No abstract

isrctn.org Identifier: ISRCTN87171227.

Circadian rhythms provide organisms with an adaptive advantage, allowing them to regulate physiological and developmental events so that they occur at the most appropriate time of day. In plants, as in other eukaryotes, multiple transcriptional feedback loops are central to clock function. In one such feedback loop, the Myb-like transcription factors CCA1 and LHY directly repress expression of the pseudoresponse regulator TOC1 by binding to an evening element (EE) in the TOC1 promoter. Another key regulatory circuit involves CCA1 and LHY and the TOC1 homologs PRR5, PRR7, and PRR9. Purification of EE–binding proteins from plant extracts followed by mass spectrometry led to the identification of RVE8, a homolog of CCA1 and LHY. Similar to these well-known clock genes, expression of RVE8 is circadian-regulated with a dawn phase of expression, and RVE8 binds specifically to the EE. However, whereas cca1 and lhy mutants have short period phenotypes and overexpression of either gene causes arrhythmia, rve8 mutants have long-period and RVE8-OX plants have short-period phenotypes. Light input to the clock is normal in rve8, but temperature compensation (a hallmark of circadian rhythms) is perturbed. RVE8 binds to the promoters of both TOC1 and PRR5 in the subjective afternoon, but surprisingly only PRR5 expression is perturbed by overexpression of RVE8. Together, our data indicate that RVE8 promotes expression of a subset of EE–containing clock genes towards the end of the subjective day and forms a negative feedback loop with PRR5. Thus RVE8 and its homologs CCA1 and LHY function close to the circadian oscillator but act via distinct molecular mechanisms.

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The circadian clock modulates expression of a large fraction of the Arabidopsis genome and affects many aspects of plant growth and development. We have discovered one way in which the circadian system regulates hormone signaling, identifying a node that links the clock and auxin networks. Auxin plays key roles in development and responses to environmental cues, in part through regulation of plant growth. We have characterized REVEILLE1 (RVE1), a Myb-like, clockregulated transcription factor that is homologous to the central clock genes CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELON-GATED HYPOCOTYL (LHY). Despite this homology, inactivation of RVE1 does not affect circadian rhythmicity but instead causes a growth phenotype, indicating this factor is a clock output affecting plant development. CCA1 regulates growth via the bHLH transcription factors PHYTOCHROME INTERACTING FACTOR4 (PIF4) and PIF5, but RVE1 acts independently of these genes. RVE1 instead controls auxin levels, promoting free auxin production during the day but having no effect during the night. RVE1 positively regulates the expression of the auxin biosynthetic gene YUCCA8 (YUC8), providing a mechanism for its growth-promoting effects. RVE1 is therefore a node that connects two important signaling networks that coordinate plant growth with rhythmic changes in the environment.growth control ͉ hypocotyl ͉ yucca C ircadian rhythms are approximately 24-h rhythms in physiology or behavior that are generated by an endogenous clock. Circadian rhythms persist in constant environmental conditions; however they can be entrained or set by environmental cues like light/dark or temperature cycles (1). In plants, these rhythms regulate myriad processes including leaf and cotyledon movement, growth, photosynthesis, and timing of the transition to flowering (2). A functional circadian clock provides an adaptive advantage, perhaps by predicting fluctuations in the external environment (3, 4). In all model systems studied, these self-sustained rhythms are generated by a cell-autonomous central oscillator which regulates the expression of many genes involved in metabolic and physiological functions (1). Microarray studies show that nearly one third of Arabidopsis genes are circadian regulated, with peak expression at different times of day (5, 6).In plants, the central oscillator is composed of interlocking feedback loops with both positive and negative transcriptional regulators (1). The central loop is thought to consist of three genes, CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), LATE ELON-GATED HYPOCOTYL (LHY), and TIMING OF CAB EXPRES-SION (TOC1). CCA1 and LHY are morning-phased transcription factors with a single Myb-like domain containing a distinctive SHAQKYF motif (7,8). They bind a motif, termed the evening element (EE; AAAATATCT), in the TOC1 promoter to negatively regulate TOC1 expression. TOC1, an evening-phased gene, in turn positively regulates the expression of CCA1 and LHY through an unknown mechanism, thus forming the core clock negative feedback loop (7...

