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Darragh, Kathy ; Orteu, Anna ; Black, Daniella ; Byers, Kelsey J. R. P. ; Szczerbowski, Daiane ; Warren, Ian A. ; Rastas, Pasi ; Pinharanda, Ana ; Davey, John ; Garza, Sylvia Fernanda ; Abondano Almeida, Diana ; Merrill, Richard M. ; McMillan, W. Owen ; Schulz, Stefan ; Jiggins, Chris D.
Plants and insects often use the same compounds for chemical communication, but not much is known about the genetics of convergent evolution of chemical signals. The terpene (E)-β-ocimene is a common component of floral scent and is also used by the butterfly Heliconius melpomene as an anti-aphrodisiac pheromone. While the biosynthesis of terpenes has been described in plants and microorganisms, few terpene synthases (TPSs) have been identified in insects. Here, we study the recent divergence of 2 species, H. melpomene and Heliconius cydno, which differ in the presence of (E)-β-ocimene; combining linkage mapping, gene expression, and functional analyses, we identify 2 novel TPSs. Furthermore, we demonstrate that one, HmelOS, is able to synthesise (E)-β-ocimene in vitro. We find no evidence for TPS activity in HcydOS (HmelOS ortholog of H. cydno), suggesting that the loss of (E)-β-ocimene in this species is the result of coding, not regulatory, differences. The TPS enzymes we discovered are unrelated to previously described plant and insect TPSs, demonstrating that chemical convergence has independent evolutionary origins.
Highlights
• Histone modifications alter chromatin structure and gene accessibility, allowing timely stress response, and enhancing tomato's ability to cope with environmental challenges.
• miRNAs and lncRNAs fine-tune gene expression, playing essential roles in stress tolerance, particularly in heat and drought stress responses.
• Leveraging epigenetic modifications can develop tomato varieties that maintain high productivity and quality under adverse environmental conditions.
• Detailed mapping of the tomato epigenome under various stress conditions can identify key regulatory regions and guide targeted breeding programs
Abstract
Climate change poses a major challenge to agriculture, affecting crop production through shifting weather patterns and an increase in extreme conditions such as heat waves, droughts, and floods, all of which are further compounded by biotic stress factors. Tomatoes, a vital dietary staple and significant agricultural product worldwide, are particularly susceptible to these changes. The need for developing climate-resilient tomato varieties is more urgent than ever to ensure food security. Epigenetic modifications, such as DNA methylation and histone modifications, play essential roles in gene expression regulation. These modifications can affect plant traits and responses to environmental stresses, enabling tomatoes to maintain productivity despite variable climates or disease pressures. Tomato, as a model plant, offers valuable insights into the epigenetic mechanisms underlying fruit development and responses to stress. This review provides an overview of key discoveries regarding to tomato response and resilience mechanisms related to epigenetics, highlighting their potential in breeding strategies to enhance tomato resilience against both abiotic and biotic challenges, thereby promoting sustainable agricultural practices in the context of global climate change.
Background: Bacteria of the genus Photorhabdus and Xenorhabdus are motile, Gram-negative bacteria that live in symbiosis with entomopathogenic nematodes. Due to their complex life cycle, they produce a large number of specialized metabolites (natural products) encoded in biosynthetic gene clusters (BGC). Genetic tools for Photorhabdus and Xenorhabdus have been rare and applicable to only a few strains. In the past, several tools have been developed for the activation of BGCs and the deletion of individual genes. However, these often have limited efficiency or are time consuming. Among the limitations, it is essential to have versatile expression systems and genome editing tools that could facilitate the practical work.
Results: In the present study, we developed several expression vectors and a CRISPR-Cpf1 genome editing vector for genetic manipulations in Photorhabdus and Xenorhabdus using SEVA plasmids. The SEVA collection is based on modular vectors that allow exchangeability of different elements (e.g. origin of replication and antibiotic selection markers with the ability to insert desired sequences for different end applications). Initially, we tested different SEVA vectors containing the broad host range origins and three different resistance genes for kanamycin, gentamycin and chloramphenicol, respectively. We demonstrated that these vectors are replicative not only in well-known representatives, e.g. Photorhabdus laumondii TTO1, but also in other rarely described strains like Xenorhabdus sp. TS4. For our CRISPR/Cpf1-based system, we used the pSEVA231 backbone to delete not only small genes but also large parts of BGCs. Furthermore, we were able to activate and refactor BGCs to obtain high production titers of high value compounds such as safracin B, a semisynthetic precursor for the anti-cancer drug ET-743.
Conclusions: The results of this study provide new inducible expression vectors and a CRISPR/CPf1 encoding vector all based on the SEVA (Standard European Vector Architecture) collection, which can improve genetic manipulation and genome editing processes in Photorhabdus and Xenorhabdus.
