AraLeTA: An Arabidopsis leaf expression atlas across diurnal and developmental scales.

Mature plant leaves are a composite of distinct cell types, including epidermal, mesophyll, and vascular cells. Notably, the proportion of these cells and the relative transcript concentrations within different cell types may change over time. While gene expression data at a single-cell level can provide cell-type-specific expression values, it is often too expensive to obtain these data for high-resolution time series. Although bulk RNA-seq can be performed in a high-resolution time series, RNA-seq using whole leaves measures average gene expression values across all cell types in each sample. In this study, we combined single-cell RNA-seq data with time-series data from whole leaves to assemble an atlas of cell-type-specific changes in gene expression over time for Arabidopsis (Arabidopsis thaliana). We inferred how the relative transcript concentrations of different cell types vary across diurnal and developmental timescales. Importantly, this analysis revealed 3 subgroups of mesophyll cells with distinct temporal profiles of expression. Finally, we developed tissue-specific gene networks that form a community resource: an Arabidopsis Leaf Time-dependent Atlas (AraLeTa). This allows users to extract gene networks that are confirmed by transcription factor-binding data and specific to certain cell types at certain times of day and at certain developmental stages. AraLeTa is available at https://regulatorynet.shinyapps.io/araleta/.

TIME FOR COFFEE regulates phytochrome A-mediated hypocotyl growth through dawn-phased signaling.

To enhance plant fitness under natural conditions, the circadian clock is synchronized and entrained by light via photoreceptors. In turn, the circadian clock exquisitely regulates the abundance and activity of photoreceptors via largely uncharacterized mechanisms. Here we show that the clock regulator TIME FOR COFFEE (TIC) controls the activity of the far-red light photoreceptor phytochrome A (phyA) at multiple levels in Arabidopsis thaliana. Null mutants of TIC displayed dramatically increased sensitivity to light irradiation with respect to hypocotyl growth, especially to far-red light. RNA-sequencing demonstrated that TIC and phyA play largely opposing roles in controlling light-regulated gene expression at dawn. Additionally, TIC physically interacts with the transcriptional repressor TOPLESS (TPL), which was associated with the significantly increased PHYA transcript levels in the tic-2 and tpl-1 mutants. Moreover, TIC interacts with phyA in the nucleus, thereby affecting phyA protein turnover and the formation of phyA nuclear speckles following light irradiation. Genetically, phyA was found to act downstream of TIC in regulating far red light-inhibited growth. Taken together, these findings indicate that TIC acts as a major negative regulator of phyA by integrating transcriptional and post-translational mechanisms at multiple levels.

A non-canonical function of Arabidopsis ERECTA proteins and a role of the SWI3B subunit of the SWI/SNF chromatin remodeling complex in gibberellin signaling.

The Arabidopsis ERECTA family (ERf) of leucine-rich repeat receptor-like kinases (LRR-RLKs) comprising ERECTA (ER), ERECTA-LIKE 1 (ERL1), and ERECTA-LIKE 2 (ERL2) controls epidermal patterning, inflorescence architecture, and stomata development and patterning. These proteins are reported to be plasma membrane associated. Here we show that the er/erl1/erl2 mutant exhibits impaired gibberellin (GA) biosynthesis and perception alongside broad transcriptional changes. The ERf kinase domains were found to localize to the nucleus where they interact with the SWI3B subunit of the SWI/SNF chromatin remodeling complex (CRCs). The er/erl1/erl2 mutant exhibits reduced SWI3B protein level and affected nucleosomal chromatin structure. Similar to swi3c and brm plants with inactivated subunits of SWI/SNF CRCs, it also does not accumulate DELLA RGA and GAI proteins. The ER kinase phosphorylates SWI3B in vitro, and the inactivation of all ERf proteins leads to the decreased phosphorylation of SWI3B protein in vivo. The identified correlation between DELLA overaccumulation and SWI3B proteasomal degradation, and the physical interaction of SWI3B with DELLA proteins indicate an important role of SWI3B-containing SWI/SNF CRCs in gibberellin signaling. Co-localization of ER and SWI3B on GID1 (GIBBERELLIN INSENSITIVE DWARF 1) DELLA target gene promoter regions and abolished SWI3B binding to GID1 promoters in er/erl1/erl2 plants supports the conclusion that ERf-SWI/SNF CRC interaction is important for transcriptional control of GA receptors. Thus, the involvement of ERf proteins in the transcriptional control of gene expression, and observed similar features for human HER2 (epidermal growth family receptor member), indicate an exciting target for further studies of evolutionarily conserved non-canonical functions of eukaryotic membrane receptors.