Phototropins (phot1 and phot2) are plasma membrane-associated receptor kinases that respond specifically to blue and UV wavelengths. In addition to a C-terminal Ser/Thr kinase domain, phototropins contain two N-terminal chromophore binding LOV domains that function as photoswitches to regulate a wide range of enzymatic activities in prokaryotes and eukaryotes. Through domain swapping, we show that the photochemical properties of Arabidopsis thaliana phot1 rely on interactions between LOV1 and LOV2, which are facilitated by their intervening linker sequence. Functional analysis of domain-swap proteins supports a mechanism whereby LOV2 acts as a dark-state repressor of phot1 activity both in vitro and in vivo. Moreover, we find a photoactive role for LOV1 in arresting chloroplast accumulation at high light intensities. Unlike LOV2, LOV1 cannot operate as a dark-state repressor, resulting in constitutive receptor autophosphorylation and accelerated internalization from the plasma membrane. Coexpression of active and inactive forms of phot1 demonstrates that autophosphorylation can occur intermolecularly, independent of LOV1, via light-dependent receptor dimerization in vivo. Indeed, transphosphorylation is sufficient to promote phot1 internalization through a clathrin-dependent endocytic pathway triggered primarily by phosphorylation of Ser-851 within the kinase activation loop. The mechanistic implications of these findings in regard to light-driven receptor activation and trafficking are discussed.

Circadian clocks are near-ubiquitous molecular oscillators that coordinate biochemical, physiological, and behavioral processes with environmental cues, such as dawn and dusk. Circadian timing mechanisms are thought to have arisen multiple times throughout the evolution of eukaryotes but share a similar overall structure consisting of interlocking transcriptional and posttranslational feedback loops. Recent work in both plants and animals has also linked modification of histones to circadian clock function. Now, using data from published microarray experiments, we have identified a histone demethylase, jumonji domain containing 5 (JMJD5), as a previously undescribed participant in both the human and Arabidopsis circadian systems. Arabidopsis JMJD5 is coregulated with evening-phased clock components and positively affects expression of clock genes expressed at dawn. We found that both Arabidopsis jmjd5 mutant seedlings and mammalian cell cultures deficient for the human ortholog of this gene have similar fastrunning circadian oscillations compared with WT. Remarkably, both the Arabidopsis and human JMJD5 orthologs retain sufficient commonality to rescue the circadian phenotype of the reciprocal system. Thus, JMJD5 plays an interchangeable role in the timing mechanisms of plants and animals despite their highly divergent evolutionary paths.C ircadian rhythms are endogenous oscillations that attune the behavior and physiology of organisms to regular changes in their environment, such as dusk and dawn. It is estimated that 1/10th of mammalian genes and one-third of Arabidopsis genes are regulated in a circadian fashion (1, 2). Not surprisingly, the circadian clock modulates a broad range of processes, ranging from the regulation of growth and flowering time in plants to the control of body temperature and the sleep-wake cycle in mammals (3, 4). The coordination of these molecular and physiological processes with the external environment has been shown to provide an adaptive advantage in organisms as diverse as cyanobacteria, higher plants, and insects (5-7).Although it is thought that circadian clocks have arisen multiple times during eukaryotic evolution (8), molecular oscillators in a diverse range of lineages share a broad overall structure consisting of interlocking transcriptional and posttranslational feedback loops (3, 4). In plants, the morning-phased Myb-like transcription factors CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) repress the expression of TIMING OF CAB1 EXPRESSION 1 (TOC1) by binding to a conserved motif within its promoter known as the evening element (9-11). TOC1, in turn, promotes expression of CCA1 and LHY via an indirect mechanism, forming a negative feedback loop (12). This central CCA1/LHY/TOC1 loop interlocks with additional morningand evening-phased circuits to provide more robustness and flexibility to the circadian mechanism (reviewed in 3, 13).Because genes that act together are often coregulated (14), we have characterized a gene that is coexpressed with TO...

Phototropins (phot1 and phot2) are blue-light receptor kinases controlling a range of responses that optimize the photosynthetic efficiency of plants. Light sensing is mediated by two flavin-binding motifs, known as LOV1 and LOV2, located within the N-terminal region of the protein. Photoexcitation via LOV2 leads to activation of the C-terminal kinase domain and consequently receptor autophosphorylation. However, knowledge of the in-vivo phosphorylation sites for Arabidopsis phototropins is lacking and has impeded progress in elucidating the functional significance of receptor phosphorylation. We have purified phot1 from Arabidopsis and identified the in-vivo sites of receptor phosphorylation by liquid chromatography tandem mass spectrometry. Arabidopsis-derived phot1 binds flavin mononucleotide as chromophore and is phosphorylated at four major sites located upstream of LOV2 (Ser(58), Ser(85), Ser(350), and Ser(410)), three of which are induced by blue light. Nevertheless, structure-function analysis indicates that the biological activity of phot1 can be attributed to a modular unit comprising the LOV2-kinase region of the protein. Thus, peptide regions upstream of LOV2, including the sites of receptor phosphorylation identified here, do not appear to be important for receptor signaling. By contrast, these regions may be necessary for maximizing stomatal performance and possibly light-induced relocalization of phot1.