REVEILLE2 thermosensitive splicing: a molecular basis for the integration of nocturnal temperature information by the Arabidopsis circadian clock.

Cold stress is one of the major environmental factors that limit growth and yield of plants. However, it is still not fully understood how plants account for daily temperature fluctuations, nor how these temperature changes are integrated with other regulatory systems such as the circadian clock. We demonstrate that REVEILLE2 undergoes alternative splicing after chilling that increases accumulation of a transcript isoform encoding a MYB-like transcription factor. We explore the biological function of REVEILLE2 in Arabidopsis thaliana using a combination of molecular genetics, transcriptomics, and physiology. Disruption of REVEILLE2 alternative splicing alters regulatory gene expression, impairs circadian timing, and improves photosynthetic capacity. Changes in nuclear gene expression are particularly apparent in the initial hours following chilling, with chloroplast gene expression subsequently upregulated. The response of REVEILLE2 to chilling extends our understanding of plants immediate response to cooling. We propose that the circadian component REVEILLE2 restricts plants responses to nocturnal reductions in temperature, thereby enabling appropriate responses to daily environmental changes.

Taser barb penetration causing phalangeal fracture.

We present an unusual case of phalangeal fracture resulting from direct penetration by the barb of a conducted electrical weapon (Taser). When a Taser is triggered, compressed gas propels two barbs with trailing insulated wires which deliver a pulsed electrical discharge on contact. A 51-year-old man presented with a single barb of the Taser embedded in the diaphysis of the proximal phalanx and an associated open fracture. The barb was removed under local anaesthesia. The fracture was stable and was mobilised in a flexible splint. Oral antibiotics were commenced in recognition of the risk of flexor sheath and bone inoculation. While the most severe complications associated with Taser are related to the electrical component, the most common injuries are associated with falls and barb penetrations. Clinicians must be mindful of the risk of fracture, infection and soft tissue injury when such a foreign body penetrates a phalanx.

Shades of green: untying the knots of green photoperception.

The development of economical LED technology has enabled the application of different light qualities and quantities to control plant growth. Although we have a comprehensive understanding of plants' perception of red and blue light, the lack of a dedicated green light sensor has frustrated our utilization of intermediate wavelengths, with many contradictory reports in the literature. We discuss the contribution of red and blue photoreceptors to green light perception and highlight how green light can be used to improve crop quality. Importantly, our meta-analysis demonstrates that green light perception should instead be considered as a combination of distinct 'green' and 'yellow' light-induced responses. This distinction will enable clearer interpretation of plants' behaviour in response to green light as we seek to optimize plant growth and nutritional quality in horticultural contexts.

Arabidopsis JMJD5/JMJ30 Acts Independently of LUX ARRHYTHMO Within the Plant Circadian Clock to Enable Temperature Compensation.

The circadian system ensures that plants respond appropriately to environmental change by predicting regular transitions that occur during diel cycles. In order to be most useful, the circadian system needs to be compensated against daily and seasonal changes in temperature that would otherwise alter the pace of this biological oscillator. We demonstrate that an evening-phased protein, the putative histone demethylase JMJD5, contributes to temperature compensation. JMJD5 is co-expressed with components of the Evening Complex, an agglomeration of proteins including EARLY FLOWERING3 (ELF3), ELF4, and LUX ARRHYTHYMO (LUX), which also integrates temperature changes into the molecular clockwork. One role of the Evening Complex is to regulate expression of PSEUDORESPONSE REGULATOR9 (PRR9) and PRR7, important components of the temperature compensation mechanism. Surprisingly we find that LUX, but not other Evening Complex components, is dispensable for clock function at low temperatures. Further genetic analysis suggests JMJD5 acts in a parallel pathway to LUX within the circadian system. Although an intact JMJD5 catalytic domain is required for its function within the clock, our findings suggest JMJD5 does not directly regulate H3K36 methylation at circadian loci. Such data refine our understanding of how JMDJ5 acts within the Arabidopsis circadian system.

Retrograde signalling as an informant of circadian timing.