Reflections on Several Landmark Advances in Circadian Biology.

Circadian Biology intersects with diverse scientific domains, intricately woven into the fabric of organismal physiology and behavior. The rhythmic orchestration of life by the circadian clock serves as a focal point for researchers across disciplines. This retrospective examination delves into several of the scientific milestones that have fundamentally shaped our contemporary understanding of circadian rhythms. From deciphering the complexities of clock genes at a cellular level to exploring the nuances of coupled oscillators in whole organism responses to stimuli. The field has undergone significant evolution lately guided by genetics approaches. Our exploration here considers key moments in the circadian-research landscape, elucidating the trajectory of this discipline with a keen eye on scientific advancements and paradigm shifts.

Timely Questions Emerging in Chronobiology: The Circadian Clock Keeps on Ticking.

Chronobiology investigations have revealed much about cellular and physiological clockworks but we are far from having a complete mechanistic understanding of the physiological and ecological implications. Here we present some unresolved questions in circadian biology research as posed by the editorial staff and guest contributors to the Journal of Circadian Rhythms. This collection of ideas is not meant to be comprehensive but does reveal the breadth of our observations on emerging trends in chronobiology and circadian biology. It is amazing what could be achieved with various expected innovations in technologies, techniques, and mathematical tools that are being developed. We fully expect strengthening mechanistic work will be linked to health care and environmental understandings of circadian function. Now that most clock genes are known, linking these to physiological, metabolic, and developmental traits requires investigations from the single molecule to the terrestrial ecological scales. Real answers are expected for these questions over the next decade. Where are the circadian clocks at a cellular level? How are clocks coupled cellularly to generate organism level outcomes? How do communities of circadian organisms rhythmically interact with each other? In what way does the natural genetic variation in populations sculpt community behaviors? How will methods development for circadian research be used in disparate academic and commercial endeavors? These and other questions make it a very exciting time to be working as a chronobiologist.

Cellular localization of Arabidopsis EARLY FLOWERING3 is responsive to light quality.

Circadian clocks facilitate the coordination of physiological and developmental processes to changing daily and seasonal cycles. A hub for environmental signaling pathways in the Arabidopsis (Arabidopsis thaliana) circadian clock is the evening complex (EC), a protein complex composed of EARLY FLOWERING3 (ELF3), ELF4, and LUX ARRYTHMO (LUX). Formation of the EC depends on ELF3, a scaffold protein that recruits the other components of the EC and chromatin remodeling enzymes to repress gene expression. Regulating the cellular distribution of ELF3 is thus an important mechanism in controlling its activity. Here, we determined that the cellular and sub-nuclear localization of ELF3 is responsive to red (RL) and blue light and that these two wavelengths have apparently competitive effects on where in the cell ELF3 localizes. We further characterized the RL response, revealing that at least two RL pathways influence the cellular localization of ELF3. One of these depends on the RL photoreceptor phytochrome B (phyB), while the second is at least partially independent of phyB activity. Finally, we investigated how changes in the cellular localization of ELF3 are associated with repression of EC target-gene expression. Our analyses revealed a complex effect whereby ELF3 is required for controlling RL sensitivity of morning-phased genes, but not evening-phased genes. Together, our findings establish a previously unknown mechanism through which light signaling influences ELF3 activity.

Optimizing Flap Harvest in Auricular Reconstruction.

