The mycorrhizal root-shoot axis elicits Coffea arabica growth under low phosphate conditions.

Coffee is one of the most traded commodities world-wide. As with 70% of land plants, coffee is associated with arbuscular mycorrhizal (AM) fungi, but the molecular bases of this interaction are unknown. We studied the mycorrhizal phenotype of two commercially important Coffea arabica cultivars ('Typica National' and 'Catimor Amarillo'), upon Funnelliformis mosseae colonisation grown under phosphorus limitation, using an integrated functional approach based on multi-omics, physiology and biochemistry. The two cultivars revealed a strong biomass increase upon mycorrhization, even at low level of fungal colonisation, improving photosynthetic efficiency and plant nutrition. The more important iconic markers of AM symbiosis were activated: We detected two gene copies of AM-inducible phosphate (Pt4), ammonium (AM2) and nitrate (NPF4.5) transporters, which were identified as belonging to the C. arabica parental species (C. canephora and C. eugenioides) with both copies being upregulated. Transcriptomics data were confirmed by ions and metabolomics analyses, which highlighted an increased amount of glucose, fructose and flavonoid glycosides. In conclusion, both coffee cultivars revealed a high responsiveness to the AM fungus along their root-shoot axis, showing a clear-cut re-organisation of the major metabolic pathways, which involve nutrient acquisition, carbon fixation, and primary and secondary metabolism.

Databases and Tools to Investigate Protein-Metabolite Interactions.

Protein-metabolite interactions (PMIs) are directly responsible for the regulation of numerous processes. From the direct regulation of enzymes to complex developmental processes intermediated by hormones, PMIs are central to understanding the molecular mechanisms of important physiological phenomena. Still, proving such interactions experimentally has proven an arduous task. We discuss here some of the current technologies contributing to expand our knowledge on PMIs, with particular emphasis on platforms and databases to explore the highly heterogenous nature of characterized PMIs, which is likely to be an essential resource on the development of new computational approaches to predict and validate interactions based on large-scale PMI screenings.

The AtMYB60 transcription factor regulates stomatal opening by modulating oxylipin synthesis in guard cells.

Stomata are epidermal pores formed by pairs of specialized guard cells, which regulate gas exchanges between the plant and the atmosphere. Modulation of transcription has emerged as an important level of regulation of stomatal activity. The AtMYB60 transcription factor was previously identified as a positive regulator of stomatal opening, although the details of its function remain unknown. Here, we propose a role for AtMYB60 as a negative modulator of oxylipins synthesis in stomata. The atmyb60-1 mutant shows reduced stomatal opening and accumulates increased levels of 12-oxo-phytodienoic acid (12-OPDA), jasmonic acid (JA) and jasmonoyl-L-isoleucine (JA-Ile) in guard cells. We provide evidence that 12-OPDA triggers stomatal closure independently of JA and cooperatively with abscisic acid (ABA) in atmyb60-1. Our study highlights the relevance of oxylipins metabolism in stomatal regulation and indicates AtMYB60 as transcriptional integrator of ABA and oxylipins responses in guard cells.

Global mapping of protein-metabolite interactions in Saccharomyces cerevisiae reveals that Ser-Leu dipeptide regulates phosphoglycerate kinase activity.

Protein-metabolite interactions are of crucial importance for all cellular processes but remain understudied. Here, we applied a biochemical approach named PROMIS, to address the complexity of the protein-small molecule interactome in the model yeast Saccharomyces cerevisiae. By doing so, we provide a unique dataset, which can be queried for interactions between 74 small molecules and 3982 proteins using a user-friendly interface available at https://promis.mpimp-golm.mpg.de/yeastpmi/ . By interpolating PROMIS with the list of predicted protein-metabolite interactions, we provided experimental validation for 225 binding events. Remarkably, of the 74 small molecules co-eluting with proteins, 36 were proteogenic dipeptides. Targeted analysis of a representative dipeptide, Ser-Leu, revealed numerous protein interactors comprising chaperones, proteasomal subunits, and metabolic enzymes. We could further demonstrate that Ser-Leu binding increases activity of a glycolytic enzyme phosphoglycerate kinase (Pgk1). Consistent with the binding analysis, Ser-Leu supplementation leads to the acute metabolic changes and delays timing of a diauxic shift. Supported by the dipeptide accumulation analysis our work attests to the role of Ser-Leu as a metabolic regulator at the interface of protein degradation and central metabolism.

