Characterization of extracellular secondary metabolites in Oudemansiella canarii BRM-044600 displaying antifungal activity against the phytopathogen Sclerotinia sclerotiorum.

White mold disease, caused by the phytopathogen Sclerotinia sclerotiorum, provokes severe productivity losses in several economically important crops. Biocontrol agents, especially antagonist filamentous fungi, are environmentally friendly alternatives to the chemical fungicides used in white mold management. The objective of this study was to screen for basidiomycete fungi capable of inhibiting S. sclerotiorum and investigate their bioactive metabolites responsible for antifungal activities. Two out of 17 tested basidiomycete isolates inhibited the mycelial growth of S. sclerotiorum in pair culture experiments on agar plates, namely Oudemansiella canarii BRM-044600 and Laetisaria arvalis ATCC52088. O. canarii BRM-044600 liquid culture filtrate exhibited the greatest antifungal activity and was selected for further investigation. UHPLC-MS analysis suggests that six putative strobilurins, including strobilurin A and/or stereoisomers of this compound (m/z 259.1299, [M + H]+) and three putative strobilurins with m/z 257.1184 ([M + H]+) are likely responsible for the antifungal activity observed in the culture filtrate. For the first time, this work demonstrated the potential of O. canarii for white mold biocontrol and strobilurin production.

Metabolic fingerprinting analysis of oil palm reveals a set of differentially expressed metabolites in fatal yellowing symptomatic and non-symptomatic plants.

INTRODUCTION: Oil palm (E. guineensis), the most consumed vegetable oil in the world, is affected by fatal yellowing (FY), a condition that can lead to the plant's death. Although studies have been performed since the 1980s, including investigations of biotic and abiotic factors, FY's cause remains unknown and efforts in researches are still necessary. OBJECTIVES: This work aims to investigate the metabolic expression in plants affected by FY using an untargeted metabolomics approach. METHOD: Metabolic fingerprinting analysis of oil palm leaves was performed using ultra high liquid chromatography-electrospray ionization-mass spectrometry (UHPLC-ESI-MS). Chemometric analysis, using principal component analysis (PCA) and partial least square discriminant analysis (PLS-DA), was applied to data analysis. Metabolites identification was performed by high resolution mass spectrometry (HRMS), MS/MS experiments and comparison with databases and literature. RESULTS: Metabolomics analysis based on MS detected more than 50 metabolites in oil palm leaf samples. PCA and PLS-DS analysis provided group segregation and classification of symptomatic and non-symptomatic FY samples, with a great external validation of the results. Nine differentially expressed metabolites were identified as glycerophosphorylcholine, arginine, asparagine, apigenin 6,8-di-C-hexose, tyramine, chlorophyllide, 1,2-dihexanoyl-sn-glycero-3-phosphoethanolamine, proline and malvidin 3-glucoside-5-(6″-malonylglucoside). Metabolic pathways and biological importance of those metabolites were assigned. CONCLUSION: Nine metabolites were detected in a higher concentration in non-symptomatic FY plants. Seven are related to stress factors i.e. plant defense and nutrient absorption, which can be affected by the metabolic depression of these compounds. Two of those metabolites (glycerophosphorylcholine and 1,2-dihexanoyl-sn-glycero-3-phosphoethanolamine) are presented as potential biomarkers, since they have no known direct relation to plant stress.

Osmoprotectants play a major role in the Portulaca oleracea resistance to high levels of salinity stress-insights from a metabolomics and proteomics integrated approach.

