Circadian Clock in Arabidopsis thaliana Determines Flower Opening Time Early in the Morning and Dominantly Closes Early in the Afternoon.

Many plant species exhibit diurnal flower opening and closing, which is an adaptation influenced by the lifestyle of pollinators and herbivores. However, it remains unclear how these temporal floral movements are modulated. To clarify the role of the circadian clock in flower movement, we examined temporal floral movements in Arabidopsis thaliana. Wild-type (accessions; Col-0, Ler-0 and Ws-4) flowers opened between 0.7 and 1.4 h in a 16-h light period and closed between 7.5 and 8.3 h in a diurnal light period. In the arrhythmic mutants pcl1-1 and prr975, the former flowers closed slowly and imperfectly and the latter ones never closed. Under continuous light conditions, new flowers emerged and opened within a 23-26 h window in the wild-type, but the flowers in pcl1-1 and prr975 developed straight petals, whose curvatures were extremely small. Anti-phasic circadian gene expression of CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), LATE ELONGATED HYPOCOTYLE (LHY) and TIMING OF CAB EXPRESSION 1 (TOC1) occurred in wild-type flowers, but non-rhythmic expression was observed in pcl1-1 and prr975 mutants. Focusing on excised petals, bioluminescence monitoring revealed rhythmic promoter activities of genes expressed (CCA1, LHY and PHYTOCLOCK 1/LUX ARRHYTHMO, PCL1/LUX) in the morning and evening. These results suggest that the clock induces flower opening redundantly with unknown light-sensing pathways. By contrast, flower closing is completely dependent on clock control. These findings will lead to further exploration of the molecular mechanisms and evolutionary diversity of timing in flower opening and closing.

CmpR is important for circadian phasing and cell growth.

In the cyanobacterium Synechococcus elongatus PCC 7942, the circadian clock entrains to a daily light/dark cycle. The transcription factor Pex is abundant under dark conditions and represses kaiA transcription to fine-tune the KaiC-based core circadian oscillator. The transcription of pex also increases during exposure to darkness; however, its mechanism is unknown. We performed a molecular genetic study by constructing a pex expression bioluminescent reporter and screening for brightly luminescent mutants by random insertion of a drug resistance gene cassette in the reporter genome. One mutant contained an insertion of an antibiotic resistance cassette in the cmpR locus, a transcriptional regulator of inorganic carbon concentration. Insertions of the cassette in the remaining two mutant genomes were in the genes encoding flavodoxin and a putative partner of an ABC transporter with unknown function (ycf22). We further analyzed the cmpR mutant to examine whether CmpR directly or indirectly targeted pex expression. In the cmpR mutant, the pex mRNA level was 1.8-fold that of the wild type, and its circadian peak phase in bioluminescence rhythm occurred 5 h later. Moreover, a high-light stress phenotype was present in the colony. The abnormalities were complemented by ectopic induction of the native gene. However, the cmpR/pex double mutation partly suppressed the phase abnormality (2.5 h). In vitro DNA binding analysis of CmpR showed positive binding to the psbAII promoter, but not to any pex DNA. We postulate that the phenotypes of cmpR-deficient cells were attributable mainly to a feeble metabolic and/or redox status.

Expression of the circadian clock-related gene pex in cyanobacteria increases in darkness and is required to delay the clock.

The time measurement system of the unicellular cyanobacterium Synechococcus elongatus PCC 7942 is analogous to the circadian clock of eukaryotic cells. Circadian clock-related genes have been identified in this strain. The clock-related gene pex is thought to maintain the normal clock period because constitutive transcription or deficiency of this gene causes respectively longer (approximately 28 h) or shorter (approximately 24 h) circadian periods than that of the wild type (approximately 25 h). Here, the authors report other properties of pex in the circadian system. Levels of pex mRNA increased significantly in a 12-h exposure to darkness. Western blotting with a GST-Pex antibody revealed a 13.5-kDa protein band in wild-type cells that were incubated in the dark, while this protein was not detected in pex-deficient mutant cells. Therefore, the molecular weight of the Pex protein appears to be 13.5 kDa in vivo. The PadR domain, which is conserved among DNA-binding transcription factors in lactobacilli, was found in Pex. In the pex mutant, several 12-h light/12-h dark cycles reset the phase of the clock by 3 h earlier (phase advance) compared to wild-type cells. The degree of the advance in the pex mutant was proportional to the number of exposed light-dark cycles. In addition, ectopic induction of pex with an inducible Escherichia coli promoter, Ptrc, delayed the phase in the examined recombinant cells by 2.5 h (phase delay) compared to control cells. These results suggest that the dark-responsive gene expression of pex delays the circadian clock under daily light-dark cycles.

Overexpression of LSH1, a member of an uncharacterised gene family, causes enhanced light regulation of seedling development.

Light regulates plant growth and development through a network of endogenous factors. By screening Arabidopsis activation-tagged lines, we isolated a dominant mutant (light-dependent short hypocotyls 1-D (lsh1-D)) that showed hypersensitive responses to continuous red (cR), far-red (cFR) and blue (cB) light and cloned the corresponding gene, LSH1. LSH1 encodes a nuclear protein of a novel gene family that has homologues in Arabidopsis and rice. The effects of the lsh1-D mutation were tested in a series of photoreceptor mutant backgrounds. The hypersensitivity to cFR and cB light conferred by lsh1-D was abolished in a phyA null background (phyA-201), and the hypersensitivity to cR and cFR light conferred by lsh1-D was much reduced in the phytochrome-chromophore synthetic mutant, hy1-1 (long hypocotyl 1). These results indicate that LSH1 is functionally dependent on phytochrome to mediate light regulation of seedling development.

ydk1-D, an auxin-responsive GH3 mutant that is involved in hypocotyl and root elongation.