Lycopene ß-cyclase expression influences plant physiology, development, and metabolism in tobacco plants.

Carotenoids are important isoprenoids produced in the plastids of photosynthetic organisms that play key roles in photoprotection and antioxidative processes. ß-Carotene is generated from lycopene by lycopene ß-cyclase (LCYB). Previously, we demonstrated that the introduction of the Daucus carota (carrot) DcLCYB1 gene into tobacco (cv. Xanthi) resulted in increased levels of abscisic acid (ABA) and especially gibberellins (GAs), resulting in increased plant yield. In order to understand this phenomenon prior to exporting this genetic strategy to crops, we generated tobacco (Nicotiana tabacum cv. Petit Havana) mutants that exhibited a wide range of LCYB expression. Transplastomic plants expressing DcLCYB1 at high levels showed a wild-type-like growth, even though their pigment content was increased and their leaf GA1 content was reduced. RNA interference (RNAi) NtLCYB lines showed different reductions in NtLCYB transcript abundance, correlating with reduced pigment content and plant variegation. Photosynthesis (leaf absorptance, Fv/Fm, and light-saturated capacity of linear electron transport) and plant growth were impaired. Remarkably, drastic changes in phytohormone content also occurred in the RNAi lines. However, external application of phytohormones was not sufficient to rescue these phenotypes, suggesting that altered photosynthetic efficiency might be another important factor explaining their reduced biomass. These results show that LCYB expression influences plant biomass by different mechanisms and suggests thresholds for LCYB expression levels that might be beneficial or detrimental for plant growth.

Plant Single-Cell Metabolomics-Challenges and Perspectives.

Omics approaches for investigating biological systems were introduced in the mid-1990s and quickly consolidated to become a fundamental pillar of modern biology. The idea of measuring the whole complement of genes, transcripts, proteins, and metabolites has since become widespread and routinely adopted in the pursuit of an infinity of scientific questions. Incremental improvements over technical aspects such as sampling, sensitivity, cost, and throughput pushed even further the boundaries of what these techniques can achieve. In this context, single-cell genomics and transcriptomics quickly became a well-established tool to answer fundamental questions challenging to assess at a whole tissue level. Following a similar trend as the original development of these techniques, proteomics alternatives for single-cell exploration have become more accessible and reliable, whilst metabolomics lag behind the rest. This review summarizes state-of-the-art technologies for spatially resolved metabolomics analysis, as well as the challenges hindering the achievement of sensu stricto metabolome coverage at the single-cell level. Furthermore, we discuss several essential contributions to understanding plant single-cell metabolism, finishing with our opinion on near-future developments and relevant scientific questions that will hopefully be tackled by incorporating these new exciting technologies.

Antifungal Activity of a Hydroethanolic Extract From Astronium urundeuva Leaves Against Candida albicans and Candida glabrata.

We have previously reported on the activity of different extracts from Astronium sp. against Candida albicans, with the hydroethanolic extract prepared from leaves of A. urundeuva, an arboreal species widely distributed in arid environments of South America and often used in folk medicine, displaying the highest in vitro activity. Here we have further evaluated the antifungal activity of this extract against strains of C. albicans and C. glabrata, the two most common etiological agents of candidiasis. The extract was tested alone and loaded into a nanostructured lipid system (10% oil phase, 10% surfactant and 80% aqueous phase, 0.5% Poloxamer 407®). In vitro susceptibility assays demonstrated the antifungal activity of the free extract and the microemulsion against both Candida species, with increased activity against C. glabrata, including collection strains and clinical isolates displaying different levels of resistance against the most common clinically used antifungal drugs. Checkerboard results showed synergism when the free extract was combined with amphotericin B against C. albicans. Serial passage experiments confirmed development of resistance to fluconazole but not to the free extract upon prolonged exposure. Although preformed biofilms were intrinsically resistant to treatment with the extract, it was able to inhibit biofilm formation by C. albicans at concentrations comparable to those inhibiting planktonic growth. Cytotoxicity assays in different cell lines as well as an alternative model using Artemia salina L. confirmed a good safety profile of the both free and loaded extracts, and an in vivo assay demonstrated the efficacy of the free and loaded extracts when used topically in a rat model of vaginal candidiasis. Overall, these results reveal the promise of the A. urundeuva leaves extract to be further investigated and developed as an antifungal.