Highlights
• Different NADPH supply strategies are compared in Saccharomyces cerevisiae.
• Example products are d-xylitol and l-galactonate.
• ZWF1 overexpression is the most robust strategy in the diauxic batch fermentation.
• Carbon source dependencies and interferences of different strategies are explored.
Abstract
Enhancing the supply of the redox cofactor NADPH in metabolically engineered cells is a critical target for optimizing the synthesis of many product classes, such as fatty acids or terpenoids. In S. cerevisiae, several successful approaches have been developed in different experimental contexts. However, their systematic comparison has not been reported. Here, we established the reduction of xylose to xylitol by an NADPH-dependent xylose reductase as a model reaction to compare the efficacy of different NADPH supply strategies in the course of a batch fermentation, in which glucose and ethanol are sequentially used as carbon sources and redox donors. We show that strains overexpressing the glucose-6-phosphate dehydrogenase Zwf1 perform best, producing up to 16.9 g L−1 xylitol from 20 g L−1 xylose in stirred tank bioreactors. The beneficial effect of increased Zwf1 activity is especially pronounced during the ethanol consumption phase. The same notion applies to the deletion of the aldehyde dehydrogenase ALD6 gene, albeit at a quantitatively lower level. Reduced expression of the phosphoglucose isomerase Pgi1 and heterologous expression of the NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase Gdp1 from Kluyveromyces lactis acted synergistically with ZWF1 overexpression in the presence of glucose, but had a detrimental effect after the diauxic shift. Expression of the mitochondrial NADH kinase Pos5 in the cytosol likewise improved the production of xylitol only on glucose, but not in combination with enhanced Zwf1 activity. To demonstrate the generalizability of our observations, we show that the most promising strategies – ZWF1 overexpression and deletion of ALD6 - also improve the production of l-galactonate from d-galacturonic acid. Therefore, we expect that these findings will provide valuable guidelines for engineering not only the production of xylitol but also of diverse other pathways that require NADPH.
Highlights
• Determination of styrene-butadiene rubber as tire constituent using TED-GC/MS.
• Determination of zinc content as tire constituent using ICP-OES.
• Representative sampling strategy with large-volume mixed samples.
• Tire wear content is decreasing with increasing sampling depth and distance to road.
• Deposited tire wear particles are mainly present in soil fraction <100 μm.
Abstract
Tire wear (TW) constitutes a significant source of microplastic in terrestrial ecosystems. It is known that particles emitted by roads can have an effect up to 100 m into adjacent areas. Here, we apply for the first-time thermal extraction desorption gas chromatography-mass spectrometry (TED-GC/MS) to determine TW in soil samples by detection of thermal decomposition products of styrene-butadiene rubber (SBR), without additional enrichment. Additionally, zinc contents were determined as an elemental marker for TW. Mixed soil samples were taken along three transects along a German motorway in 0.3, 2.0, and 5.0 m distance from the road. Sampling depths were 0–2, 2–5, 5–10, and 10–20 cm. Four fine fractions, 1 000–500, 500–100, 100–50, and <50 μm, were analyzed.
TW contents based on SBR ranged from 155 to 15 898 mg kg−1. TW contents based on zinc were between 413 and 44 812 mg kg−1. Comparison of individual values of SBR and zinc reveals SBR as a more specific marker. Results confirm that most TW ends up in the topsoil within a 2 m distance.
The sampling strategy resulted in representative data for a larger area. Standard deviations of quadruple TED-GC/MS determination of SBR were <10% for all grain size fractions. TED-GC/MS is a suitable analytical tool for determining TW in soil samples without the use of toxic chemicals, enrichment, or special sample preparation.
The main goal of this work is to contribute to the existing knowledge of soil micro-fungi in Panama and Germany. Studies about soil degradation and its influents in the soil fungi diversity have not been investigated as extensively in these countries. This is an extensive and challenging topic to examine since there is an immense phenotypic and genetic diversity in the soil fungal community and relating this community together with factors of soil degradation is an extensive task. For this reason, the present thesis studies the species identified in the study areas, in other words, the soil fungal diversity in relation to environmental factors in the Taunus Mountain range in Frankfurt, Germany, and in the Majagua valley in Chiriquí, Panama. Two complementary objectives were achieved, the first was the development of a theoretical irrigation model for degraded soils. The second was the development of a mobile application to facilitate laboratory work in the cultivation of soil micro-fungi.
The design of the methodology was based on identifying the species and relating the diversity found to soil factors. Soil samples were taken in both countries: the Taunus Mountain range was sampled eight times from January to November 2012 and the Majagua valley was sampled on three occasions between February and July 2012. In both studies, the areas included three different vegetation types (forest, grassland, and bare soil). Samples were separated for two purposes: the assessment of fungal diversity by molecular and morphological methods and soil characterization.
