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.

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.

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 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.

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.

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.

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.

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.

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.

Domain swapping to assess the mechanistic basis of Arabidopsis phototropin 1 receptor kinase activation and endocytosis by blue light.

Phototropins (phot1 and phot2) are plasma membrane-associated receptor kinases that respond specifically to blue and UV wavelengths. In addition to a C-terminal Ser/Thr kinase domain, phototropins contain two N-terminal chromophore binding LOV domains that function as photoswitches to regulate a wide range of enzymatic activities in prokaryotes and eukaryotes. Through domain swapping, we show that the photochemical properties of Arabidopsis thaliana phot1 rely on interactions between LOV1 and LOV2, which are facilitated by their intervening linker sequence. Functional analysis of domain-swap proteins supports a mechanism whereby LOV2 acts as a dark-state repressor of phot1 activity both in vitro and in vivo. Moreover, we find a photoactive role for LOV1 in arresting chloroplast accumulation at high light intensities. Unlike LOV2, LOV1 cannot operate as a dark-state repressor, resulting in constitutive receptor autophosphorylation and accelerated internalization from the plasma membrane. Coexpression of active and inactive forms of phot1 demonstrates that autophosphorylation can occur intermolecularly, independent of LOV1, via light-dependent receptor dimerization in vivo. Indeed, transphosphorylation is sufficient to promote phot1 internalization through a clathrin-dependent endocytic pathway triggered primarily by phosphorylation of Ser-851 within the kinase activation loop. The mechanistic implications of these findings in regard to light-driven receptor activation and trafficking are discussed.

REVEILLE1, a Myb-like transcription factor, integrates the circadian clock and auxin pathways.

The circadian clock modulates expression of a large fraction of the Arabidopsis genome and affects many aspects of plant growth and development. We have discovered one way in which the circadian system regulates hormone signaling, identifying a node that links the clock and auxin networks. Auxin plays key roles in development and responses to environmental cues, in part through regulation of plant growth. We have characterized REVEILLE1 (RVE1), a Myb-like, clock-regulated transcription factor that is homologous to the central clock genes CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY). Despite this homology, inactivation of RVE1 does not affect circadian rhythmicity but instead causes a growth phenotype, indicating this factor is a clock output affecting plant development. CCA1 regulates growth via the bHLH transcription factors PHYTOCHROME INTERACTING FACTOR4 (PIF4) and PIF5, but RVE1 acts independently of these genes. RVE1 instead controls auxin levels, promoting free auxin production during the day but having no effect during the night. RVE1 positively regulates the expression of the auxin biosynthetic gene YUCCA8 (YUC8), providing a mechanism for its growth-promoting effects. RVE1 is therefore a node that connects two important signaling networks that coordinate plant growth with rhythmic changes in the environment.

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.

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.

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.

Synthesis of macrocyclic beta-strand templates by ring closing metathesis.

We report the synthesis of macrocycles 1-6 via ring closing metathesis of dienes 7-12. Addition of chlorodicyclohexylborane to thermal and microwave assisted RCM of dienes 8 and 9 markedly improves the yield. The geometric isomers of macrocycles 1-3 and 5 have been assigned using X-ray crystallography and NMR.

Effects of skeletal deformities on swimming performance and recovery from exhaustive exercise in triploid Atlantic salmon.

The occurrence of spinal deformity in aquaculture can be considerable, and a high rate of deformity has been suggested in triploid smolts in Tasmania. However, the physiological performance of fish with skeletal deformities has not been addressed. The swimming performance and oxygen consumption of triploid Atlantic salmon smolts with either a vertebral fusion (platyspondyly) or multifocal scoliosis were compared to normal (non-deformed) triploid smolts. Fish with vertebral fusion attained swim speeds similar to normal fish, whereas scoliotic fish were unable to attain comparable swim speeds. Routine and maximum oxygen consumption was higher for deformed fish compared with normal fish, translating into apparent increased routine metabolic scope in vertebral fusion fish, and equivocal scope in scoliotic fish compared with normal controls. Deformed fish developed a lower excess post-exercise oxygen consumption compared to non-deformed fish, suggesting they are either incapable of sustained anaerobic activity or possess an increased recovery capacity. These data suggest that skeletal deformity has differential effects on swimming performance depending upon the type of deformity but imposes a significant metabolic cost on salmon smolts.

Molecular modeling, synthesis, and biological evaluation of macrocyclic calpain inhibitors.

The design and elaboration of a series of macrocyclic templates that exhibit a propensity to adopt a beta-strand-like peptide-backbone conformation led to potent and selective inhibitors of calpain 2. Macrocycle 1 retarded calcium-induced opacification in an ovine-lens culture assay and is a lead compound for the development of a drug for cataract treatment. Cbz=carbobenzyloxy.

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.

Synthesis, biological evaluation and molecular modelling of N-heterocyclic dipeptide aldehydes as selective calpain inhibitors.

A series of N-heterocyclic dipeptide aldehydes 4-13 have been synthesised and evaluated as inhibitors of ovine calpain 1 (o-CAPN1) and ovine calpain 2 (o-CAPN2). 5-Formyl-pyrrole 9 (IC(50) values of 290 and 25nM against o-CAPN1 and o-CAPN2, respectively) was the most potent and selective o-CAPN2 inhibitor, displaying >11-fold selectivity. The amino acid sequences of o-CAPN1 and o-CAPN2 have been determined. Because of the lack of available structural information on the ovine calpains, in silico homology models of the active site cleft of o-CAPN1 and o-CAPN2 were developed based on human calpain 1 (h-CAPN1) X-ray crystal structure (PDB code 1ZCM). These models were used to rationalise the observed SAR for compounds 4-13 and the selectivity observed for 9. The o-CAPN2 selective inhibitor 9 (CAT0059) was assayed in an in vitro ovine lens culture system and shown to successfully protect the lens from calcium-induced opacification.

Evaluation of a novel calpain inhibitor as a treatment for cataract.

PURPOSE: The aim of this study is to evaluate the therapeutic potential of a newly synthesized calpain inhibitor, CAT0059, using a naturally occurring in vivo sheep cataract model. METHODS: The selectivity of CAT0059 was investigated by an in vitro protease assay. The efficacy of CAT0059 in preventing proteolysis of lens cytoskeletal proteins by calpain 2 was investigated using a lens-based cell-free method. The cytotoxicity and stability of CAT0059 in physiological conditions were examined using cultured sheep lenses. Protein binding of CAT0059 by ocular proteins was assessed and quantified by a modified high-performance liquid chromatography assay. CAT0059 was formulated in an eye drop solution and as an eye ointment. These were applied in vivo daily to one eye of the cataract lambs, over a 67- and 97-day trial period, respectively. The progression of cataracts in the treated and untreated eyes was assessed by an independent veterinary ophthalmologist using a slit-lamp microscope. RESULTS: In vitro assays revealed that CAT0059 was selective for cysteine proteases and also protected lens cytoskeletal proteins from degradation. CAT0059 was stable in physiological conditions and non-toxic to the lens. Only 15% of CAT0059 is bound to proteins in the aqueous humour but >90% bound to lens homogenate. The 67-day CAT0059 eye drop treatment was not effective in slowing the rate of cataract development. However, application of CAT0059 in an eye ointment initially slowed cataract development compared with the untreated eye. This effect was temporary. CONCLUSIONS: In vitro assays confirmed CAT0059 to be a potent calpain inhibitor. The two in vivo trials addressed the ability of CAT0059 to reach the lens and established its limitations as a therapeutic molecule for cataract treatment.