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Abstract: Soil salinity and drought are among the most serious agricultural and environmental problems of today. Therefore, investigations of plant resistance to abiotic stress have received a lot of attention in recent years. In this study, we identified the complete coding sequence of a 3′-phosphoadenosine-5′-phosphatase protein, ApHal2, from the halotolerant yeast Aureobasidium pullulans. Expression of the ApHAL2 gene in a Saccharomyces cerevisiae hal2 mutant complemented the mutant auxotrophy for methionine, and rescued the growth of the hal2 mutant in media with high NaCl concentrations. A 21-amino-acids-long region of the ApHal2 enzyme was inserted into the Arabidopsis thaliana homologue of Hal2, the SAL1 phosphatase. The inserted sequence included the META motif, which has previously been implicated in increased sodium tolerance of the Hal2 homologue from a related fungal species. Transgenic Arabidopsis plants overexpressing this modified SAL1 (mSAL1) showed improved halotolerance and drought tolerance. In a medium with an elevated salt concentration, mSAL1-expressing plants were twice as likely to have roots in a higher length category in comparison with the wild-type Arabidopsis and with plants overexpressing the native SAL1, and had 5% to 10% larger leaf surface area under moderate and severe salt stress, respectively. Similarly, after moderate drought exposure, the mSAL1-expressing plants showed 14% increased dry weight after revitalisation, with no increase in dry weight of the wild-type plants. With severe drought, plants overexpressing native SAL1 had the worst rehydration success, consistent with the recently proposed role of SAL1 in severe drought. This was not observed for plants expressing mSAL1. Therefore, the presence of this fungal META motif sequence is beneficial under conditions of increased salinity and moderate drought, and shows no drawbacks for plant survival under severe drought. This demonstrates that adaptations of extremotolerant fungi should be considered as a valuable resource for improving stress-tolerance in plant breeding in the future.
Keywords: soil salinity and drought, plant resistance, abiotic stress
Published in DiRROS: 02.08.2024; Views: 304; Downloads: 280
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Abstract: Endothelial tip cells are leading cells at the tips of vascular sprouts coordinating multiple processes during angiogenesis. In the developing retina, tip cells play a tightly controlled, timely role in angiogenesis. In contrast, excessive numbers of tip cells are a characteristic of the chaotic pathological blood vessels in proliferative retinopathies. Tip cells control adjacent endothelial cells in a hierarchical manner to form the stalk of the sprouting vessel, using, among others, the VEGF-DLL-Notch signaling pathway, and recruit pericytes. Tip cells are guided toward avascular areas by signals from the local extracellular matrix that are released by cells from the neuroretina such as astrocytes. Recently, tip cells were identified in endothelial cell cultures, enabling identification of novel molecular markers and mechanisms involved in tip cell biology. These mechanisms are relevant for understanding proliferative retinopathies. Agents that primarily target tip cells can block pathological angiogenesis in the retina efficiently and safely without adverse effects. A striking example is platelet-derived growth factor, which was recently shown to be an efficacious additional target in the treatment of retinal neovascularization. Here we discuss these and other tip cell-based strategies with respect to their potential to treat patients with ocular diseases dominated by neovascularization.
Keywords: angiogenesis, endothelial tip cell, proliferative retinopathy, anti-angiogenesis therapy, retinal neovascularization, vascular sprouts, endothelial stalk cell, molecular mediators of angiogenesis, pericytes
Published in DiRROS: 02.08.2024; Views: 287; Downloads: 176
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Abstract: Background Erythromycin is a medically important antibiotic, biosynthesized by the actinomycete Saccharopolyspora erythraea. Genes encoding erythromycin biosynthesis are organized in a gene cluster, spanning over 60 kbp of DNA. Most often, gene clusters encoding biosynthesis of secondary metabolites contain regulatory genes. In contrast, the erythromycin gene cluster does not contain regulatory genes and regulation of its biosynthesis has therefore remained poorly understood, which has for a long time limited genetic engineering approaches for erythromycin yield improvement. Results We used a comparative proteomic approach to screen for potential regulatory proteins involved in erythromycin biosynthesis. We have identified a putative regulatory protein SACE_5599 which shows significantly higher levels of expression in an erythromycin high-producing strain, compared to the wild type S. erythraea strain. SACE_5599 is a member of an uncharacterized family of putative regulatory genes, located in several actinomycete biosynthetic gene clusters. Importantly, increased expression of SACE_5599 was observed in the complex fermentation medium and at controlled bioprocess conditions, simulating a high-yield industrial fermentation process in the bioreactor. Inactivation of SACE_5599 in the high-producing strain significantly reduced erythromycin yield, in addition to drastically decreasing sporulation intensity of the SACE_5599-inactivated strains when cultivated on ABSM4 agar medium. In contrast, constitutive overexpression of SACE_5599 in the wild type NRRL23338 strain resulted in an increase of erythromycin yield by 32%. Similar yield increase was also observed when we overexpressed the bldD gene, a previously identified regulator of erythromycin biosynthesis, thereby for the first time revealing its potential for improving erythromycin biosynthesis. Conclusions SACE_5599 is the second putative regulatory gene to be identified in S. erythraea which has positive influence on erythromycin yield. Like bldD, SACE_5599 is involved in morphological development of S. erythraea, suggesting a very close relationship between secondary metabolite biosynthesis and morphological differentiation in this organism. While the mode of action of SACE_5599 remains to be elucidated, the manipulation of this gene clearly shows potential for improvement of erythromycin production in S. erythraea in industrial setting. We have also demonstrated the applicability of the comparative proteomics approach for identifying new regulatory elements involved in biosynthesis of secondary metabolites in industrial conditions.
