The evolutionarily conserved OsPRR quintet: rice pseudo-response regulators implicated in circadian rhythm

In Arabidopsis thaliana, a number of circadian-associated factors have been identified, including TOC1 (TIMING OF CAB EXPRESSION 1) that 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 REGULATORS (APRR1/TOC1, APRR3, APRR5, APRR7, and APRR9). As demonstrated previously, these APRR1/TOC1 quintet members are crucial for a better understanding of the molecular links between circadian rhythms, control of flowering time through photoperiodic pathways, and also photosensory signal transduction in this dicotyledonous plant. In this respect, both the dicotyledonous (e.g. A. thaliana) and monocotyledonous (e.g. Oryza sativa) plants might share the evolutionarily conserved molecular mechanism underlying the circadian rhythm. Based on such an assumption, and as the main objective of this study, we asked the question of whether rice also has a set of pseudo-response regulators, and if so, whether or not they are associated with the circadian rhythm. Here we showed that rice has five members of the OsPRR family (Oryza sativa Pseudo-Response Regulator), and also that the expressions of these OsPRR genes are under the control of circadian rhythm. They are expressed in a diurnal and sequential manner in the order of OsPRR73 (OsPRR37)-->OsPRR95 (OsPRR59)-->OsPRR1, which is reminiscent of the circadian waves of the APRR1/TOC1 quintet in A. thaliana. These and other results of this study suggested that the OsPRR quintet, including the ortholog of APRR1/TOC1, might play important roles within, or close to, the circadian clock of rice.     

Comparative genetic studies on the APRR5 and APRR7 genes belonging to the APRR1/TOC1 quintet implicated in circadian rhythm, control of flowering time, and early photomorphogenesis

In Arabidopsis thaliana, a number of circadian-associated factors have been identified. Among those, 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 REGULATORS (APRR1/TOC1, APRR3, APRR5, APRR7, and APRR9). Nonetheless, it is not very clear whether or not the APRR family members other than APRR1/TOC1 are also implicated in the mechanisms underlying the circadian rhythm. To address this issue further, here we characterized a set of T-DNA insertion mutants, each of which is assumed to have a severe lesion in each one of the quintet genes (i.e. APRR5 and APRR7). For each of these mutants (aprr5-11 and aprr7-11) we demonstrate that a given mutation singly, if not directly, affects the circadian-associated biological events simultaneously: (i) flowering time in the long-day photoperiod conditions, (ii) red light sensitivity of seedlings during the early photomorphogenesis, and (iii) the period of free-running rhythms of certain clock-controlled genes including CCA1 and APRR1/TOC1 in constant white light. These results suggest that, although the quintet members other than APRR1/TOC1 may not be directly integrated into the framework of the central oscillator, they are crucial for a better understanding of the molecular mechanisms underlying the Arabidopsis circadian clock.     

Characterization of circadian-associated APRR3 pseudo-response regulator belonging to the APRR1/TOC1 quintet in Arabidopsis thaliana

In higher plants, there are wide ranges of biological processes that are controlled through the circadian clock. In this connection, we have been characterizing a small family of proteins, designated as ARABIDOPSIS PSEUDO-RESPONSE REGULATORS (APRR1, APRR3, APRR5, APRR7, and APRR9), among which APRR1 is identical to TOC1 (TIMING OF CAB EXPRESSION1) that is believed to be a component of the central oscillator. Through previous genetic studies, several lines of evidence have already been provided to support the view that, not only APRR1/TOC1, but also other APRR1/TOC1 quintet members are important for a better understanding of the molecular links between circadian rhythm, control of flowering time, and also photomorphogenesis. However, the least characterized one was APRR3 in that no genetic study has been conducted to see if APRR3 also plays an important role in the circadian-associated biological events. Here we show that APRR3-overexpressing transgenic plants (APRR3-ox) exhibited: (i). a phenotype of longer period (and/or delayed phase) of rhythms of certain circadian-controlled genes under continuous white light, (ii). a phenotype of late flowering under long-day photoperiod conditions, (iii). a phenotype of hypo-sensitiveness to red light during early photomorphogenesis of de-etiolated seedlings, supporting the current idea as to the APRR1/TOC1 quintet described above.     

Characterization of plant circadian rhythms by employing Arabidopsis cultured cells with bioluminescence reporters

Recent intensive studies have begun to shed light on the molecular mechanisms underlying the plant circadian clock in Arabidopsis thaliana. During the course of these previous studies, the most powerful technique, elegantly adopted, was a real-time bioluminescence monitoring system of circadian rhythms in intact plants carrying a luciferase (LUC) fusion transgene. We previously demonstrated that Arabidopsis cultured cells also retain an ability to generate circadian rhythms, at least partly. To further improve the cultured cell system for studies on circadian rhythms, here we adopted a bioluminescence monitoring system by establishing the cell lines carrying appropriate reporter genes, namely, CCA1::LUC and APRR1::LUC, with which CCA1 (CIRCADIAN CLOCK-ASSOCIATED1) and APRR1 (or TOC1) (ARABIDOPSIS PSEUDO-RESPONSE REGULATORS1 or TIMING OF CAB EXPRESSION1) are believed to be the components of the central oscillator. We report the results that consistently supported the view that the established cell lines, equipped with such bioluminescence reporters, might provide us with an advantageous means to characterize the plant circadian clock.     

