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More than a decade has passed since the conclusion of the Minnesota tobacco trial and the signing of the Master Settlement Agreement (MSA) by 46 US State Attorneys General and the US tobacco industry. The Minnesota settlement exposed the tobacco industry's long history of deceptive marketing, advertising, and research and ultimately forced the industry to change its business practices. The provisions for public document disclosure that were included in the Minnesota settlement and the MSA have resulted in the release of approximately 70 million pages of documents and nearly 20,000 other media materials. No comparable dynamic, voluminous, and contemporaneous document archive exists. Only a few single events in the history of public health have had as dramatic an effect on tobacco control as the public release of the tobacco industry's previously secret internal documents. This review highlights the genesis of the release of these documents, the history of the document depositories created by the Minnesota settlement, the scientific and policy output based on the documents, and the use of the documents in furthering global public health strategies. [ABSTRACT FROM AUTHOR]

Unionid mussels deposit growth rings (annuli) within the shell, which can be used to estimate age and growth. Thin-sectioning is a common technique for counting annuli, wherein a cross-section of a shell valve is taken and evaluated by multiple readers. Correctly identifying annuli can be challenging because ambiguous annuli can bias growth estimates. Staining with calcein, a fluorescent chemical, is a technique that has been used with marine and freshwater species to improve accuracy of growth estimates. This method chelates calcium, causing a permanent mark that fluoresces under ultraviolet light. Calcein has seen limited testing on unionid mussels so it remains unclear if this method has adverse effects on survival and growth. We evaluated calcein against 2 concentrations (125 mg L -1 and 250 mg L -1 ) at 2 exposure times (12 and 24 h) on Cyclonaias pustulosa, a common North American unionid. Survivorship remained above 80% 6 months post-immersion. Mark quality and retention for 250 mg L -1 were high for both 12- and 24-h immersions, although historical annuli were not highlighted. These findings corroborate studies indicating calcein immersion is generally safe and effective in juveniles and adults and suggest it may be useful in validating new growth., (© 2024. The Author(s).)

This study compared early weaning (EW; 150 days) with conventional weaning (CW; 240 days) in Nellore young bulls, evaluating performance, carcass characteristics, and meat quality. A total of 74 non-castrated male calves were divided into two weaning strategies: EW ( n = 37) and CW ( n = 37). During the growth phase, which lasted 454 ± 14 d for EW calves and 359 ± 16 d for CW calves, animals received a protein-energy supplement at a ratio of 5 g per kg of body weight while grazing Brachiaria brizantha cv. Marandu. The animals were managed for an 87d finishing phase in three collective feedlot pens, with a 3-week adaptation protocol, starting with corn silage to a concentrate ratio of 55:45 and reaching a ratio of 30:70 in the final diet. Body weight, average daily gain (ADG), dry matter intake (DMI), feed efficiency (FE), carcass characteristics, and meat quality were evaluated. The EW group was approximately 44 kg lighter than the CW at the time of conventional weaning ( p < 0.001). However, this weight difference did not influence ADG, DMI, and FE in the finishing phase. No significant differences were observed in carcass characteristics such as yield percentage, loin area, subcutaneous fat thickness, and meat quality, except for the weight of primal cuts, which was greater in the CW group ( p < 0.001). Thus, although calves weaned early are lighter throughout subsequent production phases than those weaned conventionally, performance, efficiency, carcass yield, and meat quality are not affected.

Paternal programming is the concept that the environmental signals from the sire's experiences leading up to mating can alter semen and ultimately affect the phenotype of resulting offspring. Potential mechanisms carrying the paternal effects to offspring can be associated with epigenetic signatures (DNA methylation, histone modification and non-coding RNAs), oxidative stress, cytokines, and the seminal microbiome. Several opportunities exist for sperm/semen to be influenced during development; these opportunities are within the testicle, the epididymis, or accessory sex glands. Epigenetic signatures of sperm can be impacted during the pre-natal and pre-pubertal periods, during sexual maturity and with advancing sire age. Sperm are susceptible to alterations as dictated by their developmental stage at the time of the perturbation, and sperm and seminal plasma likely have both dependent and independent effects on offspring. Research using rodent models has revealed that many factors including over/under nutrition, dietary fat, protein, and ingredient composition (e.g., macro- or micronutrients), stress, exercise, and exposure to drugs, alcohol, and endocrine disruptors all elicit paternal programming responses that are evident in offspring phenotype. Research using livestock species has also revealed that sire age, fertility level, plane of nutrition, and heat stress can induce alterations in the epigenetic, oxidative stress, cytokine, and microbiome profiles of sperm and/or seminal plasma. In addition, recent findings in pigs, sheep, and cattle have indicated programming effects in blastocysts post-fertilization with some continuing into post-natal life of the offspring. Our research group is focused on understanding the effects of common management scenarios of plane of nutrition and growth rates in bulls and rams on mechanisms resulting in paternal programming and subsequent offspring outcomes. Understanding the implication of paternal programming is imperative as short-term feeding and management decisions have the potential to impact productivity and profitability of our herds for generations to come., Competing Interests: Conflicts of interest: The authors have no conflict of interest to declare.

