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Fruit softening in tomato (Solanum lycopersicum) is closely associated with cell wall disassembly, which is brought about through the action of a range of cell wall structure-related enzymes and other proteins such as expansins. Xyloglucan endotransglucosylase/hydrolase (XTH) (EC 2.4.1.207 and/or EC 3.2.1.151) has been proposed to be key player involved in xyloglucan metabolism. SlXTH5 showed the highest expression level among all SlXTHs during tomato ripening. In this study, the role of SlXTH5 involved in tomato softening was investigated in CRISPR-based knockout mutants of SlXTH5. Loss-of-function of SlXTH5 in transgenic tomato lines resulted in slightly firmer fruit pericarp, but significantly decreased their color index compared with azygous wild type (WT) control fruits. Increased paste viscosity was detected in CRISPR mutants, indicating that the activity of SlXTH5 is responsible for maintaining cell wall structural integrity. Immunocytochemistry studies were performed using the monoclonal antibody probe LM25 to examine the localization and distribution of xyloglucan in the pericarp cells of the CRISPR mutant fruits. The data indicated more xyloglucan was retained in the pericarp of CRISPR mutant fruit than in WT control fruit. This study revealed the link between SlXTH5 and xyloglucan metabolism and indicated the potential of manipulating SlXTH5 to regulate fruit softening. [ABSTRACT FROM AUTHOR]

Fungi represent an important source of bioactive secondary metabolites (SMs), which have wide applications in many fields, including medicine, agriculture, human health, and many other industries. The genes involved in SM biosynthesis are usually clustered adjacent to each other into a region known as a biosynthetic gene cluster (BGC). The recent advent of a diversity of genetic and genomic technologies has facilitated the identification of many cryptic or uncharacterized BGCs and their associated SMs. However, there are still many challenges that hamper the broader exploration of industrially important secondary metabolites. The recent advanced CRISPR/Cas system has revolutionized fungal genetic engineering and enabled the discovery of novel bioactive compounds. In this review, we firstly introduce fungal BGCs and their relationships with associated SMs, followed by a brief summary of the conventional strategies for fungal genetic engineering. Next, we introduce a range of state-of-the-art CRISPR/Cas-based tools that have been developed and review recent applications of these methods in fungi for research on the biosynthesis of SMs. Finally, the challenges and limitations of these CRISPR/Cas-based systems are discussed and directions for future research are proposed in order to expand their applications and improve efficiency for fungal genetic engineering. [ABSTRACT FROM AUTHOR]

Tomato (Solanum lycopersicum) is a globally important crop with an economic value in the tens of billions of dollars, and a significant supplier of essential vitamins, minerals, and phytochemicals in the human diet. Shelf life is a key quality trait related to alterations in cuticle properties and remodeling of the fruit cell walls. Studies with transgenic tomato plants undertaken over the last 20 years have indicated that a range of pectin-degrading enzymes are involved in cell wall remodeling. These studies usually involved silencing of only a single gene and it has proved difficult to compare the effects of silencing these genes across the different experimental systems. Here we report the generation of CRISPR-based mutants in the ripening-related genes encoding the pectin-degrading enzymes pectate lyase (PL), polygalacturonase 2a (PG2a), and ß-galactanase (TBG4). Comparison of the physiochemical properties of the fruits from a range of PL, PG2a, and TBG4 CRISPR lines demonstrated that only mutations in PL resulted in firmer fruits, although mutations in PG2a and TBG4 influenced fruit color and weight. Pectin localization, distribution, and solubility in the pericarp cells of the CRISPR mutant fruits were investigated using the monoclonal antibody probes LM19 to deesterified homogalacturonan, INRA-RU1 to rhamnogalacturonan I, LM5 to ß-1,4-galactan, and LM6 to arabinan epitopes, respectively. The data indicate that PL, PG2a, and TBG4 act on separate cell wall domains and the importance of cellulose microfibril-associated pectin is reflected in its increased occurrence in the different mutant lines. [ABSTRACT FROM AUTHOR]

