Plate-only open-door laminoplasty is associated with durable, significantly improved clinical outcomes and a very low reoperation rate for progressive spondylosis or adjacent segment disease: average 8-year follow-up.

BACKGROUND CONTEXT: Multilevel cervical myelopathy is a common cause of spinal cord dysfunction in adults. Surgical intervention via laminoplasty can provide satisfactory clinical outcomes by expansive decompression of the spinal cord. Traditional suture or bone graft techniques have been associated with insufficient fixation, leading to premature closure and subsequent neurological deterioration. In contrast, plated laminoplasty has been shown to provide stable fixation to maintain canal enlargement, but longer-term outcomes are lacking. PURPOSE: To evaluate longer-term clinical outcomes and reoperations associated with plate-only open-door laminoplasty. STUDY DESIGN: Retrospective review of prospectively collected data. PATIENT SAMPLE: Postoperative patients who underwent plate-only open door laminoplasty with minimum 5-year follow up. OUTCOME MEASURES: modified Japanese Orthopaedic Association (mJOA) score, Neck Disability Index (NDI), and 12-item Short Form Health Survey (SF-12). METHODS: All patients at a single academic institution who underwent plate-only open-door cervical laminoplasty from 9/1/2006 to 9/1/2016 were identified to ensure minimum 5 year follow up. Clinical outcomes included the modified Japanese Orthopaedic Association (mJOA) score, the Neck Disability Index (NDI), and the 12-item Short Form Health Survey (SF-12). The occurrence of any repeat operations on the cervical spine was evaluated, as well as its cause. The study team attempted to contact all eligible patients to achieve at least 5 years postoperative follow-up. Pairwise t tests were performed to compare clinical outcomes at preoperative, 6 months, 1-year, and final postoperative follow-up with an α level of 0.05. RESULTS: A total of 774 met the initial inclusion criteria, of which 157 were included in the study (20.3%). Most common reasons for exclusion included inability to reach after 3 attempts (49.48%), inactive phone numbers (20.28%), and patient declining (3.49%). Included patients had an average age of 60.66±10.63 and an average follow-up time of 8.37±2.57 years (minimum 5 years). mJOA scores (preoperative 11.59±2.16) improved significantly at 6-months (14.57±2.07, p<.001), 1-year (15.19±1.95, p<.001), and final follow-up (14.59±2.63, p<.001). NDI (preoperative 33.89±18.54) improved significantly at 6 months (27.89±19.72, p=.03), 1-year (25.96±19.79, p=.01) and final follow-up (17.88±17.17, p<.001). SF-12 MCS (preoperative 44.73) improved significantly at 6 months (52.01, p=.001), 1-year (51.62, p=.008), and final follow-up (52.32, p<.001). No patient underwent reoperations for plate failure or canal closure with recurrent stenosis. Reoperations for progressive spondylosis during the follow up period were rare and occurred in only three patients for new onset radiculopathy (1.9%) and two patients for myelopathy (1.3%) at an average of 3.2 years postoperative. There were no reoperations performed for adjacent segment disease. CONCLUSIONS: At a minimum of 5 years and an average of more than 8 years postoperative, laminoplasty was associated with significant and sustained improvements in mJOA, NDI, and SF-12 MCS. Importantly, no patients underwent revision surgery for plate failure or recurrent canal closure. Reoperations for new onset radiculopathy and myelopathy were also very rare over the 8-year average follow-up period, with no reoperations for adjacent segment disease. Plate-only laminoplasty is a durable means of treating multilevel myelopathy with excellent longer-term outcomes and a very low risk of reoperation, either for premature closure or the inevitable spondylotic changes that occur over time in patients with similar baseline characteristics to the study population.

Compartmentation of photosynthesis gene expression in C4 maize depends on time of day.

Compared with the ancestral C3 state, C4 photosynthesis occurs at higher rates with improved water and nitrogen use efficiencies. In both C3 and C4 plants, rates of photosynthesis increase with light intensity and are maximal around midday. We determined that in the absence of light or temperature fluctuations, photosynthesis in maize (Zea mays) peaks in the middle of the subjective photoperiod. To investigate the molecular processes associated with these temporal changes, we performed RNA sequencing of maize mesophyll and bundle sheath strands over a 24-h time course. Preferential expression of C4 cycle genes in these cell types was strongest between 6 and 10 h after dawn when rates of photosynthesis were highest. For the bundle sheath, DNA motif enrichment and gene coexpression analyses suggested members of the DNA binding with one finger (DOF) and MADS (MINICHROMOSOME MAINTENANCE FACTOR 1/AGAMOUS/DEFICIENS/Serum Response Factor)-domain transcription factor families mediate diurnal fluctuations in C4 gene expression, while trans-activation assays in planta confirmed their ability to activate promoter fragments from bundle sheath expressed genes. The work thus identifies transcriptional regulators and peaks in cell-specific C4 gene expression coincident with maximum rates of photosynthesis in the maize leaf at midday.