Freshwater mussels are particularly sensitive to hydrologic changes, including streamflow and temperature, resulting in global decline. The Devils River in south-central Texas harbors the endangered freshwater mussel Popenaias popeii (Unionidae; Texas hornshell). There is concern that water withdrawals from the underlying aquifer may be negatively impacting this species. To assess this risk, we evaluated upper thermal tolerances (LT05 and LT50) of larvae (glochidia) and juveniles from two sites. After being acclimated to 27 °C, glochidia were subjected to five experimental temperatures (30, 32, 34, 36, and 38 °C) and non-acclimated control (20 °C) for 12-h and 24-h while juveniles were subjected to three experimental temperatures (30, 32, and 36 °C) and non-acclimated control (20 °C) for 96-h. We overlaid tolerance estimates against in situ water temperature and discharge data to evaluate thermal exceedances. Additionally, we reviewed upper thermal tolerances of P. popeii's presumed host fish (Carpiodes carpio, Cyprinellas lutrensis, and Moxostoma congestum) and their congeners. Stream temperatures only occasionally exceeded mussel LT05/50 and fish CLMax/LTMax, likely due to the Devils River's large spring input, highlighting the importance of protecting spring flows. We provide a practical framework for assessing hydrological needs of aquatic ectotherms, including the parasite-host relationship, which can be used to optimize environmental management., (© 2023. The Author(s).)

We aimed to evaluate the effects of early weaning on the productive and reproductive characteristics of Nelore heifer progeny. Ninety-five calves from primiparous (PRI) and multiparous (MUL) dams were assigned to one of two weaning strategies; 1) early weaning at 150 d (149 ± 1.97) of age (EW; n = 16 from PRI and 31 from MUL); or 2) conventional weaning at 240 d (247 ± 2.41) of age (CW; 16 from PRI and 32 from MUL). All heifers received ~5 g/kg of body weight (BW) of creep-feed as fed from 90 d of age until weaning. After weaning, each group of heifers was transferred to a Brachiaria spp. paddock and received 5 g/kg of BW of a protein-energy supplement until 12 mo of age. Then, heifers were confined and fed a diet with a ratio of 79:31 (corn silage: concentrate) for 4 mo, during which they were submitted to a hormonal protocol to induce puberty and timed artificial insemination (TAI). Reproductive tract score (RTS, 1 to 5 scale: 1 being infantile and 5 being cyclic) and endometrial thickness were determined at 12 mo of age, rump fat thickness (RFT), and BW every 28 ± 4 d through the breeding season, and plasma concentrations of IGF-I were evaluated at 12, 14, and 16 mo. At 15.6 mo of age heifers were submitted to a P4/E2 protocol for TAI at day 0 (D0), and a second TAI was performed at D22 in nonpregnant heifers. Ultrasound was used to determine the presence of corpus luteum on D10 and dominant follicle (DF) diameter and blood perfusion on D2 and D0. Data were analyzed using SAS by ANOVA or logistic regression. Though heifers from EW were lighter (P < 0.05) than CW at postweaning time points and CW presented a greater (P = 0.002) RFT than EW heifers from 11 to 15 mo, weaning strategy did not affect (P > 0.1) body condition score at TAI. Concentrations of IGF-I did not differ (P > 0.1) between heifers weaned at 150 and 240 d. The proportion of pubertal heifers, endometrium tone and thickness, and RTS at 16 mo did not differ (P > 0.1) between EW and CW groups. The diameter of DF on D2 and D0 and follicular blood perfusion on D0 were greater (P < 0.05) for heifers in the CW group than EW group, but P/AI at first and second TAI did not differ (P > 0.1) between groups. In conclusion, early weaning in Nelore heifers moderately reduces postweaning growth but does not affect puberty and reproductive performance before the breeding season when submitted to confinement., (© The Author(s) 2023. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

