Effects of different light conditions on morphological, anatomical, photosynthetic and biochemical parameters of Cypripedium macranthos Sw.

In this study, the morphological (plant height, leaf length and width, stem diameter and leaf number), anatomical (epidermal cell density and thickness, Stomatal length and width), photosynthetic (net photosynthetic rate, transpiration rate, stomatal conductance, intercellular CO2 concentration, relative humidity, leaf temperature and chlorophyll fluorescence parameters) and biochemical parameters (the content of soluble sugar, soluble protein, proline, malondialdehyde and electrical conductivity) of Cypripedium macranthos Sw. in Changbai Mountain were determined under different light conditions (L10, L30, L50, L100). The results showed that morphological values including plant height, leaf area, stem diameter and leaf number of C. macranthos were smaller under the condition of full light at L100. The epidermal cell density and epidermal thickness of C. macranthos were the highest under L30 and L50 treatments, respectively. It had the highest net photosynthetic rate (Pn) and chlorophyll content under L50 treatment. Meanwhile, correlation analysis indicated that photosynthetically active radiation (PAR) and water use efficiency (WUE) were the main factors influencing Pn. C. macranthos accumulated more soluble sugars and soluble proteins under L100 treatment, while the degree of membrane peroxidation was the highest and the plant was severely damaged. In summary, the adaptability of C. macranthos to light conditions is ranked as follows L50 > L30 > L10 > L100. Appropriate light conditions for C. macranthos are 30%-50% of full light, which should be taken into account in protection and cultivation.

Comparative proteomic analysis of retinal hypoxia-ischemia in an acute ocular hypertension model using tandem mass tag-based quantitative proteomics.

The main symptom of acute glaucoma is acute ocular hypertension (AOH), which leads to the death of retinal ganglion cells (RGCs) and permanent loss of vision. However, effective treatments for these conditions are lacking. This study aimed to identify major regulators and overall protein changes involved in AOH-induced RGC death. Proteomic patterns of the retinal protein extracts from the AOH and sham groups were analyzed using mass spectrometry (MS), followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. Proteomic analysis revealed 92 proteins in the AOH group compared to the control group; 58 proteins were upregulated and 34 were downregulated. Alterations in fatty acid-binding protein 7 (FABP7) and caveolin-1 (Cav-1), which are related to fatty acid metabolism and ocular inflammatory signaling, were detected using western blotting and biochemical assays. Variations in the expression of galectin-1 (Gal-1), S100 calcium-binding protein A6 (S100a6), and visinin-like protein-1 (VILIP) have been associated with neuronal ischemia. Our investigation demonstrates that neuroinflammation and fatty acid metabolism are involved in retinal impairment following AOH, suggesting a possible treatment approach for acute glaucoma.

Efficacy of anti-VEGF intravitreal injection in traumatic submacular hemorrhage: a retrospective study.

OBJECTIVE: In this study we investigated the efficacy of short-term intravitreal injections of anti-vascular endothelial growth factors (anti-VEGF) in treating traumatic submacular hemorrhage. METHODS: A total of 115 patients were diagnosed with submacular hemorrhage between 2018 and 2022 at Shenzhen Eye Hospital. In a retrospective analysis, we examined 13 of these patients who presented with submacular hemorrhage and choroidal rupture due to ocular trauma. Eight patients were treated with intravitreal anti-VEGF injection and 5 with oral drugs. We systematically analyzed changes in their ocular conditions pre and post-treatment. The evaluations encompassed best-corrected visual acuity (BCVA), optical coherence tomography, fundus fluorescein angiography, and retinal imaging. RESULTS: The 13 patients diagnosed with submacular hemorrhage comprised of 10 males and 3 female, with their age ranging between 27 and 64 years, with an average age of 38.1 years (standard deviation [SD]: 11.27). A statistically significant reduction in central foveal thickness (CFT) was observed following intravitreal injections of anti-VEGF drugs (P = 0.03). In control group, the CFT was reduced without statistical significance (P = 0.10). The BCVA of the patients in treatment group improved significantly from 1.15 (SD: 0.62. Range: 0.4-2) to 0.63 (SD: 0.59. Range: 0.1-1.6), indicating an average increase of 4.13 lines (SD: 3.36. Range: 0-9) as measured by the visual acuity test using an eye chart (P = 0.01). The difference between baseline visual acuity and final visual acuity was not statistically significant in control group (P = 0.51). CONCLUSION: Short-term administration of anti-VEGF drugs exhibited significant efficacy in reducing submacular hemorrhage following ocular trauma and enhancing visual acuity.

