Complex epistatic interactions between ELF3, PRR9, and PRR7 regulate the circadian clock and plant physiology.

Circadian clocks are endogenous timekeeping mechanisms that coordinate internal physiological responses with the external environment. EARLY FLOWERING3 (ELF3), PSEUDO RESPONSE REGULATOR (PRR9), and PRR7 are essential components of the plant circadian clock and facilitate entrainment of the clock to internal and external stimuli. Previous studies have highlighted a critical role for ELF3 in repressing the expression of PRR9 and PRR7. However, the functional significance of activity in regulating circadian clock dynamics and plant development is unknown. To explore this regulatory dynamic further, we first employed mathematical modeling to simulate the effect of the prr9/prr7 mutation on the elf3 circadian phenotype. These simulations suggested that simultaneous mutations in prr9/prr7 could rescue the elf3 circadian arrhythmia. Following these simulations, we generated all Arabidopsis elf3/prr9/prr7 mutant combinations and investigated their circadian and developmental phenotypes. Although these assays could not replicate the results from the mathematical modeling, our results have revealed a complex epistatic relationship between ELF3 and PRR9/7 in regulating different aspects of plant development. ELF3 was essential for hypocotyl development under ambient and warm temperatures, while PRR9 was critical for root thermomorphogenesis. Finally, mutations in prr9 and prr7 rescued the photoperiod-insensitive flowering phenotype of the elf3 mutant. Together, our results highlight the importance of investigating the genetic relationship among plant circadian genes.

Multiple metals influence distinct properties of the Arabidopsis circadian clock.

Circadian rhythms coordinate endogenous events with external signals, and are essential to biological function. When environmental contaminants affect these rhythms, the organism may experience fitness consequences such as reduced growth or increased susceptibility to pathogens. In their natural environment plants may be exposed to a wide range of industrial and agricultural soil pollutants. Here, we investigate how the addition of various metal salts to the root-interaction environment can impact rhythms, measured via the promoter:luciferase system. The consequences of these environmental changes were found to be varied and complex. Therefore, in addition to traditional Fourier-based analyses, we additionally apply novel wavelet-based spectral hypothesis testing and clustering methodologies to organize and understand the data. We are able to classify broad sets of responses to these metal salts, including those that increase, and those that decrease, the period, or which induce a lack of precision or disrupt any meaningful periodicity. Our methods are general, and may be applied to discover common responses and hidden structures within a wide range of biological time series data.

Chemical Perturbation of Chloroplast-Related Processes Affects Circadian Rhythms of Gene Expression in Arabidopsis: Salicylic Acid Application Can Entrain the Clock.

The plant circadian system reciprocally interacts with metabolic processes. To investigate entrainment features in metabolic-circadian interactions, we used a chemical approach to perturb metabolism and monitored the pace of nuclear-driven circadian oscillations. We found that chemicals that alter chloroplast-related functions modified the circadian rhythms. Both vitamin C and paraquat altered the circadian period in a light-quality-dependent manner, whereas rifampicin lengthened the circadian period under darkness. Salicylic acid (SA) increased oscillatory robustness and shortened the period. The latter was attenuated by sucrose addition and was also gated, taking place during the first 3 h of the subjective day. Furthermore, the effect of SA on period length was dependent on light quality and genotype. Period lengthening or shortening by these chemicals was correlated to their inferred impact on photosynthetic electron transport activity and the redox state of plastoquinone (PQ). Based on these data and on previous publications on circadian effects that alter the redox state of PQ, we propose that the photosynthetic electron transport and the redox state of PQ participate in circadian periodicity. Moreover, coupling between chloroplast-derived signals and nuclear oscillations, as observed in our chemical and genetic assays, produces traits that are predicted by previous models. SA signaling or a related process forms a rhythmic input loop to drive robust nuclear oscillations in the context predicted by the zeitnehmer model, which was previously developed for Neurospora. We further discuss the possibility that electron transport chains (ETCs) are part of this mechanism.

Physiological and Genetic Dissection of Sucrose Inputs to the Arabidopsis thaliana Circadian System.