INTRODUCTION: The temporoparietal fascial (TPF) and occipital cranial fascial (OCP) flaps are the mainstay of implant coverage in alloplastic auricular reconstruction. Their optimal design is critical for elevating a robust flap that ultimately leads to favorable outcomes. MATERIALS AND METHODS: Sixteen TPF and OCP dissections were performed on 8 cadaveric specimens. Vascular anatomy and key landmarks were documented. The minimum flap size that incorporated ideal vasculature and would appropriately cover a porous polyethylene implant was measured. RESULTS: The minimum flap dimensions (length × width × base width) to cover a standard PPE auricular implants were on average 11×8.3×6.4 cm for TPF and 13.1×8.6×6.5 cm for OCP. The average axial length of the superficial temporal artery and occipital artery were 12.51 and 13.2 cm, respectively. An "occipital elbow" was located on average 8.2 cm posterior to the external acoustic canal. The postauricular fascia contained additional contributions from the occipital artery and mastoid emissary vein, which was located on average 5.9 cm posterior to the superficial temporal artery. CONCLUSIONS: This study highlights the anatomic features behind optimal TPF and OCP flap design for auricular reconstruction. Contributions to axial length and anatomic relationships of their primary arterial supply, significance of the occipital elbow as a reliable landmark for fascial dissection, and importance of the postauricular fascia and its vascular supply for flap viability are emphasized. Ultimately, the authors provide minimal dimensions for both TPF and OCP flaps to obtain adequate alloplastic implant coverage.

Contemporary application of microbubble technology in water treatment.

Microbubble (MB) technology constitutes a suite of promising low-cost technologies with potential applications in various sectors. Microbubbles (MBs) are tiny gas bubbles with diameters in the micrometre range of 10-100 µm. Along with their small size, they share special characteristics like slow buoyancy, large gas-liquid interfacial area and high mass-transfer efficiency. Initially, the review examines the key dissimilarities among the different types of microbubble generators (MBG) towards economic large-scale production of MBs. The applications of MBs to explore their effectiveness at different stages of wastewater treatment extending from aeration, separation/ flotation, ozonation, disinfection and other processes are investigated. A summary of the recent advances of MBs in real and synthetic wastewater treatment, existing research gaps, and limitations in upscaling of the technology, conclusion and future recommendations is detailed. A critical analysis of the energetics and treatment cost of combined approaches of MB technology with other advanced oxidation processes (AOPs) is carried out highlighting the potential applicability of hybrid technology in large-scale wastewater treatment.

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.

Temperature robustness in Arabidopsis circadian clock models is facilitated by repressive interactions, autoregulation, and three-node feedbacks.

The biological interactions underpinning the Arabidopsis circadian clock have been systematically uncovered and explored by biological experiments and mathematical models. This is captured by a series of published ordinary differential equation (ODE) models, which describe plant clock dynamics in response to light/dark conditions. However, understanding the role of temperature in resetting the clock (entrainment) and the mechanisms by which circadian rhythms maintain a near-24 h period over a range of temperatures (temperature compensation) is still unclear. Understanding entrainment and temperature compensation may elucidate the principles governing the structure of the circadian clock network. Here we explore the design principles of the Arabidopsis clock and its responses to changes in temperature. We analyse published clock models of Arabidopsis, spanning a range of complexity, and incorporate temperature-dependent dynamics into the parameters of translation rates in these models, to discern which regulatory patterns may best explain clock function and temperature compensation. We additionally construct three minimal clock models and explore what key features govern their rhythmicity and temperature robustness via a series of random parameterisations. Results show that the highly repressive interactions between the components of the plant clock, together with autoregulation patterns and three-node feedback loops, are associated with circadian function of the clock in general, and enhance its robustness to temperature variation in particular. However, because the networks governing clock function vary with time due to light and temperature conditions, we emphasise the importance of studying plant clock functionality in its entirety rather than as a set of discrete regulation patterns.

Diurnal patterns of growth and transient reserves of sink and source tissues are affected by cold nights in barley.

Barley is described to mostly use sucrose for night carbon requirements. To understand how the transient carbon is accumulated and utilized in response to cold, barley plants were grown in a combination of cold days and/or nights. Both daytime and night cold reduced growth. Sucrose was the main carbohydrate supplying growth at night, representing 50-60% of the carbon consumed. Under warm days and nights, starch was the second contributor with 26% and malate the third with 15%. Under cold nights, the contribution of starch was severely reduced, due to an inhibition of its synthesis, including under warm days, and malate was the second contributor to C requirements with 24-28% of the total amount of carbon consumed. We propose that malate plays a critical role as an alternative carbon source to sucrose and starch in barley. Hexoses, malate, and sucrose mobilization and starch accumulation were affected in barley elf3 clock mutants, suggesting a clock regulation of their metabolism, without affecting growth and photosynthesis however. Altogether, our data suggest that the mobilization of sucrose and malate and/or barley growth machinery are sensitive to cold.

Circadian oscillator proteins across the kingdoms of life: structural aspects.