Development of Low-Pressure He-MC-MIP-MS and Its Application for Oxygen Isotopic Analysis.

In Ar-based inductively coupled plasma mass spectrometry (MS), Ar-related interference and the low ionization capacity of the Ar-ion source prevent facile and precise determination of certain elements. To address this problem, we investigated the application of microwave-induced plasma (MIP), and we improved its ionization capacity using He as the working gas. The MIP ion source was connected to a multicollector mass spectrometry (MC-MS) apparatus to improve the accuracy and precision of the isotopic analysis. A vacuum pump was used to achieve a low pressure (200-300 Pa) at the interface. The analytical figures of merit were discussed and evaluated by measuring the oxygen isotopes in oxygen. With the application of low-pressure He-MC-MIP-MS, the degree of ionization of oxygen could be significantly improved with He plasma. The interference of oxygen from the atmosphere could also be eliminated with low-pressure plasma, and the determination precision of oxygen isotopes could be improved with the application of MC-MS. Subsequently, using this method, 16O18O/16O16O was applied as the analytical ratio to investigate the interference, sensitivity, and precision. With this constructed method, the obtained long-term producibility of δ18O was 0.16‰ (2 SD), and the measured result for oxygen was consistent with that obtained by MAT 253 within the uncertainty limit. The development of low-pressure He-MC-MIP-MS can pave the way for the accurate measurement of nonmetal isotopes and easily interfered isotopes in Ar plasma.

PPAR-γ promotes the polarization of rat retinal microglia to M2 phenotype by regulating the expression of CD200-CD200R1 under hypoxia.

BACKGROUND: Recent reports suggest that peroxisome proliferator-activated receptor-γ (PPAR-γ) could promote microglial M2 polarization to inhibit inflammation. However, the specific molecular mechanisms that trigger PPAR-γ's anti-inflammatory ability in microglia are yet to be expounded. Thus, in this study, we aimed to explore the molecular mechanisms behind the anti-inflammatory effects of PPAR-γ in hypoxia-stimulated rat retinal microglial cells. METHODS AND RESULTS: We used shRNA expressing lentivirus to knock down PPAR-γ and CD200 genes, and we assessed hypoxia-induced polarization markers release - M1 (iNOS, IL-1ß, IL-6, and TNF-α) and M2 (Arg-1, YM1, IL-4, and IL-10) by RT-PCR. We also monitored PPAR-γ-related signals (PPAR-γ, PPAR-γ in cytoplasm or nucleus, CD200, and CD200Rs) by Western blot and RT-PCR. Our results showed that hypoxia enhanced PPAR-γ and CD200 expressions in microglial cells. Moreover, PPAR-γ agonist 15d-PGJ2 elevated CD200 and CD200R1 expressions, whereas sh-PPAR-γ had the opposite effect. Following hypoxia, expressions of M1 markers increased significantly, while those of M2 markers decreased, and the above effects were attenuated by 15d-PGJ2. Conversely, knocking down PPAR-γ or CD200 inhibited the polarization of microglial cells to M2 phenotype. CONCLUSION: Our findings demonstrated that PPAR-γ performed an anti-inflammatory function in hypoxia-stimulated microglial cells by promoting their polarization to M2 phenotype via the CD200-CD200R1 pathway.

Starch and Sucrose Metabolism and Plant Hormone Signaling Pathways Play Crucial Roles in Aquilegia Salt Stress Adaption.

