Overcoming diverse homologous recombinations and single chimeric guide RNA competitive inhibition enhances Cas9-based cyclical multiple genes coediting in filamentous fungi.

Deciphering the complex cellular behaviours and advancing the biotechnology applications of filamentous fungi increase the requirement for genetically manipulating a large number of target genes. The current strategies cannot cyclically coedit multiple genes simultaneously. In this study, we firstly revealed the existence of diverse homologous recombination (HR) types in marker-free editing of filamentous fungi, and then, demonstrated that sgRNA efficiency-mediated competitive inhibition resulted in the low integration of multiple genetic sites during coediting, which are the two major obstacles to limit the efficiency of cyclically coediting of multiple genes. To overcome these obstacles, we developed a biased cutting strategy by Cas9 to greatly enhance the desired HR type and applied a new selection marker labelling strategy for multiple donor DNAs, in which only the donor DNA with the lowest sgRNA efficiency was labelled. Combined with these strategies, we successfully developed a convenient method for cyclically coediting multiple genes in different filamentous fungi. In addition, diverse HRs resulted in a useful and convenient one-step approach for gene functional study combining both gene disruption and complementation. This research provided both a useful one-step approach for gene functional study and an efficient strategy for cyclically coediting multiple genes in filamentous fungi.

Genome-wide identification and characterization of PIN-FORMED (PIN) and PIN-LIKES (PILS) gene family reveals their role in adventitious root development in tea nodal cutting (Camellia Sinensis).

Auxin affects all aspects of plant growth and development, including morphogenesis and adaptive responses. Auxin transmembrane transport is promoted by PIN formation (PIN) and a structurally similar PIN-like (PILS) gene family, which jointly controls the directional transport of the auxin between plant cells, and the accumulation of intracellular auxin. At present, there is no study investigating the roles of CslPIN and CslPILS gene family in root development in the tea plant (Camellia sinensis). In this study, 8 CslPIN and 10 CslPILS genes were identified in the tea plant, and their evolutionary relationships, physical and chemical properties, conserved motifs, cis-acting elements, chromosome location, collinearity, and expression characteristics were analyzed. The mechanism of CslPIN and CslPILS in the formation of tea adventitious roots (ARs) was studied by the AR induction system. Through functional verification, the regulation of CslPIN3 gene on root growth and development of tea plant was studied by over-expression of CslPIN3 in Arabidopsis thaliana and in situ hybridization in Camellia sinensis. The results confirmed CslPIN3 was involved in the regulation of root growth and development as well as auxin accumulation. This study provides a better insight into the regulatory mechanism of CslPIN and CslPILS gene family on the formation of AR in tea plant.

Genome-wide analysis of long non-coding RNAs (lncRNAs) in tea plants (Camellia sinensis) lateral roots in response to nitrogen application.

Tea (Camellia sinensis) is one of the significant cash crops in China. As a leaf crop, nitrogen supply can not only increase the number of new shoots and leaves but also improve the tenderness of the former. However, a conundrum remains in science, which is the molecular mechanism of nitrogen use efficiency, especially long non-coding RNA (lncRNA). In this study, a total of 16,452 lncRNAs were identified through high-throughput sequencing analysis of lateral roots under nitrogen stress and control conditions, of which 9,451 were differentially expressed lncRNAs (DE-lncRNAs). To figure out the potential function of nitrogen-responsive lncRNAs, co-expression clustering was employed between lncRNAs and coding genes. KEGG enrichment analysis revealed nitrogen-responsive lncRNAs may involve in many biological processes such as plant hormone signal transduction, nitrogen metabolism and protein processing in endoplasmic reticulum. The expression abundance of 12 DE-lncRNAs were further verified by RT-PCR, and their expression trends were consistent with the results of RNA-seq. This study expands the research on lncRNAs in tea plants, provides a novel perspective for the potential regulation of lncRNAs on nitrogen stress, and valuable resources for further improving the nitrogen use efficiency of tea plants.

The Role of Green Tea on the Regulation of Gut Microbes and Prevention of High-Fat Diet-Induced Metabolic Syndrome in Mice.

