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The mitochondrial-homing drug MA-5 ameliorates the pathophysiology of human mitochondrial-disease fibroblasts and Caenorhabditis elegans DMD and PD disease models. Here, we found that MA-5 could suppress the age-related decline in motor performance, loss of muscle mitochondria, and degeneration of dopaminergic neurons associated with mitochondrial Ca2+ overload in C. elegans. These suggest that MA-5 may act as an anti-aging agent against a wide range of neuromuscular dysfunctions in metazoan.

Life on Earth has evolved in a form suitable for the gravitational force of 1 ×g. Although the pivotal role of gravity in gene expression has been revealed by multiomics approaches in space-flown samples and astronauts, the molecular details of how mammalian cells harness gravity have remained unclear. Here, we show that mitochondria utilize gravity to activate protein synthesis within the organelle. Genome-wide ribosome profiling unveiled reduced mitochondrial translation in mammalian cells andCaenorhabditis elegansunder microgravity in the International Space Station and under simulated microgravity in the 3D-clinostat on the ground. In addition, we found that cell adhesion through laminin–integrin interaction, which is attenuated by microgravity, and the downstream FAK, RAC1, PAK1, BAD, and Bcl-2 family proteins relay the signals for mitochondrial protein synthesis. Consistent with the role of integrin as a mechanosensor, we observed the decrease in mitochondrial translation by minimization of mechanical stress in mouse skeletal muscle. Our work provides mechanistic insights into how cells convert gravitational and mechanical forces into translation in an energy-producing organelle.

Resistance exercise training (RET) can counteract negative features of muscle ageing but older age associates with reduced adaptive capacity to RET. Altered muscle protein networks likely contribute to ageing RET adaptation; therefore, associated proteome-wide responses warrant exploration. We employed quantitative sarcoplasmic proteomics to compare age-related proteome and phosphoproteome responses to RET. Thigh muscle biopsies were collected from eight young (25 ± 1.1 years) and eight older (67.5 ± 2.6 years) adults before and after 20 weeks supervised RET. Muscle sarcoplasmic fractions were pooled for each condition and analysed using Isobaric Tags for Relative and Absolute Quantification (iTRAQ) labelling, tandem mass spectrometry and network-based hub protein identification. Older adults displayed impaired RET-induced adaptations in whole-body lean mass, body fat percentage and thigh lean mass (P 0.05). iTRAQ identified 73 differentially expressed proteins with age and/or RET. Despite possible proteomic stochasticity, RET improved ageing profiles for mitochondrial function and glucose metabolism (top hub; PYK (pyruvate kinase)) but failed to correct altered ageing expression of cytoskeletal proteins (top hub; YWHAZ (14-3-3 protein zeta/delta)). These ageing RET proteomic profiles were generally unchanged or oppositely regulated post-RET in younger muscle. Similarly, RET corrected expression of 10 phosphoproteins altered in ageing, but these responses were again different vs. younger adults. Older muscle is characterised by RET-induced metabolic protein profiles that, whilst not present in younger muscle, improve untrained age-related proteomic deficits. Combined with impaired cytoskeletal adhesion responses, these results provide a proteomic framework for understanding and optimising ageing muscle RET adaptation.

SummarySarcopenia is a geriatric syndrome characterized by an age-related decline in skeletal muscle mass and strength. Here, we show that suppression of mitochondrial calcium uniporter (MCU)-mediated Ca2+ influx into mitochondria in the body wall muscles of the nematode Caenorhabditis elegans improved the sarcopenic phenotypes, blunting movement and mitochondrial structural and functional decline with age. We found that normally aged muscle cells exhibited elevated resting mitochondrial Ca2+ levels and increased mitophagy to eliminate damaged mitochondria. Similar to aging muscle, we found that suppressing MCU function in muscular dystrophy improved movement via reducing elevated resting mitochondrial Ca2+ levels. Taken together, our results reveal that elevated resting mitochondrial Ca2+ levels contribute to muscle decline with age and in muscular dystrophy. Further, modulation of MCU activity may act as a potent pharmacological target in various conditions involving muscle loss.

