A complex genetic interaction between Arabidopsis thaliana TOC1 and CCA1/LHY in driving the circadian clock and in output regulation

It has been proposed that CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) together with TIMING OF CAB EXPRESSION 1 (TOC1) make up the central oscillator of the Arabidopsis thaliana circadian clock. These genes thus drive rhythmic outputs, including seasonal control of flowering and photomorphogenesis. To test various clock models and to disclose the genetic relationship between TOC1 and CCA1/LHY in floral induction and photomorphogenesis, we constructed the cca1 lhy toc1 triple mutant and cca1 toc1 and lhy toc1 double mutants and tested various rhythmic responses and circadian output regulation. Here we report that rhythmic activity was dramatically attenuated in cca1 lhy toc1. Interestingly, we also found that TOC1 regulates the floral transition in a CCA1/LHY-dependent manner while CCA1/LHY functions upstream of TOC1 in regulating a photomorphogenic process. This suggests to us that TOC1 and CCA1/LHY participate in these two processes through different strategies. Collectively, we have used genetics to provide direct experimental support of previous modeling efforts where CCA1/LHY, along with TOC1, drives the circadian oscillator and have shown that this clock is essential for correct output regulation.     

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.     

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.     

拟南芥生物钟分子机制研究进展

本文主要概述了目前拟南芥生物钟分子机制的研究进展。生物钟通过调控导引节律的相位来调节植物的生理活动。拟南芥生物钟由CCA1、LHY和TOC1 3个主要基因构成了一个稳定的负反馈环,来调节昼夜节律中各个基因如APRR/TOC1 5重奏的作用, 从而调控昼夜节律的相位。在开花的光周期调控中, 提出了外协和模型, 其中的关键基因是CO , 它与拟南芥的开花时间直接相关。     

Differential expression of genes important for adaptation in Capsella bursa-pastoris (Brassicaceae)

Understanding the genetic basis of natural variation is of primary interest for evolutionary studies of adaptation. In Capsella bursa-pastoris, a close relative of Arabidopsis (Arabidopsis thaliana), variation in flowering time is correlated with latitude, suggestive of an adaptation to photoperiod. To identify pathways regulating natural flowering time variation in C. bursa-pastoris, we have studied gene expression differences between two pairs of early- and late-flowering C. bursa-pastoris accessions and compared their response to vernalization. Using Arabidopsis microarrays, we found a large number of significant differences in gene expression between flowering ecotypes. The key flowering time gene FLOWERING LOCUS C (FLC) was not differentially expressed prior to vernalization. This result is in contrast to those in Arabidopsis, where most natural flowering time variation acts through FLC. However, the gibberellin and photoperiodic flowering pathways were significantly enriched for gene expression differences between early- and late-flowering C. bursa-pastoris. Gibberellin biosynthesis genes were down-regulated in late-flowering accessions, whereas circadian core genes in the photoperiodic pathway were differentially expressed between early- and late-flowering accessions. Detailed time-series experiments clearly demonstrated that the diurnal rhythm of CIRCADIAN CLOCK-ASSOCIATED1 (CCA1) and TIMING OF CAB EXPRESSION1 (TOC1) expression differed between flowering ecotypes, both under constant light and long-day conditions. Differential expression of flowering time genes was biologically validated in an independent pair of flowering ecotypes, suggesting a shared genetic basis or parallel evolution of similar regulatory differences. We conclude that genes involved in regulation of the circadian clock, such as CCA1 and TOC1, are strong candidates for the evolution of adaptive flowering time variation in C. bursa-pastoris.     

Conserved expression profiles of circadian clock-related genes in two Lemna species showing long-day and short-day photoperiodic flowering responses

The Lemna genus is a group of monocotyledonous plants with tiny, floating bodies. Lemna gibba G3 and L. paucicostata 6746 were once intensively analyzed for physiological timing systems of photoperiodic flowering and circadian rhythms since they showed obligatory and sensitive photoperiodic responses of a long-day and a short-day plant, respectively. We attempted to approach the divergence of biological timing systems at the molecular level using these plants. We first employed molecular techniques to study their circadian clock systems. We developed a convenient bioluminescent reporter system to monitor the circadian rhythms of Lemna plants. As in Arabidopsis, the Arabidopsis CCA1 promoter produced circadian expression in Lemna plants, though the phases and the sustainability of bioluminescence rhythms were somewhat diverged between them. Lemna homologs of the Arabidopsis clock-related genes LHY/CCA1, GI, ELF3 and PRRs were then isolated as candidates for clock-related genes in these plants. These genes showed rhythmic expression profiles that were basically similar to those of Arabidopsis under light-dark conditions. Results from co-transfection assays using the bioluminescence reporter and overexpression effectors suggested that the LHY and GI homologs of Lemna can function in the circadian clock system like the counterparts of Arabidopsis. All these results suggested that the frame of the circadian clock appeared to be conserved not only between the two Lemna plants but also between monocotyledons and dicotyledons. However, divergence of gene numbers and expression profiles for LHY/CCA1 homologs were found between Lemna, rice and Arabidopsis, suggesting that some modification of clock-related components occurred through their evolution.