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The circadian clock regulates the daily pattern of temporal gene expression. In Arabidopsis, aging is associated with a shortening of the endogenous period of circadian rhythms under circadian conditions. However, the functional link between the circadian clock and aging under diurnal conditions and its physiological relevance remain elusive. In this study, we investigate and characterize the effect of aging on the waveforms of rhythmic gene expression patterns under light/dark cycles. Our analysis revealed that the diurnal rhythmic patterns of core clock genes undergo significant rhythmic alteration with phase shift and change of waveforms in aged plants compared to younger plants. Transcriptomic analysis indicated that this age-dependent rhythmic alteration occurs not only in core clock genes but also globally. Due to the rhythmic alteration patterns of the diurnal rhythmic gene expression, aged plants experience subjectively a shorter day and longer night. We also observed that genetic mutants of core clock component genes exhibited broadly yet distinctively altered changes in diurnal rhythmic gene expression patterns as aging progresses. Collectively, our findings support that age-dependent rhythmic alteration of diurnal gene expression rhythms reprograms the timetable of daily gene expression, leading to the physiological changes required for plant senescence., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Jung, Kim, Lee, Kang, Kim, Kim, Lim and Nam.)

Leaf senescence is a crucial process throughout evolution, vital for plant fitness as it facilitates the gradual shift of energy allocation between photosynthesis and catabolism overtime. This onset is influenced by a complex interplay of genetic and environmental factors, making senescence a key adaptation mechanism for plants in their natural habitats. Our study investigated the genetic mechanism underlying age-induced leaf senescence in Arabidopsis natural populations. Using a phenome high-throughput investigator, we comprehensively analyzed senescence responses across 234 Arabidopsis accessions and identified that environmental factors (e.g., ambient temperature) and physiological factors (e.g., defense responses) are substantially linked to senescence phenotypes. Through genome-wide association mapping, we identified the ACCELERATED CELL DEATH 6 (ACD6) locus as a potential regulator of senescence variation among natural accessions. Knocking out ACD6 in accessions with early and delayed senescence phenotypes resulted in varying degrees of delay in age-induced senescence, highlighting the accession-dependent regulatory role of ACD6 in leaf senescence. Furthermore, our findings suggest ACD6's involvement in senescence regulation via the salicylic acid signaling pathway. In summary, our study sheds light on the genetic regulation of leaf senescence in Arabidopsis natural populations, with the discovery of ACD6 as a potential candidate for genetic modification to enhance plant adaptation and survival., (© 2024 Scandinavian Plant Physiology Society.)

Aging triggers spinal degeneration, including common spinal stenosis, which causes back and leg pain in older individuals, significantly impacting their quality of life. Here, we explored aging traits in turquoise killifish spines, potentially offering a model for age-linked spinal stenosis in humans. Aged turquoise killifish exhibited body shape deformation and increased vertebral collapse, which was further accelerated by spawning. High-resolution CT scans revealed suppressed cortical bone thickness and hemal arch area in vertebrae due to spawning, and osteophyte formation was observed in both aged and breeding fish populations. Scale mineralization mirrored these changes, increasing with age but being suppressed by spawning. The expression of sp7 , sox9b , axin1 , and wnt4a/b genes can be utilized to monitor age- and reproduction-dependent spine deformation. This study demonstrates that turquoise killifish and humans share certain phenotypes of age-related vertebral abnormalities, suggesting that turquoise killifish could serve as a potential model for studying human spinal stenosis., Competing Interests: The authors declare no competing interests., (© 2023 The Author(s).)

Ultradian rhythms have been proved to be critical for diverse biological processes. However, comprehensive understanding of the short-period rhythms remains limited. Here, we discover that leaf excision triggers a gene expression rhythm with ~3-h periodicity, named as the excision ultradian rhythm (UR), which is regulated by the plant hormone auxin. Promoter-luciferase analyses showed that the spatiotemporal patterns of the excision UR were positively associated with de novo root regeneration (DNRR), a post-embryonic developmental process. Transcriptomic analysis indicated more than 4,000 genes including DNRR-associated genes were reprogramed toward ultradian oscillation. Genetic studies showed that EXCISION ULTRADIAN RHYTHM 1 (EUR1) encoding ENHANCER OF ABSCISIC ACID CO-RECEPTOR1 (EAR1), an abscisic acid signaling regulator, was required to generate the excision ultradian rhythm and enhance root regeneration. The eur1 mutant exhibited the absence of auxin-induced excision UR generation and partial failure during rescuing root regeneration. Our results demonstrate a link between the excision UR and adventitious root formation via EAR1/EUR1 , implying an additional regulatory layer in plant regeneration., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Vu, Song, Park, Xu, Nam and Hong.)

Plants use sophisticated mechanisms of gene expression to control senescence in response to environmental stress or aging. ORE1 (Arabidopsis thaliana NAC092) is a master regulator of senescence that belongs to the plant-specific NAC transcription factor protein family. ORE1 has been reported to bind to multiple DNA targets to orchestrate leaf senescence, yet the mechanistic basis for recognition of the cognate gene sequence remains unclear. Here, we report the crystal structure of the ORE1-NAC domain alone and its DNA-binding form. The structure of DNA-bound ORE1-NAC revealed the molecular basis for nucleobase recognition and phosphate backbone interactions. We show that local versatility in the DNA-binding site, in combination with domain flexibility of the ORE-NAC homodimer, is crucial for the maintenance of binding to intrinsically flexible DNA. Our results provide a platform for understanding other plant-specific NAC protein-DNA interactions as well as insight into the structural basis of NAC regulators in plants of agronomic and scientific importance., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Non-invasive measurement of circadian rhythms is important for longitudinal assays of the rhythmic swimming behavior of the turquoise killifish (Nothobranchius furzeri). Here, we introduce a custom-built, video-based system for non-invasive circadian rhythm measurement. We describe imaging tank setup, video recording and editing, and fish movement tracking. We then detail circadian rhythm analysis. This protocol can be used for repetitive and longitudinal analysis of circadian rhythms in the same fish with minimal stress and can be applied to other fish species. For complete details on the use and execution of this protocol, please refer to Lee et al.. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Nicotianamine (NA) is produced by NA synthase (NAS), which contains three genes in rice and is responsible for chelating metals such as iron (Fe) and zinc (Zn), as well as preserving metal homeostasis. In this study, we generated a transgenic plant ( 23D ) that shows simultaneous activation of OsNAS2 and OsNAS3 by crossing two previously identified activation-tagged mutants, OsNAS2-D1 ( 2D ) and OsNAS3-D1 ( 3D ). Concomitant activation of both genes resulted in the highest Fe and Zn concentrations in shoots and roots of the 23D plants grown under normal conditions and Fe and Zn limited growth conditions. Expression of genes for the biosynthesis of mugineic acid family phytosiderophores (MAs) and Fe and Zn uptake were enhanced in 23D roots. Additionally, 23D plants displayed superior growth to other plants at higher pH levels. Importantly, 23D seeds had NA and 2'-deoxymugineic acid (DMA) concentrations that were 50.6- and 10.0-fold higher than those of the WT. As a result, the mature grain Fe and Zn concentrations of the 23D plant were 4.0 and 3.5 times greater, respectively, than those of the WT. Furthermore, 23D plants exhibited the greatest resistance to excess metals. Our research suggests that simultaneous activation of OsNAS2 and OsNAS3 can enhance Fe and Zn accumulation in rice grains while also increasing plant tolerance to growing situations with metal deficiency and excess metal availability.

