An Arabidopsis gene regulatory network for secondary cell wall synthesis.

The plant cell wall is an important factor for determining cell shape, function and response to the environment. Secondary cell walls, such as those found in xylem, are composed of cellulose, hemicelluloses and lignin and account for the bulk of plant biomass. The coordination between transcriptional regulation of synthesis for each polymer is complex and vital to cell function. A regulatory hierarchy of developmental switches has been proposed, although the full complement of regulators remains unknown. Here we present a protein-DNA network between Arabidopsis thaliana transcription factors and secondary cell wall metabolic genes with gene expression regulated by a series of feed-forward loops. This model allowed us to develop and validate new hypotheses about secondary wall gene regulation under abiotic stress. Distinct stresses are able to perturb targeted genes to potentially promote functional adaptation. These interactions will serve as a foundation for understanding the regulation of a complex, integral plant component.

The DIURNAL project: DIURNAL and circadian expression profiling, model-based pattern matching, and promoter analysis.

The DIURNAL project ( http://diurnal.cgrb.oregonstate.edu/ ) provides a graphical interface for mining and viewing diurnal and circadian microarray data for Arabidopsis thaliana, poplar, and rice. The database is searchable and provides access to several user-friendly Web-based data-mining tools with easy-to-understand output. The associated tools include HAYSTACK ( http://haystack.cgrb.oregonstate.edu/ ) and ELEMENT ( http://element.cgrb.oregonstate.edu/ ). HAYSTACK is a model-based pattern-matching algorithm for identifying genes that are coexpressed and potentially coregulated. HAYSTACK can be used to analyze virtually any large-scale microarray data set and provides an alternative method for clustering microarray data from any experimental system by grouping together genes whose expression patterns match the same or similar user-defined patterns. ELEMENT is a Web-based program for identifying potential cis-regulatory elements in the promoters of coregulated genes in Arabidopsis, poplar, and rice. Together, DIURNAL, HAYSTACK, and ELEMENT can be used to facilitate cross-species comparisons among the plant species supported and to accelerate functional genomics efforts in the laboratory.

A role for LKP2 in the circadian clock of Arabidopsis.

A third member of the ZTL gene family was identified in the Arabidopsis genome and was named LKP2 (for LOV kelch protein2). A cDNA was isolated corresponding to this gene, and plants overexpressing LKP2 were generated. The overexpression of LKP2 resulted in arrhythmic phenotypes for a number of circadian clock outputs in both constant light and constant darkness, long hypocotyls under multiple fluences of both red and blue light, and a loss of photoperiodic control of flowering time. The LKP2 mRNA is not regulated by the circadian clock and was detected in all tissues examined. These results suggest that LKP2 functions either within or very close to the circadian oscillator in Arabidopsis. A model is presented for its mode of action.

Molecular bases of circadian rhythms.

Circadian rhythms are found in most eukaryotes and some prokaryotes. The mechanism by which organisms maintain these roughly 24-h rhythms in the absence of environmental stimuli has long been a mystery and has recently been the subject of intense research. In the past few years, we have seen explosive progress in the understanding of the molecular basis of circadian rhythms in model systems ranging from cyanobacteria to mammals. This review attempts to outline these primarily genetic and biochemical findings and encompasses work done in cyanobacteria, Neurospora, higher plants, Drosophila, and rodents. Although actual clock components do not seem to be conserved between kingdoms, central clock mechanisms are conserved. Somewhat paradoxically, clock components that are conserved between species can be used in diverse ways. The different uses of common components may reflect the important role that the circadian clock plays in adaptation of species to particular environmental niches.

Signaling networks in the plant circadian system.

Significant advances have been made during the past year in the genetic and molecular dissection of the plant circadian system. Several proteins involved in circadian clock regulation have been identified and the way that their interactions contribute to temporal organization is starting to emerge. In addition, genomic approaches have identified hundreds of genes under clock control, providing a molecular basis to our understanding of how the clock coordinates plant physiology and development with daily and seasonal environmental cycles.

Time zones: a comparative genetics of circadian clocks.