In legume-rhizobia symbiosis, partner recognition and the initiation of symbiosis processes require the mutual exchange of chemical signals. Chemicals, generally (iso)flavonoids, in the root exudates of the host plant induce the expression of nod genes in rhizobia, and, thus, are called nod gene inducers. The expression of nod genes leads to the production of lipochitooligosaccharides (LCOs) called Nod factors. Natural nod gene inducer(s) in Lotus japonicus-Mesorhizobium symbiosis remain unknown. Therefore, we developed an LCO detection method based on ultra-high-performance liquid chromatography-tandem-quadrupole mass spectrometry (UPLC-TQMS) to identify these inducers and used it herein to screen 40 phenolic compounds and aldonic acids for their ability to induce LCOs in Mesorhizobium japonicum MAFF303099. We identified five phenolic acids with LCO-inducing activities, including p-coumaric, caffeic, and ferulic acids. The induced LCOs caused root hair deformation, and nodule numbers in L. japonicus inoculated with M. japonicum were increased by these phenolic acids. The three phenolic acids listed above induced the expression of the nodA, nodB, and ttsI genes in a strain harboring a multicopy plasmid encoding NodD1, but not that encoding NodD2. The presence of p-coumaric and ferulic acids in the root exudates of L. japonicus was confirmed by UPLC-TQMS, and the induction of ttsI::lacZ in the strain harboring the nodD1 plasmid was detected in the rhizosphere of L. japonicus. Based on these results, we propose that phenolic acids are a novel type of nod gene inducer in L. japonicus-Mesorhizobium symbiosis.

Mechanical stimulation is well known to be important for maintaining tissue and organ homeostasis. Here, we found that hydrostatic pressure induced nuclear translocation of a forkhead box O (FOXO) transcription factor DAF-16, in C. elegans within minutes, whereas the removal of this pressure resulted in immediate export of DAF-16 to the cytoplasm. We also monitored DAF-16-dependent transcriptional changes by exposure to 1 MPa pressure for 5 min, and found significant changes in collagen and other genes in a DAF-16 dependent manner. Lifespan was markedly prolonged with exposure to cyclic pressure treatment (1 MPa once a day for 5 min from L1 larvae until death). Furthermore, age-dependent decline in locomotor activity was suppressed by the treatment. In contrast, the nuclear translocation of the yes-associated protein YAP-1 was not induced under the same pressure conditions. Thus, moderate hydrostatic pressure improves ageing progression through activation of DAF-16/FOXO in C. elegans.

The division of labour among DNA polymerase underlies the accuracy and efficiency of replication. However, the roles of replicative polymerases have not been directly established in human cells. We developed polymerase usage sequence (Pu-seq) in HCT116 cells and mapped Polε and Polα usage genome wide. The polymerase usage profiles show Polε synthesises the leading strand and Polα contributes mainly to lagging strand synthesis. Combining the Polε and Polα profiles, we accurately predict the genome-wide pattern of fork directionality plus zones of replication initiation and termination. We confirm that transcriptional activity contributes to the pattern of initiation and termination and, by separately analysing the effect of transcription on both co-directional and converging forks, demonstrate that coupled DNA synthesis of leading and lagging strands in both co- directional and convergent forks is compromised by transcription. Polymerase uncoupling is particularly evident in the vicinity of large genes, including the two most unstable common fragile sites, FRA3B and FRA3D, thus linking transcription-induced polymerase uncoupling to chromosomal instability.

Progressive neuromuscular decline in microgravity is a prominent health concern preventing interplanetary human habitation. We establish functional dopamine-mediated impairments as a consistent feature across multiple spaceflight exposures and during simulated microgravity in

形態: カラー図版あり, Physical characteristics: Original contains color illustrations, Accepted: 2021-08-03, 資料番号: PA2210078000

