A modern circadian clock in the common angiosperm ancestor of monocots and eudicots

The circadian clock enhances fitness through temporal organization of plant gene expression, metabolism and physiology. Two recent studies, one in BMC Evolutionary Biology, demonstrate through phylogenetic analysis of the CCA1/LHY and TOC1/PRR gene families that the common ancestor of monocots and eudicots had components sufficient to construct a circadian clock consisting of multiple interlocked feedback loops.     

Dynamic localization of the actin-bundling protein cortexillin I during cell migration

The circadian clock in plants regulates many important physiological and biological processes, including leaf movement. We have used an imaging system to genetically screen Arabidopsis seedlings for altered leaf movement with the aim of identifying a circadian clock gene. A total of 285 genes were selected from publicly available microarrays that showed an expression pattern similar to those of the Arabidopsis core oscillator genes. We subsequently isolated 42 homozygous recessive mutants and analyzed their leaf movements. We also analyzed leaf movements of activation tagging mutants that showed altered flowering time. We found that agl6-1D plants, in which AGAMOUS-LIKE 6 (AGL6) was activated by the 35S enhancer, showed a shortened period of leaf movement as well as a high level of ZEITLUPE (ZTL) expression, reduced amplitude of LATE ELONGATED HYPOCOTYL (LHY) expression, and arrhythmic TIMING OF CAB EXPRESSION1 (TOC1)/CIRCADIAN CLOCK ASSOCIATED1 (CCA1) expression. A shortened period of leaf movement was also seen in 35S-AGL6-myc plants, although 35S-amiRAGL6 plants, transgenic plants overexpressing an artificial miRNA (amiR) targeting AGL6, showed unaltered leaf movement. The amplitude of CHLOROPHYLL A/B BINDING PROTEIN 2 (CAB2) expression, a circadian output gene, was also reduced in agl6-1D plants. Taken together, these results suggest that AGL6 plays a potential role in the regulation of the circadian clock by regulating ZTL mRNA level in Arabidopsis.     

Accumulation of maize γ-zein and γ-zein: KDEL to high levels in tobacco leaves and differential increase of BiP synthesis in transformants

Two gene constructs (pROK.TG1L and pROK.TG1LK) were utilized to achieve accumulation of maize γ-zein to high levels in tobacco (Nicotiana tabacum L.) leaves. Both the chimaeric genes contained the γ-zein-coding region preceded by the 5′untranslated leader from the coat protein mRNA of TMV, but one of them (pROK.TG1LK) was modified in its protein-coding region by the addition of the ER retention signal KDEL. The accumulation of γ-zein and γ-zein:KDEL in leaves was compared with heterologous protein accumulation in tobacco plants previously transformed with a γ-zein cDNA harbouring a native 5′UTR. Replacement of γ-zein 5′UTR with the TMV leader dramatically increased γ-zein production. Furthermore, γ-zein:KDEL-expressing plants, on average, accumulated twice as much foreign protein in their leaves as pROK.TG1L plants. The two-fold increase in the level of γ-zein:KDEL can probably be attributed to an improvement in the mechanism for ER retention of zeins in the transgenic cells. Transformants also showed increased production of BiP, though to a lesser extent in γ-zein:KDEL-expressing plants compared with pROK.TG1L plants. It is therefore likely that γ-zein:KDEL retention is made less dependent on the chaperone assistance of BiP by the presence of the KDEL signal on the γ-zein mutant. Received: 15 October 1999 / Accepted: 28 February 2000     

Isolation and Characterization of a Curcin Promoter from <Emphasis Type="Italic">Jatropha curcas</Emphasis> L. and Its Regulation of Gene Expression in Transgenic Tobacco Plants

Ribosome-inactivating proteins (RIPs) represent those proteins that universally depurinate conserved α-sarcin loops of large rRNAs. In this study, a 0.6-kb fragment of a 5′ flanking region preceding a curcin gene, encoding a type I RIP curcin, of Jatropha curcas L. endosperm was cloned, and its regulation of expression of the β-glucuronidase (GUS) reporter gene was investigated in transgenic tobacco. Analysis of GUS activities showed that the 0.6-kb flanking fragment of the curcin gene was sufficient to drive the GUS reporter gene expression in tobacco seed. The activity of this flanking fragment was analyzed at different stages of seed development. Histochemical localization of GUS activity indicated that the promoter was specifically active in the endosperm tissue of the dicotyledonous tobacco embryo. Moreover, this activity was first initiated at the heart-shaped embryonic stage during seed development.     

