Anti-MAPK1 monoclonal antibody, clone 4G9

Seed-flooding stress is one of the most important constraints that affects the yield and quality of soybean. However, very little is known about the molecular mechanisms underlying seed-flooding tolerance. Hence, the present study investigated the transcriptome profile in root tissues of two contrasting soybean genotypes viz., PI342618B/SFT-tolerant, and NN86-4/SFS-sensitive under seed-flooding stress using RNA-Seq approach. A total of 1563 and 1958 differentially expressed genes (DEGs) were identified in SFT-tolerant and SFS-sensitive, respectively suggesting that the former is less affected by flooding stress. Both Gene ontology (GO) enrichment and MapMan pathway analyses revealed that response to seed-flooding stress was mostly enriched in the DEGs that are involved in the cell wall, antioxidant activity, catalytic and transcription factor activities as well as protein metabolism and signaling. Based on gene annotation, GO enrichment and protein-protein interaction (PPI) network analysis, a total of 807 DEGs were screened from the above terms related to the stress including response to stress, defense response and response to stimulus for candidate gene prediction analysis. Out of them, 51 genes revealing significant opposite gene expression pattern between tolerant and sensitive genotypes along with GmERFVII1, GmERFVII2 & GmERFVII3 and MAPK1 were predicted as the possible candidate genes for further analysis. Using quantitative real-time RT-PCR (qRT-PCR) and sequencing analysis, five of the randomly selected ten genes viz., Glyma.01G231200, Glyma.08G083300, Glyma.06G045400, Glyma.05G215900, and Glyma.15G015100 revealed both significant opposite expression pattern and nucleotide differences, respectively between SFT-tolerant and SFS-sensitive. Hence, these genes will be of great value for breeding seed-flooding tolerance in soybean after their proper functional validation. Overall, the transcriptome profiling indicates that all the three major processes viz., biological process (BP), cellular components (CC), and molecular functions (MF) are altered in the soybean in response to seed-flooding stress. In conclusion, these findings provide in-depth insights that will be of great utility for the exploration of candidate gene functions for seed-flooding tolerance in soybean.

Macroautophagy/autophagy is a lysosome-dependent catabolic process for the turnover of proteins and organelles in eukaryotes. Autophagy plays an important role in immunity and inflammation, as well as metabolism and cell survival. Diverse immune and inflammatory signals induce autophagy in macrophages through pattern recognition receptors, such as toll-like receptors (TLRs). However, the physiological role of autophagy and its signaling mechanisms in microglia remain poorly understood. Microglia are phagocytic immune cells that are resident in the central nervous system and share many characteristics with macrophages. Here, we show that autophagic flux and expression of autophagy-related (Atg) genes in microglia are significantly suppressed upon TLR4 activation by lipopolysaccharide (LPS), in contrast to their stimulation by LPS in macrophages. Metabolomics analysis of the levels of phosphatidylinositol (PtdIns) and its 3-phosphorylated form, PtdIns3P, in combination with bioinformatics prediction, revealed an LPS-induced reduction in the synthesis of PtdIns and PtdIns3P in microglia but not macrophages. Interestingly, inhibition of PI3K, but not MTOR or MAPK1/3, restored autophagic flux with concomitant dephosphorylation and nuclear translocation of FOXO3. A constitutively active form of FOXO3 also induced autophagy, suggesting FOXO3 as a downstream target of the PI3K pathway for autophagy inhibition. LPS treatment impaired phagocytic capacity of microglia, including MAP1LC3B/LC3-associated phagocytosis (LAP) and amyloid (A) clearance. PI3K inhibition restored LAP and degradation capacity of microglia against A. These findings suggest a unique mechanism for the regulation of microglial autophagy and point to the PI3K-FOXO3 pathway as a potential therapeutic target to regulate microglial function in brain disorders.Abbreviations: Atg: autophagy-related gene; A: amyloid-; BafA1: bafilomycin A(1); BECN1: beclin 1, autophagy related; BMDM: bone marrow-derived macrophage; CA: constitutively active; CNS: central nervous system; ZFYVE1/DFCP1: zinc finger, FYVE domain containing 1; FOXO: forkhead box O; ELISA:enzyme-linked immunosorbent assay; HBSS: Hanks balanced salt solution; LAP: LC3-associated phagocytosis; MAP1LC3B: microtubule-associated protein 1 light chain 3; LPS: lipopolysaccharide; LY: LY294002; MTOR: mechanistic target of rapamycin kinase; Pam(3)CSK(4): N-palmitoyl-S-dipalmitoylglyceryl Cys-Ser-(Lys)(4); PtdIns: phosphatidylinositol; PtdIns3P: phosphatidylinositol-3-phosphate; PLA: proximity ligation assay; Poly(I:C): polyinosinic-polycytidylic acid; qRT-PCR: quantitative real-time polymerase chain reaction; RPS6KB1: ribosomal protein S6 kinase, polypeptide 1; TLR: Toll-like receptor; TNF: tumor necrosis factor; TFEB: transcription factor EB; TSPO: translocator protein.