Impairment of ovarian follicular development caused by titanium dioxide nanoparticles exposure involved in the TGF-ß/BMP/Smad pathway.

Titanium dioxide nanoparticles (TiO2 NPs) have been shown to induce reproductive system damages in animals. To better underline how TiO2 NPs act in reproductive system, female mice were exposed to 2.5, 5, or 10 mg/kg TiO2 NPs by gavage administration for 60 days, the ovary injuries, follicle stimulating hormone (FSH) and luteinizing hormone (LH) levels as well as ovarian follicular development-related molecule expression were investigated. The results showed that TiO2 NPs exposure resulted in reduction of ovary weight and inhibition of ovarian follicular development. Furthermore, the suppression of follicular development was demonstrated to be closely related to higher FSH and LH levels, and higher expression of activin, follistatin, BMP2, BMP4, TGF-ß1, Smad2, Smad3, and Smad4 as well as decreased inhibin-α expression in mouse ovary in a dose-dependent manner. It implies that the impairment of ovarian follicular development caused by TiO2 NPs exposure may be mediated by TGF-ß signal pathway.

Mutation in OsFWL7 Affects Cadmium and Micronutrient Metal Accumulation in Rice.

Micronutrient metals, such as Mn, Cu, Fe, and Zn, are essential heavy metals for plant growth and development, while Cd is a nonessential heavy metal that is highly toxic to both plants and humans. Our understanding of the molecular mechanisms underlying Cd and micronutrient metal accumulation in plants remains incomplete. Here, we show that OsFWL7, an FW2.2-like (FWL) family gene in Oryza sativa, is preferentially expressed in the root and encodes a protein localized to the cell membrane. The osfwl7 mutation reduces both the uptake and the root-to-shoot translocation of Cd in rice plants. Additionally, the accumulation of micronutrient metals, including Mn, Cu, and Fe, was lower in osfwl7 mutants than in the wildtype plants under normal growth conditions. Moreover, the osfwl7 mutation affects the expression of several heavy metal transporter genes. Protein interaction analyses reveal that rice FWL proteins interact with themselves and one another, and with several membrane microdomain marker proteins. Our results suggest that OsFWL7 is involved in Cd and micronutrient metal accumulation in rice. Additionally, rice FWL proteins may form oligomers and some of them may be located in membrane microdomains.

Damage to the Blood Brain Barrier Structure and Function from Nano Titanium Dioxide Exposure Involves the Destruction of Key Tight Junction Proteins in the Mouse Brain.

Numerous studies have proven that nano titanium dioxide (nano TiO2) can accumulate in animal brains, where it damages the blood brain barrier (BBB); however, whether this process involves destruction of tight junction proteins in the mouse brain has not been adequately investigated. In this study, mice were exposed to nano TiO2 for 30 consecutive days, and then we used transmission electron microscopy to observe the BBB ultrastructure and the Evans blue assay to evaluate the permeability of the BBB. Our data suggested that nano TiO2 damaged the BBB ultrastructure and increased BBB permeability. Furthermore, we used immunofluorescence and Western blotting to examine the expression of key tight junction proteins, including Occludin, ZO-1, and Claudin-5 in the mouse brain. Our data showed that nano TiO2 reduced Occludin, ZO-1 and Claudin-5 expression. Taken together, nano TiO2-induced damage to the BBB structure and function may involve the destruction of key tight junction proteins.

Molecular mechanism of mice gastric oxidative damage induced by nanoparticulate titanium dioxide.

BACKGROUND: Nanoparticulate titanium dioxide (Nano-TiO2) has been widely used in food industry, and it has been demonstrated to have adverse effects on mice and human stomach, but its mechanism is rarely concerned. The aim of this study is to determine the effects of nano-TiO2 on the stomach and confirm the role of oxidative stress and apoptosis in the mice gastric damage caused by nano-TiO2, as well as its molecular mechanisms. METHODS: Mice were continuously exposed to nano-TiO2 with 1.25, 2.5 and 5 mg/kg bw by intragastric administration for 9 months in the present study. The ultrastructure, levels of reactive oxygen species (ROS) and peroxides, activities of antioxidant enzymes and mitochondria-related enzymes, ATP contents as well as apoptosis-related factors expression in mice stomach were examined. RESULTS: Oxidative stress, apoptosis and nano-TiO2 aggregation were found in gastric mucosal smooth muscle cells after nano-TiO2 exposure. Nano-TiO2 exposure also resulted in the over-production of ROS and peroxides, decrease of ATP production and activities of antioxidant enzymes and mitochondria-related ATPases, upregulation of apoptosis-related factors including γH2AX, Cyt c, caspase 3, and p-JNK expression, and down-regulation of Bcl-2 expression in mice stomach. CONCLUSIONS: The gastric toxicity of mice induced by chronic exposure to low dose nano-TiO2 may be associated with oxidative stress and mitochondria-mediated apoptosis in mice.