Introduction: Purslane (Portulaca oleracea L.) is a non-conventional food plant used extensively in folk medicine and classified as a multipurpose plant species, serving as a source of features of direct importance to the agricultural and agri-industrial sectors. This species is considered a suitable model to study the mechanisms behind resistance to several abiotic stresses including salinity. The recently achieved technological developments in high-throughput biology opened a new window of opportunity to gain additional insights on purslane resistance to salinity stress-a complex, multigenic, and still not well-understood trait. Only a few reports on single-omics analysis (SOA) of purslane are available, and only one multi-omics integration (MOI) analysis exists so far integrating distinct omics platforms (transcriptomics and metabolomics) to characterize the response of purslane plants to salinity stress. Methods: The present study is a second step in building a robust database on the morpho-physiological and molecular responses purslane to salinity stress and its subsequent use in attempting to decode the genetics behind its resistance to this abiotic stress. Here, the characterization of the morpho-physiological responses of adult purslane plants to salinity stress and a metabolomics and proteomics integrative approach to study the changes at the molecular level in their leaves and roots is presented. Results and discussion: Adult plants of the B1 purslane accession lost approximately 50% of the fresh and dry weight (from shoots and roots) whensubmitted to very high salinity stress (2.0 g of NaCl/100 g of the substrate). The resistance to very high levels of salinity stress increases as the purslane plant matures, and most of the absorbed sodium remains in the roots, with only a part (~12%) reaching the shoots. Crystal-like structures, constituted mainly by Na+, Cl-, and K+, were found in the leaf veins and intercellular space near the stoma, indicating that this species has a mechanism of salt exclusion operating on the leaves, which has its role in salt tolerance. The MOI approach showed that 41 metabolites were statistically significant on the leaves and 65 metabolites on the roots of adult purslane plants. The combination of the mummichog algorithm and metabolomics database comparison revealed that the glycine, serine, and threonine, amino sugar and nucleotide sugar, and glycolysis/gluconeogenesis pathways were the most significantly enriched pathways when considering the total number of occurrences in the leaves (with 14, 13, and 13, respectively) and roots (all with eight) of adult plants; and that purslane plants employ the adaptive mechanism of osmoprotection to mitigate the negative effect of very high levels of salinity stress; and that this mechanism is prevalent in the leaves. The multi-omics database built by our group underwent a screen for salt-responsive genes, which are now under further characterization for their potential to promote resistance to salinity stress when heterologously overexpressed in salt-sensitive plants.

Multi-omics Analysis of Young Portulaca oleracea L. Plants' Responses to High NaCl Doses Reveals Insights into Pathways and Genes Responsive to Salinity Stress in this Halophyte Species.

Soil salinity is among the abiotic stressors that threaten agriculture the most, and purslane (Portulaca oleracea L.) is a dicot species adapted to inland salt desert and saline habitats that hyper accumulates salt and has high phytoremediation potential. Many researchers consider purslane a suitable model species to study the mechanisms of plant tolerance to drought and salt stresses. Here, a robust salinity stress protocol was developed and used to characterize the morphophysiological responses of young purslane plants to salinity stress; then, leaf tissue underwent characterization by distinct omics platforms to gain further insights into its response to very high salinity stress. The salinity stress protocol did generate different levels of stress by gradients of electrical conductivity at field capacity and water potential in the saturation extract of the substrate, and the morphological parameters indicated three distinct stress levels. As expected from a halophyte species, these plants remained alive under very high levels of salinity stress, showing salt crystal-like structures constituted mainly by Na+, Cl-, and K+ on and around closed stomata. A comprehensive and large-scale metabolome and transcriptome single and integrated analyses were then employed using leaf samples. The multi-omics integration (MOI) system analysis led to a data-set of 51 metabolic pathways with at least one enzyme and one metabolite differentially expressed due to salinity stress. These data sets (of genes and metabolites) are valuable for future studies aimed to deepen our knowledge on the mechanisms behind the high tolerance of this species to salinity stress. In conclusion, besides showing that this species applies salt exclusion already in young plants to support very high levels of salinity stress, the initial analysis of metabolites and transcripts data sets already give some insights into other salt tolerance mechanisms used by this species to support high levels of salinity stress. Supplementary Information: The online version contains supplementary material available at 10.1007/s43657-022-00061-2.

Antioxidant action of mangrove polyphenols against gastric damage induced by absolute ethanol and ischemia-reperfusion in the rat.

Rhizophora mangle, the red mangrove, has long been known as a traditional medicine. Its bark has been used as astringent, antiseptic, hemostatic, with antifungic and antiulcerogenic properties. In this paper, we aimed to evaluate the antioxidant properties of a buthanolic fraction of the R. mangle bark extract (RM) against experimental gastric ulcer in rats. Unib-Wh rats received pretreatment of R. mangle after the induction of gastric injury with absolute ethanol and ischemia-reperfusion. Gastric tissues from both methods were prepared to the enzymatic assays, the levels of sulfhydril compounds (GSH), lipid peroxides (LPO), and the activities of glutathione reductase (GR), glutathione peroxidase (GPx), superoxide dismutase (SOD) and myeloperoxidase (MPO) were measured. The RM protected the gastric mucosa in both methods used, ethanol-induced gastric ulcer and ischemia-reperfusion, probably, by modulating the activities of the enzymes SOD, GPx, and GR and increasing or maintaining the levels of GSH; in addition, LPO levels were reduced. The results suggest that the RM antioxidant activity leads to tissue protection; thus one of the antiulcer mechanisms present on the pharmacological effects of R. mangle is the antioxidant property.

Deep Untargeted Metabolomics Analysis to Further Characterize the Adaptation Response of Gliricidia sepium (Jacq.) Walp. to Very High Salinity Stress.