Green tea is a popular non-alcoholic beverage consumed worldwide and has been shown to be beneficial for human health. However, further exploration is needed to fully understand its function in reducing obesity and regulating gut microbes. Here, we investigated the modulatory effects of green tea and its functional components on high-fat diet (HF)-induced metabolic alterations and gut microbiota in obese mice. Our results showed that 1%, 2%, and 4% of green tea promotes weight loss, with the 2% and 4% groups exhibiting distinct gut microflora clusters compared to the HF group. These results were comparable to those observed in the tea polyphenols (TPP)-treated group, suggesting the TPP in green tea plays a crucial role in body weight control and gut microbiota regulation. Additionally, 32 bacteria were identified as potential obesity markers via 16S rRNA gene sequencing. The 16SrDNA gene is a chromosomal gene present in all bacterial species, highly conserved in structure and function, that can reflect the differences between different taxa. The 16S rRNA-based analysis revealed that Akkermansia, a gut-beneficial bacteria, significantly increased in the TPP group.

Effects of leaf-spreading on the volatile aroma components of green tea under red light of different intensities.

Spreading is an indispensable process in the aroma formation of green tea. The application of exogenous red-light spreading in tea processing has been verified to significantly improve the aroma of green tea, and endow tea with freshness, sweet flavor, and mellow taste. However, there were no previous studies investigating the effects of spreading with different red-light intensities on the aroma components of green tea. The aim of the present study was to evaluate the effect of the relationship between the aroma component and spreading with different red-light intensities (300 µmol∙m-2∙s-1, 150 µmol∙m-2∙s-1 and 75 µmol∙m-2∙s-1). As a result, a total of ninety-one volatile components were identified in this study. The orthogonal partial least squares discriminant analysis (OPLS-DA) model clearly distinguished the volatile components of green tea between different red-light intensities and obtained thirty-three differential volatile compounds. Combined with odor activity value (OAV > 1) analysis revealed that eleven volatile components were the key volatile compounds of green tea under different light conditions. Among them, 3-methyl-butanal, (E)-nerolidol, and linalool were the sources of chestnut-like aroma in green tea and were significantly accumulated under medium (MRL) and low intensity (LRL) red light. The results of the present study provided a theoretical basis that could guide green tea processing with red-light intensities to increase the aroma quality components of green tea.

Mycorrhizal Colonization Enhanced Sorghum bicolor Tolerance under Soil Water Deficit Conditions by Coordination of Proline and Reduced Glutathione (GSH).

Drought stress is an important limiting factor in crop production. Arbuscular mycorrhizal fungi (AMF) enhance plant drought tolerance through antioxidant activities. However, the coordination of nonenzymatic antioxidants against drought remains unclear. Here, we investigated the AMF symbiosis in drought tolerance of Sorghum bicolor by increasing proline and reducing glutathione (GSH). Glomus mosseae inoculation increased grain yield, biochemical content, and bioactivities of millets. Under drought conditions, seedlings inoculated with G. mosseae had higher SOD, POD, CAT, PPO, proline, and GSH activities compared to noninoculated controls. Meanwhile, a lower accumulation of MDA and H2O2 was observed in the G. mosseae seedlings. Furthermore, genes attributed to nonenzymatic antioxidants, such as GST29, P5CS2, FD3, GST, and GAD, were significantly up-regulated by G. mosseae under drought conditions. In conclusion, G. mosseae inoculation enhanced the drought tolerance of S. bicolor by improving reactive oxygen species (ROS) scavengers, including proline and GSH, that regulate ROS production and prevent oxidative damage.

Inhibitory Effect of Polypeptides Produced by Brevibacillus brevis on Ochratoxigenic Fungi in the Process of Pile-Fermentation of Post-Fermented Tea.