PRD1, a homologous recombination initiation factor, is involved in spindle assembly in rice meiosis.

The bipolar spindle structure in meiosis is essential for faithful chromosome segregation. PUTATIVE RECOMBINATION INITIATION DEFECT 1 (PRD1) previously has been shown to participate in the formation of DNA double strand breaks (DSBs). However, the role of PRD1 in meiotic spindle assembly has not been elucidated. Here, we reveal by both genetic analysis and immunostaining technology that PRD1 is involved in spindle assembly in rice (Oryza sativa) meiosis. We show that DSB formation and bipolar spindle assembly are disturbed in prd1 meiocytes. PRD1 signals display a dynamic pattern of localization from covering entire chromosomes at leptotene to congregating at the centromere region after leptotene. Centromeric localization of PRD1 signals depends on the organization of leptotene chromosomes, but not on DSB formation and axis establishment. PRD1 exhibits interaction and co-localization with several kinetochore components. We also find that bi-orientation of sister kinetochores within a univalent induced by mutation of REC8 can restore bipolarity in prd1. Furthermore, PRD1 directly interacts with REC8 and SGO1, suggesting that PRD1 may play a role in regulating the orientation of sister kinetochores. Taken together, we speculate that PRD1 promotes bipolar spindle assembly, presumably by modulating the orientation of sister kinetochores in rice meiosis.

Molecular mechanisms associated with oxidative damage in the mouse testis induced by LaCl3.

China is the world's largest rare earth producer and exporter, previous studies have shown that rare earth elements can cause oxidative damage in animal testis. However, the molecular mechanisms underlying these observations have yet to be elucidated. In this paper, male mice were fed with different doses (10, 20, and 40 mg/kg BW) of LaCl3 for 90 consecutive days, regulatory role of nuclear factor erythroid-2 related factor 2 (Nrf-2)/antioxidant response element (ARE) pathway in testicular oxidative stress induced by LaCl3 were investigated. Analysis showed that LaCl3 exposure could lead to severe testicular pathological changes and apoptosis in spermatogenic cells, it up-regulated the peroxidation of lipids, proteins and DNA, and induced the excessive levels of reactive oxygen species (ROS) production in mouse testis, reduced the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and glutathione S epoxide transferase (GST) as well as the glutathione (GSH) content. Furthermore, exposure to LaCl3 also downregulated the expression of Nrf2 and its target gene products, including heme oxygenase 1 (HO-1), glutamate-cysteine ligase catalytic subunit (GCLC), NAD(P)H dehydrogenase [quinine] 1(NQO1), protein kinase C (PKC), and phosphatidylinositol 3-kinase (PI3K), but upregulated the expression of Kelch-like ECH-related protein 1 (Keap1) in damaged mouse testes. Collectively, our data imply that the oxidative damage induced by LaCl3 in testis was related to inhibition of the Nrf-2/AREs pathway activation.

Liver Inflammation and Fibrosis Induced by Long-Term Exposure to Nano Titanium Dioxide (TiO2) Nanoparticles in Mice and Its Molecular Mechanism.