The multipurpose tree Gliricidia sepium (Jacq.) Walp. adapts to a very high level of salt stress (≥20 dS m-1) and resumes the production of new leaves around 2 weeks after losing all leaves due to abrupt salinity stress. The integration of metabolome and transcriptome profiles from gliricidia leaves points to a central role of the phenylpropanoid biosynthesis pathway in the short-term response to salinity stress. In this study, a deeper untargeted metabolomics analysis of the leaves and roots of young gliricidia plants was conducted to characterize the mechanism(s) behind this adaptation response. The polar and lipidic fractions from leaf and root samples were extracted and analyzed on a UHPLC.ESI.Q-TOF.HRMS system. Acquired data were analyzed using the XCMS Online, and MetaboAnalyst platforms, via three distinct and complementary strategies. Together, the results obtained first led us to postulate that these plants are salt-excluding plants, which adapted to high salinity stress via two salt-excluding mechanisms, starting in the canopy-severe defoliation-and concluding in the roots-limited entry of Na. Besides that, it was possible to show that the phenylpropanoid biosynthesis pathway plays a role throughout the entire adaptation response, starting in the short term and continuing in the long one. The roots metabolome analysis revealed 11 distinct metabolic pathways affected by salt stress, and the initial analysis of the two most affected ones-steroid biosynthesis and lysine biosynthesis-led us also to postulate that the accumulation of lignin and some phytosterols, as well as lysine biosynthesis-but not degradation, play a role in promoting the adaptation response. However, additional studies are necessary to investigate these hypotheses.

Insights from a Multi-Omics Integration (MOI) Study in Oil Palm (Elaeis guineensis Jacq.) Response to Abiotic Stresses: Part One-Salinity.

Oil palm (Elaeis guineensis Jacq.) is the number one source of consumed vegetable oil nowadays. It is cultivated in areas of tropical rainforest, where it meets its natural condition of high rainfall throughout the year. The palm oil industry faces criticism due to a series of practices that was considered not environmentally sustainable, and it finds itself under pressure to adopt new and innovative procedures to reverse this negative public perception. Cultivating this oilseed crop outside the rainforest zone is only possible using artificial irrigation. Close to 30% of the world's irrigated agricultural lands also face problems due to salinity stress. Consequently, the research community must consider drought and salinity together when studying to empower breeding programs in order to develop superior genotypes adapted to those potential new areas for oil palm cultivation. Multi-Omics Integration (MOI) offers a new window of opportunity for the non-trivial challenge of unraveling the mechanisms behind multigenic traits, such as drought and salinity tolerance. The current study carried out a comprehensive, large-scale, single-omics analysis (SOA), and MOI study on the leaves of young oil palm plants submitted to very high salinity stress. Taken together, a total of 1239 proteins were positively regulated, and 1660 were negatively regulated in transcriptomics and proteomics analyses. Meanwhile, the metabolomics analysis revealed 37 metabolites that were upregulated and 92 that were downregulated. After performing SOA, 436 differentially expressed (DE) full-length transcripts, 74 DE proteins, and 19 DE metabolites underwent MOI analysis, revealing several pathways affected by this stress, with at least one DE molecule in all three omics platforms used. The Cysteine and methionine metabolism (map00270) and Glycolysis/Gluconeogenesis (map00010) pathways were the most affected ones, each one with 20 DE molecules.

Integration of metabolomics and transcriptomics data to further characterize Gliricidia sepium (Jacq.) Kunth under high salinity stress.

Soil salinity is one abiotic stress that threatens agriculture in more than 100 countries. Gliricidia [Gliricidia sepium (Jacq.) Kunth] is a multipurpose tree known for its ability to adapt to a wide range of soils; however, its tolerance limits and responses to salt stress are not yet well understood. In this study, after characterizing the morphophysiological responses of young gliricidia plants to salinity stress, leaf metabolic and transcription profiles were generated and submitted to single and integrated analyses. RNA from leaf samples were subjected to RNA sequencing using an Illumina HiSeq platform and the paired-end strategy. Polar and lipidic fractions from leaf samples were extracted and analyzed on an ultra-high-performance liquid chromatography (UHPLC) coupled with electrospray ionization quadrupole time-of-flight high-resolution mass spectrometry (MS) system. Acquired data were analyzed using the OmicsBox, XCMS Online, MetaboAnalyst, and Omics Fusion platforms. The substrate salinization protocol used allowed the identification of two distinct responses to salt stress: tolerance and adaptation. Single analysis on transcriptome and metabolome data sets led to a group of 5,672 transcripts and 107 metabolites differentially expressed in gliricidia leaves under salt stress. The phenylpropanoid biosynthesis was the most affected pathway, with 15 metabolites and three genes differentially expressed. Results showed that the differentially expressed metabolites and genes from this pathway affect mainly short-term salt stress (STS). The single analysis of the transcriptome identified 12 genes coding for proteins that might play a role in gliricidia response at both STS and long-term salt stress (LTS). Further studies are needed to reveal the mechanisms behind the adaptation response.