Wheat EARLY FLOWERING 3 affects heading date without disrupting circadian oscillations.

Plant breeders have indirectly selected for variation at circadian-associated loci in many of the world's major crops, when breeding to increase yield and improve crop performance. Using an eight-parent Multiparent Advanced Generation Inter-Cross (MAGIC) population, we investigated how variation in circadian clock-associated genes contributes to the regulation of heading date in UK and European winter wheat (Triticum aestivum) varieties. We identified homoeologues of EARLY FLOWERING 3 (ELF3) as candidates for the Earliness per se (Eps) D1 and B1 loci under field conditions. We then confirmed a single-nucleotide polymorphism within the coding region of TaELF3-B1 as a candidate polymorphism underlying the Eps-B1 locus. We found that a reported deletion at the Eps-D1 locus encompassing TaELF3-D1 is, instead, an allele that lies within an introgression region containing an inversion relative to the Chinese Spring D genome. Using Triticum turgidum cv. Kronos carrying loss-of-function alleles of TtELF3, we showed that ELF3 regulates heading, with loss of a single ELF3 homoeologue sufficient to alter heading date. These studies demonstrated that ELF3 forms part of the circadian oscillator; however, the loss of all homoeologues was required to affect circadian rhythms. Similarly, loss of functional LUX ARRHYTHMO (LUX) in T. aestivum, an orthologue of a protein partner of Arabidopsis (Arabidopsis thaliana) ELF3, severely disrupted circadian rhythms. ELF3 and LUX transcripts are not co-expressed at dusk, suggesting that the structure of the wheat circadian oscillator might differ from that of Arabidopsis. Our demonstration that alterations to ELF3 homoeologues can affect heading date separately from effects on the circadian oscillator suggests a role for ELF3 in cereal photoperiodic responses that could be selected for without pleiotropic deleterious alterations to circadian rhythms.

Effect of Obesity on Patient-Perceived Outcomes After First Metatarsophalangeal Joint Arthrodesis.

BACKGROUND: While first metatarsophalangeal joint (MTPJ) arthrodesis is a common and effective procedure, there is a paucity of studies examining obesity's effect on outcomes of 1st MTPJ arthrodesis. This study's purpose was to evaluate patient-reported outcomes following 1st MTPJ arthrodesis in obese versus non-obese patients. METHODS: A retrospective cohort study of 94 patients undergoing first MTPJ fusion over the age of 18 with a diagnosis of hallux valgus or hallux rigidus was performed. Surgical and postoperative outcomes were examined preoperatively and at 6 and 12 months follow-up via Visual Analog Pain scale (VAS), and Short Form 36 (SF-36) surveys, and data were stratified into 2 patient groups: BMI < 30 (n = 62, mean age 63.9 ± 9.1 and ≥ 30 (n = 32, mean age 61.9 ± 8.4). RESULTS: Average overall VAS and SF-36 physical component scores improved significantly at 6 months (P < .001, .006) and 1 year postoperative visits (P < .001, .007) with no differences in survey scores, outcomes, or complications between weight groups. CONCLUSION: Our study showed first MTPJ fusion improves short-term pain and physical quality-of-life in arthritic obese and non-obese patients without differences in nonunion, complications, or patient-reported measures. LEVEL OF EVIDENCE: Level III, Prognostic, Case-Control Study.

Overexpression of the WAPO-A1 gene increases the number of spikelets per spike in bread wheat.

Two homoeologous QTLs for number of spikelets per spike (SPS) were mapped on chromosomes 7AL and 7BL using two wheat MAGIC populations. Sets of lines contrasting for the QTL on 7AL were developed which allowed for the validation and fine mapping of the 7AL QTL and for the identification of a previously described candidate gene, WHEAT ORTHOLOG OF APO1 (WAPO1). Using transgenic overexpression in both a low and a high SPS line, we provide a functional validation for the role of this gene in determining SPS also in hexaploid wheat. We show that the expression levels of this gene positively correlate with SPS in multiple MAGIC founder lines under field conditions as well as in transgenic lines grown in the greenhouse. This work highlights the potential use of WAPO1 in hexaploid wheat for further yield increases. The impact of WAPO1 and SPS on yield depends on other genetic and environmental factors, hence, will require a finely balanced expression level to avoid the development of detrimental pleiotropic phenotypes.