Titanium dioxide (TiO2) and nano-sized titanium dioxide (nano-TiO2), which are used in food production, may be harmful to the body. Long-term exposure to nano-TiO2 can lead to hepatic injury; however, the effect of nano-TiO2 on liver fibrosis and the underlying mechanism remain unclear. The TGF-ß/Smad/MAPK/Wnt signaling pathway is important for tissue fibrosis. In this study, mice were fed nano-TiO2 (2.5, 5, and 10 mg/kg body weight) for nine consecutive months to investigate its effect on liver fibrosis. Nano-TiO2 induced hepatic inflammatory cell infiltration and hepatic fibrosis and upregulated the expression of HIF-1α (+75-fold to +2.38-fold), Wnt3 (+12% to +135%), Wnt4 (1.33-fold to 6-fold), NF-κB (+3.13% to +34.38%), TGF-ß1 (+1307-fold to +1.85-fold), TGF-ß1R (+0.8-fold to 1.33-fold), Smad-2 (+0.58-fold to +1.58-fold), ILK (+0.43-fold to +1.19-fold), ECM (+1.82-fold to 2.36-fold), calpain 2 (+0.11-fold to +0.78-fold), α-SMA (+0.63-fold to +1.56-fold), c-Myc (+0.27-fold to +0.46-fold), and collagen I (+8% to +36%), and increased the phosphorylation level of p38MAPK (+66.67% to +153.33%) in inflammatory and fibrotic liver tissues, whereas it downregulated cyclin D (-6.25% to -43.75%) and decreased the phosphorylation levels of GSK-3ß (-3.12% to -46.88%) and ß-catenin (-19.57% to -45.65%). These results indicate that hepatic fibrosis induced by nano-TiO2 is mediated by the TGF-ß/Smads/MAPK/Wnt signaling pathway. This study provides insight into the mechanism underlying hepatic toxicity induced by nano-TiO2 .

Nanostructured Titanium Dioxide (TiO2) Reduces Sperm Concentration Involving Disorder of Meiosis and Signal Pathway.

Nano-titanium dioxide (nano-TiO2) has been widely used in food and cosmetic industries, and the medical sector. However, nano-TiO2 is potentially toxic to the reproductive system. Previous research has shown that nano-TiO2 can reduce sperm concentration but do not yet known whether this effect occurs because of dysfunctional meiosis in spermatogenic cells. In the present paper, we demonstrate that Nano-TiO2 can penetrate through the blood-testis barrier of a mouse model and enter the testicular tissue, thus causing damage to the testis and epididymis. This reduced the number of developing sperm; we demonstrated that the mechanism underlying this effect was the inhibition or destruction of meiosis in spermatogenic cells, particularly during meiosis I. We also found that the inhibition of meiosis I caused by nano-TiO2 exposure was related to dysfunctional meiosis and that the abnormal expression of meiosis-related factors. Therefore, our data demonstrate that nano-TiO2 reduces sperm concentration by disrupting meiosis and related signaling pathways.

Targeted Mutagenesis of the Rice FW 2.2-Like Gene Family Using the CRISPR/Cas9 System Reveals OsFWL4 as a Regulator of Tiller Number and Plant Yield in Rice.

The FW2.2-like (FWL) genes encode cysteine-rich proteins with a placenta-specific 8 domain. They play roles in cell division and organ size control, response to rhizobium infection, and metal ion homeostasis in plants. Here, we target eight rice FWL genes using the CRISPR/Cas9 system delivered by Agrobacterium-mediated transformation. We successfully generate transgenic T0 lines for 15 of the 16 targets. The targeted mutations are detected in the T0 lines of all 15 targets and the average mutation rate is found to be 81.6%. Transfer DNA (T-DNA) truncation is a major reason for the failure of mutagenesis in T0 plants. T-DNA segregation analysis reveals that the T-DNA inserts in transgenic plants can be easily eliminated in the T1 generation. Of the 30 putative off-target sites examined, unintended mutations are detected in 13 sites. Phenotypic analysis reveals that tiller number and plant yield of OsFWL4 gene mutants are significantly greater than those of the wild type. Flag leaves of OsFWL4 gene mutants are wider than those of the wild type. The increase in leaf width of the mutants is caused by an increase in cell number. Additionally, grain length of OsFWL1 gene mutants is higher than that of the wild type. Our results suggest that transgene-free rice plants with targeted mutations can be produced in the T1 generation using the Agrobacterium-mediated CRISPR/Cas9 system and that the OsFWL4 gene is a negative regulator of tiller number and plant yield.

Respiratory exposure to nano-TiO2 induces pulmonary toxicity in mice involving reactive free radical-activated TGF-ß/Smad/p38MAPK/Wnt pathways.