Iron (Fe) and zinc (Zn) are essential micronutrients needed by virtually all living organisms, including plants and humans, for proper growth and development. Due to its capacity to easily exchange electrons, Fe is important for electron transport in mitochondria and chloroplasts. Fe is also necessary for chlorophyll synthesis. Zn is a cofactor for several proteins, including Zn-finger transcription factors and redox metabolism enzymes such as copper/Zn superoxide dismutases. In humans, Fe participates in oxygen transport, electron transport, and cell division whereas Zn is involved in nucleic acid metabolism, apoptosis, immunity, and reproduction. Rice ( Oryza sativa L.) is one of the major staple food crops, feeding over half of the world's population. However, Fe and Zn concentrations are low in rice grains, especially in the endosperm, which is consumed as white rice. Populations relying heavily on rice and other cereals are prone to Fe and Zn deficiency. One of the most cost-effective solutions to this problem is biofortification, which increases the nutritional value of crops, mainly in their edible organs, without yield reductions. In recent years, several approaches were applied to enhance the accumulation of Fe and Zn in rice seeds, especially in the endosperm. Here, we summarize these attempts involving transgenics and mutant lines, which resulted in Fe and/or Zn biofortification in rice grains. We review rice plant manipulations using ferritin genes, metal transporters, changes in the nicotianamine/phytosiderophore pathway (including biosynthetic genes and transporters), regulators of Fe deficiency responses, and other mutants/overexpressing lines used in gene characterization that resulted in Fe/Zn concentration changes in seeds. This review also discusses research gaps and proposes possible future directions that could be important to increase the concentration and bioavailability of Fe and Zn in rice seeds without the accumulation of deleterious elements. We also emphasize the need for a better understanding of metal homeostasis in rice, the importance of evaluating yield components of plants containing transgenes/mutations under field conditions, and the potential of identifying genes that can be manipulated by gene editing and other nontransgenic approaches., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Wairich, Ricachenevsky and Lee.)

Leaf senescence proceeds with age but is modulated by various environmental stresses and hormones. Salt stress is one of the most well-known environmental stresses that accelerate leaf senescence. However, the molecular mechanisms that integrate salt stress signalling with leaf senescence programmes remain elusive. In this study, we characterised the role of ETHYLENE RESPONSIVE FACTOR34 (ERF34), an Arabidopsis APETALA2 (AP2)/ERF family transcription factor, in leaf senescence. ERF34 was differentially expressed under various leaf senescence-inducing conditions, and negatively regulated leaf senescence induced by age, dark, and salt stress. ERF34 also promoted salt stress tolerance at different stages of the plant life cycle such as seed germination and vegetative growth. Transcriptome analysis revealed that the overexpression of ERF34 increased the transcript levels of salt stress-responsive genes including COLD-REGULATED15A (COR15A), EARLY RESPONSIVE TO DEHYDRATION10 (ERD10), and RESPONSIVE TO DESICCATION29A (RD29A). Moreover, ERF34 directly bound to ERD10 and RD29A promoters and activated their expression. Our findings indicate that ERF34 plays a key role in the convergence of the salt stress response with the leaf senescence programmes, and is a potential candidate for crop improvement, particularly by enhancing salt stress tolerance., (© 2022 John Wiley & Sons Ltd.)

An optimal size of post-embryonic root apical meristem (RAM) is achieved by a balance between cell division and differentiation. Despite extensive research, molecular mechanisms underlying the coordination of cell division and differentiation are still fragmentary. Here, we report that ORESARA 15 (ORE15), an Arabidopsis PLANT A/T-RICH SEQUENCE-AND ZINC-BINDING PROTEIN (PLATZ) transcription factor preferentially expressed in the RAM, determines RAM size. Primary root length, RAM size, cell division rate, and stem cell niche activity were reduced in an ore15 loss-of-function mutant but enhanced in an activation-tagged line overexpressing ORE15, compared with wild type. ORE15 forms mutually positive and negative feedback loops with auxin and cytokinin signalling, respectively. Collectively, our findings imply that ORE15 controls RAM size by mediating the antagonistic interaction between auxin and cytokinin signalling-related pathways., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Ribosomes are composed of a mix of ribosomal RNAs and proteins; this composition varies depending on time, condition, and organism. Here, we present an optimized protocol for preparation of intact ribosomes from the skeletal muscle of the turquoise killifish. We also detail the steps for ribosome quantification and cryo-EM grid preparation. This protocol can enable the identification of heterogeneous ribosome structures that vary by fish age or in response to specific conditions., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

MicroRNAs (miRNAs) have recently emerged as important regulators of ion channel expression. We show here that select miR-106b family members repress the expression of the KCNQ2 K + channel protein by binding to the 3'-untranslated region of KCNQ2 messenger RNA. During the first few weeks after birth, the expression of miR-106b family members rapidly decreases, whereas KCNQ2 protein level inversely increases. Overexpression of miR-106b mimics resulted in a reduction in KCNQ2 protein levels. Conversely, KCNQ2 levels were up-regulated in neurons transfected with antisense miRNA inhibitors. By constructing more specific and stable forms of miR-106b controlling systems, we further confirmed that overexpression of precursor-miR-106b-5p led to a decrease in KCNQ current density and an increase in firing frequency of hippocampal neurons, while tough decoy miR-106b-5p dramatically increased current density and decreased neuronal excitability. These results unmask a regulatory mechanism of KCNQ2 channel expression in early postnatal development and hint at a role for miR-106b up-regulation in the pathophysiology of epilepsy., Competing Interests: The authors declare no competing interest.

