines, each of which was designed to misexpress a truncated form of the PRR5 molecule, together with the original transgenic line (named PRR5-ox) that misexpresses the entire PRR5 polypeptide. The results of genetic analysis suggested that PRR5-ox seedlings showed a phenotype of hypersensitivity to red light during early photomorphogenesis in a manner dependent on red light photoreceptors (PhyA and PhyB), but independent of PRR1/TOC1. The set of newly constructed transgenic lines (named PRR5-N-ox and PRR5-C-ox) were also characterized in terms of circadian-associated phenotypes. The results suggest that the N-terminal pseudo-receiver domain of the PRR5 molecule seems to be dispensable for the misexpressed PRR5 molecule to bring about the phenotype of red light sensitivity. However, PRR5-N-ox plants, misexpressing only the pseudo-receiver domain, showed a phenotype of long period of free-running circadian rhythms of certain clock-controlled genes. Considering these and other results, we discuss the structure and function of PRR5 in the context of current views of the circadian clock in higher plants.

シロイヌナズナにおける光誘導性、時計関連遺伝子PRR9のプロモーター解析とタンパク質解析：PRR9タンパク質の存在量も概日変動し、暗条件で速やかに分解される

シロイヌナズナにおける光誘導性、時計関連遺伝子PRR9のプロモーター解析とタンパク質解析：PRR9タンパク質の存在量も概日変動し、暗条件で速やかに分解される

Comparative Overviews of Clock-Associated Genes of Arabidopsis thaliana and Oryza sativa

In higher plants, circadian rhythms are highly relevant to a wide range of biological processes. To such circadian rhythms, the clock (oscillator) is central, and recent intensive studies on the model higher plant Arabidopsis thaliana have begun to shed light on the molecular mechanisms underlying the functions of the central clock. Such representative clock-associated genes of A. thaliana are the homologous CCA1 and LHY genes, and five PRR genes that belong to a small family of pseudo-response regulators including TOC1. Others are GI, ZTL, ELF3, ELF4, LUX/PCL1, etc. In this context, a simple question arose as to whether or not the molecular picture of the model Arabidopsis clock is conserved in other higher plants. Here we made an effort to answer the question with special reference to Oryza sativa, providing experimental evidence that this model monocot also has a set of highly conserved clock-associated genes, such as those designated as OsCCA1, OsPRR-series including OsTOC1/OsPRR1, OsZTLs, OsPCL1 as well as OsGI. These results will provide us with insight into the general roles of plant circadian clocks, such as those for the photoperiodic control of flowering time that has a strong impact on the reproduction and yield in many higher plants.

Warm and cold parental reproductive environments affect seed properties, fitness, and cold responsiveness in Arabidopsis thaliana progenies

Conditions in the parental environment during reproduction can affect the performance of the progenies. The goals of this study were to investigate whether warm or cold temperatures in the parental environment during flowering and seed development affect Arabidopsis thaliana seed properties, growth performance, reproduction and stress tolerance of the progenies, and to find candidate genes for progeny-related differences in stress responsiveness. Parental plants were raised at 20 degrees C and maintained from bolting to seed maturity at warm (25 degrees C) or cold (15 degrees C) temperatures. Analysis of seed properties revealed significant increases in nitrogen in seeds from warm temperature and significant increases in lipids and in the ratio of alpha-linolenic to oleic acid in seeds from the cold parental environment. Progenies of the warm parental environment showed faster germination rates, faster root elongation growth, higher leaf biomass and increased seed production at various temperatures compared with those from the cold parental environment. This indicates that under stable environmental conditions, progenies from warm parental environments had a clear adaptive advantage over those from cold parental environments. This parental effect was presumably transmitted by the higher nitrogen content of the seeds developed in warm conditions. When offspring from parents grown at different temperatures were exposed to chilling or freezing stress, photosynthetic yield recovered faster in progenies originating from cold parental environments. Cold acclimation involved up-regulation of transcripts of flavanone 3-hydroxylase (F3H) and pseudo response regulator 9 (PRR9) and down-regulation of growth-associated transcription factors (TFs) NAP and AP2domain containing RAP2.3. NAP, a regulator of senescence, and PRR9, a temperature-sensitive modulator of the circadian clock, were probably involved in mediating parent-of-origin effects, because they showed progeny-related expression differences under chilling. Because low temperatures also delay senescence, cold responsiveness of NAP suggests that this factor is linked with the regulatory network that is important for environmental acclimation of plants.

