BACKGROUND AND PURPOSEGlutamate excitotoxicity may be involved in ischaemic injury to the CNS and some neurodegenerative diseases, such as Alzheimer's disease. Donepezil, an acetylcholinesterase (AChE) inhibitor, exerts neuroprotective effects. Here we demonstrated a novel mechanism underlying the neuroprotection induced by donepezil.EXPERIMENTAL APPROACHCell damage in primary rat neuron cultures was quantified by lactate dehydrogenase release. Morphological changes associated with neuroprotective effects of nicotine and AChE inhibitors were assessed by immunostaining. Cell surface levels of the glutamate receptor sub‐units, NR1 and NR2A, were analyzed using biotinylation. Immunoblot was used to measure protein levels of cleaved caspase‐3, total NR1, total NR2A and phosphorylated NR1. Immunoprecipitation was used to measure association of NR1 with the post‐synaptic protein, PSD‐95. Intracellular Ca²⁺concentrations were measured with fura 2‐acetoxymethylester. Caspase 3‐like activity was measured using enzyme substrate, 7‐amino‐4‐methylcoumarin (AMC)‐DEVD.KEY RESULTSLevels of NR1, a core subunit of the NMDA receptor, on the cell surface were significantly reduced by donepezil. In addition, glutamate‐mediated Ca²⁺entry was significantly attenuated by donepezil. Methyllycaconitine, an inhibitor of α7 nicotinic acetylcholine receptors (nAChR), inhibited the donepezil‐induced attenuation of glutamate‐mediated Ca²⁺entry. LY294002, a phosphatidyl inositol 3‐kinase (PI3K) inhibitor, had no effect on attenuation of glutamate‐mediated Ca²⁺entry induced by donepezil.CONCLUSIONS AND IMPLICATIONSDecreased glutamate toxicity through down‐regulation of NMDA receptors, following stimulation of α7 nAChRs, could be another mechanism underlying neuroprotection by donepezil, in addition to up‐regulating the PI3K‐Akt cascade or defensive system.

Advanced knowledge of messenger RNA (mRNA) N⁶-methyladenosine (m⁶A) and DNA N⁶-methyldeoxyadenosine (6 mA) redefine our understanding of these epigenetic modifications. Both m⁶A and 6mA carry important information for gene regulation, and the corresponding catalytic enzymes sometimes belong to the same gene family and need to be distinguished. However, a comprehensive analysis of the m⁶A gene family in tomato remains obscure. Here, 24 putative m⁶A genes and their family genes in tomato were identified and renamed according to BLASTP and phylogenetic analysis. Chromosomal location, synteny, phylogenetic, and structural analyses were performed, unravelling distinct evolutionary relationships between the MT-A70, ALKBH, and YTH protein families, respectively. Most of the 24 genes had extensive tissue expression, and 9 genes could be clustered in a similar expression trend. Besides, SlYTH1 and SlYTH3A showed a different expression pattern in leaf and fruit development. Additionally, qPCR data revealed the expression variation under multiple abiotic stresses, and LC-MS/MS determination exhibited that the cold stress decreased the level of N⁶ 2′-O dimethyladenosine (m⁶Am). Notably, the orthologs of newly identified single-strand DNA (ssDNA) 6mA writer–eraser–reader also existed in the tomato genome. Our study provides comprehensive information on m⁶A components and their family proteins in tomato and will facilitate further functional analysis of the tomato N⁶-methyladenosine modification genes.

