Dynamical differential expression (DyDE) reveals the period control mechanisms of the Arabidopsis circadian oscillator.

The circadian oscillator, an internal time-keeping device found in most organisms, enables timely regulation of daily biological activities by maintaining synchrony with the external environment. The mechanistic basis underlying the adjustment of circadian rhythms to changing external conditions, however, has yet to be clearly elucidated. We explored the mechanism of action of nicotinamide in Arabidopsis thaliana, a metabolite that lengthens the period of circadian rhythms, to understand the regulation of circadian period. To identify the key mechanisms involved in the circadian response to nicotinamide, we developed a systematic and practical modeling framework based on the identification and comparison of gene regulatory dynamics. Our mathematical predictions, confirmed by experimentation, identified key transcriptional regulatory mechanisms of circadian period and uncovered the role of blue light in the response of the circadian oscillator to nicotinamide. We suggest that our methodology could be adapted to predict mechanisms of drug action in complex biological systems.

Photosynthetic entrainment of the Arabidopsis thaliana circadian clock.

Circadian clocks provide a competitive advantage in an environment that is heavily influenced by the rotation of the Earth, by driving daily rhythms in behaviour, physiology and metabolism in bacteria, fungi, plants and animals. Circadian clocks comprise transcription-translation feedback loops, which are entrained by environmental signals such as light and temperature to adjust the phase of rhythms to match the local environment. The production of sugars by photosynthesis is a key metabolic output of the circadian clock in plants. Here we show that these rhythmic, endogenous sugar signals can entrain circadian rhythms in Arabidopsis thaliana by regulating the gene expression of circadian clock components early in the photoperiod, thus defining a 'metabolic dawn'. By inhibiting photosynthesis, we demonstrate that endogenous oscillations in sugar levels provide metabolic feedback to the circadian oscillator through the morning-expressed gene PSEUDO-RESPONSE REGULATOR 7 (PRR7), and we identify that prr7 mutants are insensitive to the effects of sucrose on the circadian period. Thus, photosynthesis has a marked effect on the entrainment and maintenance of robust circadian rhythms in A. thaliana, demonstrating that metabolism has a crucial role in regulation of the circadian clock.

The circadian oscillator gene GIGANTEA mediates a long-term response of the Arabidopsis thaliana circadian clock to sucrose.

Circadian clocks are 24-h timing devices that phase cellular responses; coordinate growth, physiology, and metabolism; and anticipate the day-night cycle. Here we report sensitivity of the Arabidopsis thaliana circadian oscillator to sucrose, providing evidence that plant metabolism can regulate circadian function. We found that the Arabidopsis circadian system is particularly sensitive to sucrose in the dark. These data suggest that there is a feedback between the molecular components that comprise the circadian oscillator and plant metabolism, with the circadian clock both regulating and being regulated by metabolism. We used also simulations within a three-loop mathematical model of the Arabidopsis circadian oscillator to identify components of the circadian clock sensitive to sucrose. The mathematical studies identified GIGANTEA (GI) as being associated with sucrose sensing. Experimental validation of this prediction demonstrated that GI is required for the full response of the circadian clock to sucrose. We demonstrate that GI acts as part of the sucrose-signaling network and propose this role permits metabolic input into circadian timing in Arabidopsis.

Correct biological timing in Arabidopsis requires multiple light-signaling pathways.

Circadian oscillators provide rhythmic temporal cues for a range of biological processes in plants and animals, enabling anticipation of the day/night cycle and enhancing fitness-associated traits. We have used engineering models to understand the control principles of a plant's response to seasonal variation. We show that the seasonal changes in the timing of circadian outputs require light regulation via feed-forward loops, combining rapid light-signaling pathways with entrained circadian oscillators. Linear time-invariant models of circadian rhythms were computed for 3,503 circadian-regulated genes and for the concentration of cytosolic-free calcium to quantify the magnitude and timing of regulation by circadian oscillators and light-signaling pathways. Bioinformatic and experimental analysis show that rapid light-induced regulation of circadian outputs is associated with seasonal rephasing of the output rhythm. We identify that external coincidence is required for rephasing of multiple output rhythms, and is therefore important in general phase control in addition to specific photoperiod-dependent processes such as flowering and hypocotyl elongation. Our findings uncover a fundamental design principle of circadian regulation, and identify the importance of rapid light-signaling pathways in temporal control.