Soil samples used in the methodology of pyrosequencing were related to global climatic factors. Morphological identification was achieved with identification keys. Micro-fungi were cultivated in different media until obtaining pure cultures. Molecular identification was performed by getting the DNA sequences using the ITS1 and ITS4 primers and comparing the sequences with other reference sequences from GenBank. This was done considering the BLAST algorithm, which considered sequences that matched 98 % or more of maximum identity as reliable identifications.
Soil characterization was carried out to measure the soil's Physico-chemical properties; those abiotic factors were compaction, temperature, pH, moisture, and soil composition.
Species richness was calculated in each study area with the estimators Chao, Jackknife, and Bootstrap. Furthermore, the species accumulation curves were performed to observe the species discovery rate and estimate sample completeness. Estimate linear regression models correlated the influence between the soil factors (temperature, moisture, pH, soil compaction, and soil composition) and the species richness. In the same way, an analysis of ecological distance was undertaken based on the similarity in the species composition, compared across samples, and correlated with soil factors, using non-metric multidimensional scaling (NMDs).
Study of abundance showed differences between the bare soil abundances and the forest abundances in Germany and Panama; the grasslands in both countries work as transitional areas in the fungi abundance. The key stone species in Germany were Penicillium daleae, and Pochonia bulbillosa, whereas in Panama were Purpureocillium lilacinum and Trichoderma harzianum. Based on Pareto analysis, a theoretical irrigation model was developed to counteract the degradation effects on the abundance of micro-fungi in the soil.
Applications for mobile devices dealing with the cultivation of soil micro fungi were sought. Due to the small number of existing applications, a new App called Soil-Fungi-Cultures (SFC) was developed to facilitate data collection of cultivated soil micro fungi. App Inventor was the program used to design, program, test, and publish the application developed. The developed application was compared with other applications used in identifying bacteria cultures. The results showed that the new application needed more time to capture the records because it saves more information, the navigation flow was acceptable, the number of clicks was high, but it is due to the usefulness in data capture, and finally, the users rated it as a good application with an eight out of ten rating.
Pyrosequencing resulted in 204 Operational Taxonomic Units (OTUs) considering the two study areas (the Taunus Mountain range and the Majagua valley). The Pyrosequencing database was used to contribute to the most important study of fungal diversity globally based on OTUs, which surpasses any study of molecular and taxonomic diversity previously conducted. The principal result in this study was that the climatic factor is the best predictor of fungal richness and community composition on a global scale. However, the part of the research that focused on the local scale, that is to say, on the correlation patterns between the distribution of fungal species and abiotic factors, showed that the soil properties and degradation levels were not associated with fungal richness, diversity or soil composition in the study areas in Germany or Panama. The above confirms that there are exceptions to the way relationships between soil factors with fungal diversity are established at the local level.
In the case of soil samples used for morphological identification, 71 fungal species were obtained, 47 from Germany, and 32 from Panama.
Hoernstein, Sebastian ; Özdemir, Buğra ; Gessel, Nico ; Miniera, Alessandra A. ; Rogalla von Bieberstein, Bruno ; Nilges, Lars ; Schweikert Farinha, Joana ; Komoll, Ramona ; Glauz, Stella ; Weckerle, Tim ; Scherzinger, Friedrich ; Rodriguez‐Franco, Marta ; Müller-Schüssele, Stefanie J. ; Reski, Ralf
Reactive oxygen species (ROS) are constant by-products of aerobic life. In excess, ROS lead to cytotoxic protein aggregates, which are a hallmark of ageing in animals and linked to age-related pathologies in humans. Acylamino acid-releasing enzymes (AARE) are bifunctional serine proteases, acting on oxidized proteins. AARE are found in all domains of life, albeit under different names, such as acylpeptide hydrolase (APEH/ACPH), acylaminoacyl peptidase (AAP), or oxidized protein hydrolase (OPH). In humans, AARE malfunction is associated with age-related pathologies, while their function in plants is less clear. Here, we provide a detailed analysis of AARE genes in the plant lineage and an in-depth analysis of AARE localization and function in the moss Physcomitrella and the angiosperm Arabidopsis. AARE loss-of-function mutants have not been described for any organism so far. We generated and analysed such mutants and describe a connection between AARE function, aggregation of oxidized proteins and plant ageing, including accelerated developmental progression and reduced life span. Our findings complement similar findings in animals and humans, and suggest a unified concept of ageing may exist in different life forms.