Salt stress is one of the main abiotic stresses that strongly affects plant growth. Clarifying the molecular regulatory mechanism in ornamental plants under salt stress is of great significance for the ecological development of saline soil areas. Aquilegia vulgaris is a perennial with a high ornamental and commercial value. To narrow down the key responsive pathways and regulatory genes, we analyzed the transcriptome of A. vulgaris under a 200 mM NaCl treatment. A total of 5600 differentially expressed genes were identified. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis pointed out that starch and sucrose metabolism and plant hormone signal transduction were significantly improved. The above pathways played crucial roles when A. vulgaris was coping with salt stress, and their protein-protein interactions (PPIs) were predicted. This research provides new insights into the molecular regulatory mechanism, which could be the theoretical basis for screening candidate genes in Aquilegia.

Time-Course Transcriptome Analysis of Aquilegia vulgaris Root Reveals the Cell Wall's Roles in Salinity Tolerance.

Salt stress has a considerable impact on the development and growth of plants. The soil is currently affected by salinisation, a problem that is becoming worse every year. This means that a significant amount of salt-tolerant plant material needs to be added. Aquilegia vulgaris has aesthetically pleasing leaves, unique flowers, and a remarkable tolerance to salt. In this study, RNA-seq technology was used to sequence and analyse the transcriptome of the root of Aquilegia vulgaris seedlings subjected to 200 mM NaCl treatment for 12, 24, and 48 h. In total, 12 Aquilegia vulgaris seedling root transcriptome libraries were constructed. At the three time points of salt treatment compared with the control, 3888, 1907, and 1479 differentially expressed genes (DEGs) were identified, respectively. Various families of transcription factors (TFs), mainly AP2, MYB, and bHLH, were identified and might be linked to salt tolerance. Gene Ontology (GO) analysis of DEGs revealed that the structure and composition of the cell wall and cytoskeleton may be crucial in the response to salt stress. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of the DEGs showed a significant enrichment of the pentose and glucuronate interconversion pathway, which is associated with cell wall metabolism after 24 and 48 h of salt treatment. Based on GO and KEGG analyses of DEGs, the pentose and glucuronate interconversion pathway was selected for further investigation. AP2, MYB, and bHLH were found to be correlated with the functional genes in this pathway based on a correlation network. This study provides the groundwork for understanding the key pathways and gene networks in response to salt stress, thereby providing a theoretical basis for improving salt tolerance in Aquilegia vulgaris.

Structural insights into the CP312R protein of the African swine fever virus.

African swine fever (ASF) is a lethal hemorrhagic disease that affects domestic pigs and wild boars. There is no medication available for ASF to date. The ability to mount antigen-specific responses to viral vectored CP312R makes it a crucial potential target for designing vaccines or drugs. This study determined the crystal structure of ASFV CP312R at 2.32 Å and found it to be a monomer with a single-stranded DNA binding core domain with a clear five-strands ß-barrel OB-fold architecture. Electrophoretic mobility shift assay and size-exclusion chromatography characterization assay further confirmed the single-stranded DNA (ssDNA)-binding property of ASFV CP312R. This study revealed the structure and preliminary ssDNA interaction mechanisms of ASFV CP312R, providing new clues for developing new antiviral strategies.

miR-142-3p Regulates Tumor Cell Autophagy and Promotes Colon Cancer Progression by Targeting TP53INP2.

