A new strategy for cellulases application in high temperature industrial scenarios with syringic acid assisting

In the process of bioethanol production, more stable and active cellulase in high temperature condition is required. In this study, syringic acid was applied in cellulase hydrolysis system. At 70°C, TvEG3 activity increased 201.36%, CtBglA activity decreased 72.79% by syringic acid. With syringic acid assisting, TvEG3 thermostability was improved, CtBglA thermostability was reduced. Syringic acid scarcely affected CtCBH. In hydrolysis system with the cellulases containing TvEG3, CtCBH, and CtBglA, the reducing sugar yield improved by 28.37% with syringic acid assisting. With the molecular dynamic simulation in syringic acid system, the backbone root-mean-square deviation (RMSD) and the residue root-mean-square fluctuation (RMSF) of TvEG3, CtCBH reduced, while the RMSD and RMSF of CtBglA increased. The reduction in the number of secondary structures, especially α-helix, caused the structure of CtBglA in the presence of syringic acid to collapse at high temperature. More secondary structures in TvEG3 and more α-helix in CtCBH in the presence of syringic acid make them more stable at high temperatures. These means syringic acid can stabilize TvEG3 and CtCBH structure, destabilize CtBglA structure at high temperature. In summary, this study not only provides insight into cellulase hydrolysis at high temperature with syringic acid assisting but also demonstrates the promoting mechanism of syringic acid.     

The legacy effect of biochar application on soil nitrous oxide emissions

Existing studies suggest that biochar application can reduce soil nitrous oxide (N₂O) emissions, mainly based on short-term results. However, it remains unclear what the effects (i.e., legacy effects) and underlying mechanisms are on N₂O emissions after many years of a single application of biochar. Here, we collected intact soil columns from plots without and with biochar application in a subtropical tea plantation 7 years ago for an incubation experiment. We used the N₂O isotopocule analysis combined with ammonia oxidizer-specific inhibitors and molecular biology approaches to investigate how the legacy effect of biochar affected soil N₂O emissions. Results showed that the soil in the presence of biochar had lower N₂O emissions than the control albeit statistically insignificant. The legacy effect of biochar in decreasing N₂O emissions may be attributed to the reduced effectiveness of the soil substrate, nitrification and denitrification activities, and the promotion of the further reduction of N₂O. The legacy effect of biochar reduced the relative contribution of nitrifier denitrification/bacterial denitrification, nitrification-related N₂O production, and the relative abundance of several microorganisms involved in the nitrogen cycle. Our global meta-analysis also showed that the reduction of N₂O by biochar increased with increasing application rate but diminished and possibly even reversed with increasing experimental time. In conclusion, our findings suggest that the abatement capacity of biochar on soil N₂O emissions may weaken over time after biochar application, but this remains under further investigation.     

Transmembrane kinase 1-mediated auxin signal regulates membrane-associated clathrin in Arabidopsis roots

Clathrin-mediated endocytosis (CME) is the major endocytic pathway in eukaryotic cells that directly regulates abundance of plasma membrane proteins. Clathrin triskelia are composed of clathrin heavy chains (CHCs) and light chains (CLCs), and the phytohormone auxin differentially regulates membrane-associated CLCs and CHCs, modulating the endocytosis and therefore the distribution of auxin efflux transporter PIN-FORMED2 (PIN2). However, the molecular mechanisms by which auxin regulates clathrin are still poorly understood. Transmembrane kinase (TMKs) family proteins are considered to contribute to auxin signaling and plant development; it remains unclear whether they are involved in PIN transport by CME. We assessed TMKs involvement in the regulation of clathrin by auxin, using genetic, pharmacological, and cytological approaches including live-cell imaging and immunofluorescence. In tmk1 mutant seedlings, auxin failed to rapidly regulate abundance of both CHC and CLC and to inhibit PIN2 endocytosis, leading to an impaired asymmetric distribution of PIN2 and therefore auxin. Furthermore, TMK3 and TMK4 were shown not to be involved in regulation of clathrin by auxin. In summary, TMK1 is essential for auxin-regulated clathrin recruitment and CME. TMK1 therefore plays a critical role in the establishment of an asymmetric distribution of PIN2 and an auxin gradient during root gravitropism.     

