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Plants accumulate reserves in the daytime to support growth at night. Circadian regulation of diel reserve turnover was investigated by profiling starch, sugars, glucose 6-phosphate, organic acids, and amino acids during a light-dark cycle and after transfer to continuous light in Arabidopsis wild types and in mutants lacking dawn (lhy cca1), morning (prr7 prr9), dusk (toc1, gi), or evening (elf3) clock components. The metabolite time series were integrated with published time series for circadian clock transcripts to identify circadian outputs that regulate central metabolism. (a) Starch accumulation was slower in elf3 and prr7 prr9. It is proposed that ELF3 positively regulates starch accumulation. (b) Reducing sugars were high early in the T-cycle in elf3, revealing that ELF3 negatively regulates sucrose recycling. (c) The pattern of starch mobilization was modified in all five mutants. A model is proposed in which dawn and dusk/evening components interact to pace degradation to anticipated dawn. (d) An endogenous oscillation of glucose 6-phosphate revealed that the clock buffers metabolism against the large influx of carbon from photosynthesis. (e) Low levels of organic and amino acids in lhy cca1 and high levels in prr7 prr9 provide evidence that the dawn components positively regulate the accumulation of amino acid reserves.

Trees are carbon dioxide sinks and major producers of terrestrial biomass with distinct seasonal growth patterns. Circadian clocks enable the coordination of physiological and biochemical temporal ...

Plants use the circadian clock to sense photoperiod length. Seasonal responses like flowering are triggered at a critical photoperiod when a light-sensitive clock output coincides with light or darkness. However, many metabolic processes, like starch turnover, and growth respond progressively to photoperiod duration. We first tested the photoperiod response of 10 core clock genes and two output genes. qRT-PCR analyses of transcript abundance under 6, 8, 12 and 18 h photoperiods revealed 1-4 h earlier peak times under short photoperiods and detailed changes like rising PRR7 expression before dawn. Clock models recapitulated most of these changes. We explored the consequences for global gene expression by performing transcript profiling in 4, 6, 8, 12 and 18 h photoperiods. There were major changes in transcript abundance at dawn, which were as large as those between dawn and dusk in a given photoperiod. Contributing factors included altered timing of the clock relative to dawn, light signalling and changes in carbon availability at night as a result of clock-dependent regulation of starch degradation. Their interaction facilitates coordinated transcriptional regulation of key processes like starch turnover, anthocyanin, flavonoid and glucosinolate biosynthesis and protein synthesis and underpins the response of metabolism and growth to photoperiod.

A paradigm shift is needed and timely in moving plant modelling from largely isolated efforts to a connected community endeavour that can take full advantage of advances in computer science and in mechanistic understanding of plant processes. Plants in silico (Psi) envisions a digital representation of layered dynamic modules, linking from gene networks and metabolic pathways through to cellular organization, tissue, organ and whole plant development, together with resource capture and use efficiency in dynamic competitive environments, ultimately allowing a mechanistically rich simulation of the plant or of a community of plants in silico. The concept is to integrate models or modules from different layers of organization spanning from genome to phenome to ecosystem in a modular framework allowing the use of modules of varying mechanistic detail representing the same biological process. Developments in high-performance computing, functional knowledge of plants, the internet and open-source version controlled software make achieving the concept realistic. Open source will enhance collaboration and move towards testing and consensus on quantitative theoretical frameworks. Importantly, Psi provides a quantitative knowledge framework where the implications of a discovery at one level, for example, single gene function or developmental response, can be examined at the whole plant or even crop and natural ecosystem levels.

Summary Circadian clocks allow the temporal compartmentalization of biological processes. In Arabidopsis, circadian rhythms display organ specificity but the underlying molecular causes have not been identified. We investigated the mechanisms responsible for the similarities and differences between the clocks of mature shoots and roots in constant conditions and in light : dark cycles. We developed an imaging system to monitor clock gene expression in shoots and light- or dark-grown roots, modified a recent mathematical model of the Arabidopsis clock and used this to simulate our new data. We showed that the shoot and root circadian clocks have different rhythmic properties (period and amplitude) and respond differently to light quality. The root clock was entrained by direct exposure to low-intensity light, even in antiphase to the illumination of shoots. Differences between the clocks were more pronounced in conditions where light was present than in constant darkness, and persisted in the presence of sucrose. We simulated the data successfully by modifying those parameters of a clock model that are related to light inputs. We conclude that differences and similarities between the shoot and root clocks can largely be explained by organ-specific light inputs. This provides mechanistic insight into the developing field of organ-specific clocks.