Atmospheric-pressure plasma (APP) technology, enabling to convert air molecules into multi-functional reactive species [e.g., reactive oxygen and nitrogen species (RONS)] with electricity, has been of great interest and extensively investigated. In particular, air APP devices, working only with air and electricity, can potentially allow for ubiquitous supply of RONS, which can be applied in a wide range of fields such as medical, agricultural, environmental, and biomaterial fields [1-4]. Recently, we have developed a new composite air APP device consisting low temperature and high temperature plasma reactors, enabling to supply RONS [e.g., dinitrogen pentoxide (N2O5), ozone (O3), nitric oxides (NOx), ...] with fine control and good reproducibility [5]. Specifically, its ability to generate high density (up to 200 ppm) N2O5 with high selectivity is quite unique and could accelerate scientific and industrial N2O5 applications. In addition, the APP device can utilize room air and renewable energy sources, such as a solar cell, and thus can realize sustainable and ubiquitous N2O5 supply. N2O5 is well known as a powerful oxidizing and nitrating agent and can potentially be bioactive. However, there are no previous studies using N2O5 in bio-applications, to our best knowledge. The reason is possibly due to its ordinary synthesis methods requiring multiple hazardous raw materials (requiring handling with much care). The air APP devices is user-friendly and can easily supply N2O5 to biomaterials (e.g., amino acid, protein, cells, virus, bacteria, ...). Thus, we are exploring the inactivation effects of N2O5 exposure on pathogen and virus, and modification of amino acid by the plasma-synthesized N2O5. Using the air APP device, we have investigated the inactivation effects on C. glo eosporioides (strawberry pathogen) and Qβ phage (RNA virus). 10μL of water droplet containing C. gloeosporioides was exposed to the plasma-synthesized N2O5 gas for 60 s, and typically 8h later, the germination rate of C. gloeosporioides conidium was evaluated as an indicator of inactivation effects. As a result, the N2O5 exposure significantly decreased the germination rate and the inactivation effect was not only due to pH decrease by HNO3aq transfer into the droplet from N2O5 gas. This indicates that N2O5aq, [NO2 +][ NO3 -]aq, or NO2 + aq may contribute to the inactivation. Also, mist particles containing Qβ phage were exposed to the plasma-synthesized N2O5gas, and the N2O5gas treatment resulted in over 3-log reduction in titer of Qβ phage. In addition, there were no significant difference of the inactivation effect between water and phosphate buffer mists, suggesting unique inactivation effects of N2O5gas other than pH decrease as noted above. Furthermore, we conducted experiments on the modification of amino acids such as tyrosine by plasma-synthesized N2O5gas. Tyrosine solution was treated by N2O5gas together with several reactive species such as O3gas or NO2gas, and it is found that dopachrome and nitrotyrosine were generated by the modification of tyrosine. Interestingly, dopachrome generation rate in N2O5gas with excess O3gas was most high, and the dopachrome generation was correlated with O3gas density. Further analysis using LC-PDA (liquid chromatography-photodiode array) and MS (mass spectrometer) quantified the tyrosine and tyrosine derivatives, and tyrosine was consumed at high rate, especially under N2O5gas with O3gas treatment. Major role of NO2 + aq is considered to be strong oxidizing capacity like OH radical, and N2O5gas triggers the generation of tyrosine intermediates. The tyrosine intermediates rapidly react with other reactive species such as O3gas and NO2gas, and then, dopachrome was significantly generated in N2O5gas with O3gas treatment and nitrotyrosine was in N2O5gas with NO2gas treatment. In the presentation, the details of the various biomaterial APP processes and plasma-synthesized N2O5 reaction pathway in the gas and liquid phase will be discussed. [1] Y. Kimura, K. Takashima, S. Sasaki, and T. Kaneko: J. Phys. D. Appl. Phys. 52, 064003 (2019). [2] K. Shimada, K. Takashima, Y. Kimura, K. Nihei, H. Konishi, and T. Kaneko: Plasma Process. Polym. 17, e1900004 (2020). [3] K. Takashima, Y. Hu, T. Goto, S. Sasaki, and T. Kaneko: J. Phys. D. Appl. Phys. 53, 354004 (2020). [4] K. Takashima, A.S. bin Ahmad Nor, S. Ando, H. Takahashi, and T. Kaneko: Jpn. J. Appl. Phys. 60, 010504 (2020). [5] S. Sasaki, K. Takashima, and T. Kaneko: Ind. Eng. Chem. Res. 60, 798 (2021).

The musculoskeletal system provides the body with correct posture, support, stability, and mobility. It is composed of the bones, muscles, cartilage, tendons, ligaments, joints, and other connective tissues. Without effective countermeasures, prolonged spaceflight under microgravity results in marked muscle and bone atrophy. The molecular and physiological mechanisms of this atrophy under unloaded conditions are gradually being revealed through spaceflight experiments conducted by the Japan Aerospace Exploration Agency using a variety of model organisms, including both aquatic and terrestrial animals, and terrestrial experiments conducted under the Living in Space project of the Japan Ministry of Education, Culture, Sports, Science, and Technology. Increasing our knowledge in this field will lead not only to an understanding of how to prevent muscle and bone atrophy in humans undergoing long-term space voyages but also to an understanding of countermeasures against age-related locomotive syndrome in the elderly.

Progressive neuromuscular decline is a prominent health concern preventing the world’s ambition of interplanetary human habitation. The precise causes of this decline and effective countermeasures remain elusive. We establish faltering dopamine signalling as a consistent feature across multiple spaceflight exposures and during simulated microgravity in C. elegans , which can be prevented with dopaminetargeting treatments. Loss of mechanical contact stimuli in microgravity induces dopamine-mediated functional impairments. Reinstating physical contact events also improves dopamine signalling, neuromuscular performance and associated calcium handling. This study reveals that reduced contact stimuli in microgravity causes dopamine-induced neuromuscular failure and that targeting this system through the external environment (contact stimuli), or clinically available drugs (dopamine pathway) are realistic, viable treatment strategies to promote safe human deep space travel.