Contamination by ochratoxigenic fungi and its prevention during the pile-fermentation of post-fermented tea have always been a concern. The present study aimed to elucidate the anti-fungal effect and mechanism of polypeptides produced by B. brevis DTM05 (isolated from post-fermented tea) on ochratoxigenic fungi, and to to evaluate their use in the pile-fermentation process of post-fermented tea. The results showed that polypeptides (produced by B. brevis DTM05) with a strong antifungal effect against A. carbonarius H9 mainly had a molecular weight between 3 and 5 kDa. The Fourier-transform infrared spectra of this polypeptide extract showed that it was a mixture consisting mainly of polypeptides and small amounts of lipids and other carbohydrates. The polypeptide extracts significantly inhibited the growth of A. carbonarius H9, and its minimum inhibitory concentration (MIC) was 1.6 mg/L, which significantly reduced the survival rate of spores. The polypeptides also effectively controlled the occurrence and ochratoxin A (OTA) production of A. carbonarius H9 on the tea matrix. The lowest concentration of polypeptides that significantly inhibited the growth of A. carbonarius H9 on the tea matrix was 3.2 mg/L. The enhancement of the fluorescence staining signal in the mycelium and conidiospore showed that the polypeptides with a concentration of more than 1.6 mg/L increased the permeability of the mycelium membrane and conidial membrane of A. carbonarius H9. The significant increase in the extracellular conductivity of mycelia suggested the outward leakage of intracellular active substances, and also further indicated an increase in cell membrane permeability. Polypeptides with a concentration of 6.4 mg/L significantly down-regulated the expression level of the polyketide synthase gene related to OTA production (acpks) in A. carbonarius H9, which may be the fundamental reason why polypeptides affect OTA production. In conclusion, reasonable use of the polypeptides produced by B. brevis can destroy the structural integrity of the cell membrane, make the intracellular active substances leak outward, accelerate the death of fungal cells and down-regulate the expression level of the polyketide synthase gene in A. carbonarius; thus, they can effectively control the contamination of ochratoxigenic fungi and OTA production during the pile-fermentation of the post-fermented tea.

Pea-Tea Intercropping Improves Tea Quality through Regulating Amino Acid Metabolism and Flavonoid Biosynthesis.

Pea-tea intercropping is an excellent cultivation method that can improve tea quality. However, the underlying mechanism is still unclear. The present study was aimed at elucidating the mechanism of the effect of pea-tea intercropping on tea quality through a high-throughput method. Transcriptome and metabolome analyses were conducted to identify the changes in gene expression and metabolites changes intercropping, respectively. In addition, the amino acids and catechins were detected using the LC-MS method and quantified absolutely. The results showed that total polyphenols and catechins decreased but amino acids increased in pea intercropped tea shoots. Correspondingly, genes related to amino acid metabolism and flavonoid biosynthesis were differentially expressed. For amino acid metabolism, 11 differentially expressed genes were identified, including 5 upregulated and 6 downregulated genes. Meanwhile, three genes involved in carbohydrate transport and metabolism were upregulated in pea intercropped tea plants. These genes were also involved in amino acid metabolism. For flavonoid biosynthesis, two downregulated genes were identified, which were the flavonol synthase and anthocyanidin synthase genes and followed a similar pattern to changes in catechins and polyphenols. These advances have opened new horizons for understanding the biochemical mechanisms of amino acids and flavonoids in improving tea quality in the pea-tea intercropping cultivation model.

Glutamine Synthetases Play a Vital Role in High Accumulation of Theanine in Tender Shoots of Albino Tea Germplasm "Huabai 1".

Theanine (N-ethyl-γ-l-glutamine) is a special nonprotein amino acid that contributes to the umami taste and health function of tea. Although recent studies on tea breeding have focused on albino tea because of its umami taste, a factor of higher theanine concentration, the mechanism of biosynthesis of l-theanine is still unclear. In this study, four glutamine synthetase genes (CsGSs) were obtained and functionally characterized by overexpressing them in Arabidopsis. The enzyme activities of the purified CsGS proteins from Escherichia coli were detected. The results showed that CsGSs have a dual function in the synthesis of glutamine and theanine in vivo and in vitro. Interestingly, l-theanine was abundantly synthesized in the tender shoots of "Huabai 1". In the white tender shoots, the cytosol CsGS1.2 might exhibit increased expression to compensate for decreasing levels of chloroplast CsGS2, which plays a vital role in high accumulation of theanine in "Huabai 1". In addition, CsGS2 was most likely the key l-theanine synthases in green tissues of tea. The present findings will provide basis for and considerably broaden the scope of understanding the function of CsGSs and the mechanism of l-theanine accumulation in the tender shoots of "Huabai 1", and will be useful for breeding and screening tea with high l-theanine content.

SR18292 exerts potent antitumor effects in multiple myeloma via inhibition of oxidative phosphorylation.