Numerous studies have shown that lung injury can be caused by respiratory exposure to nanoparticulate titanium dioxide (nano-TiO2 ), but whether pulmonary inflammation and fibrosis are related to the activation of the TGF-ß/Smad/p38MAPK/Wnt pathways remains unclear. In this study, mice were administrated nano-TiO2 by nasal instillation for nine consecutive months, and the molecular mechanisms of nano-TiO2 on the pulmonary toxicity of mice were examined. The findings suggested that nano-TiO2 caused pneumonia and pulmonary fibrosis. Furthermore, the results also showed that an overproduction of reactive free radicals occurred in mouse lungs, and that the expression of TGF-ß/p38MAPK/Wnt pathway-related factors, including hypoxia-inducible factor 1α (HIF-1α), transforming growth factor-ß1 (TGF-ß1), phosphorylated p38 mitogen activated protein kinases (p-p38MAPK), small mothers against decapentaplegic homolog 2 (Smad2), extracellular matrix (ECM), Wingless/Integrated 3 (Wnt3), Wingless/Integrated 4 (Wnt4), integrin-linked kinase (ILK), ß-catenin, nuclear factor-κB (NF-κB), α-smooth muscle actin (α-SMA), c-Myc, Type I collage (collagen I), and Type collage III (collagen III) were remarkably elevated, while phosphorylated glycogen synthase kinase-3ß (p-GSK-3ß) expression was decreased. Those data implied that the pulmonary inflammation and fibrosis caused by nano-TiO2 exposure may be involved in reactive free radical-mediated activation of the TGF-ß/Smad/p38MAPK/Wnt pathways.

Nanoparticulate TiO2 Induced Suppression of Spermatogenesis is Involved in Regulatory Dysfunction of the cAMP-CREB/CREM Signaling Pathway in Mice.

Long-term exposure to nanoparticulate titanium dioxide (nano-TiO2) is known to cause reductions of sperm numbers and quality in animals, and the cAMP-dependent signaling pathway has been demonstrated to play a key role in regulating spermatogenesis. However, whether the suppression of spermatogenesis induced by nano-TiO2 is related to regulatory disturbances of the cAMP-CREB/CREM signaling pathway is not well investigated. In the current study, male mice were exposed to nano-TiO2 at doses of 1.25, 2.5, or 5 mg/kgbw via gavage instillation for 90 consecutive days and the molecular mechanisms underlying suppression of spermatogenesis caused by nano-TiO2 were investigated. Our findings showed that nano-TiO2 could cross the blood-testis barrier, and accumulated in mouse testes, thus inducing obvious pathological changes and decreasing sperm concentrations and motility, as well as increasing rate of sperm malformation. Furthermore, nano-TiO2 also induced significant reductions in protein expression including cyclic adenosine monophosphate content, protein kinase A, cAMP-responsive element modulator, p-cAMP-response element binding protein, lactate dehydrogenase-C, testis-specific protein kinase 1, and testicular specific CREM activator, and upregulation of protein expression including protein phosphatase, and transducer of regulated CREB 1, which may be associated with reductions of follicle stimulating hormone and luteinizing hormone levels. Together, the present study indicates that the reductions of FSH and LH concentrations and suppression of spermatogenesis in mice caused by nano-TiO2 may be associated with the dysfunctions of the cAMP-CREB/CREM signaling pathway.

Maternal Exposure to Nanoparticulate Titanium Dioxide Causes Inhibition of Hippocampal Development Involving Dysfunction of the Rho/NMDAR Signaling Pathway in Offspring.

