ACRYLONITRILE HAS DISTINCT HORMETIC EFFECTS ON ACETYLCHOLINESTERASE ACTIVITY IN MOUSE BRAIN AND BLOOD THAT ARE MODULATED BY ETHANOL

Acrylonitrile(AN) is a neurotoxin both in animals and humans, but its effects on acetylcholinesterase (AChE) activity remain controversial. This study aimed to determine the dose-response effects of AN on AChE activity and the modulatory role of ethanol pretreatment. A total of 144 Kunming mice were randomly divided into 18 groups: nine groups received 5% ethanol in their drinking water, and the remaining nine groups received regular tap water. One week later, both the ethanol and tap water only groups were given an intraperitoneal injection of AN at the following doses: 0 (control), 0.156, 0.3125, 0.625, 1.25, 2.5, 5, 10 or 20 mg AN/kg body weight. AChE activity was determined on whole blood and brain 24 h later. Blood AChE activity was higher in AN-injected mice than in controls at all doses. AChE activity in blood increased in a dose-dependent manner, peaking at 0.156 mg/kg, after which a gradual decrease ensued, displaying a β-typed dose-response relationship. In contrast, brain AChE activity, following a single AN injection, was consistently lower than in control mice, and continued to fall up to a dose of 0.313 mg/kg, and thereafter increased gradually with higher doses. Mice receiving a 20 mg/kg dose of AN exhibited AChE brain activity indistinguishable from that of control mice, demonstrating a typical U-typed dose-response relationship. The activity of AChE in the blood and brain of the AN + ethanol-treated groups displayed a shift to the right, and the magnitude of the decrease in AChE activity induced by AN was attenuated relative to the AN-only group. These results suggest that AN affects AChE activity in both mouse blood and brain in a hormetic manner. Pretreatment with ethanol modifies the effect of AN on AChE, indicating that parent AN has a more prominent role than its metabolites in modulating enzyme activity

Genome-Wide Identification, Evolution, and Co-expression Network Analysis of Mitogen-Activated Protein Kinase Kinase Kinases in Brachypodium distachyon

Mitogen-activated protein kinase (MAPK) cascades are the conserved and universal signal transduction modules in all eukaryotes, which play the vital roles in plant growth, development and in response to multiple stresses. In this study, we used bioinformatics methods to identify 86 MAPKKK protein encoded by 73 MAPKKK genes in Brachypodium. Phylogenetic analysis of MAPKKK family from Arabidopsis, rice and Brachypodium has classified them into three subfamilies, of which 28 belonged to MEKK, 52 to Raf and 6 to ZIK subfamily respectively. Conserved protein motif, exon-intron organization and splicing intron phase in kinase domains supported the evolutionary relationships inferred from the phylogenetic analysis. And gene duplication analysis suggested the chromosomal segment duplication happened before the divergence of the rice and Brachypodium, while all of three tandem duplicated gene pairs happened after their divergence. We further demonstrated that the MAPKKKs have evolved under strong purifying selection, implying the conservation of them. The splicing transcripts expression analysis showed that the splicesome translating longest protein tended to be adopted. Furthermore, the expression analysis of BdMAPKKKs in different organs and development stages as well as heat, virus and drought stresses revealed that the MAPKKK genes were involved in various signaling pathways. And the circadian analysis suggested there were 41 MAPKKK genes in Brachypodium showing cycled expression in at least one condition, of which seven MAPKKK genes expressed in all conditions and the promoter analysis indicated these genes possessed many cis-acting regulatory elements involved in circadian and light response. Finally, the co-expression network of MAPK, MAPKK and MAPKKK in Brachypodium was constructed using 144 microarray and RNA-seq datasets, and ten potential MAPK cascades pathway were predicted. To conclude, our study provided the important information for evolutionary and functional characterization of MAPKKK family in Brachypodium, which will facilitate the functional analysis of BdMAPKKK genes, and also will facilitate better understanding the MAPK signal pathway in Brachypodium and beyond