Keywords: erythromycin, polyketide, regulator, SACE_5599, lmbU, differentiation (biology), sporulation, strain improvement, metabolic engineering
Published in DiRROS: 02.08.2024; Views: 257; Downloads: 274
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Abstract: Background Since their first commercialization, the diversity of taxa and the genetic composition of transgene sequences in genetically modified plants (GMOs) are constantly increasing. To date, the detection of GMOs and derived products is commonly performed by PCR-based methods targeting specific DNA sequences introduced into the host genome. Information available regarding the GMOs’ molecular characterization is dispersed and not appropriately organized. For this reason, GMO testing is very challenging and requires more complex screening strategies and decision making schemes, demanding in return the use of efficient bioinformatics tools relying on reliable information. Description The GMOseek matrix was built as a comprehensive, online open-access tabulated database which provides a reliable, comprehensive and user-friendly overview of 328 GMO events and 247 different genetic elements (status: 18/07/2013). The GMOseek matrix is aiming to facilitate GMO detection from plant origin at different phases of the analysis. It assists in selecting the targets for a screening analysis, interpreting the screening results, checking the occurrence of a screening element in a group of selected GMOs, identifying gaps in the available pool of GMO detection methods, and designing a decision tree. The GMOseek matrix is an independent database with effective functionalities in a format facilitating transferability to other platforms. Data were collected from all available sources and experimentally tested where detection methods and certified reference materials (CRMs) were available. Conclusions The GMOseek matrix is currently a unique and very valuable tool with reliable information on GMOs from plant origin and their present genetic elements that enables further development of appropriate strategies for GMO detection. It is flexible enough to be further updated with new information and integrated in different applications and platforms.
Keywords: genetically modified organism, GMO, GMO screening, matrix approach, genetically modified plant
Published in DiRROS: 02.08.2024; Views: 319; Downloads: 227
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Abstract: The newly emerging cyanobacterial cytotoxin cylindrospermopsin (CYN) is increasingly found in surface freshwaters, worldwide. It poses a potential threat to humans after chronic exposure as it was shown to be genotoxic in a range of test systems and is potentially carcinogenic. However, the mechanisms of CYN toxicity and genotoxicity are not well understood. In the present study CYN induced formation of DNA double strand breaks (DSBs), after prolonged exposure (72 h), in human hepatoma cells, HepG2. CYN (0.1–0.5 µg/mL, 24–96 h) induced morphological changes and reduced cell viability in a dose and time dependent manner. No significant increase in lactate dehydrogenase (LDH) leakage could be observed after CYN exposure, indicating that the reduction in cell number was due to decreased cell proliferation and not due to cytotoxicity. This was confirmed by imunocytochemical analysis of the cell-proliferation marker Ki67. Analysis of the cell-cycle using flow-cytometry showed that CYN has an impact on the cell cycle, indicating G0/G1 arrest after 24 h and S-phase arrest after longer exposure (72 and 96 h). Our results provide new evidence that CYN is a direct acting genotoxin, causing DSBs, and these facts need to be considered in the human health risk assessment.
Keywords: cylindrospermopsin, cell-cycle, cell-proliferation, double-strand breaks, HepG2 cells
Published in DiRROS: 02.08.2024; Views: 318; Downloads: 226
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