This study aimed to evaluate the effects of early weaning (EW) on body composition, hormone concentrations and metabolites, and reproductive performance of Nelore cows in the subsequent breeding season (BS). Suckled cows that became pregnant by timed-AI (TAI) in the 2020-BS were exposed in 2021 to early weaning at 150 d (27 primiparous [PRI] and 74 multiparous [MUL]) or conventional weaning (CW) at 240 d postpartum (30 PRI and 77 MUL). Body weight (BW) and body condition score (BCS) were determined at 2020-BS, EW, CW, prepartum, and 2021-BS. Blood samples were collected at EW, CW, prepartum (54.75 ± 0.56 d prepartum), and 2021-TAI and assayed for insulin-like growth factor-1 (IGF-I), non-esterified fatty acid (NEFA), and β-hydroxybutyrate (BHB) concentrations. In 2021-BS, cows were exposed to a P4/E2-based protocol for TAI at day 0 (D0), and a second TAI was performed at D22 in females detected with luteolysis (D20) by Doppler ultrasound. The presence of corpus luteum (CL) on D10, estrous expression, and dominant follicle (DF) diameter, and blood perfusion (BP) on D2 and D0 were determined. Data were analyzed by ANOVA or logistic regression of SAS as a 2 × 2 factorial with main factors of parity (PRI or MUL) and weaning strategy (EW or CW). An interaction of parity and weaning strategy was not observed (P > 0.1), but the weight (kg) and BCS were greater (P < 0.05) in MUL cows at the five timepoints, and EW cows were heavier than CW at the moment of CW (541 vs. 493 kg; and 5.3 vs. 4.3), prepartum (551 vs. 506 kg; and 5.2 vs. 4.4) and 2021-BS (475 vs. 450 kg; and 4.5 vs. 3.7). Plasma urea concentration at 2021-BS was greater (P = 0.01) for PRI than for MUL. A parity-by-time interaction was observed (P ≤ 0.05) for concentrations of IGF-I, NEFA, and BHB. PRI cows had greater (P ≤ 0.05) concentrations of IGF-I at EW and greater (P ≤ 0.05) prepartum concentrations of NEFA and BHB than MUL cows. The proportion of cows with CL at D10 was not affected (P > 0.1) by weaning but was greater (P < 0.05) in MUL than in PRI cows (40.4 vs. 15.7%). The diameter of DF and proportion of BP on D0 were greater (P < 0.05) in EW cows than in CW cows. The pregnancy rate (P/AI, %) at the first TAI was greater (P < 0.05) in EW cows (60% vs. 45%), whereas no difference (P > 0.1) was observed at the second TAI. Cumulative P/AI (first and second TAIs) was greater (P < 0.05) in EW cows (81% vs. 63%). In conclusion, weaning at 150 d in Nelore cattle is a strategy to successfully recover the parous cow's body condition and to improve pregnancy success in the next BS, regardless of the cow's parity order., (© The Author(s) 2023. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: The daily cycling of plant physiological processes is speculated to arise from the coordinated rhythms of gene expression. However, the dynamics of diurnal 3D genome architecture and their potential functions underlying the rhythmic gene expression remain unclear., Results: Here, we reveal the genome-wide rhythmic occupancy of RNA polymerase II (RNAPII), which precedes mRNA accumulation by approximately 2 h. Rhythmic RNAPII binding dynamically correlates with RNAPII-mediated chromatin architecture remodeling at the genomic level of chromatin interactions, spatial clusters, and chromatin connectivity maps, which are associated with the circadian rhythm of gene expression. Rhythmically expressed genes within the same peak phases of expression are preferentially tethered by RNAPII for coordinated transcription. RNAPII-associated chromatin spatial clusters (CSCs) show high plasticity during the circadian cycle, and rhythmically expressed genes in the morning phase and non-rhythmically expressed genes in the evening phase tend to be enriched in RNAPII-associated CSCs to orchestrate expression. Core circadian clock genes are associated with RNAPII-mediated highly connected chromatin connectivity networks in the morning in contrast to the scattered, sporadic spatial chromatin connectivity in the evening; this indicates that they are transcribed within physical proximity to each other during the AM circadian window and are located in discrete "transcriptional factory" foci in the evening, linking chromatin architecture to coordinated transcription outputs., Conclusion: Our findings uncover fundamental diurnal genome folding principles in plants and reveal a distinct higher-order chromosome organization that is crucial for coordinating diurnal dynamics of transcriptional regulation., (© 2022. The Author(s).)

The first descriptions of circadian rhythms were of the rhythmic leaf movements of plants. Rhythmic leaf movements offer a sensitive, noninvasive, nondestructive, and non-transgenic assay of plant circadian rhythms that can be readily automated, greatly facilitating genetic studies. Rhythmic leaf movement is particularly useful for the assessment of standing variation in clock function and can be readily applied to a diverse array of dicotyledonous plants, including both wild species and domesticated crops., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Population genomics has significantly increased our ability to make inferences about microevolutionary processes and demographic histories, which have the potential to improve protection and recovery of imperiled species. Freshwater mussels (Bivalvia: Unionida) represent one of the most imperiled groups of organisms globally. Despite systemic decline of mussel abundance and diversity, studies evaluating spatiotemporal changes in distribution, demographic histories, and ecological factors that threaten long-term persistence of imperiled species remain lacking. In this study, we use genotype-by-sequencing (GBS) and mitochondrial sequence data (mtDNA) to define conservation units (CUs) for two highly imperiled freshwater mussel species, Potamilus amphichaenus and Potamilus streckersoni . We then synthesize our molecular findings with details from field collections spanning from 1901 to 2019 to further elucidate distributional trends, contemporary status, and other factors that may be contributing to population declines for our focal species. We collected GBS and mtDNA data for individuals of P. amphichaenus and P. streckersoni from freshwater mussel collections in the Brazos, Neches, Sabine, and Trinity drainages ranging from 2012 to 2019. Molecular analyses resolved disputing number of genetic clusters within P. amphichaenus and P. streckersoni ; however, we find defensible support for four CUs, each corresponding to an independent river basin. Evaluations of historical and recent occurrence data illuminated a generally increasing trend of occurrence in each of the four CUs, which were correlated with recent increases in sampling effort. Taken together, these findings suggest that P. amphichaenus and P. streckersoni are likely rare throughout their respective ranges. Because of this, the establishment of CUs will facilitate evidence-based recovery planning and ensure potential captive propagation and translocation efforts are beneficial. Our synthesis represents a case study for conservation genomic assessments in freshwater mussels and provides a model for future studies aimed at recovery planning for these highly imperiled organisms., Competing Interests: None declared., (© 2021 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

During plant domestication and improvement, farmers select for alleles present in wild species that improve performance in new selective environments associated with cultivation and use. The selected alleles become enriched and other alleles depleted in elite cultivars. One important aspect of crop improvement is expansion of the geographic area suitable for cultivation; this frequently includes growth at higher or lower latitudes, requiring the plant to adapt to novel photoperiodic environments. Many crops exhibit photoperiodic control of flowering and altered photoperiodic sensitivity is commonly required for optimal performance at novel latitudes. Alleles of a number of circadian clock genes have been selected for their effects on photoperiodic flowering in multiple crops. The circadian clock coordinates many additional aspects of plant growth, metabolism and physiology, including responses to abiotic and biotic stresses. Many of these clock-regulated processes contribute to plant performance. Examples of selection for altered clock function in tomato demonstrate that with domestication, the phasing of the clock is delayed with respect to the light-dark cycle and the period is lengthened; this modified clock is associated with increased chlorophyll content in long days. These and other data suggest the circadian clock is an attractive target during breeding for crop improvement.