An extract from Myracrodruon urundeuva inhibits matrix mineralization in human osteoblasts.

ETHNOPHARMACOLOGICAL RELEVANCE: Phytotherapy based on plant-derived compounds is an alternative medicinal strategy for the relief of symptoms and the curing of diseases. The leaves of Myracrodruon urundeuva a medicinal plant also known as "aroeira", has been used in traditional medicine as healing, antiulcer and anti-inflammatory to treat skeletal diseases in Brazil, but its role in bone cell toxicity, as well as in bone formation, remains to be established. AIM OF THE STUDY: We sought to determine the in vitro osteogenic effects of a hydroalcoholic M. urundeuva leaves extract in primary human osteoblasts. MATERIALS AND METHODS: Cell viability, reactive oxygen species (ROS) production, alkaline phosphatase (ALP) activity and matrix mineralization were evaluated by MTT assay, DCFH-DA probe, colorimetric-based enzymatic assay and Alizarin Red-staining, respectively. Besides, the matrix metalloproteinase (MMP)-2 and progressive ankylosis protein homolog (ANKH) gene expression were determined by real-time RT-qPCR and MMP-2 activity by zymography. RESULTS: Exposure of osteoblasts to M. urundeuva extract significantly decreased viability and increased reactive oxygen species (ROS) production, regardless of the extract concentration. The M. urundeuva extract at 10 µg/mL also downregulated matrix metalloproteinase (MMP)-2, while upregulating progressive ankylosis protein homolog (ANKH) gene expression. By contrast, the MMP-2 activity was unchanged. The M. urundeuva extract at 10 µg/mL also reduced alkaline phosphatase (ALP) activity and mineralization. CONCLUSIONS: Overall, our findings suggest that the inhibition of osteogenic differentiation and matrix mineralization promoted by M. urundeuva may be due more to an increase in oxidative stress than to the modulation of MMP-2 and ANKH expression.

Gas Chromatography-Mass Spectrometry-Based 13C-Labeling Studies in Plant Metabolomics.

Stable-isotope labeling analysis has been used to discover new metabolic pathways and their key regulatory points in a wide range of organisms. Given the complexity of the plant metabolic network, this analysis provides information complementary to that obtained from metabolite profiling that can be used to understand how plants cope with adverse conditions, and how metabolism varies between different cells, tissues, and organs. Here we describe the experimental procedures from sample harvesting and extraction to mass spectral analysis and interpretation that allow the researcher to perform 13C-labeling experiments. A wide range of plant material, from single cells to whole plants, can be used to investigate the metabolic fate of the 13C from a predefined tracer. Thus, a key point of this analysis is to choose the correct biological system, the substrate and the condition to be investigated; all of which implicitly relies on the biological question to be investigated. Rapid sample quenching and a careful data analysis are also critical points in such studies. By contrast to other metabolomic approaches, stable-isotope labeling can provide information concerning the fluxes through metabolic networks, which is essential for understanding and manipulating metabolic phenotypes and therefore of pivotal importance for both systems biology and plant metabolic engineering.

Current understanding of the pathways of flavonoid biosynthesis in model and crop plants.

Flavonoids are a signature class of secondary metabolites formed from a relatively simple collection of scaffolds. They are extensively decorated by chemical reactions including glycosylation, methylation, and acylation. They are present in a wide variety of fruits and vegetables and as such in Western populations it is estimated that 20-50 mg of flavonoids are consumed daily per person. In planta they have demonstrated to contribute to both flower color and UV protection. Their consumption has been suggested to presenta wide range of health benefits. Recent technical advances allowing affordable whole genome sequencing, as well as a better inventory of species-by-species chemical diversity, have greatly advanced our understanding as to how flavonoid biosynthesis pathways vary across species. In parallel, reverse genetics combined with detailed molecular phenotyping is currently allowing us to elucidate the functional importance of individual genes and metabolites and by this means to provide further mechanistic insight into their biological roles. Here we provide an inventory of current knowledge of pathways of flavonoid biosynthesis in both the model plant Arabidopsis thaliana and a range of crop species, including tomato, maize, rice, and bean.

