Previously, we screened 50 000 seedlings of Arabidopsis thaliana carrying a P GI :: LUC + bioluminescence reporter gene mutagenized with ethylmethanesulfonate for mutants with phenotypes of extensively altered circadian rhythms, and identified three loci, PHYTOCLOCK 1 ( PCL1 ), PCL2 and PCL3 , whose mutations cause arrhythmia. Here we succeeded to clone the PCL1 gene and show that the PCL1 gene encodes a novel DNA binding protein belonging to the GARP protein family and is essential for a functional clock oscillator in A. thaliana . The PCL1 gene satisfies the requirements for the clock oscillator gene: (i) pcl1 null mutations caused arrhythmia in multiple circadian outputs, including expression of potential clock genes TOC1 , CCA1 and LHY , and flowering lacked a photoperiodic response; (ii) PCL1 expression showed circadian rhythm in both constant light and constant dark; (iii) over-expression of the PCL1 gene gradually caused arrhythmicity in all the multiple circadian outputs examined; and (iv) the PCL1 gene controlled its own expression via negative feedback. Therefore, the PCL1 gene is the clock oscillator gene essential to the generation of clock oscillation in the higher plant.

Although sugar has been suggested to promote floral transition in many plant species, growth on high concentrations (5% [w/v]) of sucrose (Suc) significantly delayed flowering time, causing an increase in the number of leaves at the time of flowering in Arabidopsis. The effect of high concentrations of Suc seemed to be metabolic rather than osmotic. The delay of floral transition was due to extension of the late vegetative phase, which resulted in a delayed activation of LFY expression. In addition, growth on low concentrations (1% [w/v]) of Suc slightly inhibited flowering in wild-type plants. This delay resulted from effects on the early vegetative phase. This inhibition was more pronounced in tfl1, an early flowering mutant, than in the wild type. Although 1% (w/v) Suc was reported to promote floral transition of late-flowering mutants such as co, fca, and gi, floral transition in these mutants was delayed by a further increase in Suc concentration. These results suggest that sugar may affect floral transition by activating or inhibiting genes that act to control floral transition, depending on the concentration of sugars, the genetic background of the plants, and when the sugar is introduced. Growth on 1% (w/v) Suc did not restore the reduced expression levels of FT and SOC1/AGL20 in co or fca mutants. Rather, expression of FT and SOC1/AGL20 was repressed by 1% (w/v) Suc in wild-type background. The possible effects of sugar on gene expression to promote floral transition are discussed.

The molecular bases of circadian clocks have been studied in animals, fungi, bacteria, and plants, but not in eukaryotic algae. To establish a new model for molecular analysis of the circadian clock, here we identified a large number of components of the circadian system in the eukaryotic unicellular alga Chlamydomonas reinhardtii by a systematic forward genetic approach. We isolated 105 insertional mutants that exhibited defects in period, phase angle, and/or amplitude of circadian rhythms in bioluminescence derived from a luciferase reporter gene in their chloroplast genome. Simultaneous measurement of circadian rhythms in bioluminescence and growth rate revealed that some of these mutants had defects in the circadian clock itself, whereas one mutant had a defect in a specific process for the chloroplast bioluminescence rhythm. We identified 30 genes (or gene loci) that would be responsible for rhythm defects in 37 mutants. Classification of these genes revealed that various biological processes are involved in regulation of the chloroplast rhythmicity. Amino acid sequences of six genes that would have crucial roles in the circadian clock revealed features of the Chlamydomonas clock that have both partially plant-like and original components. The molecular bases of circadian clocks have been studied in animals, fungi, bacteria, and plants (Dunlap 1999;Harmer et al. 2001). Despite the striking biochemical features of circadian clocks (e.g., oscillation with long periodicity [∼24 h] and its temperature compensation) (Bünning 1973), their central components are not conserved between these kingdoms (Dunlap 1999;Harmer et al. 2001). To understand the nature of oscillation mechanisms and the evolutionary history of clock components, it is important to understand circadian clock systems of a wide range of organisms.Circadian rhythms of unicellular algae have been studied extensively (Mittag 2001), but no clock component of eukaryotic algae has yet been identified. Chlamydomonas reinhardtii is one of the best-studied algae in circadian rhythm research. A forward genetic approach to identify circadian clock components of Chlamydomonas was started more than three decades ago (Bruce 1970). Although several clock mutants have been isolated (Bruce 1972(Bruce , 1974Mergenhagen 1984), the genes responsible could not be identified because of limitations of tools for genetic analyses. However, since Chlamydomonas is now one of the most attractive model organisms in molecular genetics (Harris 2001), it is possible to re-establish it as a model for studying the molecular mechanism of the circadian clock. For this purpose, we previously developed bioluminescence reporter strains with a codon-optimized luciferase gene in their chloroplast genomes to enable real-time monitoring of circadian rhythms (Breton and Kay 2006;Matsuo et al. 2006).In this study, we screened ∼16,000 insertional mutants for defects in circadian rhythmicity of the chloroplast bioluminescence reporter, isolated 105 mutants, and identified 30 genes (or gene loci...

SummaryThe low-beta-amylase1 (lba1) mutant of Arabidopsis thaliana has reduced sugar-induced expression of AtbAmy and shows pleiotropic phenotypes such as early flowering; short day-sensitive growth; and seed germination that is hypersensitive to glucose and abscisic acid and resistant to mannose. lba1 was a missense mutation of UPF1 RNA helicase involved in nonsense-mediated mRNA decay (NMD), which eliminates mRNAs with premature termination codons (PTCs), and replaces highly conserved Gly 851 of UPF1 with Glu. Expression of the wild-type UPF1 in lba1 rescued not only the reduced sugar-inducible gene expression, but also early flowering and altered seed-germination phenotypes. Sugar-inducible mRNAs were over-represented among transcripts decreased in sucrose-treated lba1 compared with Col plants, suggesting that UPF1 is involved in the expression of a subset of sugar-inducible genes. On the other hand, transcripts increased in lba1, which are likely to contain direct targets of NMD, included mRNAs for many transcription factors and metabolic enzymes that play diverse functions. Among these, the level of an alternatively spliced transcript of AtTFIIIA containing PTC was 17-fold higher in lba1 compared with Col plants, and it was reduced to the level in Col by expressing the wild-type UPF1. The lba1 mutant provides a good tool for studying NMD in plants.