OBJECTIVES: Colon cancer (CC) is the third largest cancer worldwide. Investigation of the molecular mechanism of CC progression helps to explore novel therapeutic targets. We attempted to understand the modulatory mechanism of miR-142-3p in CC cell autophagy and CC progression, which will lay a theoretical groundwork for seeking potential diagnostic and therapeutic targets for CC. METHODS: Through bioinformatics methods, miRNA expression data were subjected to differential analysis for identification of target miRNA. Downstream target mRNAs were predicted, and gene set enrichment analysis was completed. qRT-PCR assessed gene expression in cells. Cell counting kit-8, cell doubling time calculation, colony formation, and flow cytometry were used to assess cellular biological functions. Dual-luciferase assay was used for targeting relationship validation of the target miRNA and mRNA. Western blot was performed to evaluate expression of proteins related to HEDGEHOG signaling pathway and autophagy. RESULTS: miR-142-3p was markedly highly expressed in CC, and high miR-142-3p expression in CC patients was implicated with relatively poor prognosis. Overexpressing miR-142-3p facilitated proliferation and inhibited apoptosis of CC cells, whereas silencing it produced an opposite result. miR-142-3p targeted and decreased TP53INP2 level. TP53INP2 overexpression suppressed the HEDGEHOG signaling pathway and induced the activation of CC cell autophagy. Rescue experiments revealed that influence of the miR-142-3p inhibitor on CC cell proliferation and apoptosis could be reversed by silencing TP53INP2. CONCLUSION: miR-142-3p hampered tumor cell autophagy and promoted CC progression via targeting TP53INP2, which will offer a fresh research orientation for the diagnosis of CC.

Structural Characteristics of Polysaccharide GP2a in Gardenia jasminoides and Its Immunomodulatory Effect on Macrophages.

Here, we elucidated the structural characteristics of a polysaccharide isolated from Gardenia jasminoides Ellis (labeled as GP2a) and its immunomodulatory activity. GP2a is an acidic polysaccharide with a molecular weight of 44.8 kDa, mostly comprising galacturonic acid. Methylation analysis revealed 4-GalpA (74.8%) to be the major sugar residue in GP2a. Nuclear magnetic resonance analysis indicated that its main chain comprised →4)-α-D-GalpA-6-OMe-(1→4)-α-D-GalpA-(1→ and →4)-α-D-GalpA-6-OMe-(1→2)-α-L-Rhap-(1→, with galactan and arabinans linked to the C-4 position of →2)-α-L-Rhap-(1→ residue as branched chains. Furthermore, GP2a showed no obvious toxicity to macrophages (RAW 264.7) while enhancing cell viability in a dose- and time-dependent manner. Compared with untreated cells, nitric oxide production and secretion of cytokines, such as tumor necrosis factor-α, interferon-γ, interleukin (IL)-1ß, IL-6, and granulocyte macrophage colony stimulating factor, in GP2a-treated cells significantly increased after 48 h. At 300 µg/mL GP2a concentration, there was no significant difference in the cytokine levels in GP2a- and lipopolysaccharide-treated cells (the positive control). In summary, GP2a is a pectic polysaccharide with homogalacturonan and rhamnogalacturonan-I structural regions in the main chain. Based on its immunomodulatory effects in vitro, GP2a may have potential uses in functional food and medicine.

The crystal structure of Arabidopsis BON1 provides insights into the copine protein family.

The Arabidopsis thaliana BON1 gene product is a member of the evolutionary conserved eukaryotic calcium-dependent membrane-binding protein family. The copine protein is composed of two C2 domains (C2A and C2B) followed by a vWA domain. The BON1 protein is localized on the plasma membrane, and is known to suppress the expression of immune receptor genes and to positively regulate stomatal closure. The first structure of this protein family has been determined to 2.5-Å resolution and shows the structural features of the three conserved domains C2A, C2B and vWA. The structure reveals the third Ca2+ -binding region in C2A domain is longer than classical C2 domains and a novel Ca2+ binding site in the vWA domain. The structure of BON1 bound to Mn2+ is also presented. The binding of the C2 domains to phospholipid (PSF) has been modeled and provides an insight into the lipid-binding mechanism of the copine proteins. Furthermore, the selectivity of the separate C2A and C2B domains and intact BON1 to bind to different phospholipids has been investigated, and we demonstrated that BON1 could mediate aggregation of liposomes in response to Ca2+ . These studies have formed the basis of further investigations into the important role that the copine proteins play in vivo.

Heterotrophic nitrification and related functional gene expression characteristics of Alcaligenes faecalis SDU20 with the potential use in swine wastewater treatment.