Numerous studies have suggested that nano-TiO2 can be translocated to the brain via the placental barrier and blood brain barrier, leading to brain damage and cognitive impairment in both mice and rat offspring. The mechanism of nanoTiO2-induced neurotoxicity is still unclear, as is its role in the inhibition of hippocampal development. In this experiment, nano-TiO2 was employed to investigate whether the inhibition of the hippocampal development of mice offspring involved the alterations in the Rho signaling pathway following consecutive gavage of female mice between 7-21 days postpartum. The results showed that nano-TiO2 particles were concentrated in the hippocampus of offspring, resulting in reduced hippocampal indices and in inhibited axonal and dendritic growth. Furthermore, nano-TiO2 downregulated expression of N-methyl-D-aspartate receptor (NR)1, NR2A, NR2B, RhoGTPase, Ras-related C1 botulinum toxin substrate (Rac1), cell division cycle42 (Cdc42), phosphorylated cAMP response element binding protein (p-CREB), p21-activated kinase (PAK) 3, and PAK1, LIMK (LIM kinase) 1, p-LIMK1, activated Cdc42 kinase (ACK), and myotonic dystrophic kinaseassociated Cdc42-binding kinase (MRCK) and increased expression of RhoA, Rho kinase (ROCK) 1 and cyclin dependent kinase (Cdk) 5 in offspring. In addition, nano-TiO2 disrupted the balance of RhoA/Rac1, RhoA/Cdc42, and Rac1/Cdc42 ratios in the hippocampus of mice offspring. Taken together, these data imply that maternal exposure to nano-TiO2 inhibited development of hippocampal axons and dendrites of offspring may be correlated with the dysfunction of the Rho pathway and that N-methyl-D-aspartate receptors (NMDAR) may also mediate nano-TiO2-Rho pathway interactions.

Retardation of Axonal and Dendritic Outgrowth Is Associated with the MAPK Signaling Pathway in Offspring Mice Following Maternal Exposure to Nanosized Titanium Dioxide.

Exposure to nanosized titanium oxide (nano-TiO2) has been proven to suppress brain growth in mouse offspring; however, whether retardation of axonal or dendritic outgrowth is associated with activation of the mitogen-activated protein kinase (MAPK) pathway remains unclear. In the present study, pregnant mice were exposed to nano-TiO2 at 1.25, 2.5, and 5 mg/kg body weight, and the molecular mechanism of axonal or dendritic outgrowth retardation was investigated. The results suggested that nano-TiO2 crossed the blood-fetal barrier and blood-brain barrier and deposited in the brain of offspring, which retarded axonal and dendritic outgrowth, including the absence of axonal outgrowth, and decreased dendritic filament length, dendritic branching number, and dendritic spine density. Importantly, maternal exposure to nano-TiO2 increased phosphorylated (p)-extracellular signal-regulated kinase1/2 (ERK1/2, +24.35% to +59.4%), p-p38 (+60.82% to 181.85%), and p-c-jun N-terminal kinase (JNK, +28.28% to 97.28%) expression in the hippocampus of the offspring. These findings suggested that retardation of axonal and dendritic outgrowth in mouse offspring caused by maternal exposure to nano-TiO2 may be related to excessive activation of the ERK1/2/MAPK signaling pathway. Therefore, the potential toxicity of nano-TiO2 is a concern, especially in pregnant woman or children who are exposed to nano-TiO2.

Suppression of ovarian follicle development by nano TiO2 is associated with TGF-ß-mediated signaling pathways.

Nanoparticulate titanium dioxide (nano TiO2 ) is extensively applied in biological tissue engineering materials, food additives, cosmetics, and sunscreens. Numerous studies to date have demonstrated that nano TiO2 penetrates through the digestive system and possibly the blood circulation, leading to accumulation in the ovary and consequent reproductive toxicity. However, the mechanisms underlying the toxic effects of nano TiO2 on the female reproductive system remain to be established. In this study, female mice were exposed to different doses of nano TiO2 (1.25, 2.5, or 5 mg/kg body weight) via intragastric administration for 60 consecutive days, followed by investigation of follicular development, regulation of TGF-ß-mediated signaling pathways, and expression of the pathway components. Subchronic exposure to nano TiO2 induced a decrease in the number of primordial, secondary, and antral follicles and corpus luteum and concomitant increase in atretic follicles. Furthermore, follicular development disorder induced by nano TiO2 was associated with upregulation of TGF-ß1, TGF-ßR1, PTEN, and Foxo3a involved in cell growth and apoptosis and downregulation of several growth factors (PI3K, AKT, p-mTOR, p70S6K, p-p70S6K1, rpS6, p-rpS6, TSC1, and TSC2) in mouse ovaries. Our data collectively implied that suppression of ovarian follicle development by nano TiO2 was triggered by dysfunction of the TGF-ß, PI3K/AKT/mTOR, and AKT/p70S6K-rpS6/TSC/mTOR pathways. The adverse effects of nano TiO2 on follicular development highlights the necessity for caution in the use of nanomaterials in the food industry. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 414-422, 2019.