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.     

What makes the Arabidopsis clock tick on time? A review on entrainment

Entrainment, the synchronization of a circadian clock with the external environment, is a crucial step in daily life. Although many signals contribute to entrainment, light and temperature are typically the strongest resetting cues. Much progress has been made concerning light resetting in the model plant Arabidopsis thaliana. Multiple photoreceptors (phytochromes, cryptochromes, LOV-domain proteins) are involved in light perception. The clock genes CCA1, LHY and TOC1 are all probable targets of light signalling, although the details of these pathways are not completely established. Temperature can entrain the clock, but little is known about the mechanism underlying this resetting; no obvious clock gene candidate for temperature resetting has been identified. Although circadian research has emphasized oscillations in free-running conditions, in the real world the circadian clock is entrained. During entrainment, short or long period mutants exhibit a 24-h period, but a mutant phenotype is often manifested as an altered phase relationship with the entraining cycle; short and long period mutants show leading and lagging phases, respectively, and this may be detrimental under some conditions. Arrhythmic CCA1-overexpressing plants display increased lethality under very short photoperiods, consistent with the circadian clock being of adaptive significance to life on a rotating world.     

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.     

Two new clock proteins, LWD1 and LWD2, regulate Arabidopsis photoperiodic flowering

The “light” signal from the environment sets the circadian clock to regulate multiple physiological processes for optimal rhythmic growth and development. One such process is the control of flowering time by photoperiod perception in plants. In Arabidopsis (Arabidopsis thaliana), the flowering time is determined by the correct interconnection of light input and signal output by the circadian clock. The identification of additional clock proteins will help to better dissect the complex nature of the circadian clock in Arabidopsis. Here, we show LIGHT-REGULATED WD1 (LWD1)/LWD2 as new clock proteins involved in photoperiod control. The lwd1lwd2 double mutant has an early-flowering phenotype, contributed by the significant phase shift of CONSTANS (CO), and, therefore, an increased expression of FLOWERING LOCUS T (FT) before dusk. Under entrainment conditions, the expression phase of oscillator (CIRCADIAN CLOCK ASSOCIATED1 [CCA1], LATE ELONGATED HYPOCOTYL [LHY], TIMING OF CAB EXPRESSION1 [TOC1], and EARLY FLOWERING4 [ELF4]) and output (GIGANTEA, FLAVIN-BINDING, KELCH REPEAT, F-BOX1, CYCLING DOF FACTOR1, CO, and FT) genes in the photoperiod pathway shifts approximately 3 h forward in the lwd1lwd2 double mutant. Both the oscillator (CCA1, LHY, TOC1, and ELF4) and output (COLD, CIRCADIAN RHYTHM, AND RNA BINDING2 and CHLOROPHYLL A/B-BINDING PROTEIN2) genes have a short period length in the lwd1lwd2 double mutant. Our data imply that LWD1/LWD2 proteins function in close proximity to or within the circadian clock for photoperiodic flowering control.     

Interaction between the Late Elongated hypocotyl (LHY) and Early flowering 3 (ELF3) genes in the Arabidopsis circadian clock

The circadian clock governs rhythms with 24 hours that allow organisms to anticipate daily changing environmental time cues. In Arabidopsis, the circadian clock is conceptually composed of three parts; input pathways for light and temperature signals, oscillators and output pathways for physiological processes including leaf movement, gene expression rhythms and flowering time. Oscillators consist of three interlocking loops, named morning, central and evening loops. Components of the central oscillator contain LHY, CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and TIMING OF CAB EXPRESSION (TOC1). The oscillator can be reset by light signals through input pathways. Genetic studies have revealed the components involved in light input pathways. The elf3 (early flowering 3) mutant was isolated by insensitivity to photoperiod showing long hypocotyls, elongated petioles and pale leaves characteristic of plants defective in light perception. Therefore the ELF3 has been proposed to act on light input pathways. The aim of this study is to test whether LHY and ELF3 encode interacting components of a circadian light input pathway. To address this possibility, lhy-1 elf3-1 (LHY overexpressing-mutant X ELF3 loss of function-mutant) and lhy-11 elf3-1 (LHY loss of function-mutant X ELF3 loss of function-mutant) double mutants were constructed. Their visual phenotypes and CAB (Chlorophyll a/b binding protein) expression patterns demonstrate that LHY may function downstream of ELF3 and that this interaction is disrupted when LHY expression is placed under the control of the 35S promoter. In addition, ELF3 is required for vigorous rhythms of LHY gene expression and LHY protein levels.