Analysis of Phase of LUCIFERASE Expression Reveals Novel Circadian Quantitative Trait Loci in Arabidopsis

In response to exogenous rhythms of light and temperature, most organisms exhibit endogenous circadian rhythms (i.e. cycles of behavior and gene expression with a periodicity of approximately 24 h). One of the defining characteristics of the circadian clock is its ability to synchronize (entrain) to an environmental rhythm. Entrainment is arguably the most salient feature of the clock in evolutionary terms. Previous quantitative trait studies of circadian characteristics in Arabidopsis (Arabidopsis thaliana) considered leaf movement under constant (free-running) conditions. This study, however, addressed the important circadian parameter of phase, which reflects the entrained relationship between the clock and the external cycle. Here it is shown that, when exposed to the same photoperiod, Arabidopsis accessions differ dramatically in phase. Variation in the timing of circadian LUCIFERASE expression was used to map loci affecting the entrained phase of the clock in a recombinant population derived from two geographically distant accessions, Landsberg erecta and Cape Verde Islands. Four quantitative trait loci (QTL) were found with major effects on circadian phase. A QTL on chromosome 5 contained SIGNALING IN RED LIGHT REDUCED 1 and PSEUDORESPONSE REGULATOR 3, both genes known to affect the circadian clock. Previously unknown polymorphisms were found in both genes, making them candidates for the effect on phase. Fine mapping of two other QTL highlighted genomic regions not previously identified in any circadian screens, indicating their effects are likely due to genes not hitherto considered part of the circadian system.

イネにおける時計関連疑似レスポンスレギュレーター因子の機能解析：AtPRR1/TOC1のオーソログOsPRR1機能解析

イネにおける時計関連疑似レスポンスレギュレーター因子の機能解析：AtPRR1/TOC1のオーソログOsPRR1機能解析

Systems biology flowering in the plant clock field

Mol Syst Biol. 2: 60 In the 18th Century, the Swedish botanist Karl von Linne designed a ‘Flower‐Clock’ by arranging a series of various plant species according to the respective time their flowers open or close every day. Watching this ‘Flower‐Clock’, one can then estimate the time of the day by noting the pattern of flower opening and closing. It has been a well‐known fact since Linne's early times that plants can open or close their flowers at a precise time of the day. However, we still do not fully understand the design principles of the molecular network that underlies the cellular circadian clock, which achieves to predict, often with exquisite precision, the cyclic changes in the environment due to the rotation of earth. In two articles currently published in Molecular Systems Biology , Millar and co‐workers (Locke et al , 2006) and Doyle and co‐workers (Zeilinger et al , 2006) propose a plausible design for the plant circadian clock. In previous work, Millar and co‐workers extended an initial ‘one‐loop model’ of the plant circadian clock into a ‘two‐loop model’ (Figure 1) (Locke et al , 2005). In the simple ‘one‐loop model’ (Figure 1 …

Experimental validation of a predicted feedback loop in the multi‐oscillator clock of Arabidopsis thaliana

Our computational model of the circadian clock comprised the feedback loop between LATE ELONGATED HYPOCOTYL (LHY), CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and TIMING OF CAB EXPRESSION 1 (TOC1), and a predicted, interlocking feedback loop involving TOC1 and a hypothetical component Y. Experiments based on model predictions suggested GIGANTEA (GI) as a candidate for Y. We now extend the model to include a recently demonstrated feedback loop between the TOC1 homologues PSEUDO-RESPONSE REGULATOR 7 (PRR7), PRR9 and LHY and CCA1. This three-loop network explains the rhythmic phenotype of toc1 mutant alleles. Model predictions fit closely to new data on the gi;lhy;cca1 mutant, which confirm that GI is a major contributor to Y function. Analysis of the three-loop network suggests that the plant clock consists of morning and evening oscillators, coupled intracellularly, which may be analogous to coupled, morning and evening clock cells in Drosophila and the mouse.

The Pseudo-Response Regulator Ppd-H1 Provides Adaptation to Photoperiod in Barley

Plants commonly use photoperiod (day length) to control the timing of flowering during the year, and variation in photoperiod response has been selected in many crops to provide adaptation to different environments and farming practices. Positional cloning identified Ppd-H1, the major determinant of barley photoperiod response, as a pseudo-response regulator, a class of genes involved in circadian clock function. Reduced photoperiod responsiveness of the ppd-H1 mutant, which is highly advantageous in spring-sown varieties, is explained by altered circadian expression of the photoperiod pathway gene CONSTANS and reduced expression of its downstream target, FT, a key regulator of flowering.

Positive and Negative Factors Confer Phase-Specific Circadian Regulation of Transcription in Arabidopsis

The circadian clock exerts a major influence on transcriptional regulation in plants and other organisms. We have previously identified a motif called the evening element (EE) that is overrepresented in the promoters of evening-phased genes. Here, we demonstrate that multimerized EEs are necessary and sufficient to confer evening-phased circadian regulation. Although flanking sequences are not required for EE function, they can modulate EE activity. One flanking sequence, taken from the PSEUDORESPONSE REGULATOR 9 promoter, itself confers dawn-phased rhythms and has allowed us to define a new clock promoter motif (the morning element [ME]). Scanning mutagenesis reveals that both activators and repressors of gene expression act through the ME and EE. Although our experiments confirm that CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) are likely to act as repressors via the EE, they also show that they have an unexpected positive effect on EE-mediated gene expression as well. We have identified a clock-regulated activity in plant extracts that binds specifically to the EE and has a phase consistent with it being an activator of expression through the EE. This activity is reduced in CCA1/LHY null plants, suggesting it may itself be part of a circadian feedback loop and perhaps explaining the reduction in EE activity in these double mutant plants.