13CO2 Labeling and Mass Spectral Analysis of Photorespiration.

Photorespiratory metabolism is compartmented over the chloroplast, peroxisome, cytosol, and mitochondria, and due to its complex structure it is often the case that metabolite levels alone are not able to fully describe photorespiration. Metabolic fluxes represent a more meaningful biological description of metabolism, adding to metabolite levels and often revealing different aspects of the system such as the presence of inactive metabolic pools of photorespiratory intermediates. We describe here a protocol for the 13CO2 feeding of Arabidopsis and tracing of 13C enriched metabolites for metabolic fluxes estimation, which allows high throughput analysis of labeling pattern on different metabolites involved in photorespiration and downstream processes.

"Aroeira" (Myracrodruon urundeuva) methanol extract: the relationship between chemical compounds and cellular effects.

CONTEXT: "Aroeira" [Myracrodruon urundeuva Allemão (Anacardiaceae)] is a tree whose leaves have been studied for therapeutic purposes in medicine and dentistry. OBJECTIVE: The study chemically identifies the leaf extract of aroeira and determines its effect on human gingival fibroblasts. MATERIALS AND METHODS: An 80% methanol leave extract was obtained by maceration and chemically identified through flow-injection analysis-electrospray ionization-ion trap-tandem mass spectrometry (FIA-ESI-IT-MSn). Cytotoxicity of the aroeira's methanol extract was evaluated in lineage of fibroblasts. Adherent cells were treated with different concentrations of aroeira's methanol extract in the medium: 0.1, 1, 10, 100 and 1000 µg/mL. Control cells were cultivated in the medium only. Analyses were done at 24, 48, 72 and 96 h of culture by neutral red assay; and at 24, 48 and 96 h by crystal violet assay. RESULTS: FIA-ESI-IT-MS analysis determined the presence of compounds, for the first time in the species: quercetin-O-glucuronide and quercetin-O-deoxyhexose-O-glucose in the extract. On one hand, neutral red and crystal violet assay showed a reduction (to 50% up until 100%) of cellular viability of groups of 100 and 1000 µg/mL compared with control at 96 h (p < 0.05). On the other hand, lower concentrations (0.1; 1 and 10 µg/mL) of the extract were similar to that of the control at 96 h (p < 0.05), in general. CONCLUSIONS: In view of the results, we can conclude that the extract of aroeira presents tannins and flavonoids. Furthermore, the extract is capable of modulating the viability of human gingival fibroblasts according to its concentration.

Nanostructured lipid system as a strategy to improve the anti-Candida albicans activity of Astronium sp.

The genus Astronium (Anacardiaceae) includes species, such as Astronium fraxinifolium, Astronium graveolens, and Astronium urundeuva, which possess anti-inflammatory, anti-ulcerogenic, healing, and antimicrobial properties. Nanostructured lipid systems are able to potentiate the action of plant extracts, reducing the required dose and side effects and improving antimicrobial activity. This work aims to evaluate a nanostructured lipid system that was developed as a strategy to improve the anti-Candida albicans activity of hydroethanolic extracts of stems and leaves from Astronium sp. The antifungal activity against C. albicans (ATCC 18804) was evaluated in vitro by a microdilution technique. In addition to the in vitro assays, the Astronium sp. that showed the best antifungal activity and selectivity index was submitted to an in vivo assay using a model of vulvovaginal candidiasis infection. In these assays, the extracts were either used alone or were incorporated into the nanostructured lipid system (comprising 10% oil phase, 10% surfactant, and 80% aqueous phase). The results indicated a minimal inhibitory concentration of 125.00 µg/mL before incorporation into the nanostructured system; this activity was even more enhanced when this extract presented a minimal inhibitory concentration of 15.62 µg/mL after its incorporation. In vivo assay dates showed that the nanostructure-incorporated extract of A. urundeuva leaves was more effective than both the unincorporated extract and the antifungal positive control (amphotericin B). These results suggest that this nanostructured lipid system can be used in a strategy to improve the in vitro and in vivo anti-C. albicans activity of hydroethanolic extracts of Astronium sp.