Circadian entrainment in Arabidopsis.

Circadian clocks coordinate physiology and development as an adaption to the oscillating day/night cycle caused by the rotation of Earth on its axis and the changing length of day and night away from the equator caused by orbiting the sun. Circadian clocks confer advantages by entraining to rhythmic environmental cycles to ensure that internal events within the plant occur at the correct time with respect to the cyclic external environment. Advances in determining the structure of circadian oscillators and the pathways that allow them to respond to light, temperature, and metabolic signals have begun to provide a mechanistic insight to the process of entrainment in Arabidopsis (Arabidopsis thaliana). We describe the concepts of entrainment and how it occurs. It is likely that a thorough mechanistic understanding of the genetic and physiological basis of circadian entrainment will provide opportunities for crop improvement.

ZEITLUPE Promotes ABA-Induced Stomatal Closure in Arabidopsis and Populus.

Plants balance water availability with gas exchange and photosynthesis by controlling stomatal aperture. This control is regulated in part by the circadian clock, but it remains unclear how signalling pathways of daily rhythms are integrated into stress responses. The serine/threonine protein kinase OPEN STOMATA 1 (OST1) contributes to the regulation of stomatal closure via activation of S-type anion channels. OST1 also mediates gene regulation in response to ABA/drought stress. We show that ZEITLUPE (ZTL), a blue light photoreceptor and clock component, also regulates ABA-induced stomatal closure in Arabidopsis thaliana, establishing a link between clock and ABA-signalling pathways. ZTL sustains expression of OST1 and ABA-signalling genes. Stomatal closure in response to ABA is reduced in ztl mutants, which maintain wider stomatal apertures and show higher rates of gas exchange and water loss than wild-type plants. Detached rosette leaf assays revealed a stronger water loss phenotype in ztl-3, ost1-3 double mutants, indicating that ZTL and OST1 contributed synergistically to the control of stomatal aperture. Experimental studies of Populus sp., revealed that ZTL regulated the circadian clock and stomata, indicating ZTL function was similar in these trees and Arabidopsis. PSEUDO-RESPONSE REGULATOR 5 (PRR5), a known target of ZTL, affects ABA-induced responses, including stomatal regulation. Like ZTL, PRR5 interacted physically with OST1 and contributed to the integration of ABA responses with circadian clock signalling. This suggests a novel mechanism whereby the PRR proteins-which are expressed from dawn to dusk-interact with OST1 to mediate ABA-dependent plant responses to reduce water loss in time of stress.

Treatment modalities and outcomes following acetabular fractures in the elderly: a systematic review.

PURPOSE: The treatment of geriatric acetabular fractures remains controversial. Treatment options include nonoperative management, open reduction and internal fixation (ORIF), total hip arthroplasty (THA) with or without internal fixation, and closed reduction with percutaneous pinning (CRPP). There is currently no consensus on the optimal treatment strategy for geriatric patients with acetabular fractures. The purpose of this study is to compare adverse event rates, functional and radiographic outcomes, and intraoperative results between the various treatment modalities in order to help guide surgical decision making. METHODS: We performed a systematic review (registration number CRD42019124624) of observational and comparative studies including patients aged ≥ 55 with acetabular fractures. RESULTS: Thirty-eight studies including 3,928 patients with a mean age of 72.6 years (range 55-99 years) and a mean follow-up duration of 29.4 months met our eligibility criteria. The pooled mortality rate of all patients was 21.6% (95% confidence interval [CI] 20.9-22.4%) with a mean time to mortality of 12.6 months, and the pooled non-fatal complication rate was 24.7% (95% CI 23.9-25.5%). Patients treated with ORIF had a significantly higher non-fatal complication rate than those treated with ORIF + THA, THA alone, CRPP, or nonoperative management (odds ratios [ORs] 1.87, 2.24, 2.15, and 4.48, respectively; p < 0.01). Patients that underwent ORIF were significantly less likely to undergo subsequent THA than these treated with CRPP (OR 0.49, 95% CI 0.32-0.77) but were more likely to require THA than patients treated nonoperatively (OR 6.81, 95% CI 4.63-10.02). CONCLUSION: Elderly patients with acetabular fractures tend to have favorable functional outcomes but suffer from high rates of mortality and complications. In patients treated with internal or percutaneous fixation, there was a high rate of conversion to THA. When determining surgical treatment in this population, THA alone or concurrent with ORIF should be considered given the significantly lower rate of non-fatal complications and similar mortality rate. Nonoperative management remains a viable option and was associated with the lowest non-fatal complication rate. LEVEL OF EVIDENCE: This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Player age and initial helmet contact among American football players.