The globally important crop Brassica rapa, a close relative of Arabidopsis, is an excellent system for modeling our current knowledge of plant growth on a morphologically diverse crop. The long history of B. rapa domestication across Asia and Europe provides a unique collection of locally adapted varieties that span large climatic regions with various abiotic and biotic stress-tolerance traits. This diverse gene pool provides a rich source of targets with the potential for manipulation toward the enhancement of productivity of crops both within and outside the Brassicaceae. To expand the genetic resources available to study natural variation in B. rapa, we constructed an Advanced Intercross Recombinant Inbred Line (AI-RIL) population using B. rapa subsp. trilocularis (Yellow Sarson) R500 and the B. rapa subsp. parachinensis (Cai Xin) variety L58. Our current understanding of genomic structure variation across crops suggests that a single reference genome is insufficient for capturing the genetic diversity within a species. To complement this AI-RIL population and current and future B. rapa genomic resources, we generated a de novo genome assembly of the B. rapa subsp. trilocularis (Yellow Sarson) variety R500, the maternal parent of the AI-RIL population. The genetic map for the R500 x L58 population generated using this de novo genome was used to map Quantitative Trait Loci (QTL) for seed coat color and revealed the improved mapping resolution afforded by this new assembly., Competing Interests: The authors declare no conflict of interest associated with the work described in this manuscript., (© 2020 The Authors. Plant Direct published by American Society of Plant Biologists, Society for Experimental Biology and John Wiley & Sons Ltd.)

Environmental variation along an elevational gradient can yield phenotypic differentiation resulting from varying selection pressures on plant traits related to seasonal responses. Thus, genetic clines can evolve in a suite of traits, including the circadian clock, that drives daily cycling in varied traits and that shares its genetic background with adaptation to seasonality. We used populations of annual Mimulus laciniatus from different elevations in the Sierra Nevada in California to explore among-population differentiation in the circadian clock, flowering responses to photoperiod, and phenological traits (days to cotyledon emergence, days to flowering, and days to seed ripening) in controlled common-garden conditions. Further, we examined correlations of these traits with environmental variables related to temperature and precipitation. We observed that the circadian period in leaf movement was differentiated among populations sampled within about 100 km, with population means varying by 1.6 h. Significant local genetic variation occurred within 2 populations in which circadian period among families varied by up to 1.8 h. Replicated treatments with variable ecologically relevant photoperiods revealed marked population differentiation in critical day length for flowering that ranged from 11.0 to 14.1 h, corresponding to the time period between late February and mid-May in the wild. Flowering time varied among populations in a 14-h photoperiod. Regardless of this substantial population-level diversity, obvious linear clinality in trait variability across elevations could not be determined based on our genotypic sample; it is possible that more complex spatial patterns of variation arise in complex terrains such as those in the Sierra Nevada. Moreover, we did not find statistically significant bivariate correlations between population means of different traits. Our research contributes to the understanding of genetic variation in the circadian clock and in seasonal responses in natural populations, highlighting the need for more comprehensive investigations on the association between the clock and other adaptive traits in plants.

An important challenge of crop improvement strategies is assigning function to paralogs in polyploid crops. Here we describe the circadian transcriptome in the polyploid crop Brassica rapa . Strikingly, almost three-quarters of the expressed genes exhibited circadian rhythmicity. Genetic redundancy resulting from whole genome duplication is thought to facilitate evolutionary change through sub- and neo-functionalization among paralogous gene pairs. We observed genome-wide expansion of the circadian expression phase among retained paralogous pairs. Using gene regulatory network models, we compared transcription factor targets between B. rapa and Arabidopsis circadian networks to reveal evidence for divergence between B. rapa paralogs that may be driven in part by variation in conserved non-coding sequences (CNS). Additionally, differential drought response among retained paralogous pairs suggests further functional diversification. These findings support the rapid expansion and divergence of the transcriptional network in a polyploid crop and offer a new approach for assessing paralog activity at the transcript level., Competing Interests: KG, RS, SZ, PL, TM, CM No competing interests declared, (© 2020, Greenham et al.)

Major geological processes have shaped biogeographical patterns of riverine biota. The Edwards Plateau of central Texas, USA, exhibits unique aquatic communities and endemism, including several species of freshwater mussels. Lampsilis bracteata (Gould, 1855) is endemic to the Edwards Plateau region; however, its phylogenetic relationship with other species in the Gulf coastal rivers and Mississippi River basin is unknown. We evaluated phylogenetic relationships, shell morphologies and soft anatomy characters of L. bracteata and a closely related congener, Lampsilis hydiana (Lea, 1838) throughout their ranges. Our results showed the presence of an undescribed species: Lampsilis bergmanni sp.n. Lampsilis bracteata and L. bergmanni sp.n. share similar shell morphologies and soft anatomy characters; however, they are genetically distinct. Geological processes, such as faulting and sea-level changes during the Miocene to Pliocene, are likely to have facilitated diversification of Lampsilis species, resulting in isolation of L. bracteata on the Edwards Plateau and diversification between L. bergmanni sp.n. and L. hydiana. We conclude that L. bracteata range is restricted to the Colorado River basin, whereas L. bergmanni sp.n. occurs only in upstream reaches of the Guadalupe River basin. Conservation actions are warranted for both species due to their restricted distributions and potential anthropogenic threats., (© The Willi Hennig Society 2019.)