Circadian oscillators are networks of biochemical feedback loops that generate 24-hour rhythms in organisms from bacteria to animals. These periodic rhythms result from a complex interplay among clock components that are specific to the organism, but share molecular mechanisms across kingdoms. A full understanding of these processes requires detailed knowledge, not only of the biochemical properties of clock proteins and their interactions, but also of the three-dimensional structure of clockwork components. Posttranslational modifications and protein-protein interactions have become a recent focus, in particular the complex interactions mediated by the phosphorylation of clock proteins and the formation of multimeric protein complexes that regulate clock genes at transcriptional and translational levels. This review covers the structural aspects of circadian oscillators, and serves as a primer for this exciting realm of structural biology.

Focusing on the nuclear and subnuclear dynamics of light and circadian signalling.

Circadian clocks provide organisms the ability to synchronize their internal physiological responses with the external environment. This process, termed entrainment, occurs through the perception of internal and external stimuli. As with other organisms, in plants, the perception of light is a critical for the entrainment and sustainment of circadian rhythms. Red, blue, far-red, and UV-B light are perceived by the oscillator through the activity of photoreceptors. Four classes of photoreceptors signal to the oscillator: phytochromes, cryptochromes, UVR8, and LOV-KELCH domain proteins. In most cases, these photoreceptors localize to the nucleus in response to light and can associate to subnuclear structures to initiate downstream signalling. In this review, we will highlight the recent advances made in understanding the mechanisms facilitating the nuclear and subnuclear localization of photoreceptors and the role these subnuclear bodies have in photoreceptor signalling, including to the oscillator. We will also highlight recent progress that has been made in understanding the regulation of the nuclear and subnuclear localization of components of the plant circadian clock.

Heat the Clock: Entrainment and Compensation in Arabidopsis Circadian Rhythms.

The circadian clock is a biological mechanism that permits some organisms to anticipate daily environmental variations. This clock generates biological rhythms, which can be reset by environmental cues such as cycles of light or temperature, a process known as entrainment. After entrainment, circadian rhythms typically persist with approximately 24 hours periodicity in free-running conditions, i.e. in the absence of environmental cues. Experimental evidence also shows that a free-running period close to 24 hours is maintained across a range of temperatures, a process known as temperature compensation. In the plant Arabidopsis, the effect of light on the circadian system has been widely studied and successfully modelled mathematically. However, the role of temperature in periodicity, and the relationship between entrainment and compensation, are not fully understood. Here we adapt recent models to incorporate temperature dependence by applying Arrhenius equations to the parameters of the models that characterize transcription, translation, and degradation rates. We show that the resulting models can exhibit thermal entrainment and temperature compensation, but that these phenomena emerge from physiologically different sets of processes. Further simulations combining thermal and photic forcing in more realistic scenarios clearly distinguish between the processes of entrainment and compensation, and reveal temperature compensation as an emergent property which can arise as a result of multiple temperature-dependent interactions. Our results consistently point to the thermal sensitivity of degradation rates as driving compensation and entrainment across a range of conditions.

Circadian rhythms are associated with shoot architecture in natural settings.

Circadian rhythms are key regulators of diverse biological processes under controlled settings. Yet, the phenotypic and fitness consequences of quantitative variation in circadian rhythms remain largely unexplored in the field. As with other pathways, phenotypic characterization of circadian outputs in the field may reveal novel clock functions. Across consecutive growing seasons, we test for associations between clock variation and flowering phenology, plant size, shoot architecture, and fruit set in clock mutants and segregating progenies of Arabidopsis thaliana expressing quantitative variation in circadian rhythms. Using structural equation modeling, we find that genotypic variation in circadian rhythms within a growing season is associated directly with branching, which in turn affects fruit production. Consistent with direct associations between the clock and branching in segregating progenies, cauline branch number is lower and rosette branch number higher in a short-period mutant relative to wild-type and long-period genotypes, independent of flowering time. Differences in branching arise from variation in meristem fate as well as leaf production rate before flowering and attendant increases in meristem number. Our results suggest that clock variation directly affects shoot architecture in the field, suggesting a novel clock function and means by which the clock affects performance.

Circadian rhythms are associated with variation in photosystem II function and photoprotective mechanisms.