Deep space exploration by humans has become more realistic, with planned returns to the Moon, travel to Mars, and beyond. Space radiation with a low dose rate would be a constant risk for space travelers. The combined effects of space radiation and partial gravity such as on the Moon and Mars are unknown. The difficulty for such research is that there are no good simulating systems on the ground to investigate these combined effects. To address this knowledge gap, we developed the Simulator of the environments on the Moon and Mars with Neutron irradiation and Gravity change (SwiNG) for in vitro experiments using disposable closed cell culture chambers. The device simulates partial gravity using a centrifuge in a three-dimensional clinostat. Six samples are exposed at once to neutrons at a low dose rate (1 mGy/day) using Californium-252 in the center of the centrifuge. The system is compact including two SwiNG devices in the incubator, one with and one without radiation source, with a cooling function. This simulator is highly convenient for ground-based biological experiments because of limited access to spaceflight experiments. SwiNG can contribute significantly to research on the combined effects of space radiation and partial gravity.

Bradyrhizobium elkanii, a rhizobium with a relatively wide host range, possesses a functional type III secretion system (T3SS) that is involved in symbiotic incompatibility against Rj4-genotype soybean (Glycine max) and some accessions of mung bean (Vigna radiata). To expand our knowledge on the T3SS-mediated partner selection mechanism in the symbiotic legume-rhizobia association, we inoculated three Lotus experimental accessions with wild-type and T3SS-mutant strains of B. elkanii USDA61. Different responses were induced by T3SS in a host genotype-dependent manner. Lotus japonicus Gifu inhibited infection; L. burttii allowed infection, but inhibited nodule maturation at the post-infection stage; and L. burttii and L. japonicus MG-20 both displayed a nodule early senescence-like response. By conducting inoculation tests with mutants of previously reported and newly identified effector protein genes of B. elkanii USDA61, we identified NopF as the effector protein triggering the inhibition of infection, and NopM as the effector protein triggering the nodule early senescence-like response. Consistent with these results, the B. elkanii USDA61 gene for NopF introduced into the Lotus symbiont Mesorhizobium japonicum induced infection inhibition in L. japonicus Gifu, but did not induce any response in L. burttii or L. japonicus MG-20. These results suggest that Lotus accessions possess at least three checkpoints to eliminate unfavorable symbionts, including the post-infection stage, by recognizing different T3SS effector proteins at each checkpoint.

Glucocorticoids are the drugs most commonly used to manage inflammatory diseases. However, they are prone to inducing muscle atrophy by increasing muscle proteolysis and decreasing protein synthesis. Various studies have demonstrated that antioxidants can mitigate glucocorticoid-induced skeletal muscle atrophy. Here, we investigated the effect of a potent antioxidative natural flavonoid, morin, on the muscle atrophy and oxidative stress induced by dexamethasone (Dex) using mouse C2C12 skeletal myotubes. Dex (10 μM) enhanced the production of reactive oxygen species (ROS) in C2C12 myotubes via glucocorticoid receptor. Moreover, Dex administration reduced the diameter and expression levels of the myosin heavy chain protein in C2C12 myotubes, together with the upregulation of muscle atrophy-associated ubiquitin ligases, such as muscle atrophy F-box protein 1/atrogin-1, muscle ring finger protein-1, and casitas B-lineage lymphoma proto-oncogene-b. Dex also significantly decreased phosphorylated Foxo3a and increased total Foxo3a expression. Interestingly, Dex-induced ROS accumulation and Foxo3a expression were inhibited by morin (10 μM) pretreatment. Morin also prevented the Dex-induced reduction of myotube thickness, together with muscle protein degradation and suppression of the upregulation of atrophy-associated ubiquitin ligases. In conclusion, our results suggest that morin effectively prevents glucocorticoid-induced muscle atrophy by reducing oxidative stress.