The circadian clock is known to regulate plant innate immunity but the underlying mechanism of this regulation remains largely unclear. We show here that mutations in the core clock component LUX ARRHYTHMO (LUX) disrupt circadian regulation of stomata under free running and Pseudomonas syringae challenge conditions as well as defense signaling mediated by SA and JA, leading to compromised disease resistance. RNA-seq analysis reveals that both clock- and defense-related genes are regulated by LUX. LUX binds to clock gene promoters that have not been shown before, expanding the clock gene networks that require LUX function. LUX also binds to the promoters of EDS1 and JAZ5, likely acting through these genes to affect SA- and JA-signaling. We further show that JA signaling reciprocally affects clock activity. Thus, our data support crosstalk between the circadian clock and plant innate immunity and imply an important role of LUX in this process.

It has been nearly 300 years since the first scientific demonstration of a self-sustaining circadian clock in plants. It has become clear that plants are richly rhythmic, and many aspects of plant biology, including photosynthetic light harvesting and carbon assimilation, resistance to abiotic stresses, pathogens, and pests, photoperiodic flower induction, petal movement, and floral fragrance emission, exhibit circadian rhythmicity in one or more plant species. Much experimental effort, primarily, but not exclusively in Arabidopsis thaliana , has been expended to characterize and understand the plant circadian oscillator, which has been revealed to be a highly complex network of interlocked transcriptional feedback loops. In addition, the plant circadian oscillator has employed a panoply of post-transcriptional regulatory mechanisms, including alternative splicing, adjustable rates of translation, and regulated protein activity and stability. This review focuses on our present understanding of the regulatory network that comprises the plant circadian oscillator. The complexity of this oscillatory network facilitates the maintenance of robust rhythmicity in response to environmental extremes and permits nuanced control of multiple clock outputs. Consistent with this view, the clock is emerging as a target of domestication and presents multiple targets for targeted breeding to improve crop performance.

In plants, seasonal inputs such as photoperiod and temperature modulate the plant's internal genetic program to regulate the timing of the developmental transition from vegetative to reproductive growth. This regulation of the floral transition involves chromatin remodeling, including covalent modification of histones. Here, we report that HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE 15 (HOS15), a WD40 repeat protein, associates with a histone deacetylase complex to repress transcription of the GIGANTEA ( GI )-mediated photoperiodic flowering pathway in Arabidopsis ( Arabidopsis thaliana ). Loss of function of HOS15 confers early flowering under long-day conditions because elevated GI expression. LUX ARRHYTHMO (LUX), a DNA binding transcription factor and component of the Evening Complex (EC), is important for the binding of HOS15 to the GI promoter. In wild type, HOS15 associates with the EC components LUX, EARLY FLOWERING 3 (ELF3), and ELF4 and the histone deacetylase HDA9 at the GI promoter, resulting in histone deacetylation and reduced GI expression. In the hos15-2 mutant, the levels of histone acetylation are elevated at the GI promoter, resulting in increased GI expression. Our data suggest that the HOS15-EC-HDA9 histone-modifying complex regulates photoperiodic flowering via the transcriptional repression of GI ., (© 2019 American Society of Plant Biologists. All rights reserved.)

The circadian clock coordinates the daily cyclic rhythm of numerous biological processes by regulating a large portion of the transcriptome. In animals, the circadian clock is involved in aging and senescence, and circadian disruption by mutations in clock genes frequently accelerates aging. Conversely, aging alters circadian rhythmicity, which causes age-associated physiological alterations. However, interactions between the circadian clock and aging have been rarely studied in plants. Here, we investigated potential roles for the circadian clock in the regulation of leaf senescence in plants. Members of the evening complex in Arabidopsis circadian clock, EARLY FLOWERING 3 (ELF3), EARLY FLOWERING 4 (ELF4), and LUX ARRHYTHMO (LUX), as well as the morning component PSEUDO-RESPONSE REGULATOR 9 (PRR9), affect both age-dependent and dark-induced leaf senescence. The circadian clock regulates the expression of several senescence-related transcription factors. In particular, PRR9 binds directly to the promoter of the positive aging regulator ORESARA1 ( ORE1 ) gene to promote its expression. PRR9 also represses miR164 , a posttranscriptional repressor of ORE1 Consistently, genetic analysis revealed that delayed leaf senescence of a prr9 mutant was rescued by ORE1 overexpression. Thus, PRR9, a core circadian component, is a key regulator of leaf senescence via positive regulation of ORE1 through a feed-forward pathway involving posttranscriptional regulation by miR164 and direct transcriptional regulation. Our results indicate that, in plants, the circadian clock and leaf senescence are intimately interwoven as are the clock and aging in animals., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

This article comments on: Entrainment of Arabidopsis roots to the light:dark cycle by light piping., (© 2018 John Wiley & Sons Ltd.)

Competing Interests: The authors declare no conflict of interest.