Insights from a Multi-Omics Integration (MOI) Study in Oil Palm (Elaeis guineensis Jacq.) Response to Abiotic Stresses: Part Two-Drought.

Drought and salinity are two of the most severe abiotic stresses affecting agriculture worldwide and bear some similarities regarding the responses of plants to them. The first is also known as osmotic stress and shows similarities mainly with the osmotic effect, the first phase of salinity stress. Multi-Omics Integration (MOI) offers a new opportunity for the non-trivial challenge of unraveling the mechanisms behind multigenic traits, such as drought and salinity resistance. The current study carried out a comprehensive, large-scale, single-omics analysis (SOA) and MOI studies on the leaves of young oil palm plants submitted to water deprivation. After performing SOA, 1955 DE enzymes from transcriptomics analysis, 131 DE enzymes from proteomics analysis, and 269 DE metabolites underwent MOI analysis, revealing several pathways affected by this stress, with at least one DE molecule in all three omics platforms used. Moreover, the similarities and dissimilarities in the molecular response of those plants to those two abiotic stresses underwent mapping. Cysteine and methionine metabolism (map00270) was the most affected pathway in all scenarios evaluated. The correlation analysis revealed that 91.55% of those enzymes expressed under both stresses had similar qualitative profiles, corroborating the already known fact that plant responses to drought and salinity show several similarities. At last, the results shed light on some candidate genes for engineering crop species resilient to both abiotic stresses.

Metabolic effect of drought stress on the leaves of young oil palm (Elaeis guineensis) plants using UHPLC-MS and multivariate analysis.

The expansion of the oil palm in marginal areas can face challenges, such as water deficit, leading to an impact on palm oil production. A better understanding of the biological consequences of abiotic stresses on this crop can result from joint metabolic profiling and multivariate analysis. Metabolic profiling of leaves was performed from control and stressed plants (7 and 14 days of stress). Samples were extracted and analyzed on a UHPLC-ESI-Q-TOF-HRMS system. Acquired data were processed using XCMS Online and MetaboAnalyst for multivariate and pathway activity analysis. Metabolism was affected by drought stress through clear segregation between control and stressed groups. More importantly, metabolism changed through time, gradually from 7 to 14 days. The pathways most affected by drought stress were: starch and sucrose metabolism, glyoxylate and dicarboxylate metabolism, alanine, aspartate and glutamate metabolism, arginine and proline metabolism, and glycine, serine and threonine metabolism. The analysis of the metabolic profile were efficient to correlate and differentiate groups of oil palm plants submitted to different levels of drought stress. Putative compounds and their affected pathways can be used in future multiomics analysis.

Targeted Metabolomics of Xylose-Fermenting Yeasts Based on Mass Spectrometry.

Mass spectrometry is a sensitive and selective analytical technique that enables detection and quantitation of low abundance compounds in a complex sample matrix. Targeted metabolomics allows for quantitative analysis of metabolites, providing kinetic information of production and consumption rates, an essential step to investigate microbial metabolism. Here, we describe a targeted metabolomics protocol for yeast samples, from sample preparation to mass spectrometry analysis, which enables the identification of metabolic fluxes after xylose consumption. Sample preparation methods were optimized for quenching of yeast metabolism followed by intracellular metabolite extraction, using cold methanol and boiling ethanol protocols. Ultrahigh performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) methods using ion pair chromatography (IPC) and hydrophilic interaction liquid chromatography (HILIC) allowed for the quantitation of 18 metabolites involved in central carbon metabolism (glycolysis, pentose phosphate pathway, and tricarboxylic acid cycle). The protocol here described was successfully applied to quantify metabolites in Scheffersomyces stipitis, Spathaspora passalidarum, Spathaspora arborariae, and Candida tenuis samples after xylose consumption.

Rhamnolipids production from sucrose by engineered Saccharomyces cerevisiae.