AIMS: Multiple myeloma (MM) was recently reported to rely on increased oxidative phosphorylation (OXPHOS) for survival, providing a potential opportunity for MM therapy. Herein, we aimed to propose a novel targeted drug for MM treatment, followed by the exploration of reason for OXPHOS enhancement in MM cells. MATERIALS AND METHODS: The expression of OXPHOS genes and peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) was analyzed using bioinformatics analyses, followed by verification in MM cell lines. The effects of SR18292 on OXPHOS were measured by qRT-PCR, Western blot, transmission electron microscopy, oxygen consumption rate and so on. The proliferation and apoptosis were evaluated by CCK-8, flow cytometry and Western blot. The efficiency and safety of SR18292 were assessed in a mouse model of MM. KEY FINDINGS: The OXPHOS genes were generally overexpressed in MM cells, which was associated with poorer prognosis of MM patients. PGC-1α, a transcriptional coactivator, was upregulated in MM cells, and MM patients with higher PGC-1α expression exhibited increased enrichment of the OXPHOS gene set. Treatment with SR18292 (an inhibitor of PGC-1α) significantly impaired the proliferation and survival of MM cells due to OXPHOS metabolism dysfunction, which leads to energy exhaustion and oxidative damage. Besides, SR18292 potently inhibited tumor growth at a well-tolerated dose in MM model mice. SIGNIFICANCE: The overexpression of OXPHOS gene set mediated by upregulated PGC-1α provides a structural basis for enhanced OXPHOS in MM cells, and SR18292 (a PGC-1α inhibitor) exerts potent antimyeloma effects, offering a potential tangible avenue for MM therapy.

Pterostilbene inhibits nutrient metabolism and induces apoptosis through AMPK activation in multiple myeloma cells.

Multiple myeloma (MM) cells are characterized by an abnormal nutrient metabolism that is distinct from normal plasma cells. Pterostilbene (PTE), a bioactive component of blueberries, has been demonstrated to induce apoptosis in multiple types of cancer cell. The present study evaluated whether PTE treatment affected the survival of MM cells from a metabolic perspective, and the potential mechanisms of this. It was observed that the administration of PTE induced apoptosis, which was mediated by the increased activation of AMP­activated protein kinase (AMPK). Once activated, AMPK decreased the expression and/or activity of key lipogenic enzymes, including fatty acid synthase and acetyl­CoA carboxylase. In addition, the activation of AMPK suppressed the downstream substrate, mechanistic target of rapamycin, which dephosphorylated eukaryotic initiation factor 4E­binding protein 1, leading to a general decrease in mRNA translation. Pre­treatment with the AMPK inhibitor compound C prior to PTE treatment compromised the anti­myeloma apoptosis effect, suggesting the critical role of AMPK in mediating PTE­induced cell toxicity. Consistent results were obtained in vivo. Finally, autophagy was adaptively upregulated subsequent to PTE treatment; the pro­apoptotic efficacy of PTE was potentiated once autophagic flux was inhibited by 3­methyladenine. Taken together, these data demonstrated that PTE exerts anti­tumor effects on MM cells via AMPK­induced nutrient suppression.

Adipocytes secreted leptin is a pro-tumor factor for survival of multiple myeloma under chemotherapy.

Accumulating evidences have shown that adipokines secreted from adipocytes contributes to tumor development, especially leptin. However, underlying mechanisms remain unclear. This study aims to explore the effect of leptin on development and chemoresistance in multiple myeloma cells and the potential mechanism. Analysis of levels of adipokines including leptin and adiponectin in 28 multiple myeloma patients identified significantly higher leptin compared with 28 normal controls(P < 0.05), and leptin level was positively correlated with clinical stage, IgG, ER, and ß2MG. Next, by using co-culture system of myeloma and adipocytes, and pharmacologic enhancement of leptin, we found that increased growth of myeloma cells and reduced toxicity of bortezomib were best observed at 50 ng/ml of leptin, along with increased expression of cyclinD1, Bcl-2 and decreased caspase-3 expression. We also found that phosphorylated AKT and STAT3 but not the proteins expression reached peak after 1h and 6h treatment of leptin, respectively. By using AG490, an agent blocking the phosphorylation of AKT and ERK, the proliferation of myeloma cells was inhibited, as well as the phosphorylation of AKT and STAT3, even adding leptin. Taken together, our study demonstrated that up-regulated leptin could stimulate proliferation of myeloma and reduce the anti-tumor effect of chemotherapy possibly via activating AKT and STAT3 pathways, and leptin might be one of the potential therapeutic targets for treating myeloma.