Correct identification of sex is an important component of wildlife management because changes in sex ratios can affect population viability. Identification of sex often relies on external morphology, which can be biased by intermediate or nondistinctive morphotypes and observer experience. For unionid mussels, research has demonstrated that species misidentification is common but less attention has been given to the reliability of sex identification. To evaluate whether this is an issue, we surveyed 117 researchers on their ability to correctly identify sex of Lampsilis teres (Yellow Sandshell), a wide ranging, sexually dimorphic species. Personal background information of each observer was analyzed to identify factors that may contribute to misidentification of sex. We found that median misidentification rates were ~20% across males and females and that observers falsely identified the number of female specimens more often (~23%) than males (~10%). Misidentification rates were partially explained by geographic region of prior mussel experience and where observers learned how to identify mussels, but there remained substantial variation among observers after controlling for these factors. We also used three morphometric methods (traditional, geometric, and Fourier) to investigate whether sex could be more correctly identified statistically and found that misidentification rates for the geometric and Fourier methods (which characterize shape) were less than 5% (on average 7% and 2% for females and males, respectively). Our results show that misidentification of sex is likely common for mussels if based solely on external morphology, which raises general questions, regardless of taxonomic group, about its reliability for conservation efforts.

The previously published version of this Article contained errors in Figure 5. In panel c, the second and fourth blot images were incorrectly labeled 'α-Myc' and should have been labelled 'α-HA'. These errors have been corrected in both the PDF and HTML versions of the Article.

The ability to perform laboratory testing near the patient and with smaller blood volumes would benefit patients and physicians alike. We describe our design of a miniaturized clinical laboratory system with three components: a hardware platform (ie, the miniLab) that performs preanalytical and analytical processing steps using miniaturized sample manipulation and detection modules, an assay-configurable cartridge that provides consumable materials and assay reagents, and a server that communicates bidirectionally with the miniLab to manage assay-specific protocols and analyze, store, and report results (i.e., the virtual analyzer). The miniLab can detect analytes in blood using multiple methods, including molecular diagnostics, immunoassays, clinical chemistry, and hematology. Analytical performance results show that our qualitative Zika virus assay has a limit of detection of 55 genomic copies/ml. For our anti-herpes simplex virus type 2 immunoglobulin G, lipid panel, and lymphocyte subset panel assays, the miniLab has low imprecision, and method comparison results agree well with those from the United States Food and Drug Administration-cleared devices. With its small footprint and versatility, the miniLab has the potential to provide testing of a range of analytes in decentralized locations.

In Arabidopsis thaliana, CONSTANS (CO) plays an essential role in the regulation of photoperiodic flowering under long-day conditions. CO protein is stable only in the afternoon of long days, when it induces the expression of FLOWERING LOCUS T (FT), which promotes flowering. The blue-light photoreceptor FLAVIN-BINDING, KELCH REPEAT, F-BOX1 (FKF1) interacts with CO and stabilizes it by an unknown mechanism. Here, we provide genetic and biochemical evidence that FKF1 inhibits CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1)-dependent CO degradation. Light-activated FKF1 has no apparent effect on COP1 stability but can interact with and negatively regulate COP1. We show that FKF1 can inhibit COP1 homo-dimerization. Mutation of the coiled-coil domain in COP1, which prevents dimer formation, impairs COP1 function in coordinating flowering time. Based on these results, we propose a model whereby the light- and day length-dependent interaction between FKF1 and COP1 controls CO stability to regulate flowering time.

The dynamics of local climates make development of agricultural strategies challenging. Yield improvement has progressed slowly, especially in drought-prone regions where annual crop production suffers from episodic aridity. Underlying drought responses are circadian and diel control of gene expression that regulate daily variations in metabolic and physiological pathways. To identify transcriptomic changes that occur in the crop Brassica rapa during initial perception of drought, we applied a co-expression network approach to associate rhythmic gene expression changes with physiological responses. Coupled analysis of transcriptome and physiological parameters over a two-day time course in control and drought-stressed plants provided temporal resolution necessary for correlation of network modules with dynamic changes in stomatal conductance, photosynthetic rate, and photosystem II efficiency. This approach enabled the identification of drought-responsive genes based on their differential rhythmic expression profiles in well-watered versus droughted networks and provided new insights into the dynamic physiological changes that occur during drought.

Many living organisms on Earth have evolved the ability to integrate environmental and internal signals to determine time and thereafter adjust appropriately their metabolism, physiology, and behavior. The circadian clock is the endogenous timekeeper critical for multiple biological processes in many organisms. A growing body of evidence supports the importance of the circadian clock for plant health. Plants activate timed defense with various strategies to anticipate daily attacks of pathogens and pests and to modulate responses to specific invaders in a time-of-day-dependent manner (gating). Pathogen infection is also known to reciprocally modulate clock activity. Such a cross talk likely reflects the adaptive nature of plants to coordinate limited resources for growth, development, and defense. This review summarizes recent progress in circadian regulation of plant innate immunity with a focus on the molecular events linking the circadian clock and defense. More and better knowledge of clock-defense cross talk could help to improve disease resistance and productivity in economically important crops.

The increasing demand for improved agricultural production will require more efficient breeding for traits that maintain yield under heterogeneous environments. The internal circadian oscillator is essential for perceiving and coordinating environmental cues such as day length, temperature, and abiotic stress responses within physiological processes. To investigate the contribution of the circadian clock to local adaptability, we have analyzed circadian period by leaf movement in natural populations of Mimulus guttatus and domesticated cultivars of Glycine max. We detected consistent variation in circadian period along a latitudinal gradient in annual populations of the wild plant and the selectively bred crop, and this provides novel evidence of natural and artificial selection for circadian performance. These findings provide new support that the circadian clock acts as a central regulator of plant adaptability and further highlight the potential of applying circadian clock gene variation to marker-assisted breeding programs in crops.