Circadian rhythms allow an organism to synchronize internal physiological responses to the external environment. Perception of external signals such as light and temperature are critical in the entrainment of the oscillator. However, sugar can also act as an entraining signal. In this work, we have confirmed that sucrose accelerates the circadian period, but this observed effect is dependent on the reporter gene used. This observed response was dependent on sucrose being available during free-running conditions. If sucrose was applied during entrainment, the circadian period was only temporally accelerated, if any effect was observed at all. We also found that sucrose acts to stabilize the robustness of the circadian period under red light or blue light, in addition to its previously described role in stabilizing the robustness of rhythms in the dark. Finally, we also found that CCA1 is required for both a short- and long-term response of the circadian oscillator to sucrose, while LHY acts to attenuate the effects of sucrose on circadian period. Together, this work highlights new pathways for how sucrose could be signaling to the oscillator and reveals further functional separation of CCA1 and LHY.

HSP90 Contributes to Entrainment of the Arabidopsis Circadian Clock via the Morning Loop.

The plant circadian clock allows the synchronization of internal physiological responses to match the predicted environment. HSP90.2 is a molecular chaperone that has been previously described as required for the proper functioning of the Arabidopsis oscillator under both ambient and warm temperatures. Here, we have characterized the circadian phenotype of the hsp90.2-3 mutant. As previously reported using pharmacological or RNA interference inhibitors of HSP90 function, we found that hsp90.2-3 lengthens the circadian period and that the observed period lengthening was more exaggerated in warm-cold-entrained seedlings. However, we observed no role for the previously identified interactors of HSP90.2, GIGANTEA and ZEITLUPPE, in HSP90-mediated period lengthening. We constructed phase-response curves (PRCs) in response to warmth pulses to identify the entry point of HSP90.2 to the oscillator. These PRCs revealed that hsp90.2-3 has a circadian defect within the morning. Analysis of the cca1, lhy, prr9, and prr7 mutants revealed a role for CCA1, LHY, and PRR7, but not PRR9, in HSP90.2 action to the circadian oscillator. Overall, we define a potential pathway for how HSP90.2 can entrain the Arabidopsis circadian oscillator.

AKIN10 activity as a cellular link between metabolism and circadian-clock entrainment in Arabidopsis thaliana.

AKIN10, the catalytic subunit of the Snf1 (sucrose non-fermenting 1)-related kinase 1 (SnRK1) complex, acts as an energy sensor in plants. We showed that AKIN10-induced expression affects the pace of the circadian clock and particularly the phase of expression of GIGANTEA (GI). The AKIN10 effect on period length required TIME FOR COFFEE (TIC), a circadian-clock component with developmental and metabolic roles. Here we expand on the possible interactions between AKIN10, whose activity is involved in transcriptional reprogramming, and clock elements GI and TIC. We hypothesize how they could participate in clock entrainment through a metabolic signal derived from carbon pools and starch metabolism. Additionally, we consider further the role of cellular energy status to the clock through the formation of a hypothetical protein complex. We also demonstrate the role of AKIN10, but not its sequence-related kinase AKIN11, on clock periodicity. Altogether we present a model of action of these elements in metabolic-related clock entrainment.

Circadian rhythms vary over the growing season and correlate with fitness components.

Circadian clocks have evolved independently in all three domains of life, suggesting that internal mechanisms of time-keeping are adaptive in contemporary populations. However, the performance consequences of either discrete or quantitative clock variation have rarely been tested in field settings. Clock sensitivity of diverse segregating lines to the environment remains uncharacterized as do the statistical genetic parameters that determine evolutionary potential. In field studies with Arabidopsis thaliana, we found that major perturbations to circadian cycle length (referred to as clock period) via mutation reduce both survival and fecundity. Subtler adjustments via genomic introgression of naturally occurring alleles indicated that clock periods slightly >24 hr were adaptive, consistent with prior models describing how well the timing of biological processes is adjusted within a diurnal cycle (referred to as phase). In segregating recombinant inbred lines (RILs), circadian phase varied up to 2 hr across months of the growing season, and both period and phase expressed significant genetic variances. Performance metrics including developmental rate, size and fruit set were described by principal components (PC) analyses and circadian parameters correlated with the first PC, such that period lengths slightly >24 hr were associated with improved performance in multiple RIL sets. These experiments translate functional analyses of clock behaviour performed in controlled settings to natural ones, demonstrating that quantitative variation in circadian phase is highly responsive to seasonally variable abiotic factors. The results expand upon prior studies in controlled settings, showing that discrete and quantitative variation in clock phenotypes correlates with performance in nature.