The circadian clock is a widespread cellular mechanism that underlies diverse rhythmic functions in organisms from bacteria and fungi, to plants and animals. Intense genetic analysis during recent years has uncovered many of the components and molecular mechanisms comprising these clocks. Although autoregulatory genetic networks are a consistent feature in the design of all clocks, the weight of evidence favours their independent evolutionary origins in different kingdoms.

Reciprocal regulation between TOC1 and LHY/CCA1 within the Arabidopsis circadian clock.

The interactive regulation between clock genes is central for oscillator function. Here, we show interactions between the Arabidopsis clock genes LATE ELONGATED HYPOCOTYL (LHY), CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), and TIMING OF CAB EXPRESSION 1 (TOC1). The MYB transcription factors LHY and CCA1 negatively regulate TOC1 expression. We show that both proteins bind to a region in the TOC1 promoter that is critical for its clock regulation. Conversely, TOC1 appears to participate in the positive regulation of LHY and CCA1 expression. Our results indicate that these interactions form a loop critical for clock function in Arabidopsis.

ELF3 modulates resetting of the circadian clock in Arabidopsis.

The Arabidopsis early flowering 3 (elf3) mutation causes arrhythmic circadian output in continuous light, but there is some evidence of clock function in darkness. Here, we show conclusively that normal circadian function occurs with no alteration of period length in elf3 mutants in dark conditions and that the light-dependent arrhythmia observed in elf3 mutants is pleiotropic on multiple outputs normally expressed at different times of day. Plants overexpressing ELF3 have an increased period length in both constant blue and red light; furthermore, etiolated ELF3-overexpressing seedlings exhibit a decreased acute CAB2 response after a red light pulse, whereas the null mutant is hypersensitive to acute induction. This finding suggests that ELF3 negatively regulates light input to both the clock and its outputs. To determine whether ELF3's action is phase dependent, we examined clock resetting by using light pulses and constructed phase response curves. Absence of ELF3 activity causes a significant alteration of the phase response curve during the subjective night, and constitutive overexpression of ELF3 results in decreased sensitivity to the resetting stimulus, suggesting that ELF3 antagonizes light input to the clock during the night. The phase of ELF3 function correlates with its peak expression levels in the subjective night. ELF3 action, therefore, represents a mechanism by which the oscillator modulates light resetting.

Analysis of the function of two circadian-regulated CONSTANS-LIKE genes.

The Arabidopsis genes CONSTANS-LIKE 1 (COL1) and CONSTANS-LIKE 2 (COL2) are predicted to encode zinc finger proteins with approximately 67% amino acid identity to the protein encoded by the flowering-time gene CONSTANS (CO). We show that the circadian clock regulates expression of COL1 and COL2 with a peak in transcript levels around dawn. We analyzed transgenic plants misexpressing COL1, COL2 and CO. Unlike CO, altered expression of COL1 and COL2 in transgenic plants had little effect on flowering time. However, analysis of circadian phenotypes in the transgenic plants showed that over-expression of COL1 can shorten the period of two distinct circadian rhythms. Experiments with the highest COL1 over-expressing line indicate that its circadian defects are fluence rate-dependent, suggesting an effect on a light input pathway(s).

Regulation of the cycling of timeless (tim) RNA.