Circadian clocks have evolved independently in all three domains of life, and fitness benefits of a functional clock have been demonstrated in experimental genotypes in controlled conditions. Still, little is known about genetic variation in the clock and its fitness consequences in natural populations from heterogeneous environments. Using Wyoming populations of the Arabidopsis relative Boechera stricta as our study system, we demonstrate that genetic variation in the clock can occur at multiple levels: means of circadian period among populations sampled at different elevations differed by less than 1 h, but means among families sampled within populations varied by as much as 3.5 h. Growth traits also varied among and within populations. Within the population with the most circadian variation, we observed evidence for a positive correlation between period and growth and a negative correlation between period and root-to-shoot ratio. We then tested whether performance tradeoffs existed among families of this population across simulated seasonal settings. Growth rankings of families were similar across seasonal environments, but for root-to-shoot ratio, genotype × environment interactions contributed significantly to total variation. Therefore, further experiments are needed to identify evolutionary mechanisms that preserve substantial quantitative genetic diversity in the clock in this and other species., (© 2016 John Wiley & Sons Ltd.)

Crop selection often leads to dramatic morphological diversification, in which allocation to the harvestable component increases. Shifts in allocation are predicted to impact (as well as rely on) physiological traits; yet, little is known about the evolution of gas exchange and related anatomical features during crop diversification. In Brassica rapa, we tested for physiological differentiation among three crop morphotypes (leaf, turnip, and oilseed) and for correlated evolution of circadian, gas exchange, and phenological traits. We also examined internal and surficial leaf anatomical features and biochemical limits to photosynthesis. Crop types differed in gas exchange; oilseed varieties had higher net carbon assimilation and stomatal conductance relative to vegetable types. Phylogenetically independent contrasts indicated correlated evolution between circadian traits and both gas exchange and biomass accumulation; shifts to shorter circadian period (closer to 24 h) between phylogenetic nodes are associated with higher stomatal conductance, lower photosynthetic rate (when CO2 supply is factored out), and lower biomass accumulation. Crop type differences in gas exchange are also associated with stomatal density, epidermal thickness, numbers of palisade layers, and biochemical limits to photosynthesis. Brassica crop diversification involves correlated evolution of circadian and physiological traits, which is potentially relevant to understanding mechanistic targets for crop improvement., (© 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.)

The plant circadian clock coordinates the responses to multiple and often simultaneous environmental challenges that the sessile plant cannot avoid. These responses must be integrated efficiently into dynamic metabolic and physiological networks essential for growth and reproduction. Many of the output pathways regulated by the circadian clock feed back to modulate clock function, leading to the appreciation of the clock as a central hub in a sophisticated regulatory network. In this Review, we discuss the circadian regulation of growth, flowering time, abiotic and biotic stress responses, and metabolism, as well as why temporal 'gating' of these processes is important to plant fitness.

Background: A well characterized output of the circadian clock in plants is the daily rhythmic movement of leaves. This process has been used extensively in Arabidopsis to estimate circadian period in natural accessions as well as mutants with known defects in circadian clock function. Current methods for estimating circadian period by leaf movement involve manual steps throughout the analysis and are often limited to analyzing one leaf or cotyledon at a time., Results: In this study, we describe the development of TRiP (Tracking Rhythms in Plants), a new method for estimating circadian period using a motion estimation algorithm that can be applied to whole plant images. To validate this new method, we apply TRiP to a Recombinant Inbred Line (RIL) population in Arabidopsis using our high-throughput imaging platform. We begin imaging at the cotyledon stage and image through the emergence of true leaves. TRiP successfully tracks the movement of cotyledons and leaves without the need to select individual leaves to be analyzed., Conclusions: TRiP is a program for analyzing leaf movement by motion estimation that enables high-throughput analysis of large populations of plants. TRiP is also able to analyze plant species with diverse leaf morphologies. We have used TRiP to estimate period for 150 Arabidopsis RILs as well as 5 diverse plant species, highlighting the broad applicability of this new method.

Several factors affect the yield potential and geographical range of crops including the circadian clock, water availability, and seasonal temperature changes. In order to sustain and increase plant productivity on marginal land in the face of both biotic and abiotic stresses, we need to more efficiently generate stress-resistant crops through marker-assisted breeding, genetic modification, and new genome-editing technologies. To leverage these strategies for producing the next generation of crops, future transcriptomic data acquisition should be pursued with an appropriate temporal design and analyzed with a network-centric approach. The following review focuses on recent developments in abiotic stress transcriptional networks in economically important crops and will highlight the utility of correlation-based network analysis and applications., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

GIGANTEA (GI) was originally identified by a late-flowering mutant in Arabidopsis, but subsequently has been shown to act in circadian period determination, light inhibition of hypocotyl elongation, and responses to multiple abiotic stresses, including tolerance to high salt and cold (freezing) temperature. Genetic mapping and analysis of families of heterogeneous inbred lines showed that natural variation in GI is responsible for a major quantitative trait locus in circadian period in Brassica rapa. We confirmed this conclusion by transgenic rescue of an Arabidopsis gi-201 loss of function mutant. The two B. rapa GI alleles each fully rescued the delayed flowering of Arabidopsis gi-201 but showed differential rescue of perturbations in red light inhibition of hypocotyl elongation and altered cold and salt tolerance. The B. rapa R500 GI allele, which failed to rescue the hypocotyl and abiotic stress phenotypes, disrupted circadian period determination in Arabidopsis. Analysis of chimeric B. rapa GI alleles identified the causal nucleotide polymorphism, which results in an amino acid substitution (S264A) between the two GI proteins. This polymorphism underlies variation in circadian period, cold and salt tolerance, and red light inhibition of hypocotyl elongation. Loss-of-function mutations of B. rapa GI confer delayed flowering, perturbed circadian rhythms in leaf movement, and increased freezing and increased salt tolerance, consistent with effects of similar mutations in Arabidopsis. Collectively, these data suggest that allelic variation of GI-and possibly of clock genes in general-offers an attractive target for molecular breeding for enhanced stress tolerance and potentially for improved crop yield.