HORMETIC EFFECTS OF ACUTE METHYLMERCURY EXPOSURE ON GRP78 EXPRESSION IN RAT BRAIN CORTEX

This study aims to explore the expression of GRP78, a marker of endoplasmic reticulum (ER) stress, in the cortex of rat brains acutely exposed to methylmercury (MeHg). Thirty Sprague-Dawley (SD) rats were randomly divided into six groups, and decapitated 6 hours (h) after intraperitoneal (i.p.) injection of MeHg (2, 4, 6, 8 or 10 mg/kg body weight) or normal saline. Protein and mRNA expression of Grp78 were detected by western blotting and real-time PCR, respectively. The results showed that a gradual increase in GRP78 protein expression was observed in the cortex of rats acutely exposed to MeHg (2, 4 or 6 mg/kg). Protein levels peaked in the 6 mg/kg group (p \u3c 0.05 vs. controls), decreased in the 8 mg/kg group, and bottomed below the control level in the 10 mg/kg group. Parallel changes were noted for Grp78 mRNA expression. It may be implied that acute exposure to MeHg induced hormetic dose-dependent changes in Grp78 mRNA and protein expression, suggesting that activation of ER stress is involved in MeHg-induced neurotoxicity. Low level MeHg exposure may induce GRP78 protein expression to stimulate endogenous cytoprotective mechanisms

Barley TAPETAL DEVELOPMENT and FUNCTION1 (HvTDF1) gene reveals conserved and unique roles in controlling anther tapetum development in dicot and monocot plants

•The anther tapetum helps control microspore release and essential components for pollen wall formation. TAPETAL DEVELOPMENT and FUNCTION1 (TDF1) is an essential R2R3 MYB tapetum transcription factor in Arabidopsis thaliana; however, little is known about pollen development in the temperate monocot barley.•Here, we characterize the barley (Hordeum vulgare L.) TDF1 ortholog using reverse genetics and transcriptomics.•Spatial/temporal expression analysis indicates HvTDF1 has tapetum-specific expression during anther stage 7/8. Homozygous barley hvtdf1 mutants exhibit male sterility with retarded tapetum development, delayed tapetum endomitosis and cell wall degeneration, resulting in enlarged, vacuolated tapetum surrounding collapsing microspores. Transient protein expression and dual-luciferase assays show TDF1 is a nuclear-localized, transcription activator, that directly activates osmotin proteins. Comparison of hvtdf1 transcriptome data revealed several pathways were delayed, endorsing the observed retarded anther morphology. Arabidopsis tdf1 mutant fertility was recovered by HvTDF1, supporting a conserved role for TDF1 in monocots and dicots.•This indicates that tapetum development shares similarity between monocot and dicots; however, barley HvTDF1 appears to uniquely act as a modifier to activate tapetum gene expression pathways, which are subsequently also induced by other factors. Therefore, the absence of HvTDF1 results in delayed developmental progression rather than pathway failure, although inevitably still results in pollen degeneration

Combining phosphoproteomics and high-throughput phenotyping for the identification of gravitropism-related proteins in Arabidopsis thaliana