Pseudo-Response Regulators (PRRs) or True Oscillator Components (TOCs)

In Arabidopsis thaliana, AUTHENTIC RESPONSE REGULATORS (ARRs) act as downstream components of the His-to-Asp phosphorelay (two-component) signaling pathway that is propagated primarily by the cytokinin receptor kinases, AUTHENTIC HIS-KINASES (AHK2, AHK3 and AHK4/CRE1). Thus, this bacterial type of signaling system is essential for responses to a class of hormones in plants. Interestingly, this higher plant has also evolved its own atypical (or unique) variants of two-component signal transducers, PSEUDO-RESPONSE REGULATORS (PRRs). Several lines of recent results suggest that the functions of PRRs are closely relevant to the plant clock (oscillator) that is central to circadian rhythms, the underlying mechanisms of which have long been the subject of debate. Through an overview of recent results, the main issue addressed here is whether or not the pseudo-response regulators (PRRs) are true oscillator components (TOCs).

PSEUDO-RESPONSE REGULATORS, PRR9, PRR7 and PRR5, Together Play Essential Roles Close to the Circadian Clock of Arabidopsis thaliana

In Arabidopsis thaliana, a number of clock-associated protein components have been identified. Among them, CCA1 (CIRCADIAN CLOCK-ASSOCIATED 1)/LHY (LATE ELONGATED HYPOCOTYL) and TOC1 (TIMING OF CAB EXPRESSION 1) are believed to be the essential components of the central oscillator. CCA1 and LHY are homologous and partially redundant Myb-related DNA-binding proteins, whereas TOC1 is a member of a small family of proteins, designated as PSEUDO-RESPONSE REGULATOR. It is also believed that these two different types of clock components form an autoregulatory positive/negative feedback loop at the levels of transcription/translation that generates intrinsic rhythms. Nonetheless, it was not yet certain whether or not other PRR family members (PRR9, PRR7, PRR5 and PRR3) are implicated in clock function per se. Employing a set of prr9, prr7 and prr5 mutant alleles, here we established all possible single, double and triple prr mutants. They were examined extensively by comparing them with each other with regard to their phenotypes of circadian rhythms, photoperiodicity-dependent control of flowering time and photomorphogenic responses to red light during de-etiolation. Notably, the prr9 prr7 prr5 triple lesions in plants resulted in severe phenotypes: (i) arrhythmia in the continuous light conditions, and an anomalous phasing of diurnal oscillation of certain circadian-controlled genes even in the entrained light/dark cycle conditions; (ii) late flowering that was no longer sensitive to the photoperiodicity; and (iii) hyposensitivity (or blind) to red light in the photomorphogenic responses. The phenotypes of the single and double mutants were also characterized extensively, showing that they exhibited circadian-associated phenotypes characteristic for each. These results are discussed from the viewpoint that PRR9/PRR7/PRR5 together act as period-controlling factors, and they play overlapping and distinctive roles close to (or within) the central oscillator in which the relative, PRR1/TOC1, plays an essential role.

The Arabidopsis Pseudo-response Regulators, PRR5 and PRR7, Coordinately Play Essential Roles for Circadian Clock Function

In Arabidopsis thaliana, a number of clock-associated protein factors have been identified. Among them, TOC1 (TIMING OF CAB EXPRESSION 1) is believed to be a component of the central oscillator. TOC1 is a member of a small family of proteins, designated as ARABIDOPSIS PSEUDO-RESPONSE REGULATOR, including PRR1/TOC1, PRR3, PRR5, PRR7 and PRR9. It has not been certain whether or not other PRR family members are also implicated in clock function per se. To clarify this problem, here we constructed a double mutant line, which is assumed to have severe lesions in both the PRR5 and PRR7 genes. Resulting homozygous prr5-11 prr7-11 young seedlings showed a marked phenotype of hyposensitivity to red light during de-etiolation. In addition, they displayed a phenotype of extremely late flowering under long-day photoperiod conditions, but not short-day conditions. The rhythms at the level of transcription of certain clock-controlled genes were severely perturbed in the double mutant plants when they were released into continuous light (LL) and darkness (DD). The observed phenotype was best interpreted as 'arrhythmic in both LL and DD' and/or 'very short period with markedly reduced amplitude'. Even under the light entrainment (LD) conditions, the mutant plants showed anomalous diurnal oscillation profiles with altered amplitude and/or phase with regard to certain clock-controlled genes, including the clock component CCA1 (CIRCADIAN CLOCK-ASSOCIATED 1) gene. Such events were observed even under temperature entrainment conditions, suggesting that the prr5-11 prr7-11 lesions cannot simply be attributed to a defect in the light signal input pathway. These pleiotropic circadian-associated phenotypes of the double mutant were very remarkable, as compared with those observed previously for each single mutant. Taking these results together, we propose for the first time that PRR5 and PRR7 coordinately (or synergistically) play essential clock-associated roles close to the central oscillator.