To investigate the modification of cell wall polysaccharides in relation to aril breakdown in harvested longan fruit, three pectin fractions (WSP, water soluble pectin; CSP, CDTA-soluble pectin; ASP, alkali soluble pectin) and one hemicellulose fraction (4 M KOH-SHC, 4 M KOH-soluble hemicellulose) were extracted, and their contents, monosaccharide compositions and molecular weights were evaluated. As aril breakdown intensified, CSP content increased while ASP and 4 M KOH-SHC contents decreased, suggesting the solubilization and conversion of cell wall components. Furthermore, the molar percentage of arabinose (Ara), as the main component of the side-chains, decreased largely in CSP and ASP while that of rhamnose (Rha), as branch point for the attachment of neutral sugar side chains, increased during aril breakdown. Analysis of (Ara + Gal)/Rha ratio showed that the depolymerization of CSP and ASP happened predominantly in side-chains formed of Ara residues. For 4 M KOH-SHC, more backbones were depolymerized during aril breakdown. Moreover, it was found that the molecular weights of CSP, ASP and 4 M KOH-SHC polysaccharides tended to decrease as aril breakdown intensified. These results suggest that both enhanced depolymerization and structural modifications of polysaccharides in the CSP, ASP and 4 M KOH-SHC fractions might be responsible for aril breakdown of harvested longan fruit. [ABSTRACT FROM AUTHOR]

Abstract  Tumor hypoxia has been reported to be a negative prognostic factor in a number of tumor sites, which suggests a positive correlation between tumor hypoxia and increased metastatic efficiency. Evidence shows that vascular endothelial growth factor (VEGF) stimulates angiogenesis in tumor growth and mediates neuroprotection to prevent an apoptotic cell death. Human neuroblastoma cells (CHP126) were exposed to moderate hypoxia for different time spans to explore the molecular stress responses. Apoptotic features as an increase of Bax/Bcl-2 ratio and activation of caspase 3 were observed at early period of exposure time, but these effects were reversed with the extension of hypoxic treatment. Hypoxia also activated MAPKs signaling pathways in a time-relative manner, which were involved in the regulation of hypoxia-related resistance of CHP126 cells. Meanwhile, VEGF and its receptor KDR were found to interact with MAPKs signaling pathways except the effect of hypoxia. Furthermore, rhVEGF165 was utilized to discern that VEGF increased Bcl-2 and procaspase 3 expressions, contributing to a synergistic relationship of an angiogenic response with Bcl-2 in hypoxia via a cross talk, while the activation of ERK MAPK is important for both productions. These altered signals may be critical to predict a poor outcome; therefore, our knowledge provides new insight into apoptosis and angiogenesis control of tumor cells and suggests a strategy based on the blockade of hypoxia-induced VEGF signaling under hypoxia in neuroblastoma. [ABSTRACT FROM AUTHOR]

Hypoxia is associated with resistance to chemotherapy in a number of human cancer types; particularly in hepatocellular carcinoma (HCC), which is a highly vascularized tumor. To develop a potential combination therapy strategy that is capable of overcoming the hypoxia-induced insensitivity to chemotherapy, the HCC cell SMMC-7721 was employed to investigate the hypoxia-induced chemoresistance to etoposide. Increased levels of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) were observed when SMMC-7721 cells were exposed to hypoxia, and exposure of tumor cells to hypoxia impaired etoposide-induced DNA damage, as indicated by the failure of upregulation of γHA2X. Etoposide-induced apoptosis and cell cycle arrest of SMMC-7721 was also impaired in hypoxia. However, co-treatment with anti-VEGF significantly restored etoposide-induced cell apoptosis and cell cycle arrest, as indicated by the elimination of B-cell lymphoma 2 (Bcl-2), procaspase 3, cyclin B1 and Cdc2. Furthermore, anti-VEGF eliminated phosphorylation of AKT, ERK and IκB-α resulting from hypoxia, suggesting the involvement of VEGF in the activation of the survival pathways. In conclusion, the present study suggests a significant role of VEGF in the chemoresistance of etoposide in hypoxia. A rational chemotherapy should be developed based on a combination of etoposide and anti-VEGF. [ABSTRACT FROM AUTHOR]