Gravitropism is a fundamental process in plants that allows shoots to grow upward and roots to grow downward. Protein phosphorylation has been postulated to participate in the intricate signaling cascade of gravitropism. In order to elucidate the underlying mechanisms governing the gravitropic signaling and unearth novel protein constituents, an exhaustive investigation employing microgravity-induced phosphoproteomics was undertaken. The significantly phosphorylated proteins unraveled in this study can be effectively divided into two groups through clustering analysis. Furthermore, the elucidation of Gene Ontology (GO) enrichment analysis disclosed the conspicuous overrepresentation of these clustered phosphoproteins in cytoskeletal organization and in hormone-mediated responses intimately intertwined with the intricate phenomenon of gravitropism. Motif enrichment analysis unveiled the overrepresentation of [-pS-P-] and [-R-x-x-pS-] motifs. Notably, the [-pS-P-] motif has been suggested as the substrate for the Casein kinase II (CK II) and Cyclin-dependent kinase (CDK). Kinase-inhibitor assays confirmed the pivotal role played by CK II and CDK in root gravitropism. Mutant gravitropism assays validated the functional significance of identified phosphoproteins, with some mutants exhibiting altered bending kinetics using a custom-developed platform. The study also compared phosphoproteomics data from different platforms, revealing variations in the detected phosphopeptides and highlighting the impact of treatment differences. Furthermore, the involvement of TOR signaling in microgravity-induced phosphorylation changes was uncovered, expanding the understanding of plant gravitropism responses. To fulfill the large-scale verification of interesting candidates from the phosphoproteomics study, a novel root and hypocotyl gravitropism phenotyping platform was developed. This platform integrated cost-effective hardware, including Raspberry Pi, a high-quality camera, an Arduino board, a rotation stage (obtained from Prof. Dr. Maik Böhmer), and programmable green light (modified by Sven Plath). In addition, through collaboration with a software developer, machine-learning-based software was developed for data analysis. This platform tested the gravitropic response of candidate mutants identified in the phosphoproteomics study. Furthermore, the capabilities of this platform were expanded to investigate tropisms in other species and organs. To find novel proteins that might act as partners of a key protein that is involved in gravitropism signaling, ALTERED RESPONSE TO GRAVITY 1 (ARG1), immunoprecipitation coupled with Mass Spectrometry (IP-MS) was performed and identified ARG1-LIKE1 (ARL1) as a potential interacting protein with ARG1. This interaction was further confirmed through in vivo pull-down assays and bimolecular fluorescence complementation assays. In addition, the interaction between ARG1 and HSP70-1 was also validated. Overall, this thesis sheds light on the molecular components and signaling events involved in plant gravitropism. It contributes to existing knowledge and opens up new ways to investigate this fascinating area of plant biology.Der Gravitropismus ist ein grundlegender Prozess in Pflanzen, der es Sprossen ermöglicht, nach oben zu wachsen, und Wurzeln, nach unten zu wachsen. Die Phosphorylierung spielt eine zentrale Rolle bei der Regulierung des pflanzlichen Gravitropismus, da sie als entscheidender Mechanismus für die Übertragung und Modulation von Signalen dient, die an diesem grundlegenden biologischen Prozess beteiligt sind (Schepetilnikov et al., 2013; Van Leene et al., 2019). Mehrere Kinasen, darunter PID (Grones et al., 2018), D6PK (Tan et al., 2020), und TOR (Schepetilnikov et al., 2013) wurden als potenzielle Schlüsselfaktoren für die Vermittlung der gravitropischen Reaktion in Pflanzen identifiziert. Darüber hinaus wird die Gravitropismus-Reaktion schnell eingeleitet und tritt in der Regel innerhalb von Sekunden nach der gravitropischen Stimulation auf (Zheng et al., 2015). Um tiefere Einblicke in die molekularen Vorgänge zu gewinnen, die in den ersten Sekunden der Schwerelosigkeit auftreten, wurde in dieser Studie eine Phosphoproteomik-Studie an Arabidopsis-Keimlingen durchgeführt. Durch die Konzentration auf diese frühe Zeitspanne sollten die dynamischen Phosphorylierungsereignisse erfasst werden, die eine entscheidende Rolle bei der Reaktion der Pflanze auf die veränderten Schwerkraftbedingungen spielen. Für diese Studie wurden zwei bewährte Plattformen verwendet: der Fallturm und die Parabelflugplattform, die vom Zentrum für angewandte Raumfahrttechnologie und Mikrogravitation (ZARM) in Bremen und von Novespace in Bordeaux, Frankreich, zur Verfügung gestellt wurden. Die umfassende Analyse in dieser Studie ergab insgesamt 4.266 Phosphosites. Die in dieser Studie beobachteten Phosphorylierungsmuster und Rückstandsverhältnisse stimmten mit früheren Arabidopsis-Phosphoproteomik-Studien überein, was die Zuverlässigkeit und Reproduzierbarkeit der Ergebnisse bestätigte (Yang et al., 2020; Andrea Vega et al., 2021; Rayapuram et al., 2021). Die Analyse der signifikant unterschiedlich phosphorylierten Peptide in der Hypergravitationsbehandlung sowie in den 3 s und 22 s Mikrogravitationsbehandlungen ergab interessante Überschneidungsmuster. Insgesamt 85 Peptide wurden in allen drei Versuchsgruppen gemeinsam gefunden, was auf eine gemeinsame Reaktion unter diesen Bedingungen hindeutet. Abgesehen von den 85 Peptiden, die alle drei Gruppen gemeinsam hatten, wurden 53 Peptide sowohl in der Hypergravitations- als auch in der 3-Sekunden-Mikrogravitationsbehandlung gefunden, während weitere 53 Peptide sowohl in der Hypergravitations- als auch in der 22-Sekunden-Mikrogravitationsbehandlung zu finden waren. Darüber hinaus wurden 130 Peptide zwischen der 3-Sekunden- und der 22-Sekunden-Behandlung unter Mikrogravitation gefunden. Diese Ergebnisse deuten darauf hin, dass bestimmte Proteine konsistente Veränderungen in ihren Phosphorylierungszuständen über die verschiedenen Dauern der Mikrogravitations- und Hypergravitationsexposition aufweisen, was auf eine gemeinsame Reaktion und potenzielle Regulierungsmechanismen als Reaktion auf die Schwerkraftänderungen hindeutet. Zu den angereicherten Gene Ontology (GO)-Begriffen für die signifikant unterschiedlich phosphorylierten Proteine in der 3-Sekunden-Mikrogravitationsbehandlung gehörten "Signaltransduktion", "Mikrotubuli-Zytoskelett-Organisation" und "Protein-Targeting zur Vakuole, beteiligt am Ubiquitin-abhängigen Proteinkatabolismus über den multivesikulären Körpersortierweg". Diese Ergebnisse unterstreichen die rasche Aktivierung von Signalwegen und die Beteiligung von Zytoskelett- und Lipid-Signalproteinen in den frühen Stadien der Gravitropismus-Reaktion. Im Gegensatz dazu zeigte die 22-s-Mikrogravitationsbehandlung eine Anreicherung nur in Bezug auf biologische Prozesse, insbesondere bei der zellulären Reaktion auf Hormonreize und Salicylsäure-vermittelte Signalwege. Dies deutet darauf hin, dass eine längere Mikrogravitations-Exposition spezifische biologische Prozesse im Zusammenhang mit Hormonreaktionen und Signalübertragung auslösen kann