Non-specific phospholipase C4 hydrolyzes phosphosphingolipids and phosphoglycerolipids and promotes rapeseed growth and yield

Phosphorus is a major nutrient vital for plant growth and development, with a substantial amount of cellular phosphorus being used for the biosynthesis of membrane phospholipids. Here, we report that NON-SPECIFIC PHOSPHOLIPASE C4 (NPC4) in rapeseed (Brassica napus) releases phosphate from phospholipids to promote growth and seed yield, as plants with altered NPC4 levels showed significant changes in seed production under different phosphate conditions. Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9)-mediated knockout of BnaNPC4 led to elevated accumulation of phospholipids and decreased growth, whereas overexpression (OE) of BnaNPC4 resulted in lower phospholipid contents and increased plant growth and seed production. We demonstrate that BnaNPC4 hydrolyzes phosphosphingolipids and phosphoglycerolipids in vitro, and plants with altered BnaNPC4 function displayed changes in their sphingolipid and glycerolipid contents in roots, with a greater change in glycerolipids than sphingolipids in leaves, particularly under phosphate deficiency conditions. In addition, BnaNPC4-OE plants led to the upregulation of genes involved in lipid metabolism, phosphate release, and phosphate transport and an increase in free inorganic phosphate in leaves. These results indicate that BnaNPC4 hydrolyzes phosphosphingolipids and phosphoglycerolipids in rapeseed to enhance phosphate release from membrane phospholipids and promote growth and seed production.     

Structural insight into chitin perception by chitin elicitor receptor kinase 1 of Oryza sativa

Plants have developed innate immune systems to fight against pathogenic fungi by monitoring pathogenic signals known as pathogen-associated molecular patterns (PAMP) and have established endo symbiosis with arbuscular mycorrhizal (AM) fungi through recognition of mycorrhizal (Myc) factors. Chitin elicitor receptor kinase 1 of Oryza sativa subsp. Japonica (OsCERK1) plays a bifunctional role in mediating both chitin-triggered immunity and symbiotic relationships with AM fungi. However, it remains unclear whether OsCERK1 can directly recognize chitin molecules. In this study, we show that OsCERK1 binds to the chitin hexamer ((NAG)₆) and tetramer ((NAG)₄) directly and determine the crystal structure of the OsCERK1-(NAG)₆ complex at 2 Å. The structure shows that one OsCERK1 is associated with one (NAG)₆. Upon recognition, chitin hexamer binds OsCERK1 by interacting with the shallow groove on the surface of LysM2. These structural findings, complemented by mutational analyses, demonstrate that LysM2 is crucial for recognition of both (NAG)₆ and (NAG)₄. Altogether, these findings provide structural insights into the ability of OsCERK1 in chitin perception, which will lead to a better understanding of the role of OsCERK1 in mediating both immunity and symbiosis in rice.     

Fumarate mitigates disruption induced by fenpropathrin in the silkworm Bombyx mori (Lepidoptera): A metabolomics study

The silkworm Bombyx mori L. is a model organism of the order Lepidoptera. Understanding the mechanism of pesticide resistance in silkworms is valuable for Lepidopteran pest control. In this study, comparative metabolomics was used to analyze the metabolites of 2 silkworm strains with different pesticide resistance levels at 6, 12, and 24 h after feeding with fenpropathrin. Twenty-six of 27 metabolites showed significant differences after fenpropathrin treatment and were classified into 6 metabolic pathways: glycerophospholipid metabolism, sulfur metabolism, glycolysis, amino acid metabolism, the urea cycle, and the tricarboxylic acid (TCA) cycle. After analyzing the percentage changes in the metabolic pathways at the 3 time points, sulfur metabolism, glycolysis, and the TCA cycle showed significant responses to fenpropathrin. Confirmatory experiments were performed by feeding silkworms with key metabolites of the 3 pathways. The combination of iron(II) fumarate + folic acid (IF-FA) enhanced fenpropathrin resistance in silkworms 6.38 fold, indicating that the TCA cycle is the core pathway associated with resistance. Furthermore, the disruption of several energy-related metabolic pathways caused by fenpropathrin was shown to be recovered by IF-FA in vitro. Therefore, IF-FA may have a role in boosting silkworm pesticide resistance by modulating the equilibrium between the TCA cycle and its related metabolic pathways.     

A differential proteomics study of cerebrospinal fluid from individuals with Niemann-Pick disease,Type C1

Niemann-Pick, type C1 (NPC1) is a fatal, neurodegenerative disease, which belongs to the family of lysosomal diseases. In NPC1, endo/lysosomal accumulation of unesterified cholesterol and sphingolipids arise from improper intracellular trafficking resulting in multi-organ dysfunction. With the proximity between the brain and cerebrospinal fluid (CSF), performing differential proteomics provides a means to shed light to changes occurring in the brain. In this study, CSF samples obtained from NPC1 individuals and unaffected controls were used for protein biomarker identification. A subset of these individuals with NPC1 are being treated with miglustat, a glycosphingolipid synthesis inhibitor. Of the 300 identified proteins, 71 proteins were altered in individuals with NPC1 compared to controls including cathepsin D, and members of the complement family. Included are a report of 10 potential markers for monitoring therapeutic treatment. We observed that pro-neuropeptide Y (NPY) was significantly increased in NPC1 individuals relative to healthy controls; however, individuals treated with miglustat displayed levels comparable to healthy controls. In further investigation, NPY levels in a NPC1 mouse model corroborated our findings. We posit that NPY could be a potential therapeutic target for NPC1 due to its multiple roles in the central nervous system such as attenuating neuroinflammation and reducing excitotoxicity.