Acute onset of organ failure in heatstroke is triggered by rhabdomyolysis of skeletal muscle. Here, we showed that elevated temperature increases free cytosolic Ca2+ [Ca2+]f from RYR (ryanodine receptor)/UNC-68 in vivo in the muscles of an experimental model animal, the nematode Caenorhabditis elegans. This subsequently leads to mitochondrial fragmentation and dysfunction, and breakdown of myofilaments similar to rhabdomyolysis. In addition, treatment with an inhibitor of RYR (dantrolene) or activation of FoxO (Forkhead box O)/DAF-16 is effective against heat-induced muscle damage. Acute onset of organ failure in heatstroke is triggered by rhabdomyolysis of skeletal muscle. To gain insight into heat-induced muscle breakdown, we investigated alterations of Ca2+ homeostasis and mitochondrial morphology in vivo in body-wall muscles of C. elegans exposed to elevated temperature. Heat stress for 3 hr at 35° increased the concentration of [Ca2+]f, and led to mitochondrial fragmentation and subsequent dysfunction in the muscle cells. A similar mitochondrial fragmentation phenotype is induced in the absence of heat stress by treatment with a calcium ionophore, ionomycin. Mutation of the unc-68 gene, which encodes the ryanodine receptor that is linked to Ca2+ release from the sarcoplasmic reticulum, could suppress the mitochondrial dysfunction, muscle degeneration, and reduced mobility and life span induced by heat stress. In addition, in a daf-2 mutant, in which the DAF-16/FoxO transcription factor is activated, resistance to calcium overload, mitochondrial fragmentation, and dysfunction was observed. These findings reveal that heat-induced Ca2+ accumulation causes mitochondrial damage and consequently induces muscle breakdown.

Mitochondrial dysfunction impairs muscle health and causes subsequent muscle wasting. This study explores the role of mitochondrial dysfunction as an intramuscular signal for the extracellular matrix (ECM)–based proteolysis and, consequentially, muscle cell dystrophy. We found that inhibition of the mitochondrial electron transport chain causes paralysis as well as muscle structural damage in the nematode Caenorhabditis elegans. This was associated with a significant decline in collagen content. Both paralysis and muscle damage could be rescued with collagen IV overexpression, matrix metalloproteinase (MMP), and Furin inhibitors in Antimycin A–treated animal as well as in the C. elegans Duchenne muscular dystrophy model. Additionally, muscle cytosolic calcium increased in the Antimycin A–treated worms, and its down-regulation rescued the muscle damage, suggesting that calcium overload acts as one of the early triggers and activates Furin and MMPs for collagen degradation. In conclusion, we have established ECM degradation as an important pathway of muscle damage.—Sudevan, S., Takiura, M., Kubota, Y., Higashitani, N., Cooke, M., Ellwood, R. A., Etheridge, T., Szewczyk, N. J., Higashitani, A. Mitochondrial dysfunction causes Ca2+ overload and ECM degradation–mediated muscle damage in C. elegans.

In eukaryotes, the DNA damage bypass pathway is promoted by ubiquitylation of PCNA at the conserved lysine 164. Using CRISPR-Cas9 system, we introduced amino acid substitution at K165 of C. elegans PCNA that corresponds to K164 in other characterised organisms and examined the contribution of this residue at a variety of stages during development. In the presence of UV-induced DNA lesions, PCNA-K165 is crucial for not only the early embryonic stages but also during larval development, implicating its functions for a broad time period during animal development. We also show that, without induction of DNA damage, concomitant inhibition of PCNA ubiquitylation and checkpoint activation causes abnormal gametogenesis events and severely impairs reproduction of worms. Our findings suggest a conserved function of PCNA ubiquitylation in tolerance of UV-induced damage and also propose that PCNA ubiquitylation contributes to gametogenesis during unperturbed C. elegans development.

Proinflammatory cytokines contribute to the progression of muscle wasting caused by ubiquitin-proteasome-dependent proteolysis. We have previously demonstrated that isoflavones, such as genistein and daidzein, prevent TNF-α-induced muscle atrophy in C2C12 myotubes. In this study, we examined the effect of dietary flavonoids on the wasting of muscle. Mice were divided into the following four groups: vehicle-injected (control) mice fed the normal diet (CN); tumor-bearing mice fed the normal diet (TN); control mice fed the isoflavone diet (CI); and tumor-bearing mice fed the isoflavone diet (TI). There were no significant differences in the intake of food or body weight gain among these four groups. The wet weight and myofiber size of gastrocnemius muscle in TN significantly decreased, compared with those in CN. Interestingly, the wet weight and myofiber size of gastrocnemius muscle in TI were nearly the same as those in CN and CI, although isoflavone supplementation did not affect the increased tumor mass or concentrations of proinflammatory cytokines, such as TNF-α and IL-6, in the blood. Moreover, increased expression of muscle-specific ubiquitin ligase genes encoding MAFbx/Atrogin-1 and MuRF1 in the skeletal muscle of TN was significantly inhibited by the supplementation of isoflavones. In parallel with the expression of muscle-specific ubiquitin ligases, dietary isoflavones significantly suppressed phosphorylation of ERK in tumor-bearing mice. These results suggest that dietary isoflavones improve muscle wasting in tumor-bearing mice via the ERK signaling pathway mediated-suppression of ubiquitin ligases in muscle cells.