A new heterotrophic nitrifying bacterium was isolated from the compost of swine manure and rice husk and identified as Alcaligenes faecalis SDU20. Strain SDU20 had heterotrophic nitrification potential and could remove 99.7% of the initial NH4+-N. Nitrogen balance analysis revealed that 15.9 and 12.3% of the NH4+-N were converted into biological nitrogen and nitrate nitrogen, respectively. The remaining 71.44% could be converted into N2 or N2O. Single-factor experiments showed that the optimal conditions for ammonium removal were the carbon source of sodium succinate, C/N ratio 10, initial pH 8.0, and temperature 30 °C. Nitrification genes were determined to be upregulated when sodium succinate was used as the carbon source analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). Strain SDU20 could tolerate 4% salinity and show resistance to some heavy metal ions. Strain SDU20 removed 72.6% high concentrated NH4+-N of 2000 mg/L within 216 h. In a batch experiment, the highest NH4+-N removal efficiency of 98.7% and COD removal efficiency of 93.7% were obtained in the treatment of unsterilized swine wastewater. Strain SDU20 is promising in high-ammonium wastewater treatment.

PFBNet: a priori-fused boosting method for gene regulatory network inference.

BACKGROUND: Inferring gene regulatory networks (GRNs) from gene expression data remains a challenge in system biology. In past decade, numerous methods have been developed for the inference of GRNs. It remains a challenge due to the fact that the data is noisy and high dimensional, and there exists a large number of potential interactions. RESULTS: We present a novel method, namely priori-fused boosting network inference method (PFBNet), to infer GRNs from time-series expression data by using the non-linear model of Boosting and the prior information (e.g., the knockout data) fusion scheme. Specifically, PFBNet first calculates the confidences of the regulation relationships using the boosting-based model, where the information about the accumulation impact of the gene expressions at previous time points is taken into account. Then, a newly defined strategy is applied to fuse the information from the prior data by elevating the confidences of the regulation relationships from the corresponding regulators. CONCLUSIONS: The experiments on the benchmark datasets from DREAM challenge as well as the E.coli datasets show that PFBNet achieves significantly better performance than other state-of-the-art methods (Jump3, GEINE3-lag, HiDi, iRafNet and BiXGBoost).

Transgelin interacts with PARP1 in human colon cancer cells.

BACKGROUND: Transgelin, an actin-binding protein, is associated with cytoskeleton remodeling. Findings from our previous studies demonstrated that transgelin was up-regulated in node-positive colorectal cancer (CRC) versus node-negative disease. Over-expression of TAGLN affected the expression of 256 downstream transcripts and increased the metastatic potential of colon cancer cells in vitro and in vivo. This study aims to explore the mechanisms through which transgelin participates in the metastasis of colon cancer cells. METHODS: Immunofluorescence and immunoblotting analysis were used to determine the cellular localization of endogenous and exogenous transgelin in colon cancer cells. Co-immunoprecipitation and subsequently high-performance liquid chromatography/tandem mass spectrometry were performed to identify the proteins that were potentially interacting with transgelin. The 256 downstream transcripts regulated by transgelin were analyzed with bioinformatics methods to discriminate the specific key genes and signaling pathways. The Gene-Cloud of Biotechnology Information (GCBI) tools were used to predict the potential transcription factors (TFs) for the key genes. The predicted TFs corresponded to the proteins identified to interact with transgelin. The interaction between transgelin and the TFs was verified by co-immunoprecipitation and immunofluorescence. RESULTS: Transgelin was found to localize in both the cytoplasm and nucleus of the colon cancer cells. Approximately 297 proteins were identified to interact with transgelin. The overexpression of TAGLN led to the differential expression of 184 downstream genes. Network topology analysis discriminated seven key genes, including CALM1, MYO1F, NCKIPSD, PLK4, RAC1, WAS and WIPF1, which are mostly involved in the Rho signaling pathway. Poly (ADP-ribose) polymerase-1 (PARP1) was predicted as the unique TF for the key genes and concurrently corresponded to the DNA-binding proteins potentially interacting with transgelin. The interaction between PARP1 and transgelin in human RKO colon cancer cells was further validated by immunoprecipitation and immunofluorescence assays. CONCLUSIONS: Our results suggest that transgelin binds to PARP1 and regulates the expression of downstream key genes, which are mainly involved in the Rho signaling pathway, and thus participates in the metastasis of colon cancer.