To study the GH3 gene family of Arabidopsis, we investigated a flanking sequence database of Arabidopsis activation-tagged lines. We found a dwarf mutant, named yadokari 1-D (ydk1-D), that had a T-DNA insertion proximal to a GH3 gene. ydk1-D is dominant and has a short hypocotyl not only in light but also in darkness. Moreover, ydk1-D has a short primary root, a reduced lateral root number, and reduced apical dominance. A GH3 gene, named YDK1, was upregulated in ydk1-D, and YDK1 transgenic plants showed the ydk1-D phenotype. YDK1 gene expression was induced by exogenously applied auxin and regulated by auxin-response factor (ARF)7. In addition, YDK1 gene expression was downregulated by blue and far-red (FR) lights. Strong promoter activity of YDK1 was observed in roots and flowers. These results suggest that YDK1 may function as a negative component in auxin signaling by regulating auxin activity.

DFL2, a new member of the Arabidopsis GH3 gene family, is involved in red light-specific hypocotyl elongation.

A new GH3-related gene, designated DFL2, causes a short hypocotyl phenotype when overexpressed under red and blue light and a long hypocotyl when antisensed under red light conditions. Higher expression of this gene was observed in continuous white, blue and far-red light but the expression level was low in red light and darkness. DFL2 gene expression was induced transiently with red light pulse treatment. DFL2 transgenic plants exhibited a normal root phenotype including primary root elongation and lateral root formation, although primary root elongation was inhibited in antisense transgenic plants only under red light. The adult phenotypes of sense and antisense transgenic plants were not different from that of wild type. DFL2 promoter activity was observed in the hypocotyl. Our results suggest that DFL2 is located downstream of red light signal transduction and determines the degree of hypocotyl elongation.

Analysis of hydrogen peroxide-independent expression of the high-light-inducible ELIP2 gene with the aid of the ELIP2 promoter-luciferase fusions.

Intense and excessive light triggers the evolution of reactive oxygen species in chloroplasts, and these have the potential to cause damage. However, plants are able to respond to light stress and protect the chloroplasts by various means, including transcriptional regulation at the nucleus. Activation of light stress-responsive genes is mediated via hydrogen peroxide-dependent and -independent pathways. In this study, we characterized the Early-Light-Inducible Protein 2 (ELIP2) promoter-luciferase gene fusion (ELIP2::LUC), which responds only to the hydrogen peroxide-independent pathway. Our results show that ELIP2::LUC is expressed under nonstressful conditions in green tissue containing juvenile and developing chloroplasts. Upon light stress, expression was activated in leaves with mature as well as developing chloroplasts. In contrast to another high-light-inducible gene, APX2, which responds to the hydrogen peroxide-dependent pathway, the activation of ELIP2::LUC was cell autonomous. The activation was suppressed by application of 3-(3,4)-dichlorophenyl-1,1-dimethylurea, an inhibitor of the reduction of plastoquinone, whereas 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, an inhibitor of the oxidation of plastoquinone, gave the contrasting effect, which may suggest that the redox state of the plastoquinone plays an important role in triggering the hydrogen peroxide-independent light stress signaling.

Identification of Arabidopsis genes regulated by high light-stress using cDNA microarray.

In plants, excess light has the potential to damage the photosynthetic apparatus. The damage is caused in part by reactive oxygen species (ROS) generated by electrons leaking from the photosynthetic electron transport system. To investigate the mechanisms equipped in higher plants to reduce high light (HL) stress, we surveyed the response of 7000 Arabidopsis genes to HL, taking advantage of the recently developed microarray technology. Our analysis revealed that 110 genes had a positive response to a 3 h treatment at a light intensity of 150 W m(-2). In addition to the scavenging enzymes of ROS, the genes involved in biosynthesis of lignins and flavonoids are activated by HL and actually resulted in increased accumulation of lignins and anthocyanins. Comparing the HL-responsive genes with drought-inducible genes identified with the same microarray system revealed a dense overlap between HL- and drought-inducible genes. In addition, we have identified 10 genes that showed upregulation by HL, drought, cold and also salt stress. These genes include RD29A, ERD7, ERD10, KIN1, LEA14 and COR15a, most of which are thought to be involved in the protection of cellular components.

Bipolar localization of putative photoreceptor protein for phototaxis in thermophilic cyanobacterium Synechococcus elongatus.

We identified an open reading frame from a database of the entire genome of Synechococcus elongatus, the product of which was very similar to pixJ1, which was proposed as photoreceptor gene for phototaxis in Synechocystis sp. PCC6803 [Yoshihara et al. (2000) Plant Cell Physiol. 41: 1299]. The mRNA of S. elongatus pixJ (SepixJ) was expressed in vivo as a part of the product of an operon. SePixJ was detected exclusively in the membrane fraction after cell fractionation. Immunogold labeling of SePixJ in ultra-thin sections indicated that it existed only in both ends of the rod-shaped cell; probably bound with the cytoplasmic membrane.