Zinc (Zn) is essential for normal plant growth and development. The Zn-regulated transporter, iron-regulated transporter (IRT)-like protein (ZIP) family members are involved in Zn transport and cellular Zn homeostasis throughout the domains of life. In this study, we have characterized four ZIP transporters from Arabidopsis thaliana (IRT3, ZIP4, ZIP6, and ZIP9) to better understand their functional roles. The four ZIP proteins can restore the growth defect of a yeast Zn uptake mutant and are upregulated under Zn deficiency. Single and double mutants show no phenotypes under Zn-sufficient or Zn-limited growth conditions. In contrast, triple and quadruple mutants show impaired growth irrespective of external Zn supply due to reduced Zn translocation from root to shoot. All four ZIP genes are highly expressed during seed development, and siliques from all single and higher-order mutants exhibited an increased number of abnormal seeds and decreased Zn levels in mature seeds relative to wild type. The seed phenotypes could be reversed by supplementing the soil with Zn. Our data demonstrate that IRT3, ZIP4, ZIP6, and ZIP9 function redundantly in maintaining Zn homeostasis and seed development in A. thaliana., (© 2021 Society for Experimental Biology and John Wiley & Sons Ltd.)

Leaf senescence, the final stage of leaf development, is influenced by numerous internal and environmental signals. However, how biotic stresses such as pathogen infection regulate leaf senescence remains largely unclear. In this study, we found that the premature leaf senescence in Arabidopsis caused by the soil-borne vascular fungus Verticillium dahliae was impaired by disruption of a protein elicitor from V. dahliae 1 named PevD1. Constitutive or inducible overexpression of PevD1 accelerated Arabidopsis leaf senescence. Interestingly, a senescence-associated NAC transcription factor, ORE1, was targeted by PevD1. PevD1 could interact with and stabilize ORE1 protein by disrupting its interaction with the RING-type ubiquitin E3 ligase NLA. Mutation of ORE1 suppressed the premature senescence caused by overexpressing PevD1, whereas overexpression of ORE1 or PevD1 led to enhanced ethylene production and thereby leaf senescence. We showed that ORE1 directly binds the promoter of ACS6 and promotes its expression for mediating PevD1-induced ethylene biosynthesis. Loss-of-function of ACSs could suppress V. dahliae-induced leaf senescence in ORE1-overexpressing plants. Furthermore, we found thatPevD1 also interacts with Gossypium hirsutum ORE1 (GhORE1) and that virus-induced gene silencing of GhORE1 delays V. dahliae-triggered leaf senescence in cotton, indicating a possibly conserved mechanism in plants. Taken together, these results suggest that V. dahliae induces leaf senescence by secreting the effector PevD1 to manipulate the ORE1-ACS6 cascade, providing new insights into biotic stress-induced senescence in plants., (Copyright © 2021 The Author. Published by Elsevier Inc. All rights reserved.)

In unicellular photosynthetic organisms, circadian rhythm is tightly linked to gating of cell cycle progression, and is entrained by light signal. As several organisms obtain a fitness advantage when the external light/dark cycle matches their endogenous period, and aging alters circadian rhythms, senescence phenotypes of the microalga Euglena gracilis of different culture ages were characterized with respect to the cell division cycle. We report here the effects of prolonged-stationary-phase conditions on the cell division cycles of E. gracilis under non-24-h light/dark cycles (T-cycles). Under T-cycles, cells established from 1-month-old and 2-month-old cultures produced lower cell concentrations after cultivation in the fresh medium than cells from 1-week-old culture. This decrease was not due to higher concentrations of dead cells in the populations, suggesting that cells of different culture ages differ in their capacity for cell division. Cells from 1-week-old cultures had a shorter circadian period of their cell division cycle under shortened T-cycles than aged cells. When algae were transferred to free-running conditions after entrainment to shortened T-cycles, the young cells showed the peak growth rate at a time corresponding to the first subjective night, but the aged cells did not. This suggests that circadian rhythms are more plastic in younger E. gracilis cells.

Zinc and iron are essential micronutrients for plant growth, and their homeostasis must be tightly regulated. Previously, it has been shown that Zinc-Induced Facilitator 1 (ZIF1) is involved in basal Zn tolerance by controlling the vacuolar storage of nicotianamine (NA). However, knowledge of the functional roles of two ZIF1 paralogs, ZIF-LIKE1 (ZIFL1) and ZIFL2, in metal homeostasis remains limited. Here, we functionally characterized the roles of ZIF1, ZIFL1, and ZIFL2 in Zn and Fe homeostasis. Expression of ZIF1 and ZIFL1 was induced by both excess Zn and Fe-deficiency, and their loss-of-function led to hypersensitivity under excess Zn and Fe-deficiency, suggesting functional overlap between ZIF1 and ZIFL1. By contrast, the disruption of ZIFL2 resulted in no obvious phenotypic alteration under both conditions. Additionally, the expression of ZIFL1, but not that of ZIFL2, in the zif1 mutant partially restored the phenotype under excess Zn, suggesting that ZIF1 and ZIFL1 perform functionally redundant roles in Zn homeostasis., Competing Interests: Declaration of competing interest The author declares no conflict of interest., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Small RNAs that originate from transfer RNA (tRNA) species, tRNA-derived fragments (tRFs), play diverse biological functions but little is known for their association with aging. Moreover, biochemical aspects of tRNAs limit discovery of functional tRFs by high throughput sequencing. In particular, genes encoding tRNAs exist as multiple copies throughout genome, and mature tRNAs have various modified bases, contributing to ambiguities for RNA sequencing-based analysis of tRFs. Here, we report age-dependent changes of tRFs in Caenorhabditis elegans. We first analyzed published RNA sequencing data by using a new strategy for tRNA-associated sequencing reads. Our current method used unique mature tRNAs as a reference for the sequence alignment, and properly filtered out false positive enrichment for tRFs. Our analysis successfully distinguished de novo mutation sites from differences among homologous copies, and identified potential RNA modification sites. Overall, the majority of tRFs were upregulated during aging and originated from 5'-ends, which we validated by using Northern blot analysis. Importantly, we revealed that the major source of tRFs upregulated during aging was the tRNAs with abundant gene copy numbers. Our analysis suggests that tRFs are useful biomarkers of aging particularly when they originate from abundant homologous gene copies.