Cachexia, observed in most cancer patients, is a syndrome that includes wasting of bodily energy reserves and is characterized by muscle atrophy and fat loss. We have previously demonstrated that isoflavones, such as genistein and daidzein, prevent muscle wasting in tumor-bearing mice. In this study, we examined the effect of morin, a flavonoid, on cachexia. The wet weight and myofiber size of muscles in Lewis lung carcinoma (LLC) cell-bearing mice fed a normal diet were decreased, compared with those in control mice fed a normal diet. In contrast, intake of morin prevented the reduction of muscle wet weight and myofiber size. Moreover, the tumor weight in mice fed the morin diet was lower than that in mice fed the normal diet. Both cell viability and protein synthetic ability of LLC cells were reduced by treatment with morin, but C2C12 myotubes were not affected. Binding assay using morin-conjugated magnetic beads identified ribosomal protein S10 (RPS10) as a target protein of morin. Consistent with the result of morin treatment, knockdown of RPS10 suppressed LLC cell viability. These results suggest that morin indirectly prevents muscle wasting induced by cancer cachexia by suppressing cancer growth via binding to RPS10.

The target of rapamycin (TOR) signaling pathway is involved in starch accumulation in various eukaryotic organisms; however, the molecular mechanism behind this phenomenon in eukaryotes has not been elucidated. We report a regulatory mechanism of starch accumulation by TOR in the unicellular red alga, Cyanidioschyzon merolae. The starch content in C. merolae after TOR-inactivation by rapamycin, a TOR-specific inhibitor, was increased by approximately 10-fold in comparison with its drug vehicle, dimethyl sulfoxide. However, our previous transcriptome analysis showed that the expression level of genes related to carbohydrate metabolism was unaffected by rapamycin, indicating that starch accumulation is regulated at post-transcriptional levels. In this study, we performed a phosphoproteome analysis using liquid chromatography-tandem mass spectrometry to investigate potential post-transcriptional modifications, and identified 52 proteins as candidate TOR substrates. Among the possible substrates, we focused on the function of CmGLG1, because its phosphorylation at the Ser613 residue was decreased after rapamycin treatment, and overexpression of CmGLG1 resulted in a 4.7-fold higher starch content. CmGLG1 is similar to the priming protein, glycogenin, which is required for the initiation of starch/glycogen synthesis, and a budding yeast complementation assay demonstrated that CmGLG1 can functionally substitute for glycogenin. We found an approximately 60% reduction in the starch content in a phospho-mimicking CmGLG1 overexpression strain, in which Ser613 was substituted with aspartic acid, in comparison with the wild-type CmGLG1 overexpression cells. Our results indicate that TOR modulates starch accumulation by changing the phosphorylation status of the CmGLG1 Ser613 residue in C. merolae.

Microsporogenesis in rice (Oryza sativa) plants is susceptible to moderate low temperature (LT; approximately 19°C) that disrupts pollen development and causes severe reductions in grain yields. Although considerable research has been invested in the study of cool-temperature injury, a full understanding of the molecular mechanism has not been achieved. Here, we show that endogenous levels of the bioactive gibberellins (GAs) GA4 and GA7, and expression levels of the GA biosynthesis genes GA20ox3 and GA3ox1, decrease in the developing anthers by exposure to LT. By contrast, the levels of precursor GA12 were higher in response to LT. In addition, the expression of the dehydration-responsive element-binding protein DREB2B and SLENDER RICE1 (SLR1)/DELLA was up-regulated in response to LT. Mutants involved in GA biosynthetic and response pathways were hypersensitive to LT stress, including the semidwarf mutants sd1 and d35, the gain-of-function mutant slr1-d, and gibberellin insensitive dwarf1. The reduction in the number of sporogenous cells and the abnormal enlargement of tapetal cells occurred most severely in the GA-insensitive mutant. Application of exogenous GA significantly reversed the male sterility caused by LT, and simultaneous application of exogenous GA with sucrose substantially improved the extent of normal pollen development. Modern rice varieties carrying the sd1 mutation are widely cultivated, and the sd1 mutation is considered one of the greatest achievements of the Green Revolution. The protective strategy achieved by our work may help sustain steady yields of rice under global climate change.