The circadian clock regulates many aspects of leaf gas supply and biochemical demand for CO2 , and is hypothesized to improve plant performance. Yet the extent to which the clock may regulate the efficiency of photosystem II (PSII) and photoprotective mechanisms such as heat dissipation is less explored. Based on measurements of chlorophyll a fluorescence, we estimated the maximum efficiency of PSII in light (Fv'/Fm') and heat dissipation by nonphotochemical quenching (NPQ). We further dissected total NPQ into its main components, qE (pH-dependent quenching), qT (state-transition quenching), and qI (quenching related to photoinhibition), in clock mutant genotypes of Arabidopsis thaliana, the cognate wild-type genotypes, and a panel of recombinant inbred lines expressing quantitative variation in clock period. Compared with mutants with altered clock function, we observed that wild-type genotypes with clock period lengths of approximately 24 hr had both higher levels of Fv'/Fm', indicative of improved PSII function, and reduced NPQ, suggestive of lower stress on PSII light harvesting complexes. In the recombinant inbred lines, genetic variances were significant for Fv'/Fm' and all 3 components of NPQ, with qE explaining the greatest proportion of NPQ. Bivariate tests of association and structural equation models of hierarchical trait relationships showed that quantitative clock variation was empirically associated with Fv'/Fm' and NPQ, with qE mediating the relationship with gas exchange. The results demonstrate significant segregating variation for all photoprotective components, and suggest the adaptive significance of the clock may partly derive from its regulation of the light reactions of photosynthesis and of photoprotective mechanisms.

Cryptic dispersal of Cyanidiophytina (Rhodophyta) in non-acidic environments from Turkey.

Cyanidiophytina are a group of polyextremophilic red algae with a worldwide, but discontinuous colonization. They are restricted to widely dispersed hot springs, geothermal habitats, and also some human-altered environments. Cyanidiophytina are predominant where pH is prohibitive for the majority of eukaryotes (pH 0.5-3). Turkey is characterized by areas rich in volcanic activity separated by non-volcanic areas. Here we show that Cyanidiophycean populations are present in thermal baths located around Turkey on neutral/alkaline soils. All known genera and species within Cyanidiophytina were detected in Turkey, including Galdieria phlegrea, recorded up to now only in Italian Phlegrean Fields. By phylogenetic analyses, Turkish G. sulphuraria strains are monophyletic with Italian and Icelandic strains, and with Russian G. daedala strains. G. maxima from Turkey clustered with Icelandic, Kamchatka, and Japanese populations. The discovery of Cyanidiophytina in non-acidic Turkish soils raises new questions about the ecological boundaries of these extremophilic algae. This aids in the understanding of the dispersal abilities and distribution patterns of this ecologically and evolutionarily interesting group of algae.

Tissue-Specific MicroRNA Expression Patterns in Four Types of Kidney Disease.

MicroRNAs contribute to the development of kidney disease. Previous analyses of microRNA expression in human kidneys, however, were limited by tissue heterogeneity or the inclusion of only one pathologic type. In this study, we used laser-capture microdissection to obtain glomeruli and proximal tubules from 98 human needle kidney biopsy specimens for microRNA expression analysis using deep sequencing. We analyzed specimens from patients with diabetic nephropathy (DN), FSGS, IgA nephropathy (IgAN), membranoproliferative GN (MPGN) (n=19-23 for each disease), and a control group (n=14). Compared with control glomeruli, DN, FSGS, IgAN, and MPGN glomeruli exhibited differential expression of 18, 12, two, and 17 known microRNAs, respectively. The expression of several microRNAs also differed between disease conditions. Specifically, compared with control or FSGS glomeruli, IgAN glomeruli exhibited downregulated expression of hsa-miR-3182. Furthermore, in combination, the expression levels of hsa-miR-146a-5p and hsa-miR-30a-5p distinguished DN from all other conditions except IgAN. Compared with control proximal tubules, DN, FSGS, IgAN, and MPGN proximal tubules had differential expression of 13, 14, eight, and eight microRNAs, respectively, but expression of microRNAs did not differ significantly between the disease conditions. The abundance of several microRNAs correlated with indexes of renal function. Finally, we validated the differential glomerular expression of select microRNAs in a second cohort of patients with DN (n=19) and FSGS (n=21). In conclusion, we identified tissue-specific microRNA expression patterns associated with several kidney pathologies. The identified microRNAs could be developed as biomarkers of kidney diseases and might be involved in disease mechanisms.

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.