Circadian rhythms in Drosophila depend upon expression of the timeless (tim) and period (per) genes, which encode interacting components of the endogenous clock. These two clock genes show a robust circadian oscillation in transcription rate as well as RNA and protein levels. Transcriptional activation of both genes requires the basic helix-loop-helix (bHLH) PAS transcription factors dCLOCK (dCLK) and CYCLE (CYC), which bind E-box elements. We investigated the role of E-box elements in regulating behavioral rhythmicity and tim gene expression. We show that mutation of the upstream E-box in the tim gene prevents the rescue by tim cDNA sequences of the arrhythmic tim(01) phenotype. RNA encoded by this mutated tim transgene fails to cycle and is expressed at low levels. While a tim transgene carrying a wild-type E-box restores behavioral rhythms, tim RNA levels are intermediate to those of the mutant E-box transgenic lines and wild type, and do not display high amplitude cycling. On the other hand, high-amplitude RNA cycling was consistently obtained with a tim transgene that contains genomic, rather than cDNA, sequences. To identify additional sequences that may be required for tim cycling, we investigated the role of an E-box in the first intron of the tim gene through cell culture experiments. In these experiments, the presence of this intron did not have any effect on the activation of tim transcription by dCLK/CYC. As the upstream E-box was implicated in activation by dCLK/CYC in cell culture, we assayed sequences containing this E-box for association with proteins in fly head extracts. These studies provide the first biochemical evidence for an in vivo complex containing dCLK and CYC that binds the tim upstream sequence and is detected at all times of day. Together, these data highlight molecular mechanisms that are critical for behavioral rhythms.

Circadian photoperception.

The circadian clock is intrinsically linked to the daily cycle of day and night. A capacity for entrainment to light-dark cycles has proven to be a universal feature of the clock in all organisms examined. Here we review a wealth of recent advances that reveal more about the light input mechanisms by which the circadian clock is set to the correct time in a range of different systems. Now that we are identifying more of the molecular components of both the light input pathway and the clock mechanism itself, we are becoming increasingly less able to distinguish between the two.

Orchestrated transcription of key pathways in Arabidopsis by the circadian clock.

Like most organisms, plants have endogenous biological clocks that coordinate internal events with the external environment. We used high-density oligonucleotide microarrays to examine gene expression in Arabidopsis and found that 6% of the more than 8000 genes on the array exhibited circadian changes in steady-state messenger RNA levels. Clusters of circadian-regulated genes were found in pathways involved in plant responses to light and other key metabolic pathways. Computational analysis of cycling genes allowed the identification of a highly conserved promoter motif that we found to be required for circadian control of gene expression. Our study presents a comprehensive view of the temporal compartmentalization of physiological pathways by the circadian clock in a eukaryote.

The period E-box is sufficient to drive circadian oscillation of transcription in vivo.

The minimum element from the Drosophila period promoter capable of driving in vivo cycling mRNA is the 69 bp circadian regulatory sequence (CRS). In cell culture, an 18 bp E-box element from the period promoter is regulated by five genes that are involved in the regulation of circadian expression in flies. This E-box is a target for transcriptional activation by bHLH-PAS proteins dCLOCK (dCLK) and CYCLE (CYC), this activation is inhibited by PERIOD (PER) and TIMELESS (TIM) together, and inhibition of dCLK/CYC by PER and TIM is blocked by CRYPTOCHROME (CRY) in the presence of light. Here, the same 18 bp E-box region generated rhythmic expression of luciferase in flies under both light-dark cycling and constant conditions. Flies heterozygous for the Clke(jrk) mutation maintained rhythmic expression from the E-box although at a lower level than wild type. Homozygous mutant Clk(jrk) animals had drastically lowered and arrhythmic expression. In a per01 background, expression from the E-box was high and not rhythmic. Transcription mediated by the per E-box was restricted to the same spatial pattern as the CRS. The per E-box DNA element and cognate binding proteins can confer per-like temporal and spatial expression. This demonstrates in vivo that the known circadian genes that form the core of the circadian oscillator in Drosophila integrate their activities at a single DNA element.

Specific sequences outside the E-box are required for proper per expression and behavioral rescue.

A 69 bp circadian regulatory sequence (CRS) upstream of the per gene is sufficient to drive circadian transcription, mediate proper spatial expression, and rescue behavioral rhythmicity in per01 flies. Within the CRS, an E-box is required for transcriptional activation by two basic-helix-loop-helix (bHLH) PERARNT-SIM (PAS) transcription factors, dCLOCK (dCLK) and CYCLE (CYC). To define sequences within the CRS that are required for spatial expression, circadian expression, and behavioral rhythmicity, a series of mutants that alter blocks of 3 to 12 nucleotides across the entire CRS were used to drive lacZ or per expression in vivo. As expected, the E-box within the CRS is necessary for high-level expression and behavioral rhythmicity, but sequences outside the E-box are also required for transcriptional activation, proper spatial expression, and behavioral rhythmicity. These results indicate that the dCLK-CYC target site extends beyond the E-box and that factors other than dCLK and CYC modulate per transcription.