Distribution of cells and proteins in whole organs, such as the brain, can provide another level of insight into molecular and cellular functions. Here, we describe a whole-brain immunostaining method using the turquoise killifish, an emerging model for aging research. We optimized a protocol for tissue clearing and whole-brain immunostaining to the turquoise killifish brain. This protocol provides a comprehensive procedure from brain dissection to whole-brain imaging and image processing. For complete details on the use and execution of this protocol, please refer to Eunjeong Do (2020) and Lee et al. (2021)., Competing Interests: The authors declare no competing interests., (© 2021 The Authors.)

Leaf senescence, which is the last developmental phase of plant growth, is controlled by multiple genetic and environmental factors. Leaf yellowing is a visual indicator of senescence due to the loss of the green pigment chlorophyll. During senescence, the methodical disassembly of macromolecules occurs, facilitating nutrient recycling and translocation from the sink to the source organs, which is critical for plant fitness and productivity. Leaf senescence is a complex and tightly regulated process, with coordinated actions of multiple pathways, responding to a sophisticated integration of leaf age and various environmental signals. Many studies have been carried out to understand the leaf senescence-associated molecular mechanisms including the chlorophyll breakdown, phytohormonal and transcriptional regulation, interaction with environmental signals, and associated metabolic changes. The metabolic reprogramming and nutrient recycling occurring during leaf senescence highlight the fundamental role of this developmental stage for the nutrient economy at the whole plant level. The strong impact of the senescence-associated nutrient remobilization on cereal productivity and grain quality is of interest in many breeding programs. This review summarizes our current knowledge in rice on (i) the actors of chlorophyll degradation, (ii) the identification of stay-green genotypes, (iii) the identification of transcription factors involved in the regulation of leaf senescence, (iv) the roles of leaf-senescence-associated nitrogen enzymes on plant performance, and (v) stress-induced senescence. Compiling the different advances obtained on rice leaf senescence will provide a framework for future rice breeding strategies to improve grain yield.

Inactivation of tumor suppressor Runt-related transcription factor 3 (RUNX3) plays an important role during early tumorigenesis. However, posttranslational modifications (PTM)-based mechanism for the inactivation of RUNX3 under hypoxia is still not fully understood. Here, we demonstrate a mechanism that G9a, lysine-specific methyltransferase (KMT), modulates RUNX3 through PTM under hypoxia. Hypoxia significantly increased G9a protein level and G9a interacted with RUNX3 Runt domain, which led to increased methylation of RUNX3 at K129 and K171. This methylation inactivated transactivation activity of RUNX3 by reducing interactions with CBFβ and p300 cofactors, as well as reducing acetylation of RUNX3 by p300, which is involved in nucleocytoplasmic transport by importin-α1. G9a-mediated methylation of RUNX3 under hypoxia promotes cancer cell proliferation by increasing cell cycle or cell division, while suppresses immune response and apoptosis, thereby promoting tumor growth during early tumorigenesis. Our results demonstrate the molecular mechanism of RUNX3 inactivation by G9a-mediated methylation for cell proliferation and antiapoptosis under hypoxia, which can be a therapeutic or preventive target to control tumor growth during early tumorigenesis.

Despite the advancement of targeted therapy for pulmonary arterial hypertension (PAH), poor prognosis remains a reality. Mesenchymal stem cells (MSCs) are one of the most clinically feasible alternative treatment options. We compared the treatment effects of adipose tissue (AD)-, bone marrow (BD)-, and umbilical cord blood (UCB)-derived MSCs in the rat monocrotaline-induced pulmonary hypertension (PH) model. The greatest improvement in the right ventricular function was observed in the UCB-MSCs treated group. The UCB-MSCs treated group also exhibited the greatest improvement in terms of the largest decrease in the medial wall thickness, perivascular fibrosis, and vascular cell proliferation, as well as the lowest levels of recruitment of innate and adaptive immune cells and associated inflammatory cytokines. Gene expression profiling of lung tissue confirmed that the UCB-MSCs treated group had the most notably attenuated immune and inflammatory profiles. Network analysis further revealed that the UCB-MSCs group had the greatest therapeutic effect in terms of the normalization of all three classical PAH pathways. The intravenous injection of the UCB-MSCs, compared with those of other MSCs, showed superior therapeutic effects in the PH model for the (1) right ventricular function, (2) vascular remodeling, (3) immune/inflammatory profiles, and (4) classical PAH pathways.

Circadian rhythm is altered during aging, although the underlying molecular mechanisms remain largely unknown. Here, we used the turquoise killifish as a short-lived vertebrate model to examine the effects of aging on the major circadian network comprising the four mammalian clock protein homologs, Bmal1, Clockb, Cry1b, and Per3, which are highly conserved in the killifish with 50%-85% amino acid sequence identity to their human counterparts. The amplitude of circadian rhythm was smaller in old fish (14 weeks) than in young fish (6 weeks). In old fish brain, the Bmal1 protein level was significantly downregulated. However, the Bmal1 interaction with Clockb and chromatin binding of Bmal1 to its downstream target promoters were retained. Furthermore, Bmal1 was relatively well maintained in the pineal gland compared with other regions of the old fish brain. The results suggest that the circadian clock system in the killifish becomes spatially confined to the pineal gland upon aging., Competing Interests: The authors have no competing interests to declare., (© 2020 The Author(s).)