OBJECTIVE: Concussions and chronic traumatic encephalopathy (CTE) related to professional football has received much attention within emergency care and sports medicine. Research suggests that some of this may be due to a greater likelihood of initial helmet contact (IHC), however this association has not been studied across all age groups. This study aims to investigate the association between player age and IHC in American football. METHODS: Retrospective review of championship games between 2016 and 2018 at 6 levels of amateur tackle football as well as the National Football League (NFL). Trained raters classified plays as IHC using pre-specified criteria. A priori power analysis established the requisite impacts needed to establish non-inferiority of the incidence rate of IHC across the levels of play. RESULTS: Thirty-seven games representing 2912 hits were rated. The overall incidence of IHC was 16% across all groups, ranging from 12.6% to 18.9%. All but 2 of the non-NFL divisions had a statistically reduced risk of IHC when compared with the NFL, with relative risk ratios ranging from 0.55-0.92. IHC initiated by defensive participants were twice as high as offensive participants (RR 2.04, p < 0.01) while 6% [95% CI 5.4-7.2] of all hits were helmet-on-helmet contact. CONCLUSIONS: There is a high rate of IHC with a lower relative risk of IHC at most levels of play compared to the NFL. Further research is necessary to determine the impact of IHC; the high rates across all age groups suggests an important role for education and prevention.

Chronoculture, harnessing the circadian clock to improve crop yield and sustainability.

Human health is dependent on a plentiful and nutritious supply of food, primarily derived from crop plants. Rhythmic supply of light as a result of the day and night cycle led to the evolution of circadian clocks that modulate most plant physiology, photosynthesis, metabolism, and development. To regulate crop traits and adaptation, breeders have indirectly selected for variation at circadian genes. The pervasive impact of the circadian system on crops suggests that future food production might be improved by modifying circadian rhythms, engineering the timing of transgene expression, and applying agricultural treatments at the most effective time of day. We describe the applied research required to take advantage of circadian biology in agriculture to increase production and reduce inputs.

Magnesium maintains the length of the circadian period in Arabidopsis.

The circadian clock coordinates the physiological responses of a biological system to day and night rhythms through complex loops of transcriptional/translational regulation. It can respond to external stimuli and adjust generated circadian oscillations accordingly to maintain an endogenous period close to 24 h. However, the interaction between nutritional status and circadian rhythms in plants is poorly understood. Magnesium (Mg) is essential for numerous biological processes in plants, and its homeostasis is crucial to maintain optimal development and growth. Magnesium deficiency in young Arabidopsis thaliana seedlings increased the period of circadian oscillations of the CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1) promoter (pCCA1:LUC) activity and dampened their amplitude under constant light in a dose-dependent manner. Although the circadian period increase caused by Mg deficiency was light dependent, it did not depend on active photosynthesis. Mathematical modeling of the Mg input into the circadian clock reproduced the experimental increase of the circadian period and suggested that Mg is likely to affect global transcription/translation levels rather than a single component of the circadian oscillator. Upon addition of a low dose of cycloheximide to perturb translation, the circadian period increased further under Mg deficiency, which was rescued when sufficient Mg was supplied, supporting the model's prediction. These findings suggest that sufficient Mg supply is required to support proper timekeeping in plants.

Arabidopsis sirtuins and poly(ADP-ribose) polymerases regulate gene expression in the day but do not affect circadian rhythms.

Nicotinamide-adenine dinucleotide (NAD) is involved in redox homeostasis and acts as a substrate for NADases, including poly(ADP-ribose) polymerases (PARPs) that add poly(ADP-ribose) polymers to proteins and DNA, and sirtuins that deacetylate proteins. Nicotinamide, a by-product of NADases increases circadian period in both plants and animals. In mammals, the effect of nicotinamide on circadian period might be mediated by the PARPs and sirtuins because they directly bind to core circadian oscillator genes. We have investigated whether PARPs and sirtuins contribute to the regulation of the circadian oscillator in Arabidopsis. We found no evidence that PARPs and sirtuins regulate the circadian oscillator of Arabidopsis or are involved in the response to nicotinamide. RNA-seq analysis indicated that PARPs regulate the expression of only a few genes, including FLOWERING LOCUS C. However, we found profound effects of reduced sirtuin 1 expression on gene expression during the day but not at night, and an embryo lethal phenotype in knockouts. Our results demonstrate that PARPs and sirtuins are not associated with NAD regulation of the circadian oscillator and that sirtuin 1 is associated with daytime regulation of gene expression.