Functional interaction of phytochrome B and cryptochrome 2.

Light is a crucial environmental signal that controls many photomorphogenic and circadian responses in plants. Perception and transduction of light is achieved by at least two principal groups of photoreceptors, phytochromes and cryptochromes. Phytochromes are red/far-red light-absorbing receptors encoded by a gene family of five members (phyA to phyE) in Arabidopsis. Cryptochrome 1 (cry1), cryptochrome 2 (cry2) and phototropin are the blue/ultraviolet-A light receptors that have been characterized in Arabidopsis. Previous studies showed that modulation of many physiological responses in plants is achieved by genetic interactions between different photoreceptors; however, little is known about the nature of these interactions and their roles in the signal transduction pathway. Here we show the genetic interaction that occurs between the Arabidopsis photoreceptors phyB and cry2 in the control of flowering time, hypocotyl elongation and circadian period by the clock. PhyB interacts directly with cry2 as observed in co-immunoprecipitation experiments with transgenic Arabidopsis plants overexpressing cry2. Using fluorescent resonance energy transfer microscopy, we show that phyB and cry2 interact in nuclear speckles that are formed in a light-dependent fashion.

Circadian rhythm genetics: from flies to mice to humans.

A successful genetic dissection of the circadian regulation of behaviour has been achieved through phenotype-driven mutagenesis screens in flies and mice. Cloning and biochemical analysis of these evolutionarily conserved proteins has led to detailed molecular insight into the clock mechanism. Few behaviours enjoy the degree of understanding that exists for circadian rhythms at the genetic, cellular and anatomical levels. The circadian clock has so eagerly spilled her secrets that we may soon know the unbroken chain of events from gene to behaviour. It will likely be fruitful to wield this uncommon degree of knowledge to attack one of the most challenging problems in genetics: the basis of complex human behavioural disorders. We review here the genetic screens that provided the entreé into the heart of the circadian clock, the model of the clock mechanism that has resulted, and the prospects for using the homologues as candidate genes in studies of human circadian dysrhythmias.

Cloning of the Arabidopsis clock gene TOC1, an autoregulatory response regulator homolog.

The toc1 mutation causes shortened circadian rhythms in light-grown Arabidopsis plants. Here, we report the same toc1 effect in the absence of light input to the clock. We also show that TOC1 controls photoperiodic flowering response through clock function. The TOC1 gene was isolated and found to encode a nuclear protein containing an atypical response regulator receiver domain and two motifs that suggest a role in transcriptional regulation: a basic motif conserved within the CONSTANS family of transcription factors and an acidic domain. TOC1 is itself circadianly regulated and participates in a feedback loop to control its own expression.

Fluorescent differential display identifies circadian clock-regulated genes in Arabidopsis thaliana.

Circadian rhythms in gene expression were first observed in plants more than 13 years ago, but the underlying mechanism controlling rhythmic gene expression is still not understood. The isolation of novel circadian clock-controlled genes (ccgs) is likely to provide new tools for studying circadian rhythms. Fluorescent differential display (FDD) was used to screen Arabidopsis thaliana mRNAs for cycling transcripts. Seventy PCR primer pairs were screened, and 17 different cycling bands were observed out of an estimated 10,500 bands screened. The identities of 10 bands were determined, and the rhythmic gene expression was confirmed using northern blot analysis. The 10 cycling bands represent 7 different genes, 6 of which are present in the databases and 1 that does not match anything in current databases. The rhythmic expression of the 7 genes is composed of four distinct phases of clock regulation. The results demonstrate that FDD can be used to isolate ccgs. The genes identified in this screen range from known A. thaliana ccgs, as well as genes shown to be clock controlled in other plant species, to a novel gene that may encode a pioneer protein. Further study of these ccgs is likely to increase our understanding of circadian-regulated gene expression.