Plants undergo several important developmental transitions including flowering and senescence during their life cycle. Timing these transitions according to the environmental conditions increases plant fitness and productivity. The circadian clock senses various environmental cycles, including photoperiod, and synchronizes plant physiological processes to maximize plant fitness. Here, we propose that the cellular localization of GIGANTEA (GI), a key clock component, regulates leaf senescence and flowering in Arabidopsis thaliana . We show that GI, which connects the circadian clock with photoperiod-regulated flowering, induces leaf senescence depending on its subcellular localization. Overexpression of GI in the gi mutant rescued its delayed senescence phenotype but only when the GI protein was targeted to the nucleus, not when it was targeted to the cytosol. In the nucleus, EARLY FLOWERING 4 (ELF4) inhibited the binding of GI to ORESARA 1 ( ORE1) promoter to regulate leaf senescence. GI also positively regulated the day-peak of ORE1 expression. These results indicate that like flowering, leaf senescence is also controlled by the location of GI in the cell. Taken together, our results suggest that ELF4 and GI act together to control flowering and senescence in Arabidopsis ., (Copyright © 2020 Kim, Park, Kim and Nam.)

Fluorescent molecular rotor dyes, including Cy3, Cy5, and Alexa Fluor 555, dissolved in micron-sized aqueous droplets (microdroplets) in oil were excited, and the fluorescence intensity was recorded as function of time. We observed lengthening of the fluorescence lifetime of these dyes at the water-oil periphery, which extended several microns inward. This behavior shows that intramolecular rotation is restricted at and near the microdroplet interface. Lengthened lifetimes were observed in water microdroplets but not in microdroplets composed of organic solvents. This lifetime change was relatively insensitive to added glycerol up to 60%, suggesting that solution viscosity is not the dominant mechanism. These restricted intramolecular rotations at and near the microdroplet periphery are consistent with the reduced entropy observed in chemical reactions in microdroplets compared to the same reaction conditions in bulk solution and helps us further understand why microdroplet chemistry differs so markedly from bulk-phase chemistry.

Cells contain more than 100 mM salt ions that are typically confined to dimensions of 5 to 10 micrometers by a hydrophobic cellular membrane. We found that in aqueous microdroplets having the same size as cells and that are confined in hydrocarbon oil, negatively charged molecules were distributed rather uniformly over the interior of the microdroplet, whereas positively charged molecules were localized at and near the surface. However, the addition of salt (NaCl) to the microdroplet caused all charged molecules to be localized near the oil-water interface. This salt-induced relocalization required less salt concentration in microdroplets compared to bulk water. Moreover, the localization became more prominent as the size of the microdroplet was reduced. The relocatization also critically depended on the type of oil. Our results imply that salt ions and different hydrophobic interfaces together may govern the local distribution of charged biomolecules in confined intracellular environments., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Microglia (MG), the principal neuroimmune sentinels in the brain, continuously sense changes in their environment and respond to invading pathogens, toxins, and cellular debris, thereby affecting neuroinflammation. Microbial pathogens produce small metabolites that influence neuroinflammation, but the molecular mechanisms that determine whether pathogen-derived small metabolites affect microglial activation of neuroinflammation remain to be elucidated. We hypothesized that odorant receptors (ORs), the largest subfamily of G protein-coupled receptors, are involved in microglial activation by pathogen-derived small metabolites. We found that MG express high levels of two mouse ORs, Olfr110 and Olfr111, which recognize a pathogenic metabolite, 2-pentylfuran, secreted by Streptococcus pneumoniae. These interactions activate MG to engage in chemotaxis, cytokine production, phagocytosis, and reactive oxygen species generation. These effects were mediated through the G αs -cyclic adenosine monophosphate-protein kinase A-extracellular signal-regulated kinase and G βγ -phospholipase C-Ca 2+ pathways. Taken together, our results reveal a novel interplay between the pathogen-derived metabolite and ORs, which has major implications for our understanding of microglial activation by pathogen recognition. DATABASE: Model data are available in the PMDB database under the accession number PM0082389., (© 2020 Federation of European Biochemical Societies.)

Despite recent advances in understanding chronic inflammation remission, global analyses have not been explored to systematically discover genes or pathways underlying the resolution dynamics of chronic inflammatory diseases. Here, we performed time-course gene expression profiling of mouse synovial tissues along progression and resolution of collagen-induced arthritis (CIA) and identified genes associated with inflammation resolution. Through network analysis of these genes, we predicted 3 key secretory factors responsible for the resolution of CIA: Itgb1, Rps3, and Ywhaz. These factors were predominantly expressed by Tregs and antiinflammatory M2 macrophages, suppressing production of proinflammatory cytokines. In particular, Ywhaz was elevated in the sera of mice with arthritis resolution and in the urine of rheumatoid arthritis (RA) patients with good therapeutic responses. Moreover, adenovirus-mediated transfer of the Ywhaz gene to the affected joints substantially inhibited arthritis progression in mice with CIA and suppressed expression of proinflammatory cytokines in joint tissues, lymph nodes, and spleens, suggesting Ywhaz is an excellent target for RA therapy. Therefore, our comprehensive analysis of dynamic synovial transcriptomes provides previously unidentified antiarthritic genes, Itgb1, Rps3, and Ywhaz, which can serve as molecular markers to predict disease remission, as well as therapeutic targets for chronic inflammatory arthritis.