The characterization of the mitochondrial genome of Graptemys ouachitensis

Graptemys ouachitensis (CAGLE, 1953) belongs to the Graptemys genus, the Emydidae family, and the Testudines order. This study involved sequencing the complete mitochondrial genome (mitogenome) of G. ouachitensis using next-generation sequencing, and analyzing the essential characteristics, and phylogenetic relationship. The results revealed that the G. ouachitensis mitogenome was 16,674 bp in length (A: 34.1%, C: 26.0%, G: 13.0%, T: 26.9%) and included 22 tRNAs, 13 protein-coding genes, two ribosomal RNA genes, and a non-coding control region (GenBank accession: NC071766). The genome composition of G. ouachitensis presented a slight A + T bias (61.0%) and exhibited a positive AT skew (0.118) and a negative GC skew (–0.333). A phylogenetic analysis based on the complete mitogenome indicated that the G. ouachitensis was more closely associated with Malaclemys terrapin than the other eight known Emydidae species. Thus, our findings present a novel mitogenome at the species level. This study introduces the first complete mitogenome of G. ouachitensis, providing valuable molecular information for phylogenetic and conservation genetics analyses of G. ouachitensis

Evolution and Identification of the WRKY Gene Family in Quinoa (<i>Chenopodium quinoa</i>)

The WRKY gene family plays a unique role in plant stress tolerance. Quinoa is a cultivated crop worldwide that is known for its high stress tolerance. The WRKY gene family in quinoa has not yet been studied. Using a genome-wide search method, we identified 1226 WRKY genes in 15 plant species, seven animal species, and seven fungi species. WRKY proteins were not found in animal species and five fungi species, but were, however, widespread in land plants. A total of 92 CqWRKY genes were identified in quinoa. Based on the phylogenetic analysis, these CqWRKY genes were classified into three groups. The CqWRKY proteins have a highly conserved heptapeptide WRKYGQK with 15 conserved elements. Furthermore, a total of 25 CqWRKY genes were involved in the co-expression pathway of organ development and osmotic stress. The expression level of more than half of these CqWRKY genes showed significant variation under salt or drought stress. This study reports, for the first time, the findings of the CqWRKY gene family in quinoa at the genome-wide level. This information will be beneficial for our understanding of the molecular mechanisms of stress tolerance in crops, such as quinoa