The metabolic sensor AKIN10 modulates the Arabidopsis circadian clock in a light-dependent manner.

Plants generate rhythmic metabolism during the repetitive day/night cycle. The circadian clock produces internal biological rhythms to synchronize numerous metabolic processes such that they occur at the required time of day. Metabolism conversely influences clock function by controlling circadian period and phase and the expression of core-clock genes. Here, we show that AKIN10, a catalytic subunit of the evolutionarily conserved key energy sensor sucrose non-fermenting 1 (Snf1)-related kinase 1 (SnRK1) complex, plays an important role in the circadian clock. Elevated AKIN10 expression led to delayed peak expression of the circadian clock evening-element GIGANTEA (GI) under diurnal conditions. Moreover, it lengthened clock period specifically under light conditions. Genetic analysis showed that the clock regulator TIME FOR COFFEE (TIC) is required for this effect of AKIN10. Taken together, we propose that AKIN10 conditionally works in a circadian clock input pathway to the circadian oscillator.

Daily rhythmicity of high affinity copper transport.

A differential demand for copper (Cu) of essential cupro-proteins that act within the mitochondrial and chloroplastal electronic transport chains occurs along the daily light/dark cycles. This requires a fine-tuned spatiotemporal regulation of Cu delivery, becoming especially relevant under non-optimal growth conditions. When scarce, Cu is imported through plasma membrane-bound high affinity Cu transporters (COPTs) whose coding genes are transcriptionally induced by the SPL7 transcription factor. Temporal homeostatic mechanisms are evidenced by the presence of multiple light- and clock-responsive regulatory cis elements in the promoters of both SPL7 and its COPT targets. A model is presented here for such temporal regulation that is based on the synchrony between the basal oscillatory pattern of SPL7 and its targets, such as COPT2. Conversely, Cu feeds back to coordinate intracellular Cu availability on the SPL7-dependent regulation of further Cu acquisition. This occurs via regulation at COPT transporters. Moreover, exogenous Cu affects several circadian-clock components, such as the timing of GIGANTEA transcript abundance. Together we propose that there is a dynamic response to Cu that is integrated over diurnal time to maximize metabolic efficiency under challenging conditions.

Modulation of copper deficiency responses by diurnal and circadian rhythms in Arabidopsis thaliana.

Copper homeostasis under deficiency is regulated by the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE7 (SPL7) transcription factor. The daily oscillating expression of two SPL7-dependent copper deficiency markers, COPPER TRANSPORTER (COPT2) and IRON SUPEROXIDE DISMUTASE (FSD1), has been followed by quantitative PCR and in promoter:LUCIFERASE transgenic plants. Both genes showed circadian and diurnal regulation. Under copper deficiency, their expression decreased drastically in continuous darkness. Accordingly, total copper content was slightly reduced in etiolated seedlings under copper deficiency. The expression of SPL7 and its targets COPT2 and FSD1 was differently regulated in various light signalling mutants. On the other hand, increased copper levels reduced the amplitude of nuclear circadian clock components, such as GIGANTEA (GI). The alteration of copper homeostasis in the COPT1 overexpression line and spl7 mutants also modified the amplitude of a classical clock output, namely the circadian oscillation of cotyledon movements. In the spl7 mutant, the period of the oscillation remained constant. These results suggest a feedback of copper transport on the circadian clock and the integration of rhythmic copper homeostasis into the central oscillator of plants.

TIME FOR COFFEE is an essential component in the maintenance of metabolic homeostasis in Arabidopsis thaliana.