Circadian clocks are biological timekeepers that allow living cells to time their activity in anticipation of predictable environmental changes. Detailed understanding of the circadian network of higher plants, such as Arabidopsis thaliana, is hampered by the high number of partially redundant genes. However, the picoeukaryotic alga Ostreococcus tauri, which was recently shown to possess a small number of non-redundant clock genes, presents an attractive alternative target for detailed modelling of circadian clocks in the green lineage. Based on extensive time-series data from in vivo reporter gene assays, we developed a model of the Ostreococcus clock as a feedback loop between the genes TOC1 and CCA1. The model reproduces the dynamics of the transcriptional and translational reporters over a range of photoperiods. Surprisingly, the model is also able to predict the transient behaviour of the clock when the light conditions are altered. Despite the apparent simplicity of the clock circuit, it displays considerable complexity in its response to changing light conditions. Systematic screening of the effects of altered day length revealed a complex relationship between phase and photoperiod, which is also captured by the model. The complex light response is shown to stem from circadian gating of light-dependent mechanisms. This study provides insights into the contributions of light inputs to the Ostreococcus clock. The model suggests that a high number of light-dependent reactions are important for flexible timing in a circadian clock with only one feedback loop.

The circadian clock is a fundamental feature of eukaryotic gene regulation that is emerging as an exemplar genetic sub-network for systems biology. The circadian system in Arabidopsis plants is complex, in part due to its phototransduction pathways, which are themselves under circadian control. We therefore analysed two simpler experimental systems. Etiolated seedlings entrained by temperature cycles showed circadian rhythms in the expression of genes that are important for the clock mechanism, but only a restricted set of downstream target genes were rhythmic in microarray assays. Clock control of phototransduction pathways remained robust across a range of light inputs, despite the arrhythmic transcription of light-signalling genes. Circadian interactions with light signalling were then analysed using a single active photoreceptor. Phytochrome A (phyA) is expected to be the only active photoreceptor that can mediate far-red (FR) light input to the circadian clock. Surprisingly, rhythmic gene expression was profoundly altered under constant FR light, in a phyA-dependent manner, resulting in high expression of evening genes and low expression of morning genes. Dark intervals were required to allow high-amplitude rhythms across the transcriptome. Clock genes involved in this response were identified by mutant analysis, showing that the EARLY FLOWERING 4 gene is a likely target and mediator of the FR effects. Both experimental systems illustrate how profoundly the light input pathways affect the plant circadian clock, and provide strong experimental manipulations to understand critical steps in the plant clock mechanism.

The oxygen-atom-transfer (OAT) reactivity of [LiPrMoO2(OPh)] (1, LiPr=hydrotris(3-isopropylpyrazol-1-yl)borate) with the tertiary phosphines PEt3 and PPh2Me in acetonitrile was investigated. The first step, [LiPrMoO2(OPh)]+PR3-->[LiPrMoO(OPh)(OPR3)], follows a second-order rate law with an associative transition state (PEt3, DeltaH not equal=48.4 (+/-1.9) kJ mol-1, DeltaS not equal=-149.2 (+/-6.4) J mol-1 K-1, DeltaG not equal=92.9 kJ mol-1; PPh2Me, DeltaH not equal=73.4 (+/-3.7) kJ mol-1, DeltaS not equal=-71.9 (+/-2.3) J mol-1 K-1, DeltaG not equal=94.8 kJ mol-1). With PMe3 as a model substrate, the geometry and the free energy of the transition state (TS) for the formation of the phosphine oxide-coordinated intermediate were calculated. The latter, 95 kJ mol-1, is in good agreement with the experimental values. An unexpectedly large O-P-C angle calculated for the TS suggests that there is significant O-nucleophilic attack on the P--C sigma* in addition to the expected nucleophilic attack of the P on the Mo==O pi*. The second step of the reaction, that is, the exchange of the coordinated phosphine oxide with acetonitrile, [LiPrMoO(OPh)(OPR3)]+MeCN-->[LiPrMoO(OPh)(MeCN)]+OPR3, follows a first-order rate law in MeCN. A dissociative interchange (Id) mechanism, with activation parameters of DeltaH not equal=93.5 (+/-0.9) kJ mol-1, DeltaS not equal=18.2 (+/-3.3) J mol-1 K-1, DeltaG not equal=88.1 kJ mol-1 and DeltaH not equal=97.9 (+/-3.4) kJ mol-1, DeltaS not equal=47.3 (+/-11.8) J mol-1 K-1, DeltaG not equal=83.8 kJ mol-1, for [LiPrMoO(OPh)(OPEt3)] (2 a) and [LiPrMoO(OPh)(OPPh2Me)] (2 b), respectively, is consistent with the experimental data. Although gas-phase calculations indicate that the Mo--OPMe3 bond is stronger than the Mo--NCMe bond, solvation provides the driving force for the release of the phosphine oxide and formation of [LiPrMoO(OPh)(MeCN)] (3).