ALKBH proteins, the homologs of Escherichia coli AlkB dioxygenase, constitute a single-protein repair system that safeguards cellular DNA and RNA against the harmful effects of alkylating agents. ALKBH10B, the first discovered N⁶-methyladenosine (m⁶A) demethylase in Arabidopsis (Arabidopsis thaliana), has been shown to regulate plant growth, development, and stress responses. However, until now, the functional role of the plant ALKBH10B has solely been reported in arabidopsis, cotton, and poplar, leaving its functional implications in other plant species shrouded in mystery. In this study, we identified the AlkB homolog SlALKBH10B in tomato (Solanum lycopersicum) through phylogenetic and gene expression analyses. SlALKBH10B exhibited a wide range of expression patterns and was induced by exogenous abscisic acid (ABA) and abiotic stresses. By employing CRISPR/Cas9 gene editing techniques to knock out SlALKBH10B, we observed an increased sensitivity of mutants to ABA treatment and upregulation of gene expression related to ABA synthesis and response. Furthermore, the Slalkbh10b mutants displayed an enhanced tolerance to drought and salt stress, characterized by higher water retention, accumulation of photosynthetic products, proline accumulation, and lower levels of reactive oxygen species and cellular damage. Collectively, these findings provide insights into the negative impact of SlALKBH10B on drought and salt tolerance in tomato plant, expanding our understanding of the biological functionality of SlALKBH10B.

The tomato fruit is a complex organ and is composed of various structures from the inside out, such as columella, septum, and placenta. However, our understanding of the development and function of these internal structures remains limited. In this study, we identified a plant-specific YABBY protein, SlYABBY2a, in the tomato (Solanum lycopersicum). SlYABBY2a exhibits relatively high expression levels among the nine YABBY genes in tomatoes and shows specific expression in the septum of the fruit. Through the use of a gene-editing technique performed by CRISPR/Cas9, we noticed defects in septum development in the Slyabby2a mutant fruits, leading to the inward concavity of the fruit pericarp and delayed septum ripening. Notably, the expression levels of key genes involved in auxin (SlFZY4, SlFZY5, and SlFZY6) and ethylene (SlACS2) biosynthesis were significantly downregulated in the septum of the Slalkbh10b mutants. Furthermore, the promoter activity of SlYABBY2a was regulated by the ripening regulator, SlTAGL1, in vivo. In summary, these discoveries provide insights into the positive regulation of SlYABBY2a on septum development and ripening and furnish evidence of the coordinated regulation of the auxin and ethylene signaling pathways in the ripening process, which expands our comprehension of septum development in the internal structure of the fruit.

Fruit flavor and color are critical quality characteristics of tomatoes (Solanum lycopersicum). Numerous studies have demonstrated that tomato flavor is primarily linked to the sugar-acid content and its ratio, while fruit color is predominantly determined by the composition and concentration of carotenoids and flavonoids. To elucidate the regulatory mechanisms underlying the differences in sugar-acid and color formation during fruit ripening, transcriptome analysis was conducted for the first time on the breaker stage (Br) and mature fruit stage (MF) of the closely related strains yellow-fruited tomato (No. 19) and red-fruited tomato (No. 20). This analysis aimed to identify key regulatory genes and biosynthetic pathways associated with the formation of flavor and color in tomato fruits. The transcriptome analysis revealed that 1,546 differentially expressed genes (DEGs) were identified in the Br19_vs_Br20 comparison, of which 507 were up-regulated and 1,039 were down-regulated. In the MF19_vs_MF20 comparison, 2,178 DEGs were detected, with 1,235 up-regulated and 943 down-regulated. Upon further analysis of the DEGs, we identified several key genes in the sugar-acid metabolic pathway, including sucrose synthase (SUS), phosphofructokinase (PFK), fructose-bisphosphate aldolase (FBA), citrate synthase (CS), and succinate dehydrogenase (SuDH), which may significantly influence tomato flavor. Additionally, differential genes related to carotenoid and flavonoid metabolism, such as cytochrome P450 98A (CYP98A), caffeoyl-CoA3-O-methyltransferase (CCoAMT), carotenoid isomerase (CRTISO), lycopene beta cyclase (LCYB), zeaxanthin epoxidase (ZEP), violaxanthin deepoxidase (VDE), and 9-cis-epoxycarotenoid dioxygenase (NCED), as well as genes linked to ethylene synthesis, such as 1-aminocyclopropane-1-carboxylate synthase (ACS) and 1-aminocyclopropane-1-carboxylic acid oxidase (ACO), may play a role in the color changes observed in tomatoes. The findings of this study provide insights into the underlying mechanisms of flavor and color development in tomato fruit, offering valuable information for the genetic improvement of tomatoes.