Plants often respond to environmental changes by reprogramming metabolic and stress-associated pathways. Homeostatic integration of signaling is a central requirement for ensuring metabolic stability in living organisms. Under diurnal conditions, properly timed rhythmic metabolism provides fitness benefits to plants. TIME FOR COFFEE (TIC) is a circadian regulator known to be involved in clock resetting at dawn. Here we explored the mechanism of influence of TIC in plant growth and development, as initiated by a microarray analysis. This global profiling showed that a loss of TIC function causes a major reprogramming of gene expression that predicts numerous developmental, metabolic, and stress-related phenotypes. This led us to demonstrate that this mutant exhibits late flowering, a plastochron defect, and diverse anatomical phenotypes. We further observed a starch-excess phenotype and altered soluble carbohydrate levels. tic exhibited hypersensitivity to oxidative stress and abscisic acid, and this was associated with a striking resistance to drought. These phenotypes were connected to an increase in total glutathione levels that correlated with a readjustment of amino acids and polyamine pools. By comparatively analyzing our transcriptomic and metabolomic data, we concluded that TIC is a central element in plant homeostasis that integrates and coordinates developmental, metabolic, and environmental signals.

Environmental memory from a circadian oscillator: the Arabidopsis thaliana clock differentially integrates perception of photic vs. thermal entrainment.

The constraint of a rotating earth has led to the evolution of a circadian clock that drives anticipation of future environmental changes. During this daily rotation, the circadian clock of Arabidopsis thaliana (Arabidopsis) intersects with the diurnal environment to orchestrate virtually all transcriptional processes of the plant cell, presumably by detecting, interpreting, and anticipating the environmental alternations of light and temperature. To comparatively assess differential inputs toward phenotypic and physiological responses on a circadian parameter, we surveyed clock periodicity in a recombinant inbred population modified to allow for robust periodicity measurements after entrainment to respective photic vs. thermal cues, termed zeitgebers. Lines previously thermally entrained generally displayed reduced period length compared to those previously photically entrained. This differential zeitgeber response was also detected in a set of diverse Arabidopsis accessions. Thus, the zeitgebers of the preceding environment direct future behavior of the circadian oscillator. Allelic variation at quantitative trait loci generated significant differences in zeitgeber responses in the segregating population. These were important for periodicity variation dependent on the nature of the subsequent entrainment source. Collectively, our results provide a genetic paradigm for the basis of environmental memory of a preceding environment, which leads to the integrated coordination of circadian periodicity.

Protocol: Streamlined sub-protocols for floral-dip transformation and selection of transformants in Arabidopsis thaliana.

Generating and identifying transformants is essential for many studies of gene function. In Arabidopsis thaliana, a revolutionary protocol termed floral dip is now the most widely used transformation method. Although robust, it involves a number of relatively time-consuming and laborious steps, including manipulating an Agrobacterium tumefaciens culture and aseptic procedures for the selection of plant lines harboring antibiotic-selection markers. Furthermore, where multiple transgenes are to be introduced, achieving this by sequential transformations over multiple generations adds significantly to the time required. To circumvent these bottlenecks, we have developed three streamlined sub-protocols. First, we find that A. thaliana can be transformed by dipping directly into an A. tumefaciens culture supplemented with surfactant, eliminating the need for media exchange to a buffered solution. Next, we illustrate that A. thaliana lines possessing a double-transformation event can be readily generated by simply by floral-dipping into a mixture of two A. tumefaciens cultures harboring distinct transformation vectors. Finally, we report an alternative method of transformant selection on chromatography sand that does not require surface sterilization of seeds. These sub-protocols, which can be used separately or in combination, save time and money, and reduce the possibility of contamination.

TIME FOR COFFEE encodes a nuclear regulator in the Arabidopsis thaliana circadian clock.