Mutagenicity and chemopreventive activities of Astronium species assessed by Ames test.

In the neotropical savannah, Astronium species are used in popular medicine to treat allergies, inflammation, diarrhea and ulcers. Given that natural products are promising starting points for the discovery of novel potentially therapeutic agents, the aim of the present study was to investigate the mutagenic and antimutagenic activities of hydroalcoholic extracts of Astronium spp. The mutagenicity was determined by the Ames test on Salmonella typhimurium strains TA98, TA97a, TA100 and TA102. The antimutagenicity was tested against the direct-acting and indirect-acting mutagens. The results showed that none of the extracts induce any increase in the number of revertants, demonstrating the absence of mutagenic activity. On the other hand, the results on the antimutagenic potential showed a moderate inhibitory effect against NPD and a strong protective effect against B[a]P and AFB1. This study highlights the importance of screening species of Astronium for new medicinal compounds. The promising results obtained open up new avenues for further study and provide a better understanding the mechanisms by which these species act in protecting DNA from damage. However, further pharmacological and toxicological investigations of crude extracts of Astronium spp., as well as of its secondary metabolites, are necessary to determine the mechanism(s) of action to guarantee their safer and more effective application to human health.

Metabolomics-assisted refinement of the pathways of steroidal glycoalkaloid biosynthesis in the tomato clade.

Steroidal glycoalkaloids (SGAs) are nitrogen-containing secondary metabolites of the Solanum species, which are known to have large chemical and bioactive diversity in nature. While recent effort and development on LC/MS techniques for SGA profiling have elucidated the main pathways of SGA metabolism in tomato, the problem of peak annotation still remains due to the vast diversity of chemical structure and similar on overlapping of chemical formula. Here we provide a case study of peak classification and annotation approach by integration of species and tissue specificities of SGA accumulation for provision of comprehensive pathways of SGA biosynthesis. In order to elucidate natural diversity of SGA biosynthesis, a total of 169 putative SGAs found in eight tomato accessions (Solanum lycopersicum, S. pimpinellifolium, S. cheesmaniae, S. chmielewskii, S. neorickii, S. peruvianum, S. habrochaites, S. pennellii) and four tissue types were used for correlation analysis. The results obtained in this study contribute annotation and classification of SGAs as well as detecting putative novel biosynthetic branch points. As such this represents a novel strategy for peak annotation for plant secondary metabolites.

Genome-enabled plant metabolomics.

The grand challenge currently facing metabolomics is that of comprehensitivity whilst next generation sequencing and advanced proteomics methods now allow almost complete and at least 50% coverage of their respective target molecules, metabolomics platforms at best offer coverage of just 10% of the small molecule complement of the cell. Here we discuss the use of genome sequence information as an enabling tool for peak identity and for translational metabolomics. Whilst we argue that genome information is not sufficient to compute the size of a species metabolome it is highly useful in predicting the occurrence of a wide range of common metabolites. Furthermore, we describe how via gene functional analysis in model species the identity of unknown metabolite peaks can be resolved. Taken together these examples suggest that genome sequence information is current (and likely will remain), a highly effective tool in peak elucidation in mass spectral metabolomics strategies.

Pd(II)-catalyzed spiroketalization of ketoallylic diols.

A high-yielding stereoselective method for forming spiroketals from simple ketoallylic diols is reported. Employing catalytic [PdCl2(MeCN)2] in THF at 0 °C, these dehydrative cyclization reactions require only mild conditions to produce vinyl-substituted spiroketals in high yields after brief reaction times with water as the only byproduct. Using this method, the stereochemical information embedded at the nucleophile is transmitted "down-the-chain" and efficiently sets the stereochemistry at both the anomeric carbon atom and the newly formed allylic stereocenter.