Autophagy is one of the cellular processes that break down cellular components during senescence, starvation, and stress. The susceptibility of plant pollen development to high-temperature (HT) stress is well known, but the involvement of autophagy in HT injury is yet to be clarified. Here, we found that following transfer to 30 °C, all autophagy-deficient (atg) mutants (atg2-1, 5-1, 7-2, and 10-1) of Arabidopsis thaliana tested displayed visibly impaired pollen development and anther dehiscence. HT-induced male sterility significantly increased in the atg mutants, but the degree of HT-induced obstacles did not change between the wild type (WT) and mutants from the seedling stage to the bolting stage. Cytological analyses showed that 30 °C promoted autophagy and autolysosome formation in both anther wall cells and microspores in developing anthers of WT, but the atg5-1 mutant did not show completion of tapetum degeneration and microspore maturation. HT upregulated hydrogen peroxide and dehydroascorbate reductase 1 production in both WT and atg5-1 anthers, but the basal levels were already higher in the mutant. HT repressed expression of UNDEAD and its regulator MYB80, which are required for tapetal programmed cell death (PCD) for proper pollen development. Taken together, our results suggest that autophagy functions in tapetum degeneration and pollen development during HT-caused tapetal PCD abortion.HighlightsIn Arabidopsis, autophagy is not essential for completion of the life cycle under normal temperatures.High temperature (HT) stress induces autophagy in developing anther wall cells and microspores.Autophagy deficient atg mutants become almost completely male-sterile at moderate HT.Autophagy plays a role in tapetum degeneration and pollen development during HT-caused abortion of tapetal program cell death.

ATR is highly conserved in all eukaryotes and functions as a cell-cycle checkpoint kinase to stabilize the nuclear genome. In addition, knockout mouse models indicate that ATR is essential for viability. Here, it is shown that moderate overproduction of ATR, but not of the other phosphatidylinositol 3′ kinase-related kinases, ataxia-telangiectasia-mutated, mTOR and SMG-1, and a downstream target p53, resulted in cell death. ATR over-expression induced cellular vacuolization from 12 to 48 h after transfection, before cell death progression. A series of deletion analyses showed that overproduction of the N-terminal HEAT repeat segments of ATR was sufficient for the induction of the vacuolization. Moreover, post-transcriptional modification of LC3, a marker of autophagy, and autophagosomes with double membranes were evident in ATR-overproducing cells. The vacuolization was also suppressed in autophagy-deficient MCF7 cells. In addition, both cellular vacuolization and cell death were reduced by inhibition of Ras activity using farnesyl thiosalicylic acid. Conversely, neither inhibition of mTOR nor activation of the checkpoint system could be observed in the vacuolated cells. These results suggest that the Ras signaling pathway is involved in the autophagic response caused by ATR overproduction, and tight regulation of ATR protein expression is crucial for cell viability.

Although muscle atrophy is a serious problem during spaceflight, little is known about the sequence of molecular events leading to atrophy in response to microgravity. We carried out a spaceflight experiment using Caenorhabditis elegans onboard the Japanese Experiment Module of the International Space Station. Worms were synchronously cultured in liquid media with bacterial food for 4 days under microgravity or on a 1-G centrifuge. Worms were visually observed for health and movement and then frozen. Upon return, we analyzed global gene and protein expression using DNA microarrays and mass spectrometry. Body length and fat accumulation were also analyzed. We found that in worms grown from the L1 larval stage to adulthood under microgravity, both gene and protein expression levels for muscular thick filaments, cytoskeletal elements, and mitochondrial metabolic enzymes decreased relative to parallel cultures on the 1-G centrifuge (95% confidence interval (P less than or equal to 0.05)). In addition, altered movement and decreased body length and fat accumulation were observed in the microgravity-cultured worms relative to the 1-G cultured worms. These results suggest protein expression changes that may account for the progressive muscular atrophy observed in astronauts., 形態: カラー図版あり, Physical characteristics: Original contains color illustrations, 資料番号: PA1610036000

CDC-48/p97 is a AAA (ATPases associated with diverse cellular activities) chaperone involved in protein conformational changes such as the disassembly of protein complexes. We previously reported that Caenorhabditis elegans CDC-48.1 and CDC-48.2 (CDC-48s) are essential for the progression of meiosis I metaphase. Here, we report that CDC-48s are required for proper chromosome segregation during meiosis in C. elegans. In wild-type worms, at the diakinesis phase, phosphorylation of histone H3, one of the known substrates of aurora B kinase (AIR-2), on meiosis I chromatids correlated with AIR-2 localization at the cohesion sites of homologous chromatids. Conversely, depletion of CDC-48s resulted in a significant expansion of signals for AIR-2 and phosphorylated histone H3 over the entire length of meiotic chromosomes, leading to defective chromosome segregation, while the total amount of AIR-2 in lysates was not changed by the depletion of CDC-48s. The defective segregation of meiotic chromosomes caused by the depletion of CDC-48s was suppressed by the simultaneous depletion of AIR-2 and is similar to that observed following the depletion of protein phosphatase 1 (PP1) phosphatases. However, the amount and localization of PP1 were not changed by the depletion of CDC-48s. These results suggest that CDC-48s control the restricted localization of AIR-2 to the cohesion sites of homologous chromatids in meiosis I.