Contents Summary 1749 I. The circadian system is responsive to environmental change 1749 II. Photoassimilates regulate circadian timing 1750 III. Retrograde signals contribute to circadian timing 1750 IV. Conclusions 1752 Acknowledgements 1752 References 1752 SUMMARY: The circadian system comprises interlocking transcriptional-translational feedback loops that regulate gene expression and consequently modulate plant development and physiology. In order to maximize utility, the circadian system is entrained by changes in temperature and light, allowing endogenous rhythms to be synchronized with both daily and seasonal environmental change. Although a great deal of environmental information is decoded by a suite of photoreceptors, it is also becoming apparent that changes in cellular metabolism also contribute to circadian timing, through either the stimulation of metabolic pathways or the accumulation of metabolic intermediates as a consequence of environmental stress. As the source of many of these metabolic byproducts, mitochondria and chloroplasts have begun to be viewed as environmental sensors, and rapid advancement of this field is revealing the complex web of signalling pathways initiated by organelle perturbation. This review highlights recent advances in our understanding of how this metabolic regulation influences circadian timing.

Phototropins do not alter accumulation of evening-phased circadian transcripts under blue light.

The circadian system induces rhythmic variation in a suite of biochemical and physiological processes that serve to optimise plant growth in diel cycles. To be of greatest utility, these rhythmic behaviors are coordinated with regular environmental changes such as the rising and setting of the sun. Photoreceptors, along with metabolites produced during photosynthesis, act to synchronise the internal timing mechanism with lighting cues. We have recently shown that phototropins help maintain robust rhythms of photosynthetic operating efficiency (ϕPSII or Fq'/Fm') under blue light, although rhythmic accumulation of morning-phased circadian transcripts in the nucleus was unaffected. Here we report that evening-phased nuclear clock transcripts were also unaffected. We also observe that rhythms of nuclear clock transcript accumulation are maintained in phototropin mutant plants under a fluctuating lighting regime that induced a loss of Fq'/Fm' rhythms.

Phototropins maintain robust circadian oscillation of PSII operating efficiency under blue light.

The circadian system allows plants to coordinate metabolic and physiological functions with predictable environmental variables such as dusk and dawn. This endogenous oscillator is comprised of biochemical and transcriptional rhythms that are synchronized with a plant's surroundings via environmental signals, including light and temperature. We have used chlorophyll fluorescence techniques to describe circadian rhythms of PSII operating efficiency (Fq'/Fm') in the chloroplasts of Arabidopsis thaliana. These Fq'/Fm' oscillations appear to be influenced by transcriptional feedback loops previously described in the nucleus, and are induced by rhythmic changes in photochemical quenching over circadian time. Our work reveals that a family of blue photoreceptors, phototropins, maintain robust rhythms of Fq'/Fm' under constant blue light. As phototropins do not influence circadian gene expression in the nucleus our imaging methodology highlights differences between the modulation of circadian outputs in distinct subcellular compartments.

Analysis of cocoa flavanols and procyanidins (DP 1-10) in cocoa-containing ingredients and products by rapid resolution liquid chromatography: single-laboratory validation.

Recently, a multilaboratory validation (MLV) of AOAC Official Method 2012.24 for the determination of cocoa flavanols and procyanidins (CF-CP) in cocoa-based ingredients and products determined that the method was robust, reliable, and transferrable. Due to the complexity of the CF-CP molecules, this method required a run time exceeding 1 h to achieve acceptable separations. To address this issue, a rapid resolution normal phase LC method was developed, and a single-laboratory validation (SLV) study conducted. Flavanols and procyanidins with a degree of polymerization (DP) up to 10 were eluted in 15 min using a binary gradient applied to a diol stationary phase, detected using fluorescence detection, and reported as a total sum of DP 1-10. Quantification was achieved using (-)-epicatechin-based relative response factors for DP 2-10. Spike recovery samples and seven different types of cocoa-based samples were analyzed to evaluate the accuracy, precision, LOD, LOQ, and linearity of the method. The within-day precision of the reported content for the samples was 1.15-5.08%, and overall precision was 3.97-13.61%. Spike-recovery experiments demonstrated recoveries of over 98%. The results of this SLV were compared to those previously obtained in the MLV and found to be consistent. The translation to rapid resolution LC allowed for an 80% reduction in analysis time and solvent usage, while retaining the accuracy and reliability of the original method. The savings in both cost and time of this rapid method make it well-suited for routine laboratory use.

Determination of Flavanols and Procyanidins (DP 1-10) in Cocoa-Based Ingredients and Products by UHPLC: First Action 2013.03.