Ammonium Removal by a Newly Isolated Heterotrophic Nitrification-Aerobic Denitrification Bacteria Pseudomonas Stutzeri SDU10 and Its Potential in Treatment of Piggery Wastewater.

A strain SDU10 was isolated from swine manure compost and identified as Pseudomonas stutzeri SDU10. It demonstrated excellent capability in NH4+-N removal. Optimal conditions of NH4+-N removal were determined, which were sodium acetate as the optimal carbon source, carbon to nitrogen (C/N) ratio of 10, temperature of 30 °C, pH of 7.0. Especially, P. stutzeri SDU10 could remove high concentration NH4+-N of 1500.0 and 2000.0 mg/l in 120 h with the NH4+-N removal rates of 91.1% and 61.6%, respectively. In batch experiments, the highest NH4+-N removal rate of 97.6% and chemical oxygen demand (COD) removal rate of 94.2% were obtained at initial C/N ratio 10 during piggery wastewater treatment using P. stutzeri SDU10. Results showed that P. stutzeri SDU10 had the potential for treatment of wastewater of high NH4+-N concentration.

Molecular cloning, characterization, and expression profiling analysis of Cry toxin receptor genes from sugarcane shoot borer Chilo infuscatellus (Snellen).

The sugarcane shoot borer Chilo infuscatellus (Snellen) is known for causing severe damage to sugarcane yield in China. Methods have been developed to control this pest, including Cry toxin pesticide and transgenic Bt plants. In order to investigate the molecular mechanism of the Cry toxin binding process and provide a basis for understanding the insect's resistance mechanism, we used a high throughput sequencing platform to perform a de novo transcriptome assembly across different larval developmental stages and analyzed Cry toxin receptors based on our assembled transcripts. We cloned twelve Cry toxin receptor genes including 1 cadherin (Cad), 7 aminopeptidase-Ns (APNs), 3 alkaline phosphatases (ALPs), and 1 ATP-binding cassette transporter subfamily C2 (ABCC2), and three of them with full length. The sublethal dosage of Cry1Ac toxin was applied to sugarcane shoot borer and identified some Cry toxin receptor genes that were significantly induced after 48 h of exposure. Furthermore, quantitative RT-PCR was conducted to detect the expression profiles of these genes. Our transcriptome sequence data provided a valuable molecular resource for further study and the identified Cry toxin receptor data gave insights for improved research into the mechanism of Bt resistance.

Molecular cloning, characterization and functional analysis of GluCl from the oriental armyworm, Mythimna separata Walker.

Glutamate-gated chloride channels (GluCls) mediate inhibitory synaptic transmission in invertebrate nervous systems, and only one GluCl gene has been found in insects. Therefore, insect GluCls are one of the major targets of insecticides including avermectins. In the present study, a 1347 bp full-length cDNA encoding a 449-amino acid protein (named MsGluCl, GenBank ID: MK336885) was cloned from the oriental armyworm, Mythimna separata, and characterized two alternative splicing variants of MsGluCl. The protein shares 76.9-98.6% identity with other insect GluCl isoforms. Spatial and temporal expression analysis revealed that MsGluCl was highly expressed in the 3rd instar and adult head. Dietary ingestion of dsMsGluCl significantly reduced the mRNA level of MsGluCl and decreased abamectin mortality. Thus, our results reveal that MsGluCl could be the molecular target of abamectin and provide the basis for further understanding the resistance mechanism to abamectin in arthropods.

Effect of Ionic Composition on Physicochemical Properties of Mono-Ether Functional Ionic Liquids.