The role of the Cucumber mosaic virus 2b protein in viral movement and symptom induction.

The Cucumber mosaic virus (CMV) 2b protein is a counter-defense factor and symptom determinant. Conserved domains in the 2b protein sequence were mutated in the 2b gene of strain Fny-CMV. The effects of these mutations were assessed by infection of Nicotiana tabacum, N. benthamiana, and Arabidopsis thaliana (ecotype Col-0) with mutant viruses and by expression of mutant 2b transgenes in A. thaliana. We confirmed that two nuclear localization signals were required for symptom induction and found that the N-terminal domain was essential for symptom induction. The C-terminal domain and two serine residues within a putative phosphorylation domain modulated symptom severity. Further infection studies were conducted using Fny-CMVdelta2b, a mutant that cannot express the 2b protein and that induces no symptoms in N. tabacum, N. benthamiana, or A. thaliana ecotype Col-0. Surprisingly, in plants of A. thaliana ecotype C24, Fny-CMVdelta2b induced severe symptoms similar to those induced by the wild-type virus. However, C24 plants infected with the mutant virus recovered from disease while those infected with the wild-type virus did not. Expression of 2b transgenes from either Fny-CMV or from LS-CMV (a mild strain) in Col-0 plants enhanced systemic movement of Fny-CMVdelta2b and permitted symptom induction by Fny-CMVdelta2b. Taken together, the results indicate that the 2b protein itself is an important symptom determinant in certain hosts. However, they also suggest that the protein may somehow synergize symptom induction by other CMV-encoded factors.

Circadian oscillations of cytosolic free calcium regulate the Arabidopsis circadian clock.

In the last decade, the view of circadian oscillators has expanded from transcriptional feedback to incorporate post-transcriptional, post-translational, metabolic processes and ionic signalling. In plants and animals, there are circadian oscillations in the concentration of cytosolic free Ca2+ ([Ca2+]cyt), though their purpose has not been fully characterized. We investigated whether circadian oscillations of [Ca2+]cyt regulate the circadian oscillator of Arabidopsis thaliana. We report that in Arabidopsis, [Ca2+]cyt circadian oscillations can regulate circadian clock function through the Ca2+-dependent action of CALMODULIN-LIKE24 (CML24). Genetic analyses demonstrate a linkage between CML24 and the circadian oscillator, through pathways involving the circadian oscillator gene TIMING OF CAB2 EXPRESSION1 (TOC1).

The circadian clock rephases during lateral root organ initiation in Arabidopsis thaliana.

The endogenous circadian clock enables organisms to adapt their growth and development to environmental changes. Here we describe how the circadian clock is employed to coordinate responses to the key signal auxin during lateral root (LR) emergence. In the model plant, Arabidopsis thaliana, LRs originate from a group of stem cells deep within the root, necessitating that new organs emerge through overlying root tissues. We report that the circadian clock is rephased during LR development. Metabolite and transcript profiling revealed that the circadian clock controls the levels of auxin and auxin-related genes including the auxin response repressor IAA14 and auxin oxidase AtDAO2. Plants lacking or overexpressing core clock components exhibit LR emergence defects. We conclude that the circadian clock acts to gate auxin signalling during LR development to facilitate organ emergence.

Highly reproducible ChIP-on-chip analysis to identify genome-wide protein binding and chromatin status in Arabidopsis thaliana.

Gene activity is regulated via chromatin dynamics in eukaryotes. In plants, alterations of histone modifications are correlated with gene regulation for development, vernalization, and abiotic stress responses. Using ChIP, ChIP-on-chip, and ChIP-seq analyses, the direct binding regions of transcription factors and alterations of histone modifications can be identified on a genome-wide level. We have established reliable and reproducible ChIP and ChIP-on-chip methods that have been optimized for the Arabidopsis model system. These methods are not only useful for identifying the direct binding of transcription factors and chromatin status but also for scanning the regulatory network in Arabidopsis.

Systems analyses of circadian networks.

The circadian clock is a 24 hour timing device that co-ordinates biological activity with day/night cycles. The long history of systems analysis of circadian biology extends back to the first half of the last century when theoretical studies based on physiological experiments predicted the essential network properties, architecture and performance of circadian oscillators long before the first genetic components were isolated in the second half of the century. Systems approaches have continued to be important in analysing the circadian network in the model plant Arabidopsis thaliana and in mammals. We describe how systems analyses of transcriptional changes have led to formal mathematical models of circadian oscillators. Predictions within these mathematical models have been used to identify potential new components of circadian systems. Cross-referencing circadian regulation of transcript abundance with transcriptomic responses to abiotic and biotic signals has increased understanding of the nature of circadian clocks and their significance in regulating the daily life of plants and animals. We also highlight the need for systems analyses of the circadian regulation of proteins, metabolites and other physiological activities such as ion channel regulation.