Microphthalmia-associated transcription factor (Mitf) is a regulator for differentiation of melanoblasts that are derived from the neural crest. The mouse homozygous for the black-eyed white (Mitfmi-bw) allele is characterized by the white coat color and deafness, with black eye that is associated with the lack of melanocytes in skin and inner ear. The Mitfmi-bw mutation is an insertion of the LINE1 retrotransposable element into intron 3 of the Mitf gene that causes the selective deficiency of the melanocyte-specific Mitf isoform, Mitf-M. Here, we show the expression of Mitf-M mRNA in the trunk region of the homozygous Mitfmi-bw (bw) mouse at embryonic days (E) 11.5 and E12.5, but Mitf-M mRNA is undetectable at E13.5. In addition, using bw mouse that carries the lacZ transgene under the control of a melanoblast-specific promoter, we show that the number of migrating melanoblasts in bw embryos was less than 10% of that in control embryos at E11.5 and E12.5, and melanoblasts disappear by E13.5. The loss of melanoblasts in bw embryos was probably caused by apoptosis. Finally, forced expression of Mitf-M in the cultured neural tube of bw embryos ensured the differentiation of melanoblasts. Therefore, the correct dose of Mitf-M is required for the normal development of melanoblasts.

Many plant species are susceptible to high-temperature (HT) injury during reproductive development. We recently demonstrated that HT represses the expression of YUCCA auxin biosynthesis genes and reduces endogenous auxin in the developing anthers of barley and Arabidopsis. Here, we show that DNA proliferation in mitochondria, chloroplasts and nuclei of developing panicles is inhibited with increasing temperatures in barley. Following DNA proliferation suppression, terminal abnormalities were observed in the organelles of anther wall cells, including mitochondrial swelling and overdevelopment of chloroplasts. Comprehensive transcriptome analyses using both reproductive organs and vegetative tissues showed high and positive pairwise correlations between the expression profiles of auxin-induced genes, DNA replication-related genes and mitochondrial-related genes. In contrast, the expression profiles of auxin-repressed protein genes and photosynthesis-/chloroplast-related genes were negatively correlated with those of the previously mentioned genes. Under HT conditions, the former was repressed and the latter was up-regulated in the developing panicles. Furthermore, application of exogenous auxin promoted the expression of DNA replication-related genes under HT conditions, inducing anther cell proliferation. These suggest that compromised auxin biosynthesis/IAA level under HT condition results in nuclear and organellar DNA proliferation arrest due to co-transcriptional alterations.

With global warming, plant high temperature injury is becoming an increasingly serious problem. In wheat, barley, and various other commercially important crops, the early phase of anther development is especially susceptible to high temperatures. Activation of auxin biosynthesis with increased temperatures has been reported in certain plant tissues. In contrast, we here found that under high temperature conditions, endogenous auxin levels specifically decreased in the developing anthers of barley and Arabidopsis . In addition, expression of the YUCCA auxin biosynthesis genes was repressed by increasing temperatures. Application of auxin completely reversed male sterility in both plant species. These findings suggest that tissue-specific auxin reduction is the primary cause of high temperature injury, which leads to the abortion of pollen development. Thus, the application of auxin may help sustain steady yields of crops despite future climate change.

p97 (CDC-48 in Caenorhabditis elegans) is a ubiquitin-selective AAA (ATPases associated with diverse cellular activities) chaperone and its key function is to disassemble protein complexes. p97 functions in diverse cellular processes including endoplasmic reticulum (ER)-associated degradation, membrane fusion, and meiotic and mitotic progression. However, its cellular functions in development have not yet been clarified. Here, we present data that p97 is involved in the switch from spermatogenesis to oogenesis in the germline of the C. elegans hermaphrodite. We found that the cdc-48.1 deletion mutant produced less sperm than the wild type and thus showed a decreased brood size. The cdc-48.1 mutation suppressed the sperm-overproducing phenotypes of fbf-1 and fem-3(gf) mutants. In addition, the p97/CDC-48–UFD-1–NPL-4 complex interacted with the E3 ubiquitin ligase CUL-2 complex via NPL-4 binding to Elongin C. Furthermore, TRA-1A, which is the terminal effector of the sex determination pathway and is regulated by CUL-2-mediated proteolysis, accumulated in the cdc-48.1 mutant. Proteasome activity was also required for the brood size determination and sperm-oocyte switch. Our results demonstrate that the C. elegans p97/CDC-48–UFD-1–NPL-4 complex controls the sperm-oocyte switch by regulating CUL-2-mediated TRA-1A proteasome degradation.