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 of the circadian clock-associated genes, TOC1, CCA1 and LHY, in the photoperiodic control of flowering time in Arabidopsis thaliana

In Arabidopsis thaliana, the flowering time is regulated through the circadian clock that measures day-length and modulates the photoperiodic CO-FT output pathway in accordance with the external coincidence model. Nevertheless, the genetic linkages between the major clock-associated TOC1, CCA1 and LHY genes and the canonical CO-FT flowering pathway are less clear. By employing a set of mutants including an extremely early flowering toc1 cca1 lhy triple mutant, here we showed that CCA1 and LHY act redundantly as negative regulators of the photoperiodic flowering pathway. The partly redundant CCA1/LHY functions are largely, but not absolutely, dependent on the upstream TOC1 gene that serves as an activator. The results of examination with reference to the expression profiles of CO and FT in the mutants indicated that this clock circuitry is indeed linked to the CO-FT output pathway, if not exclusively. For this linkage, the phase control of certain flowering-associated genes, GI, CDF1 and FKF1, appears to be crucial. Furthermore, the genetic linkage between TOC1 and CCA1/LHY is compatible with the negative and positive feedback loop, which is currently believed to be a core of the circadian clock. The results of this study suggested that the circadian clock might open an exit for a photoperiodic output pathway during the daytime. In the context of the current clock model, these results will be discussed in connection with the previous finding that the same clock might open an exit for the early photomorphogenic output pathway during the night-time.     

Dual role of TOC1 in the control of circadian and photomorphogenic responses in Arabidopsis

To examine the role of the TOC1 (TIMING OF CAB EXPRESSION1) gene in the Arabidopsis circadian system, we generated a series of transgenic plants expressing a gradation in TOC1 levels. Silencing of the TOC1 gene causes arrhythmia in constant darkness and in various intensities of red light, whereas in blue light, the clock runs faster in silenced plants than in wild-type plants. Increments in TOC1 gene dosage delayed the pace of the clock, whereas TOC1 overexpression abolished rhythmicity in all light conditions tested. Our results show that TOC1 RNA interference and toc1-2 mutant plants displayed an important reduction in sensitivity to red and far-red light in the control of hypocotyl elongation, whereas increments in TOC1 gene dosage clearly enhanced light sensitivity. Furthermore, the red light-mediated induction of CCA1/LHY expression was decreased in TOC1 RNA interference and toc1-2 mutant plants, indicating a role for TOC1 in the phytochrome regulation of circadian gene expression. We conclude that TOC1 is an important component of the circadian clock in Arabidopsis with a crucial function in the integration of light signals to control circadian and morphogenic responses.     

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.     

硫化氢信号能够通过生物钟调控拟南芥对冷胁迫的响应

硫化氢(hydrogen sulfide, H_2S)是继一氧化氮(nitric oxide, NO)与一氧化碳(carbon oxide, CO)之后的第3种气体信号分子,在动植物中均发挥着重要的生理功能。生物钟是生物体的内在计时器,对动植物适应环境和生长发育至关重要。鉴于H_2S与生物钟调控的生理过程有较大的相关性,本文以拟南芥(Arabidopsis thaliana)为实验材料,对二者之间的关系进行了探索。结果发现,外源NaHS(H_2S供体)处理能够上调生物钟相关基因CCA1(circadian clock associated 1)和PRR9(pseudo-response regulator 9)的表达,而且在H_2S生成关键酶编码基因缺失的双突变体lcd/des1中,CCA1与PRR9的峰值表达时间明显滞后。CBFs(c-repeat binding factors)是受CCA1调控的冷胁迫响应基因,其表达也受H_2S的调控。lcd/des1中CBF1和CBF3的峰值表达时间延迟,同时在lcd/des1中CBF1、CBF2和CBF3都下调表达。lcd/des1幼苗对冷胁迫表现出更高的敏感性。本文也对拟南芥内源H_2S生成关键酶L-半胱氨酸脱硫基酶(L-cysteine desulfhydrase, LCD)与脱硫基酶1(desulfhydrase 1, DES1)编码基因的转录水平节律性进行了初步的探索。LCD的表达在1 d内未见明显的变化,而DES1的表达有明显的节律性,在早上8:00达到峰值。综上所述,H_2S能够通过调节CCA1与PRR9基因的表达调控生物钟,进而影响下游靶标CBFs基因的表达以增加拟南芥对冷胁迫的耐受性。