Wnt Pathway-Mediated Nano TiO2-Induced Toxic Effects on Rat Primary Cultured Sertoli Cells.

Nanosized titanium dioxide (Nano TiO2) has been widely used in daily lives, medicine, industry, and caused the potential reproduction toxicity for animals and human, however, the underlying molecular mechanisms for the reproductive toxicity of nano TiO2 are still largely unclear. In the present study, when primary cultured rat Sertoli cells (SCs) were exposed to nano TiO2, cell injury and alterations in wingless related MMTV integration site (Wnt) pathway-related factors including Wnt1, Wnt3a, Wnt5a, Wnt11, ß-catenin, and p-GSK-3ß expression were investigated. The results suggested that nano TiO2 could be translocated to cytoplasm or nucleus, and decreased cell viability, and impaired morphological structures of SCs, induced apoptosis and dead of primary cultured rat SCs. Furthermore, nano TiO2-induced the toxicity of primary cultured rat SCs was associated with increased expression of Wnt1, Wnt3a, Wnt5a, Wnt11, and ß-catenin and involved with reduced p-GSK-3ß expression. Therefore, this implies that nano TiO2-induced toxic effects on SCs may be associated with Wnt signaling pathways.

Nano-TiO2 Inhibits Development of the Central Nervous System and Its Mechanism in Offspring Mice.

Nano titanium dioxide (Nano-TiO2) has been applied in food packaging systems and food additives, but it may cause potential neurotoxicity for human and animals. In our study, the effects of nano-TiO2 exposure during pregnancy/lactation on the development of the central nervous system in offspring mice were examined and its molecular mechanism involving Rho family was investigated. Our findings showed that pregnancy/lactation exposure to nano-TiO2 resulted in thinning of cerebral and cerebellar cortex, decrease in number of neurons per unit area of cerebrum, edema and nuclear condensation, dysplasia of neurites in hippocampal pyramidal cells, thinning in pyramidal cell layer in hippocampus, and decrease in learning and memory of offspring mice. Furthermore, expressions of Rac1 and Cdc42 involved in axon and dendritic development were decreased, whereas RhoA expression and ratio of RhoA/Rac1 were increased in offspring brain. It implies that exposure to nano-TiO2 during pregnancy/lactation could result in brain retardation and cognitive impairment in offspring mice, which was closely related to alterations in the expression of Rho protein family. Therefore, application of nano-TiO2 in daily life should be performed with caution.

High Temperature Induces Expression of Tobacco Transcription Factor NtMYC2a to Regulate Nicotine and JA Biosynthesis.

Environmental stress elevates the level of jasmonic acid (JA) and activates the biosynthesis of nicotine and related pyridine alkaloids in tobacco (Nicotiana tabacum L.) by up-regulating the expression of putrescine N-methyltransferase 1 (NtPMT1), which encodes a putrescine N-methyl transferase that catalyzes nicotine formation. The JA signal suppressor JASMONATE ZIM DOMAIN 1 (NtJAZ1) and its target protein, NtMYC2a, also regulate nicotine biosynthesis; however, how these proteins interact to regulate abiotic-induced nicotine biosynthesis is poorly understood. In this study, we found that high-temperature (HT) treatment activated transcription of NtMYC2a, which subsequently stimulated the transcription of genes associated with JA biosynthesis, including Lipoxygenase (LOX), Allene oxide synthase (AOS), Allene oxide cyclase (AOC), and 12-oxophytodienodate reductase (OPR). Overexpression of NtMYC2a increased nicotine biosynthesis by enhancing its binding to the promoter of NtPMT1. Overexpression of either NtJAZ1 or proteasome-resistant NtJAZ1ΔC suppressed nicotine production under normal conditions, but overexpression only of the former resulted in low levels of nicotine under HT treatment. These data suggest that HT induces NtMYC2a accumulation through increased transcription to activate nicotine synthesis; meanwhile, HT-induced NtMYC2a can activate JA synthesis to promote additional NtMYC2a activity by degrading NtJAZ1 at the post-transcriptional level.

P31comet, a member of the synaptonemal complex, participates in meiotic DSB formation in rice.