Hoernstein, Sebastian ; Özdemir, Buğra ; Gessel, Nico ; Miniera, Alessandra A. ; Rogalla von Bieberstein, Bruno ; Nilges, Lars ; Schweikert Farinha, Joana ; Komoll, Ramona ; Glauz, Stella ; Weckerle, Tim ; Scherzinger, Friedrich ; Rodríguez-Franco, Marta ; Müller-Schüssele, Stefanie J. ; Reski, Ralf
Reactive oxygen species (ROS) are constant by-products of aerobic life. In excess, ROS lead to cytotoxic protein aggregates, which are a hallmark of ageing in animals and linked to age-related pathologies in humans. Acylamino acid-releasing enzymes (AARE) are bifunctional serine proteases, acting on oxidized proteins. AARE are found in all domains of life, albeit under different names, such as acylpeptide hydrolase (APEH/ACPH), acylaminoacyl peptidase (AAP), or oxidized protein hydrolase (OPH). In humans, AARE malfunction is associated with age-related pathologies, while their function in plants is less clear. Here, we provide a detailed analysis of AARE genes in the plant lineage and an in-depth analysis of AARE localization and function in the moss Physcomitrella and the angiosperm Arabidopsis. AARE loss-of-function mutants have not been described for any organism so far. We generated and analysed such mutants and describe a connection between AARE function, aggregation of oxidized proteins and plant ageing, including accelerated developmental progression and reduced life span. Our findings complement similar findings in animals and humans, and suggest a unified concept of ageing may exist in different life forms.
Hoernstein, Sebastian ; Özdemir, Buğra ; Gessel, Nico ; Miniera, Alessandra A. ; Rogalla von Bieberstein, Bruno ; Nilges, Lars ; Schweikert Farinha, Joana ; Komoll, Ramona ; Glauz, Stella ; Weckerle, Tim ; Scherzinger, Friedrich ; Rodríguez-Franco, Marta ; Müller-Schüssele, Stefanie J. ; Reski, Ralf
Protein oxidation results from the reaction of amino-acid side chains with reactive oxygen species (ROS) and is partly irreversible. In non-photosynthetic tissues, mitochondria are a main source of ROS, whereas plastids are the major source in photosynthetic tissues. Oxidized proteins suffer from decreased structural integrity and even loss of function, and their accumulation leads to cytotoxic aggregates. In mammals, aggregate formation correlates with aging and is linked to several age-related pathologies. Mammalian proteolytic pathways for clearance of oxidized proteins are under intensive research, while mechanistic insights into this process in plants is scarce. Acylamino acid-releasing (AARE) enzymes are ATP-independent serine proteases, presumably acting on oxidized proteins and operating in a dual exo-/endopeptidase mode. They are found in all domains of life. Here, we investigated AARE enzymes in the moss Physcomitrella and the angiosperm Arabidopsis and identified three homologous nuclear genes in Physcomitrella (PpAARE1-3) and a single nuclear gene in Arabidopsis (AtAARE). Surprisingly, we observed triple localization of the proteins AtAARE and PpAARE1 to plastids, mitochondria and the cytosol in vivo, likely conserved across the plant lineage. This represents an ATP-independent possibility for degradation of oxidized proteins in the major source organelles of ROS in plants, which is distinct to mammals. Combinatorial knockout plants and protein interaction analysis revealed specific interactions of the moss AARE isoforms and functions in progressive aging. Analysis of an AtAARE T-DNA mutant further suggests the evolutionary conservation of AARE function in age-related development.
Hoernstein, Sebastian ; Özdemir, Buğra ; Gessel, Nico ; Miniera, Alessandra A. ; Rogalla von Bieberstein, Bruno ; Nilges, Lars ; Schweikert Farinha, Joana ; Komoll, Ramona ; Glauz, Stella ; Weckerle, Tim ; Scherzinger, Friedrich ; Rodríguez-Franco, Marta ; Müller-Schüssele, Stefanie J. ; Reski, Ralf
Reactive oxygen species (ROS) are constant by-products of aerobic life. In excess, ROS lead to cytotoxic protein aggregates, which are a hallmark of ageing in animals and linked to age-related pathologies in humans. Acylamino acid-releasing enzymes (AARE) are bifunctional serine proteases, acting on oxidized proteins. AARE are found in all domains of life, albeit under different names, such as acylpeptide hydrolase (APEH/ACPH), acylaminoacyl peptidase (AAP), or oxidized protein hydrolase (OPH). In humans, AARE malfunction is associated with age-related pathologies, while their function in plants is less clear. Here, we provide a detailed analysis of AARE genes in the plant lineage and an in-depth analysis of AARE localization and function in the moss Physcomitrella and the angiosperm Arabidopsis. AARE loss-of-function mutants have not been described for any organism so far. We generated and analysed such mutants and describe a connection between AARE function, aggregation of oxidized proteins and plant ageing, including accelerated developmental progression and reduced life span. Our findings complement similar findings in animals and humans, and support a unified concept of ageing.