Isolation of circadian-associated genes in Brassica rapa by comparative genomics with Arabidopsis thaliana

Elucidation of the roles of circadian associated factors requires a better understanding of the molecular mechanisms of circadian rhythms, control of flowering time through photoperiodic pathways, and photosensory signal transduction. In Arabidopsis, the APRR1 quintet, APRRs 1, 3, 5, 7, and 9, are known as central oscillator genes. Other plants may share the molecular mechanism underlying the circadian rhythm. To identify and characterize these circadian response genes in Brassica crops whose genome was triplicated after divergence from Arabidopsis, we identified B. rapa BAC clones containing these genes by BLAST analysis of B. rapa BAC end sequences against the five corresponding Arabidopsis regions. Subsequent fingerprinting, Southern hybridization, and PCR allowed identification of five BAC clones, one for each of the five circadian-related genes. By draft shotgun sequencing of the BAC clones, we identified the complete gene sequences and cloned the five expressed B. rapa circadian-associated gene members, BrPRRs 1, 3, 5, 7, and 9. Phylogenetic analysis revealed that each BrPRR was orthologous to the corresponding APRR at the sequence level. Northern hybridization revealed that the five genes were transcribed at distinct points in the 24 hour period, and Southern hybridization revealed that they are present in 2, 1, 2, 2, and 1 copies, respectively in the B. rapa genome, which was triplicated and then diploidized during the last 15 million years.     

Insights into the regulation of the core clock component TOC1 in the green picoeukaryote Ostreococcus

Living organisms such as plants and animals have evolved endogenous clocks in order to anticipate the environmental changes associated with the earth’s rotation and to orchestrate biological processes in the course of the 24 hour daily cycle. We have recently identified clock components in the primitive green picoalga Ostreococcus tauri, a promising minimal cellular and genomic model for systems biology approaches. A homologue of the Arabidopsis core clock gene Time of CAB expression-1 (TOC1) was shown to play a central role in Ostreococcus heralding an early emergence of clock components in the green lineage. Here we report the regulation of TOC1 at dusk in response to light and dark cues.Key words: Ostreococcus, circadian, clock, plants, microalgaeThe circadian clock is an autonomous timer, which provides for living organisms a means to measure time internally. As such the clock has two fundamental properties: (1) it allows the organism to anticipate daily predictable environmental changes such as light and temperature cycles; (2) it coordinates key physiological processes during the 24 hour daily cycle. As a result, internal time given by the clock and external time given by the photoperiod exhibit a stable phase relationship for a wide range of photoperiods during the course of the year. The clock is therefore also involved in regulating annual rhythms such as flowering (also called photoperiodism) and many clock mutants have been identified on the basis of abnormalities in the timing of flowering. Other clock genes, such as TOC1 were identified through screens for defects in the rhythmic expression of circadian-regulated genes such as the Light-harvesting complex (LHCB/CAB) gene.²In plants, the clock appears as a complex circuit relying on interconnected feedback loops, which are being studied through a combination of experimental and modelling approaches.³ However, circadian studies are complicated by cell-autonomous and tissue specific clockworks in multicellular plants. We have recently implemented tools for gene function, analysis in the very simple green cell Ostreococcus tauri.⁴ Amongst known core clock genes we identified two homologues of the plant clock genes TOC1 and CCA1 (Circadian Clock Associated 1). Furthermore we found that a conserved CCA1 binding site was required for the circadian expression of TOC1. TOC1 appears to play a more central role than CCA1 in the Ostreococcus clock since only TOC1 knock-down abolishes circadian function in constant light.⁴ In plants the dark-dependent degradation of TOC1 relies on the F box protein ZEITLUPE which is stabilized by GIGANTEA in blue light.^5,6 We identified no ZEITLUPE AND GIGANTEA homologues in Ostreococcus. We chose, therefore to investigate the regulation of TOC1 in Ostreococcus at dusk since there are similarities between TOC1 functions and patterns of expression in Ostreococcus and Arabidopsis.     

Functional conservation of clock-related genes in flowering plants: overexpression and RNA interference analyses of the circadian rhythm in the monocotyledon Lemna gibba

Circadian rhythms are found in organisms from cyanobacteria to plants and animals. In flowering plants, the circadian clock is involved in the regulation of various physiological phenomena, including growth, leaf movement, stomata opening, and floral transitions. Molecular mechanisms underlying the circadian clock have been identified using Arabidopsis (Arabidopsis thaliana); the functions and genetic networks of a number of clock-related genes, including CIRCADIAN CLOCK ASSOCIATED1, LATE ELONGATED HYPOCOTYL (LHY), TIMING OF CAB EXPRESSION1, GIGANTEA (GI), and EARLY FLOWERING3 (ELF3), have been analyzed. The degree to which clock systems are conserved among flowering plants, however, is still unclear. We previously isolated homologs for Arabidopsis clock-related genes from monocotyledon Lemna plants. Here, we report the physiological roles of these Lemna gibba genes (LgLHYH1, LgLHYH2, LgGIH1, and LgELF3H1) in the circadian system. We studied the effects of overexpression and RNA interference (RNAi) of these genes on the rhythmic expression of morning- and evening-specific reporters. Overexpression of each gene disrupted the rhythmicity of either or both reporters, suggesting that these four homologs can be involved in the circadian system. RNAi of each of the genes except LgLHYH2 affected the bioluminescence rhythms of both reporters. These results indicated that these homologs are involved in the circadian system of Lemna plants and that the structure of the circadian clock is likely to be conserved between monocotyledons and dicotyledons. Interestingly, RNAi of LgGIH1 almost completely abolished the circadian rhythm; because this effect appeared to be much stronger than the phenotype observed in an Arabidopsis gi loss-of-function mutant, the precise role of each clock gene may have diverged in the clock systems of Lemna and Arabidopsis.