Carotenoids are essential components of photosynthetic organisms including land plants, algae, cyanobacteria, and photosynthetic bacteria. Although the light-mediated regulation of carotenoid biosynthesis, including the light/dark cycle as well as the dependence of carotenoid biosynthesis-related gene translation on light wavelength, has been investigated in land plants, these aspects have not been studied in microalgae. Here, we investigated carotenoid biosynthesis in Euglena gracilis and found that zeaxanthin accumulates in the dark. The major carotenoid species in E. gracilis, namely β-carotene, neoxanthin, diadinoxanthin and diatoxanthin, accumulated corresponding to the duration of light irradiation under the light/dark cycle, although the translation of carotenoid biosynthesis genes hardly changed. Irradiation with either blue or red-light (3 μmol photons m -2  s -1 ) caused a 1.3-fold increase in β-carotene content compared with the dark control. Blue-light irradiation (300 μmol photons m -2  s -1 ) caused an increase in the cellular content of both zeaxanthin and all trans-diatoxanthin, and this increase was proportional to blue-light intensity. In addition, pre-irradiation with blue-light of 3 or 30 μmol photons m -2  s -1 enhanced the photosynthetic activity and tolerance to high-light stress. These findings suggest that the accumulation of β-carotene is regulated by the intensity of light, which may contribute to the acclimation of E. gracilis to the light environment in day night conditions., Competing Interests: Declaration of Competing Interest The authors have no conflicts of interest to declare., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Leaf senescence is a developmental process by which a plant actively remobilizes nutrients from aged and photosynthetically inefficient leaves to young growing ones by disassembling organelles and degrading macromolecules. Senescence is accelerated by age and environmental stresses such as prolonged darkness. Phytochrome B (phyB) inhibits leaf senescence by inhibiting phytochrome-interacting factor 4 (PIF4) and PIF5 in prolonged darkness. However, it remains unknown whether phyB mediates the temperature signal that regulates leaf senescence. We found the light-activated form of phyB (Pfr) remains active at least four days after a transfer to darkness at 20°C but is inactivated more rapidly at 28°C. This faster inactivation of Pfr further increases PIF4 protein levels at the higher ambient temperature. In addition, PIF4 mRNA levels rise faster after the transfer to darkness at high ambient temperature via a mechanism that depends on ELF3 but not phyB. Increased PIF4 protein then binds to the ORE1 promoter and activates its expression together with ABA and ethylene signaling, accelerating leaf senescence at high ambient temperature. Our results support a role for the phy-PIF signaling module in integrating not only light signaling but also temperature signaling in the regulation of leaf senescence.

All living organisms are unavoidably exposed to various endogenous and environmental stresses that trigger potentially fatal DNA damage, including double-strand breaks (DSBs). Although a growing body of evidence indicates that DNA damage is one of the prime drivers of aging in animals, little is known regarding the importance of DNA damage and its repair on lifespan control in plants. We found that the level of DSBs increases but DNA repair efficiency decreases as Arabidopsis leaves age. Generation of DSBs by inducible expression of I-PpoI leads to premature senescence phenotypes. We examined the senescence phenotypes in the loss-of-function mutants for 13 key components of the DNA repair pathway and found that deficiency in ATAXIA TELANGIECTASIA MUTATED (ATM), the chief transducer of the DSB signal, results in premature senescence in Arabidopsis. ATM represses DSB-induced expression of senescence-associated genes, including the genes encoding the WRKY and NAC transcription factors, central components of the leaf senescence process, via modulation of histone lysine methylation. Our work highlights the significance of ATM in the control of leaf senescence and has significant implications for the conservation of aging mechanisms in animals and plants., (© 2020 The Authors. New Phytologist © 2020 New Phytologist Trust.)

Nitrogen (N) is a major factor for plant development and productivity. However, the application of nitrogenous fertilizers generates environmental and economic problems. To cope with the increasing global food demand, the development of rice varieties with high nitrogen use efficiency (NUE) is indispensable for reducing environmental issues and achieving sustainable agriculture. Here, we report that the concomitant activation of the rice (Oryza sativa) Ammonium transporter 1;2 (OsAMT1;2) and Glutamate synthetase 1 (OsGOGAT1) genes leads to increased tolerance to nitrogen limitation and to better ammonium uptake and N remobilization at the whole plant level. We show that the double activation of OsAMT1;2 and OsGOGAT1 increases plant performance in agriculture, providing better N grain filling without yield penalty under paddy field conditions, as well as better grain yield and N content when plants are grown under N llimitations in field conditions. Combining OsAMT1;2 and OsGOGAT1 activation provides a good breeding strategy for improving plant growth, nitrogen use efficiency and grain productivity, especially under nitrogen limitation, through the enhancement of both nitrogen uptake and assimilation., (© 2020 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

A predominant physiological change that occurs during leaf senescence is a decrease in photosynthetic efficiency. An optimal organization of photosynthesis complexes in plant leaves is critical for efficient photosynthesis. However, molecular mechanisms for regulating photosynthesis complexes during leaf senescence remain largely unknown. Here we tracked photosynthesis complexes alterations during leaf senescence in Arabidopsis thaliana. Grana stack is significantly thickened and photosynthesis complexes were disassembled in senescing leaves. Defects in STN7 and CP29 led to an altered chloroplast ultrastructure and a malformation of photosynthesis complex organization in stroma lamella. Both CP29 phosphorylation by STN7 and CP29 fragmentation are highly associated with the photosynthesis complex disassembly. In turn, CP29 functions as a molecular glue to facilitate protein complex formation leading phosphorylation cascade and to maintain photosynthetic efficiency during leaf senescence. These data suggest a novel molecular mechanism to modulate leaf senescence via CP29 phosphorylation and fragmentation, serving as an efficient strategy to control photosynthesis complexes.

Increased grain yield will be critical to meet the growing demand for food, and could be achieved by delaying crop senescence. Here, via quantitative trait locus (QTL) mapping, we uncover the genetic basis underlying distinct life cycles and senescence patterns of two rice subspecies, indica and japonica. Promoter variations in the Stay-Green (OsSGR) gene encoding the chlorophyll-degrading Mg ++ -dechelatase were found to trigger higher and earlier induction of OsSGR in indica, which accelerated senescence of indica rice cultivars. The indica-type promoter is present in a progenitor subspecies O. nivara and thus was acquired early during the evolution of rapid cycling trait in rice subspecies. Japonica OsSGR alleles introgressed into indica-type cultivars in Korean rice fields lead to delayed senescence, with increased grain yield and enhanced photosynthetic competence. Taken together, these data establish that naturally occurring OsSGR promoter and related lifespan variations can be exploited in breeding programs to augment rice yield.