The plant circadian clock is required for daily anticipation of the diurnal environment. Mutation in Arabidopsis thaliana TIME FOR COFFEE (TIC) affects free-running circadian rhythms. To investigate how TIC functions within the circadian system, we introduced markers for the evening and morning phases of the clock into tic and measured evident rhythms. The phases of evening clock genes in tic were all advanced under light/dark cycles without major expression level defects. With regard to morning-acting genes, we unexpectedly found that TIC has a closer relationship with LATE ELONGATED HYPOCOTYL (LHY) than with CIRCADIAN CLOCK ASSOCIATED1, as tic has a specific LHY expression level defect. Epistasis analysis demonstrated that there were no clear rhythms in double mutants of tic and evening-acting clock genes, although double mutants of tic and morning-acting genes exhibited a similar free-running period as tic. We isolated TIC and found that its mRNA expression is continuously present over the diurnal cycle, and the encoded protein appears to be strictly localized to the nucleus. Neither its abundance nor its cellular distribution was found to be clock regulated. We suggest that TIC encodes a nucleus-acting clock regulator working close to the central oscillator.

Signaling pathways in ischemic preconditioning.

Ischemic preconditioning renders the heart resistant to infarction from ischemia/reperfusion. Over the past two decades a great deal has been learned about preconditioning's mechanism. Adenosine, bradykinin, and opioids act in parallel to trigger the preconditioned state and do so by activating PKC. While adenosine couples directly to PKC through the phospholipases, bradykinin and opioids do so through a complex pathway that includes in order: phosphatidylinositol 3-kinase (PI3-kinase), Akt, nitric oxide synthase, guanylyl cyclase, PKG, opening of mitochondrial K(ATP) channels, and activation of PKC by redox signaling. There are even differences between the opioid and bradykinin coupling as the former activates PI3-kinase through transactivation of the epidermal growth factor receptor while the latter has an unknown coupling mechanism. Protection stems from inhibition of formation of mitochondrial permeability transition pores early in reperfusion through activation of the survival kinases, Akt and ERK. These kinases are activated as a result of PKC somehow promoting signaling from adenosine A(2) receptors early in reperfusion. The survival kinases are thought to inhibit pore formation by phosphorylating GSK-3beta. The reperfused heart requires the support of the protective signals for only about an hour after which the ischemic injury is repaired and the signals are no longer needed.

Postconditioning protects rabbit hearts through a protein kinase C-adenosine A2b receptor cascade.

OBJECTIVE: Ischemic postconditioning protects the reperfused heart from infarction, and this protection is dependent on the occupancy of adenosine receptors. We further explored the role of adenosine receptors in this salvage. METHODS: In situ rabbit hearts underwent 30 min of regional ischemia and 3 h of reperfusion, and postconditioning was effected with four cycles of 30-s reperfusion/30-s coronary artery occlusion at the end of ischemia. RESULTS: Postconditioning reduced infarct size from 40.2+/-3.4% of the risk zone in untreated hearts to 15.5+/-2.5%. Protection by postconditioning was blocked by either the non-selective adenosine receptor blocker 8-p-(sulfophenyl)theophylline or the A2b-selective antagonist MRS 1754, injected intravenously 5 min before reperfusion. The protein kinase C (PKC) antagonist chelerythrine also aborted postconditioning's salvage, indicating a PKC-dependent mechanism. Neither the A1-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine nor the A2a-selective antagonist 8-(13-chlorostyryl)caffeine had an effect on protection. The non-selective but A2b-potent adenosine agonist 5'-(N-ethylcarboxamido)adenosine (NECA) infused from 5 min before to 1h after reperfusion mimicked postconditioning's effect on infarct size (17.2+/-2.7% infarction) and MRS 1754 blocked the NECA-induced cardioprotection, confirming that A2b activation was protective. The PKC activator phorbol 12-myristate 13-acetate delivered just before reperfusion also duplicated the protective effect of postconditioning (16.3+/-4.1% infarction), and co-administration of the PKC antagonist chelerythrine aborted PMA's protection, confirming that the protection was the result of PKC activation. NECA's protective effect was not affected by chelerythrine, but rather MRS 1754 blocked PMA's salutary effect (42.8+/-1.0% infarction), suggesting that the A2b receptor's effect is under control of PKC. Finally, wortmannin, a blocker of phosphatidylinositol 3-kinase, also abrogated protection by PMA. CONCLUSIONS: Salvage of ischemic myocardium by postconditioning is dependent on activation of A2b receptors, which in turn depends on activation of PKC. It is still unclear why PKC activation is required to make the heart's adenosine become protective.