Standardized Platelet Rich Plasma Injections for Osteoarthritis of the Knee.

Objective Platelet-rich plasma (PRP) and adipose-derived stem cells (ADSC) injections are non-surgical treatments for knee osteoarthritis (OA). The purpose of this study is to assess the effectiveness of serial PRP with or without ADSC injections in the treatment of refractory OA of the knee.  Design Patients who failed to achieve pain relief with conventional non-surgical treatments, with Kellgren-Lawrence grade 3 or 4 knee OA, were recruited from a private outpatient clinic. Over 67 patients were elected to receive serial PRP injections and 22 patients were elected to receive an ADSC+PRP injection. These patients completed Western Ontario and McMaster Universities Arthritis Index (WOMAC) surveys prior to each treatment and at follow-up appointments. These surveys were retrospectively reviewed to assess changes in functional status and pain over time. Results Twenty-nine patients from the PRP group and eight patients in the ADSC+PRP group had adequate follow-up for inclusion in the analysis. The PRP group had an improvement in WOMAC scores by 34.30%, 60.2%, and 58.5% for patients reporting at 1-3, 4-6, and >6 months of follow-up. The ADSC+PRP group experienced an improvement of 51% at an average of 4.66 months of follow-up. Conclusions Serial PRP injections and a single ADSC+PRP injection yield improved and sustained functional outcome scores for patients with severe, refractory OA of the knee. Future studies should consider consistent orthobiologic preparation protocols to ensure reproducibility.

Calcium Binding by Arabinogalactan Polysaccharides Is Important for Normal Plant Development.

Arabinogalactan proteins (AGPs) are a family of plant extracellular proteoglycans involved in many physiological events. AGPs are often anchored to the extracellular side of the plasma membrane and are highly glycosylated with arabinogalactan (AG) polysaccharides, but the molecular function of this glycosylation remains largely unknown. The ß-linked glucuronic acid (GlcA) residues in AG polysaccharides have been shown in vitro to bind to calcium in a pH-dependent manner. Here, we used Arabidopsis (Arabidopsis thaliana) mutants in four AG ß-glucuronyltransferases (GlcAT14A, -B, -D, and -E) to understand the role of glucuronidation of AG. AG isolated from glcat14 triple mutants had a strong reduction in glucuronidation. AG from a glcat14a/b/d triple mutant had lower calcium binding capacity in vitro than AG from wild-type plants. Some mutants had multiple developmental defects such as reduced trichome branching. glcat14a/b/e triple mutant plants had severely limited seedling growth and were sterile, and the propagation of calcium waves was perturbed in roots. Several of the developmental phenotypes were suppressed by increasing the calcium concentration in the growth medium. Our results show that AG glucuronidation is crucial for multiple developmental processes in plants and suggest that a function of AGPs might be to bind and release cell-surface apoplastic calcium.

Differential Effects of Day/Night Cues and the Circadian Clock on the Barley Transcriptome.

The circadian clock is a complex transcriptional network that regulates gene expression in anticipation of the day/night cycle and controls agronomic traits in plants. However, in crops, how the internal clock and day/night cues affect the transcriptome remains poorly understood. We analyzed the diel and circadian leaf transcriptomes in the barley (Hordeum vulgare) cultivar 'Bowman' and derived introgression lines harboring mutations in EARLY FLOWERING3 (ELF3), LUX ARRHYTHMO1 (LUX1), and EARLY MATURITY7 (EAM7). The elf3 and lux1 mutants exhibited abolished circadian transcriptome oscillations under constant conditions, whereas eam7 maintained oscillations of ≈30% of the circadian transcriptome. However, day/night cues fully restored transcript oscillations in all three mutants and thus compensated for a disrupted oscillator in the arrhythmic barley clock mutants elf3 and lux1 Nevertheless, elf3, but not lux1, affected the phase of the diel oscillating transcriptome and thus the integration of external cues into the clock. Using dynamical modeling, we predicted a structure of the barley circadian oscillator and interactions of its individual components with day/night cues. Our findings provide a valuable resource for exploring the function and output targets of the circadian clock and for further investigations into the diel and circadian control of the barley transcriptome.