Leaf senescence is a developmental process designed for nutrient recycling and relocation to maximize growth competence and reproductive capacity of plants. Thus, plants integrate developmental and environmental signals to precisely control senescence. To genetically dissect the complex regulatory mechanism underlying leaf senescence, we identified an early leaf senescence mutant, rse1 . RSE1 encodes a putative glycosyltransferase. Loss-of-function mutations in RSE1 resulted in precocious leaf yellowing and up-regulation of senescence marker genes, indicating enhanced leaf senescence. Transcriptome analysis revealed that salicylic acid (SA) and defense signaling cascades were up-regulated in rse1 prior to the onset of leaf senescence. We found that SA accumulation was significantly increased in rse1. The rse1 phenotypes are dependent on SA-INDUCTION DEFICIENT 2 ( SID2 ), supporting a role of SA in accelerated leaf senescence in rse1 . Furthermore, RSE1 protein was localized to the cell wall, implying a possible link between the cell wall and RSE1 function. Together, we show that RSE1 negatively modulates leaf senescence through an SID2 -dependent SA signaling pathway., (Copyright © 2020 Lee, Kim, Lee, Jeon, Kwak and Kim.)

Physiological stresses, such as pathogen infection, are detected by "cellular Surveillance Activated Detoxification and Defenses" (cSADD) systems that trigger host defense responses. Aging is associated with physiological stress, including impaired mitochondrial function. Here, we investigated whether an endogenous cSADD pathway is activated during aging in C. elegans . We provide evidence that the transcription factor ZIP-2, a well-known immune response effector in C. elegans , is activated in response to age-associated mitochondrial dysfunction. ZIP-2 mitigates multiple aging phenotypes, including mitochondrial disintegration and reduced motility of the pharynx and intestine. Importantly, our data suggest that ZIP-2 is activated during aging independently of bacterial infection and of the transcription factors ATFS-1 and CEBP-2. Thus, ZIP-2 is a key component of an endogenous pathway that delays aging phenotypes in C. elegans . Our data suggest that aging coopted a compensatory strategy for regulation of aging process as a guarded process rather than a simple passive deterioration process.

Internode elongation is one of the key agronomic traits determining a plant's height and biomass. However, our understanding of the molecular mechanisms controlling internode elongation is still limited in crop plant species. Here, we report the functional identification of an atypical basic helix-loop-helix transcription factor ( OsbHLH073 ) through gain-of-function studies using overexpression ( OsbHLH073-OX ) and activation tagging ( osbhlh073-D ) lines of rice. The expression of OsbHLH073 was significantly increased in the osbhlh073-D line. The phenotype of osbhlh073-D showed semi-dwarfism due to deficient elongation of the first internode and poor panicle exsertion. Transgenic lines overexpressing OsbHLH073 confirmed the phenotype of the osbhlh073-D line. Exogenous gibberellic acid (GA 3 ) treatment recovered the semi-dwarf phenotype of osbhlh073-D plants at the seedling stage. In addition, quantitative expression analysis of genes involving in GA biosynthetic and signaling pathway revealed that the transcripts of rice ent-kaurene oxidases 1 and 2 ( OsKO1 and OsKO2 ) encoding the GA biosynthetic enzyme were significantly downregulated in osbhlh073-D and OsbHLH073-OX lines. Yeast two-hybrid and localization assays showed that the OsbHLH073 protein is a nuclear localized-transcriptional activator. We report that OsbHLH073 participates in regulating plant height, internode elongation, and panicle exsertion by regulating GA biosynthesis associated with the OsKO1 and OsKO2 genes.

Stomata in the plant epidermis play a critical role in growth and survival by controlling gas exchange, transpiration, and immunity to pathogens. Plants modulate stomatal cell fate and patterning through key transcriptional factors and signaling pathways. MicroRNAs (miRNAs) are known to contribute to developmental plasticity in multicellular organisms; however, no miRNAs appear to target the known regulators of stomatal development. It remains unclear as to whether miRNAs are involved in stomatal development. Here, we report highly dynamic, developmentally stage-specific miRNA expression profiles from stomatal lineage cells. We demonstrate that stomatal lineage miRNAs positively and negatively regulate stomatal formation and patterning to avoid clustered stomata. Target prediction of stomatal lineage miRNAs implicates potential cellular processes in stomatal development. We show that miR399-mediated PHO2 regulation, involved in phosphate homeostasis, contributes to the control of stomatal development. Our study demonstrates that miRNAs constitute a critical component in the regulatory mechanisms controlling stomatal development., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Carotenoids are the most universal and most widespread pigments in nature. They have played pivotal roles in the evolution of photosensing mechanisms in microbes and of vision in animals. Several groups of phytoflagellates developed a photoreceptive organelle called the eyespot apparatus (EA) consisting of two separable components: the eyespot, a cluster of carotenoid-rich globules that acts as a reflector device, and actual photoreceptors for photobehaviors. Unlike other algal eyespots, the eyespot of Euglenophyta lacks reflective properties and is generally considered to act as a shading device for the photoreceptor (paraflagellar body, PFB) for major photomovements. However, the function of the eyespot of Euglenophyta has not yet been fully proven. Here, we report that the blocking carotenoid biosynthesis in Euglena gracilis by suppressing the phytoene synthase gene (crtB) caused a defect in eyespot function resulting in a loss of phototaxis. Raman spectroscopy and transmission electron microscopy suggested that EgcrtB-suppressed cells formed eyespot globules but had a defect in the accumulation of carotenoids in those packets. Motion analysis revealed the loss of phototaxis in EgcrtB-suppressed cells: a defect in the initiation of turning movements immediately after a change in light direction, rather than a defect in the termination of cell turning at the appropriate position due to a loss of the shading effect on the PFB. This study revealed that carotenoids are essential for light perception by the EA for the initiation of phototactic movement by E. gracilis, suggesting one possible photosensory role of carotenoids in the EA for the phototaxis., (© 2019 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Complex diseases involve dynamic perturbations of pathophysiological processes during disease progression. Transcriptional programs underlying such perturbations are unknown in many diseases. Here, we present core transcriptional regulatory circuits underlying early and late perturbations in prion disease. We first identified cellular processes perturbed early and late using time-course gene expression data from three prion-infected mouse strains. We then built a transcriptional regulatory network (TRN) describing regulation of early and late processes. We found over-represented feed-forward loops (FFLs) comprising transcription factor (TF) pairs and target genes in the TRN. Using gene expression data of brain cell types, we further selected active FFLs where TF pairs and target genes were expressed in the same cell type and showed correlated temporal expression changes in the brain. We finally determined core transcriptional regulatory circuits by combining these active FFLs. These circuits provide insights into transcriptional programs for early and late pathophysiological processes in prion disease.