Single-laboratory validation data were reviewed by the Expert Review Panel (ERP) of the Stakeholder Panel on Strategic Food Analytical Methods at the AOAC Mid-Year Meeting, March 12-14, 2013, in Rockville, MD. The ERP determined that the data presented met established standard method performance requirements and adopted a method for determination of flavanols and procyanidins (DP 1-10) in cocoa-based ingredients and products by ultra-HPLC as AOAC Official First Action Method 2013.03 on March 14, 2013. The flavanols and procyanidins (DP 1-10) are eluted using a binary gradient (solvents A and B) consisting of 98 + 2 (v/v) acetonitrile-glacial acetic acid (A) and 95 + 3 + 2 (v/v/v) methanol-water-glacial acetic acid (B). The mobile phase is applied to a diol stationary phase. Detection occurs using fluorescence detection. Recovery of flavanols and procyanidins (DP 1-10) from both high- and low-fat matrixes was 98.4-99.8%. Precision was determined for seven different sample types (cocoa extract, cocoa nib, natural cocoa powder, cocoa liquor, alkalized cocoa powder, dark chocolate, and milk chocolate).

Unanticipated regulatory roles for Arabidopsis phytochromes revealed by null mutant analysis.

In view of the extensive literature on phytochrome mutants in the Ler accession of Arabidopsis, we sought to secure a phytochrome-null line in the same genetic background for comparative studies. Here we report the isolation and phenotypic characterization of phyABCDE quintuple and phyABDE quadruple mutants in the Ler background. Unlike earlier studies, these lines possess a functional allele of FT permitting measurements of photoperiod-dependent flowering behavior. Comparative studies of both classes of mutants establish that phytochromes are dispensable for completion of the Arabidopsis life cycle under red light, despite the lack of a transcriptomic response, and also indicate that phyC is nonfunctional in the absence of other phytochromes. Phytochrome-less plants can produce chlorophyll for photosynthesis under continuous red light, yet require elevated fluence rates for survival. Unexpectedly, our analyses reveal both light-dependent and -independent roles for phytochromes to regulate the Arabidopsis circadian clock. The rapid transition of these mutants from vegetative to reproductive growth, as well as their insensitivity to photoperiod, establish a dual role for phytochromes to arrest and to promote progression of plant development in response to the prevailing light environment.

A macrocyclic calpain inhibitor slows the development of inherited cortical cataracts in a sheep model.

PURPOSE: We used sheep with an autosomal dominant gene for cortical cataract as an animal model to evaluate novel macrocyclic calpain inhibitors with potential for the medical treatment of human cataract. METHODS: The macrocyclic aldehyde, CAT811, identified previously as a calpain inhibitor that prevents calcium-induced opacification in cultured sheep lenses, was tested for its ability to protect cytoskeletal proteins from calpain proteolysis. CAT811 and its alcohol analogue, CAT505, were formulated separately into ointments, and each was applied twice daily to the right eye of sheep with early cataracts for five months. Progress of cataracts in the sheep was determined by ophthalmologic examination and comparison with a matched sample of sheep treated similarly with ointment that did not contain the active ingredient. RESULTS: The novel macrocyclic aldehyde, CAT811, was able to inhibit calpain proteolysis of lens cytoskeletal proteins at micromolar concentrations. When applied topically to the eyes of sheep, CAT811 was able to slow cataract development by 27% in the initial three months of treatment (P < 0.05). Its alcohol analogue, CAT505, was not able to slow cataractogenesis significantly. CONCLUSIONS: The inherited sheep cataract provides a reproducible model of cortical cataract over a time scale of several months. The data reported here, using this model, demonstrated the potential of the macrocyclic calpain inhibitor, CAT811, to act as a therapeutic for treatment of cortical cataract.

New tripeptide-based macrocyclic calpain inhibitors formed by N-alkylation of histidine.

Two new series of 15-membered macrocyclic peptidomimetics, in which the P1 and P3 residues of the peptide backbone are linked by a bridge containing a 1,4-disubstituted 1H-imidazole, are reported. The structure with an aldehyde at the C-terminus and the imidazole at P3, i.e., 4c, shows significant inhibitory activity against calpain 2, with an IC(50) value of 238 nM. The macrocyclic aldehyde with the imidazole at the alternative P1 position, i.e., 5c, is significantly less active. The relative activities are linked to the ability of the component macrocycles to mimic a ß-strand geometry that is known to favor active-site binding. This ability is defined by conformational searches and docking studies with calpain.

Mutation of Arabidopsis spliceosomal timekeeper locus1 causes circadian clock defects.