Recent advances in understanding regulation of the Arabidopsis circadian clock by local cellular environment.

Circadian clocks have evolved to synchronise an organism's physiology with the environmental rhythms driven by the Earth's rotation on its axis. Over the past two decades, many of the genetic components of the Arabidopsis thaliana circadian oscillator have been identified. The interactions between these components have been formulized into mathematical models that describe the transcriptional translational feedback loops of the oscillator. More recently, focus has turned to the regulation and functions of the circadian clock. These studies have shown that the system dynamically responds to environmental signals and small molecules. We describe advances that have been made in discovering the cellular mechanisms by which signals regulate the circadian oscillator of Arabidopsis in the context of tissue-specific regulation.

Combining GAL4 GFP enhancer trap with split luciferase to measure spatiotemporal promoter activity in Arabidopsis.

In multicellular organisms different types of tissues have distinct gene expression profiles associated with specific function or structure of the cell. Quantification of gene expression in whole organs or whole organisms can give misleading information about levels or dynamics of expression in specific cell types. Tissue- or cell-specific analysis of gene expression has potential to enhance our understanding of gene regulation and interactions of cell signalling networks. The Arabidopsis circadian oscillator is a gene network which orchestrates rhythmic expression across the day/night cycle. There is heterogeneity between cell and tissue types of the composition and behaviour of the oscillator. In order to better understand the spatial and temporal patterns of gene expression, flexible tools are required. By combining a Gateway®-compatible split luciferase construct with a GAL4 GFP enhancer trap system, we describe a tissue-specific split luciferase assay for non-invasive detection of spatiotemporal gene expression in Arabidopsis. We demonstrate the utility of this enhancer trap-compatible split luciferase assay (ETSLA) system to investigate tissue-specific dynamics of circadian gene expression. We confirm spatial heterogeneity of circadian gene expression in Arabidopsis leaves and describe the resources available to investigate any gene of interest.

Circadian gating of dark-induced increases in chloroplast- and cytosolic-free calcium in Arabidopsis.

Changes in the spatiotemporal concentration of free Ca2+ ([Ca2+ ]) in different organelles of the cell contribute to responses of plants to physiological and environmental stimuli. One example are [Ca2+ ] increases in the stroma of chloroplasts during light-to-dark transitions; however, the function and mechanisms responsible are unknown, in part because there is a disagreement in the literature concerning whether corresponding dark-induced changes in cytosolic [Ca2+ ] ([Ca2+ ]cyt ) can be detected. We have measured changes in [Ca2+ ]cyt upon darkness in addition to the already known dark-induced increases in [Ca2+ ]stroma in the aerial part of the Arabidopsis thaliana plant. These [Ca2+ ]cyt transients depend on the photoperiod and time of day, peaking at anticipated dusk, and are superimposed on daily 24 h oscillations in [Ca2+ ]cyt . We also find that the magnitude of the dark-induced increases in Ca2+ in both the cytosol and chloroplasts are gated by the nuclear circadian oscillator. The modulation of the magnitude of dark-induced increases in [Ca2+ ]stroma and [Ca2+ ]cyt by transcriptional regulators in the nucleus that are part of the circadian oscillator demonstrates a new role for the circadian system in subcellular Ca2+ signalling, in addition to its role in driving circadian oscillations of [Ca2+ ] in the cytosol and chloroplasts.

TTG1 proteins regulate circadian activity as well as epidermal cell fate and pigmentation.

The Arabidopsis genome contains three genes encoding proteins of the TRANSPARENT TESTA GLABRA 1 (TTG1) WD-repeat (WDR) subfamily. TTG1 is a known regulator of epidermal cell differentiation and pigment production, while LIGHT-REGULATED WD1 and LIGHT-REGULATED WD2 are known regulators of the circadian clock. Here, we discovered a new central role for TTG1 WDR proteins as regulators of the circadian system, as evidenced by the lack of detectable circadian rhythms in a triple lwd1 lwd2 ttg1 mutant. This shows that there has been subfunctionalization via protein changes within the angiosperms, with some TTG1 WDR proteins developing a stronger role in circadian clock regulation while losing the protein characteristics essential for pigment production and epidermal cell specification, and others weakening their ability to drive circadian clock regulation. Our work shows that even where proteins are very conserved, small changes can drive big functional differences.