Biosurfactants are biological tensioactive agents that can be used in the cosmetic and food industries. Rhamnolipids are glycolipid biosurfactants naturally produced by Pseudomonas aeruginosa and are composed of one or two rhamnose molecules linked to beta-hydroxy fatty acid chains. These compounds are green alternatives to petrochemical surfactants, but their large-scale production is still in its infancy, hindered due to pathogenicity of natural producer, high substrate and purification costs and low yields and productivities. This study, for the first time, aimed at producing mono-rhamnolipids from sucrose by recombinant GRAS Saccharomyces cerevisiae strains. Six enzymes from P. aeruginosa involved in mono-rhamnolipid biosynthesis were functionally expressed in the yeast. Furthermore, its SUC2 invertase gene was disrupted and a sucrose phosphorylase gene from Pelomonas saccharophila was also expressed to reduce the pathway's overall energy requirement. Two strains were constructed aiming to produce mono-rhamnolipids and the pathway's intermediate dTDP-L-rhamnose. Production of both molecules was analyzed by confocal microscopy and mass spectrometry, respectively. These strains displayed, for the first time as a proof of concept, the potential of production of these molecules by a GRAS eukaryotic microorganism from an inexpensive substrate. These constructs show the potential to further improve rhamnolipids production in a yeast-based industrial bioprocess.

New Protocol Based on UHPLC-MS/MS for Quantitation of Metabolites in Xylose-Fermenting Yeasts.

Xylose fermentation is a bottleneck in second-generation ethanol production. As such, a comprehensive understanding of xylose metabolism in naturally xylose-fermenting yeasts is essential for prospection and construction of recombinant yeast strains. The objective of the current study was to establish a reliable metabolomics protocol for quantification of key metabolites of xylose catabolism pathways in yeast, and to apply this protocol to Spathaspora arborariae. Ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) was used to quantify metabolites, and afterwards, sample preparation was optimized to examine yeast intracellular metabolites. S. arborariae was cultivated using xylose as a carbon source under aerobic and oxygen-limited conditions. Ion pair chromatography (IPC) and hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) were shown to efficiently quantify 14 and 5 metabolites, respectively, in a more rapid chromatographic protocol than previously described. Thirteen and eleven metabolites were quantified in S. arborariae under aerobic and oxygen-limited conditions, respectively. This targeted metabolomics protocol is shown here to quantify a total of 19 metabolites, including sugars, phosphates, coenzymes, monosaccharides, and alcohols, from xylose catabolism pathways (glycolysis, pentose phosphate pathway, and tricarboxylic acid cycle) in yeast. Furthermore, to our knowledge, this is the first time that intracellular metabolites have been quantified in S. arborariae after xylose consumption. The results indicated that fine control of oxygen levels during fermentation is necessary to optimize ethanol production by S. arborariae. The protocol presented here may be applied to other yeast species and could support yeast genetic engineering to improve second generation ethanol production. Graphical Abstract ᅟ.

Fingerprinting of propolis by easy ambient sonic-spray ionization mass spectrometry.

Chemical profiles of a representative set of 49 propolis ethanolic extracts collected worldwide (North and South America, Europe, Asia and Oceania) were obtained via easy ambient sonic-spray ionization mass spectrometry (EASI-MS). This simple and easily implemented fingerprinting technique analyses directly (without any pre-separation or sample manipulation) a tiny droplet of the ethanolic extract placed on a inert surface under ambient conditions. Data acquisition took about a minute per sample with no substantial sample carry-over. Extraction of propolis with ethanol by using an ultrasonic bath method gave EASI-MS data similar to the traditional maceration method, reducing total analysis time for the crude propolis resin from a week to just ca 1h. Principal component analysis of the EASI-MS data is shown to group samples according to the plant sources of their resins, which characterizes their geographical origin.

Chemical characterization of Citrus sinensis grafted on C. limonia and the effect of some isolated compounds on the growth of Xylella fastidiosa.

Citrus sinensis grafted on C. limonia produces a considerable number of compounds that are not common in both plants developed from germination of seeds. The chemical profile of scion and rootstock differ notably for absence in the form of flavonoids and coumarins containing C5 prenyl groups attached to the carbon atoms of aromatic and heterocyclic systems or to oxygen. Only linear pyranocoumarins xanthyletin and xanthoxyletin were found in scion. This observation indicates that the prenylated compounds once biosynthesized in the roots could have been translocated to other organs. Xylella fastidiosa colonizes the xylem of plants causing diseases on several economically important crops such as citrus variegated chlorosis (CVC). A number of flavonoids, coumarins, alkaloids, dihydrocinnamic acid derivative, anacardic acid, triterpenes, and limonoids were tested for in vitro activity on the growth of Xylella fastidiosa. Azadirachtin A was the most active. Hesperidin, which occurs in great amounts in cells of the mesophyll of the affected leaves with CVC, showed a moderate activity suggesting that it can act as a good barrier for small-size colonies from X. fastidiosa.