The circadian clock plays a crucial role in coordinating plant metabolic and physiological functions with predictable environmental variables, such as dusk and dawn, while also modulating responses to biotic and abiotic challenges. Much of the initial characterization of the circadian system has focused on transcriptional initiation, but it is now apparent that considerable regulation is exerted after this key regulatory step. Transcript processing, protein stability, and cofactor availability have all been reported to influence circadian rhythms in a variety of species. We used a genetic screen to identify a mutation within a putative RNA binding protein (spliceosomal timekeeper locus1 [STIPL1]) that induces a long circadian period phenotype under constant conditions. STIPL1 is a homolog of the spliceosomal proteins TFP11 (Homo sapiens) and Ntr1p (Saccharomyces cerevisiae) involved in spliceosome disassembly. Analysis of general and alternative splicing using a high-resolution RT-PCR system revealed that mutation of this protein causes less efficient splicing of most but not all of the introns analyzed. In particular, the altered accumulation of circadian-associated transcripts may contribute to the observed mutant phenotype. Interestingly, mutation of a close homolog of STIPL1, STIP-LIKE2, does not cause a circadian phenotype, which suggests divergence in function between these family members. Our work highlights the importance of posttranscriptional control within the clock mechanism.

Method for the determination of catechin and epicatechin enantiomers in cocoa-based ingredients and products by high-performance liquid chromatography: single-laboratory validation.

A single-laboratory validation study was performed for an HPLC method to identify and quantify the flavanol enantiomers (+)- and (-)-epicatechin and (+)- and (-)-catechin in cocoa-based ingredients and products. These compounds were eluted isocratically with an ammonium acetate-methanol mobile phase applied to a modified beta-cyclodextrin chiral stationary phase and detected using fluorescence. Spike recovery experiments using appropriate matrix blanks, along with cocoa extract, cocoa powder, and dark chocolate, were used to evaluate accuracy, repeatability, specificity, LOD, LOQ, and linearity of the method as performed by a single analyst on multiple days. In all samples analyzed, (-)-epicatechin was the predominant flavanol and represented 68-91% of the total monomeric flavanols detected. For the cocoa-based products, within-day (intraday) precision for (-)-epicatechin was between 1.46-3.22%, for (+)-catechin between 3.66-6.90%, and for (-)-catechin between 1.69-6.89%; (+)-epicatechin was not detected in these samples. Recoveries for the three sample types investigated ranged from 82.2 to 102.1% at the 50% spiking level, 83.7 to 102.0% at the 100% spiking level, and 80.4 to 101.1% at the 200% spiking level. Based on performance results, this method may be suitable for routine laboratory use in analysis of cocoa-based ingredients and products.

Molecular modeling: a search for a calpain inhibitor as a new treatment for cataractogenesis.

Studies of 17 analoges of 3 (SJA6017) in an in silico calpain model are reconciled to measured IC(50) values against ovine calpain. The studies validate the potential of the "model" and criteria established for inhibition as a tool to select structures for synthesis to test as calpain inhibitors. Using this screening methodology of virtual libraries led us to synthesize several inhibitors including macrocycle 33, which in vitro sheep eye lens culture experiments showed to substantially slow opacification.

REVEILLE8 and PSEUDO-REPONSE REGULATOR5 form a negative feedback loop within the Arabidopsis circadian clock.

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.

JMJD5 Functions in concert with TOC1 in the arabidopsis circadian system.

The circadian clock modulates the expression of approximately one-third of Arabidopsis genes and as such plays a central role in the regulation of plant metabolism and responses to the environment. We have recently identified a novel component of the Arabidopsis circadian clock, JMJD5, based on its coexpression with TOC1, an evening-phased component of the molecular oscillator. We now examine the genetic interaction between TOC1 and JMJD5 in greater detail and demonstrate that toc1 is not epistatic to jmjd5, suggesting that these two proteins act in closely linked but parallel genetic pathways. The human homolog of JMJD5, KDM8, has been shown to have histone demethylation activity and is able to partially rescue the plant jmjd5 circadian phenotype. The potential role of JMJD5 as a histone demethylase within the circadian clock is discussed.

Jumonji domain protein JMJD5 functions in both the plant and human circadian systems.

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 fast-running 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.

N-heterocyclic dipeptide aldehyde calpain inhibitors.

A series of Val-Leu based peptidic aldehydes containing either a furan or thiophene at the N-terminus was prepared and assayed against ovine m-calpain. In general, potency is favoured by a 2-substituted (rather than 3-substituted) heterocycle, a thiophene rather than a furan, and a shorter chain length at the N-terminus. Molecular docking experiments provide some rationale for these observations.