Interactions between circadian and hormonal signalling in plants.

Growth and development of plants is controlled by external and internal signals. Key internal signals are those generated by hormones and the circadian clock. We highlight interactions between the circadian clock and hormonal signalling networks in regulating the physiology and growth of plants. Microarray analysis has shown that a significant proportion of transcripts involved in hormonal metabolism, catabolism, perception and signalling are also regulated by the circadian clock. In particular, there are interactions between the clock and abscisic acid, auxin, cytokinin and ethylene signalling. We discuss the role of circadian modulation ('gating') of hormonal signals in preventing temporally inappropriate responses. A consideration of the daily changes in physiology provides evidence that circadian gating of hormonal signalling couples the rhythmic regulation of carbon and water utilisation to rhythmic patterns of growth.

Selective targeting of miRNA-regulated plant development by a viral counter-silencing protein.

The cucumber mosaic virus (CMV) 2b protein suppresses RNA silencing and determines viral symptoms. Among Arabidopsis thaliana lines expressing 2b proteins from mild (LS and Q CMV) or severe (Fny CMV) strains, only Fny 2b-transgenic plants displayed strong symptom-like phenotypes in leaves, stems and flowers, together with stunting of main root growth and increased emergence of lateral roots. However, LS and Fny 2b proteins both enhanced lateral root length. Micro (mi)RNA-mediated cellular mRNA turnover was inhibited in Fny 2b-transgenic plants, but there was no evidence for this in LS 2b-transgenic plants. Both 2b proteins efficiently suppressed small interfering (si)RNA-mediated RNA silencing, suggesting that 2b proteins can target the siRNA pathway without disrupting miRNA-regulated RNA turnover. Thus, symptom induction is not an inevitable consequence of RNA silencing suppression. For CMV, strain-specific differences between the 2b silencing proteins determine whether only one or both small RNA-guided RNA destruction pathways are disrupted.

The Arabidopsis circadian clock incorporates a cADPR-based feedback loop.

Transcriptional feedback loops are a feature of circadian clocks in both animals and plants. We show that the plant circadian clock also incorporates the cytosolic signaling molecule cyclic adenosine diphosphate ribose (cADPR). cADPR modulates the circadian oscillator's transcriptional feedback loops and drives circadian oscillations of Ca2+ release. The effects of antagonists of cADPR signaling, manipulation of cADPR synthesis, and mathematical simulation of the interaction of cADPR with the circadian clock indicate that cADPR forms a feedback loop within the plant circadian clock.

ARABIDOPSIS CRINKLY4 function, internalization, and turnover are dependent on the extracellular crinkly repeat domain.

The study of the regulation and cellular dynamics of receptor kinase signaling in plants is a rapidly evolving field that promises to give enormous insights into the molecular control of signal perception. In this study, we have analyzed the behavior of the L1-specific receptor kinase ARABIDOPSIS CRINKLY4 (ACR4) from Arabidopsis thaliana in planta and have shown it to be present in two distinct compartments within cells. These represent protein export bodies and a population of internalized vesicles. In parallel, deletion analysis has shown that a predicted beta-propeller-forming extracellular domain is necessary for ACR4 function. Nonfunctional ACR4 variants with deletions or point mutations in this domain behave differently to wild-type fusion protein in that they are not internalized to the same extent. In addition, in contrast with functional ACR4, which appears to be rapidly turned over, they are stabilized. Thus, for ACR4, internalization and turnover are linked and depend on functionality, suggesting that ACR4 signaling may be subject to damping down via internalization and degradation. The observed rapid turnover of ACR4 sets it apart from other recently studied plant receptor kinases. Finally, ACR4 kinase activity is not required for protein function, leading us to propose, by analogy to animal systems, that ACR4 may hetero-oligomerize with a kinase-active partner during signaling. Plant and animal receptor kinases have distinct evolutionary origins. However, with other recent work, our study suggests that there has been considerable convergent evolution between mechanisms used to regulate their activity.