Tunable properties prompt the development of different "tailor-made" functional ionic liquids (FILs) for specific tasks. FILs with an ether group are good solvents for many organic compounds and enzymatic reactions. However, ionic composition influences the solubility by affecting the physiochemical properties of these FILs. To address the structure effect, a series of novel FILs with a mono-ether group (ME) based on imidazole were prepared through cationic functionalization and anionic exchange reactions, and characterized by NMR, mass spectroscopy, and Thermogravimetric analysis (TGA). The effect of ionic composition (cationic structure and anions) on density, viscosity, ionic conductivity, electrochemical window, and thermal properties of these ME-FILs were systematically investigated. In general, the viscosity and heat capacity increases with the bigger cationic volume of ME-FILs; in particular, the 2-alkyl substitution of imidazolium enhances the viscosity remarkably, whereas the density and conductivity decrease on the condition of the same [NTf2]- anion; For these ME-FILs with the same cations, the density follows the order of [NTf2]- > [PF6]- > [BF4]-. The viscosity follows the order of [PF6]- > [BF4]- > [NTf2]-. Ion conductivity follows the order of [NTf2]- ≈ [BF4]- > [PF6]-. It is noted that the dynamic density has a good linear relationship with the temperature, and the slopes are the same for all ME-FILs. Furthermore, these ME-FILs have broad electrochemical windows and glass transition temperatures in addition to a cold crystallization and a melt temperature for ME-FIL7. Therefore, the cationic structure and counter anion affect the physicochemical properties of these ME-FILs together.

Evidence for a specific and critical role of mitogen-activated protein kinase 20 in uni-to-binucleate transition of microgametogenesis in tomato.

Mitogen-activated protein kinases (MAPKs) regulate diverse aspects of plant growth. However, their potential role in reproductive development remains elusive. Here, we discovered an unique role of SlMPK20, a plant-specific group D MAPK, in pollen development in tomato. RNAi-mediated suppression of SlMPK20 or its knockout using CRISPR/Cas9 significantly reduced or completely abolished pollen viability, respectively, with no effects on maternal fertility. Cell biology and gene expression analyses established that SlMPK20 exerts its role specifically at the uni-to-binucleate transition during microgametogenesis. This assertion is based on the findings that the transgenic pollen was largely arrested at the binucleate stage with the appearance of subcellular abnormality at the middle uninucleate microspore stage; and SlMPK20 mRNA and SlMPK20-GUS signals were localized in the tetrads, uninuclear microspores and binuclear pollen grains but not in microspore mother cells or mature pollen grains. Transcriptomic and proteomic analyses revealed that knockout of SlMPK20 significantly reduced the expression of a large number of genes controlling sugar and auxin metabolism and signaling in anthers. Finally, protein-protein interaction assays identified SlMYB32 as a putative target protein of SlMPK20. We conclude that SlMPK20 specifically regulates post-meiotic pollen development through modulating sugar and auxin metabolism and signaling.

Silence of ryanodine receptor gene decreases susceptibility to chlorantraniliprole in the oriental armyworm, Mythimna separata Walker.

The ryanodine receptors of insects are the main target sites of diamide insecticides, which show highly selective insecticidal activity relative to toxicity in mammals and provide a novel option for managing lepidopteran pests. The oriental armyworm, Mythimna separata (Walker), is a destructive pest of agricultural crops, and great efforts have been undertaken to control this pest including repeated insecticide applications. In this study, full-length cDNA of a ryanodine receptor gene from M. separata (MsRyR) was cloned and characterized. The cDNA of MsRyR had a 15,372 bp open reading frame and encoded 5124 amino acids (GenBank ID: MG712298). MsRyR shares 78-97% identity with RyR isoforms of other insects, and <50% identities with Homo sapiens RyRs 1-3. Temporal and spatial expression analysis detected MsRyR at all developmental stages and in all tissues. The highest relative levels of MsRyR were detected in the second instar and head. Exposure to chlorantraniliprole after 24 h significantly increased the expression levels of whole body MsRyR mRNA. In addition, dietary ingestion of dsMsRyR significantly reduced the mRNA level of MsRyR and greatly decreased chlorantraniliprole-induced mortality. Our results revealed that the MsRyR could be the molecular target of chlorantraniliprole, and provided the basis for further understanding the resistance mechanism of chlorantraniliprole.