Intermediates in the oxygen atom transfer from Mo V I to P I I I , [Tp i P r MoOX(OPR 3 )] (Tp i P r =hydrotris(3-isopropylpyrazol-l-yl)borate; X= Cl - , phenolates, thiolates), have been isolated from the reactions of [Tp i P r -MoO 2 X] with phosphines (PEt 3 , PMePh 2 , PPh 3 ). The green, diamagnetic oxomolybdenum(iv) complexes possess local C 1 symmetry (by NMR spectroscopy) and exhibit IR bands assigned to ν(Mo=O) (approximately 950 cm - 1 ) and ν(P=O) (1140-1083 cm - 1 ) vibrations. The X-ray crystal structures of [Tp i P r MoOX(OPEt 3 )] (X=OC 6 H 4 -2-sBu, SnBu), [Tp i P r MoO(OPh)-(OPMePh 2 )], and [Tp i P r MoOCl-(OPPh 3 )] have been determined. The monomeric complexes exhibit distorted octahedral geometries, with coordination spheres composed of tridentate fac-Tp i P r and mutually cis monodentate terminal oxo, phosphoryl (phosphine oxide), and monoanionic X ligands. The electronic structures and stabilities of the complexes have been probed by computational methods, with the three-dimensional energy surfaces confirming the existence of a low-energy steric pocket that restricts the conformational freedom of the phosphoryl ligand and inhibits complete oxygen atom transfer. The reactivity of the complexes is also briefly described.

Phytochrome B (phyB) is a major phytochrome active in light-grown plants. The circadian clock controls the expression of the PHYB gene. We have used the luciferase reporter gene (LUC) to monitor the rhythmic expression of PHYB in photoreceptor and clock-associated mutant backgrounds. Surprisingly, we found that PHYB and CAB expression have different free-running periods, indicating that separate circadian clocks control these genes. The effects of mutations show that the clocks share common components. This suggests that they are copies of the same clock mechanism in different locations, most likely in different cell layers. Furthermore, we show that phyB is required for a negative feedback loop that strongly antagonises the expression of PHYB. Compared to a system with only one clock, this regulatory complexity might allow the phase of peak expression for one clock-controlled gene to alter, relative to other genes or to changing environmental conditions.

Summary The phytochrome-induced expression pattern of the chlorophyll a/b binding protein (cab) gene was studied using a cab2::luciferase reporter transgene in the tobacco cotyledon. The role of developmentally regulated competence, cooperativity among cells and signal propagation was investigated by red-light microbeam irradiation of distinct areas of the cotyledon. Even with a minimal fluence, the response was not restricted to the irradiated cells. Following irradiation of the cotyledon base, the luciferase activity revealed a robust propagation of activating signals to areas that had not received a light stimulus. The acropetal outspread formed distinct expression patterns depending on the site of the irradiation. The combination of imaging luciferase activity in living seedlings and microbeam microscopy provides significant experimental evidence of how cellular light perception and intercellular signalling contribute to the cab gene expression pattern.

A wide range of biological processes, in all eukaryotes and in some prokaryotes, are controlled by rhythms with a period close to 24 hours. The circadian oscillator, which is responsible for generating these rhythms, is controlled by light signals that maintain its synchrony with the environmental day/night cycle. Higher plants exhibit many circadian rhythms, including rhythms in the transcription of specific genes. Molecular tools derived from such clock-controlled genes have led to the identification of several circadian rhythm mutants in the genetic model, Arabidopsis thaliana. The extensive understanding of photoperception in this species will make it a powerful system with which to investigate the light regulation of circadian rhythms. We compare Arabidopsis rhythms to the results from other systems, and discuss these data with respect to the current phototransduction models.

Recent findings are incorporated into a new mathematical model of the plant circadian clock, revealing a complex circuit structure comprised of multiple negative feedback loops, and predicting a repressive role for a key regulator, TOC1, which the authors confirm experimentally., The feedback structure of the plant clock's evening loop was reconstructed based on multiple data, and is now represented by the evening complex (ELF3–ELF4–LUX), which represses transcription from the ELF4 and LUX promoters. Computational analysis of timeseries data from mutant plants predicts that TOC1 is a repressor of the key morning genes LHY and CCA1, not an activator. Analysis of LHY and CCA1 expression in TOC1 gain- and loss-of-function plants confirms this prediction. Light induction of LHY and CCA1 expression is predicted to determine the clock's response to brief light pulses, matching the observed phase-response curve. The evening complex controls LHY and CCA1 expression by a double-negative connection, rather than direct activation, forming part of a three-component repressilator circuit, which is itself only part of the more complex circuit of the clock system., Circadian clocks synchronise biological processes with the day/night cycle, using molecular mechanisms that include interlocked, transcriptional feedback loops. Recent experiments identified the evening complex (EC) as a repressor that can be essential for gene expression rhythms in plants. Integrating the EC components in this role significantly alters our mechanistic, mathematical model of the clock gene circuit. Negative autoregulation of the EC genes constitutes the clock's evening loop, replacing the hypothetical component Y. The EC explains our earlier conjecture that the morning gene PSEUDO-RESPONSE REGULATOR 9 was repressed by an evening gene, previously identified with TIMING OF CAB EXPRESSION1 (TOC1). Our computational analysis suggests that TOC1 is a repressor of the morning genes LATE ELONGATED HYPOCOTYL and CIRCADIAN CLOCK ASSOCIATED1 rather than an activator as first conceived. This removes the necessity for the unknown component X (or TOC1mod) from previous clock models. As well as matching timeseries and phase-response data, the model provides a new conceptual framework for the plant clock that includes a three-component repressilator circuit in its complex structure.