Alzheimer's disease (AD) is an age-related neurodegenerative disease. The most common pathological hallmarks are amyloid plaques and neurofibrillary tangles in the brain. In the brains of patients with AD, pathological tau is abnormally accumulated causing neuronal loss, synaptic dysfunction, and cognitive decline. We found a histone deacetylase 6 (HDAC6) inhibitor, CKD-504, changed the tau interactome dramatically to degrade pathological tau not only in AD animal model (ADLP APT ) brains containing both amyloid plaques and neurofibrillary tangles but also in AD patient-derived brain organoids. Acetylated tau recruited chaperone proteins such as Hsp40, Hsp70, and Hsp110, and this complex bound to novel tau E3 ligases including UBE2O and RNF14. This complex degraded pathological tau through proteasomal pathway. We also identified the responsible acetylation sites on tau. These dramatic tau-interactome changes may result in tau degradation, leading to the recovery of synaptic pathology and cognitive decline in the ADLP APT mice., (© 2019 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

Seed germination is a developmental process regulated by numerous internal and external cues. Our previous studies have shown that calcium influx mediated by the Arabidopsis glutamate receptor homolog 3.5 (AtGLR3.5) modulates the expression of the ABSCISIC ACID INSENSITIVE 4 (ABI4) transcription factor during germination and that L-methionine (L-Met) activates AtGLR3.1/3.5 Ca2+ channels in guard cells. However, it is not known whether L-Met participates in regulation of germination and what cellular mechanism is responsible for Met production during germination. Here, we describe Arabidopsis methionine synthase 1 (AtMS1), which acts in the final step of Met biosynthesis, synthesizes the Met required for the activation of AtGLR3.5 Ca2+ channels whose expression is up-regulated during germination, leading to the regulation of seed germination. We show that exogenous L-Met promotes germination in an AtGRL3.5-dependent manner. We also demonstrate that L-Met directly regulates the AtGLR3.5-mediated increase in cytosolic Ca2+ level in seedlings. We provide pharmacological and genetic evidence that Met synthesized via AtMS1 acts upstream of the AtGLR3.5-mediated Ca2+ signal and regulates the expression of ABI4, a major regulator in the abscisic acid response in seeds. Overall, our results link AtMS1, L-Met, the AtGLR3.5 Ca2+ channel, Ca2+ signals, and ABI4, and shed light on the physiological role and molecular mechanism of L-Met in germination., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Shoot branching is an essential agronomic trait that impacts on plant architecture and yield. Shoot branching is determined by two independent steps: axillary meristem formation and axillary bud outgrowth. Although several genes and regulatory mechanism have been studied with respect to shoot branching, the roles of chromatin-remodeling factors in the developmental process have not been reported in rice. We previously identified a chromatin-remodeling factor OsVIL2 that controls the trimethylation of histone H3 lysine 27 (H3K27me3) at target genes. In this study, we report that loss-of-function mutants in OsVIL2 showed a phenotype of reduced tiller number in rice. The reduction was due to a defect in axillary bud (tiller) outgrowth rather than axillary meristem initiation. Analysis of the expression patterns of the tiller-related genes revealed that expression of OsTB1 , which is a negative regulator of bud outgrowth, was increased in osvil2 mutants. Chromatin immunoprecipitation assays showed that OsVIL2 binds to the promoter region of OsTB1 chromatin in wild-type rice, but the binding was not observed in osvil2 mutants. Tiller number of double mutant osvil2 ostb1 was similar to that of ostb1 , suggesting that osvil2 is epistatic to ostb1 . These observations indicate that OsVIL2 suppresses OsTB1 expression by chromatin modification, thereby inducing bud outgrowth.

RNA quality control (RQC) and post-transcriptional gene silencing (PTGS) target and degrade aberrant endogenous RNAs and foreign RNAs, contributing to homeostasis of cellular RNAs. In plants, RQC and PTGS compete for foreign and selected endogenous RNAs; however, little is known about the mechanism interconnecting the two pathways. Using a reporter system designed for monitoring PTGS, we revealed that the 26S proteasome subunit RPT2a enhances transgene PTGS by promoting the accumulation of transgene-derived short interfering RNAs without affecting their biogenesis. RPT2a physically associated with a subset of RQC components and downregulated the protein level. Overexpression of the RQC components interfered with transgene silencing, and impairment of the RQC machinery reinforced transgene PTGS attenuated by rpt2a. Overall, we demonstrate that the 26S proteasome subunit RPT2a promotes PTGS by repressing the RQC machinery to control foreign RNAs.

Alzheimer's disease (AD) is the most common age-associated dementia. Many studies have sought to predict cerebral amyloid deposition, the major pathological hallmark of AD, using body fluids such as blood or cerebral spinal fluid (CSF). The use of blood in diagnostic procedures is widespread in medicine; however, existing blood biomarkers for AD remain unreliable. We sought to discover blood biomarkers that discriminate Aβ deposition status in the brain. This study used 107 individuals who were cognitively normal (CN), 107 patients with mild cognitive impairment (MCI), and 40 AD patients with Pittsburg compound B positron emission tomography (PiB-PET) amyloid imaging data available. We found five plasma biomarker candidates via mass spectrometry (MS) based-proteomic analysis and validated these proteins using enzyme-linked immunosorbent assay (ELISA). Our integrated models were highly predictive of brain amyloid deposition, exhibiting 0.871 accuracy with 79% sensitivity and 84% specificity overall, and 0.836 accuracy with 68% sensitivity and 90% specificity in patients with MCI. These results indicated that a combination of proteomic-based blood proteins might be a possible biomarker set for predicting cerebral amyloid deposition., (Copyright © 2019 Elsevier Ltd. All rights reserved.)