Transcriptional feedback loops are central to the architecture of eukaryotic circadian clocks. Models of the Arabidopsis thaliana circadian clock have emphasized transcriptional repressors, but recently, Myb-like REVEILLE (RVE) transcription factors have been established as transcriptional activators of central clock components, including PSEUDO-RESPONSE REGULATOR5 (PRR5) and TIMING OF CAB EXPRESSION1 (TOC1). We show here that NIGHT LIGHT-INDUCIBLE AND CLOCK-REGULATED1 (LNK1) and LNK2, members of a small family of four LNK proteins, dynamically interact with morning-expressed oscillator components, including RVE4 and RVE8. Mutational disruption of LNK1 and LNK2 function prevents transcriptional activation of PRR5 by RVE8. The LNKs lack known DNA binding domains, yet LNK1 acts as a transcriptional activator in yeast and in planta. Chromatin immunoprecipitation shows that LNK1 is recruited to the PRR5 and TOC1 promoters in planta. We conclude that LNK1 is a transcriptional coactivator necessary for expression of the clock genes PRR5 and TOC1 through recruitment to their promoters via interaction with bona fide DNA binding proteins such as RVE4 and RVE8., (© 2014 American Society of Plant Biologists. All rights reserved.)

Hybrid plants and animals often show increased levels of growth and fitness, a phenomenon known as hybrid vigor or heterosis. Circadian rhythms optimize physiology and metabolism in plants and animals. In plant hybrids and polyploids, expression changes of the genes within the circadian regulatory network, such as CIRCADIAN CLOCK ASSOCIATED1 (CCA1), lead to heterosis. However, the relationship between allelic CCA1 expression and heterosis has remained elusive. Here, we show a parent-of-origin effect on altered circadian rhythms and heterosis in Arabidopsis thaliana F1 hybrids. This parent-of-origin effect on biomass heterosis correlates with altered CCA1 expression amplitudes, which are associated with methylation levels of CHH (where H = A, T, or C) sites in the promoter region. The direction of rhythmic expression and hybrid vigor is reversed in reciprocal F1 crosses involving mutants that are defective in the RNA-directed DNA methylation pathway (argonaute4 and nuclear RNA polymerase D1a) but not in the maintenance methylation pathway (methyltransferase1 and decrease in DNA methylation1). This parent-of-origin effect on circadian regulation and heterosis is established during early embryogenesis and maintained throughout growth and development., (© 2014 American Society of Plant Biologists. All rights reserved.)

The onset of flowering, the change from vegetative to reproductive development, is a major life history transition in flowering plants. Recent work suggests that mutations in cis-regulatory mutations should play critical roles in the evolution of this (as well as other) important adaptive traits, but thus far there has been little evidence that directly links regulatory mutations to evolutionary change at the species level. While several genes have previously been shown to affect natural variation in flowering time in Arabidopsis thaliana, most either show protein-coding changes and/or are found at low frequency (<5%). Here we identify and characterize natural variation in the cis-regulatory sequence in the transcription factor CONSTANS that underlies flowering time diversity in Arabidopsis. Mutation in this regulatory motif evolved recently and has spread to high frequency in Arabidopsis natural accessions, suggesting a role for these cis-regulatory changes in adaptive variation of flowering time.

The circadian clock allows organisms to temporally coordinate their biology with the diurnal oscillation of the environment, which enhances plant performance. Accordingly, a fuller understanding of the circadian clock mechanism may contribute to efforts to optimize plant performance. One recurring theme in clock mechanism is coupled transcription-translation feedback loops. To date, the majority of plant transcription factors constituting these loops, including the central oscillator components CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), LATE ELONGATED HYPOCOTYL (LHY), and TIMING OF CAB2 EXPRESSION 1 (TOC1), and the related PSEUDO-RESPONSE REGULATORS (PRRs), are transcriptional repressors, leading to a model of the clock emphasizing repressive interactions. Recent work, however, has revealed that a subset of the REVEILLE (RVE) family of Myb transcription factors closely related to CCA1 and LHY are transcriptional activators in novel feedback transcription-translation feedback loops. Other recently identified transcriptional activators that contribute to clock function include LIGHT-REGULATED WD 1 (LWD1) and LWD2 and night light-inducible and clock-regulated transcription factors NIGHT LIGHT-INDUCIBLE AND CLOCK-REGULATED1 (LNK1) and LNK2. Collectively, these advances permit a substantial reconfiguration of the clock model.

Firefly luciferase (LUC) is a sensitive and versatile reporter for the analysis of gene expression. Transgenic plants carrying CLOCK GENE promoter:LUC fusions can be assayed with high temporal resolution. LUC measurement is sensitive, noninvasive, and nondestructive and can be readily automated, greatly facilitating genetic studies. For these reasons, LUC fusion analysis is a mainstay in the study of plant circadian clocks.