The human mitotic arrest-deficient 2 (Mad2) binding protein p31(comet) participates in the spindle checkpoint and coordinates cell cycle events in mitosis although its function in meiosis remains unknown in all organisms. Here, we reveal P31(comet) as a synaptonemal complex (SC) protein in rice (Oryza sativa L.). In p31(comet), homologous pairing and synapsis are eliminated, leading to the homologous nondisjunction and complete sterility. The failure in loading of histone H2AX phosphorylation (γH2AX) in p31(comet), together with the suppressed chromosome fragmentation in rice completion of meiotic recombination 1 (com1) p31(comet) and radiation sensitive 51c (rad51c) p31(comet) double mutants, indicates that P31(comet) plays an essential role in double-strand break (DSB) formation. Interestingly, the dynamic colocalization pattern between P31(comet) and ZEP1 (a transverse filament protein of SC) by immunostaining, as well as the interaction between P31(comet) and CENTRAL REGION COMPONENT 1 (CRC1) in yeast two-hybrid assays, suggests possible involvement of P31(comet) in SC installation. Together, these data indicate that P31(comet) plays a key role in DSB formation and SC installation, mainly through its cooperation with CRC1.

The Exonuclease Homolog OsRAD1 Promotes Accurate Meiotic Double-Strand Break Repair by Suppressing Nonhomologous End Joining.

During meiosis, programmed double-strand breaks (DSBs) are generated to initiate homologous recombination, which is crucial for faithful chromosome segregation. In yeast, Radiation sensitive1 (RAD1) acts together with Radiation sensitive9 (RAD9) and Hydroxyurea sensitive1 (HUS1) to facilitate meiotic recombination via cell-cycle checkpoint control. However, little is known about the meiotic functions of these proteins in higher eukaryotes. Here, we characterized a RAD1 homolog in rice (Oryza sativa) and obtained evidence that O. sativa RAD1 (OsRAD1) is important for meiotic DSB repair. Loss of OsRAD1 led to abnormal chromosome association and fragmentation upon completion of homologous pairing and synapsis. These aberrant chromosome associations were independent of OsDMC1. We found that classical nonhomologous end-joining mediated by Ku70 accounted for most of the ectopic associations in Osrad1 In addition, OsRAD1 interacts directly with OsHUS1 and OsRAD9, suggesting that these proteins act as a complex to promote DSB repair during rice meiosis. Together, these findings suggest that the 9-1-1 complex facilitates accurate meiotic recombination by suppressing nonhomologous end-joining during meiosis in rice.

Carbon Monoxide Interacts with Auxin and Nitric Oxide to Cope with Iron Deficiency in Arabidopsis.

To clarify the roles of carbon monoxide (CO), nitric oxide (NO), and auxin in the plant response to iron deficiency (-Fe), and to establish how the signaling molecules interact to enhance Fe acquisition, we conducted physiological, genetic, and molecular analyses that compared the responses of various Arabidopsis mutants, including hy1 (CO deficient), noa1 (NO deficient), nia1/nia2 (NO deficient), yuc1 (auxin over-accumulation), and cue1 (NO over-accumulation) to -Fe stress. We also generated a HY1 over-expression line (named HY1-OX) in which CO is over-produced compared to wild-type. We found that the suppression of CO and NO generation using various inhibitors enhanced the sensitivity of wild-type plants to Fe depletion. Similarly, the hy1, noa1, and nia1/nia2 mutants were more sensitive to Fe deficiency. By contrast, the yuc1, cue1, and HY1-OX lines were less sensitive to Fe depletion. The hy1 mutant with low CO content exhibited no induced expression of the Fe uptake-related genes FIT1 and FRO2 as compared to wild-type plants. On the other hand, the treatments of exogenous CO and NO enhanced Fe uptake. Likewise, cue1 and HY1-OX lines with increased endogenous content of NO and CO, respectively, also exhibited enhanced Fe uptake and increased expression of bHLH transcriptional factor FIT1as compared to wild-type plants. Furthermore, we found that CO affected auxin accumulation and transport in the root tip by altering the PIN1 and PIN2 proteins distribution that control lateral root structure under -Fe stress. Our results demonstrated the integration of CO, NO, and auxin signaling to cope with Fe deficiency in Arabidopsis.