The biosynthetic pathway of the hallucinogen mescaline and its heterologous reconstruction.

Mescaline, among the earliest identified natural hallucinogens, holds great potential in psychotherapy treatment. Nonetheless, despite the existence of a postulated biosynthetic pathway for more than half a century, the specific enzymes involved in this process are yet to be identified. In this study, we investigated the cactus Lophophora williamsii (Peyote), the largest known natural producer of the phenethylamine mescaline. We employed a multi-faceted approach, combining de novo whole-genome and transcriptome sequencing with comprehensive chemical profiling, enzymatic assays, molecular modeling, and pathway engineering for pathway elucidation. We identified four groups of enzymes responsible for the six catalytic steps in the mescaline biosynthetic pathway, and an N-methyltransferase enzyme that N-methylates all phenethylamine intermediates, likely modulating mescaline levels in Peyote. Finally, we reconstructed the mescaline biosynthetic pathway in both Nicotiana benthamiana plants and yeast cells, providing novel insights into several challenges hindering complete heterologous mescaline production. Taken together, our study opens up avenues for exploration of sustainable production approaches and responsible utilization of mescaline, safeguarding this valuable natural resource for future generations.

Fatty Acid Profiling of Grapevine Extracellular Compartment.

The apoplast is the plant compartment present between the plasma membrane and the cuticle, comprised of the cell wall and the extracellular spaces where the "secretomes" are released and where the apoplastic fluid circulates. Within the many functions attributed to this compartment, its role in plant-pathogen interactions is irrefutable. It is the major point where plant and pathogen secretomes come in contact and several plant and pathogenic secreted proteins and small molecules present in this compartment are already cataloged in the literature. In plant-pathogen interactions, fatty acids and lipid molecules were shown to play a crucial role in the activation of plant immunity; however, the lipid composition of the apoplast is still a black box. Most of the studies performed to understand apoplast dynamics have used proteomic-based techniques; however, knowledge about apoplastic proteins involved in lipid metabolism and transport is still severely limited. In grapevine, only three studies have been published so far focusing on the characterization of this compartment and only one of them deals with grapevine-pathogen interaction. Here we refer to our recently established method for grapevine leaves' apoplastic fluid isolation and describe a direct methylation protocol for the analysis of apoplastic fluid fatty acids. We also point out a novel intracellular marker that may be used to assess apoplastic fluid purity.

More than Just Wine: The Nutritional Benefits of Grapevine Leaves.

The domesticated species Vitis vinifera L. harbours many cultivars throughout the world that present distinctive phenology and grape quality. Not only have the grapes been used for human consumption, but the leaves are also used as a source of bioactive compounds and are present in the diets of several Mediterranean countries. We have selected seven different cultivars and performed elemental, fatty acid (FA) and pigment profiling. Total reflection X-ray fluorescence (TXRF) enabled the identification of 21 elements. Among them, Na, Ca and K were highly represented in all the cultivars and Zn was prevalent in V. vinifera cv. 'Pinot noir' and 'Cabernet sauvignon'. Through gas chromatography, six FAs were identified, including omega-3 and omega-6 FA, the most abundant mainly in V. vinifera cv. 'Tinta barroca', 'Pinot noir' and 'Cabernet sauvignon'. FA composition was used to determine nutritional quality parameters, namely atherogenic, thrombogenic, hypocholesterolemic/hypercholesterolemic and peroxidisability indexes as well as oxidability and oxidative susceptibility. Grapevine leaves were highlighted as a suitable source of health-promoting lipids. Given the popularity of "green" diets, we have also performed a leaf pigment analysis. Seventeen pigments including chlorophylls, trans-lutein, b-carotene and zeaxanthins were detected. 'C19' presented the highest content of most of the detected pigments.

Fatty Acid Desaturases: Uncovering Their Involvement in Grapevine Defence against Downy Mildew.

Grapevine downy mildew, caused by the biotrophic oomycete Plasmopara viticola, is one of the most severe and devastating diseases in viticulture. Unravelling the grapevine defence mechanisms is crucial to develop sustainable disease control measures. Here we provide new insights concerning fatty acid's (FA) desaturation, a fundamental process in lipid remodelling and signalling. Previously, we have provided evidence that lipid signalling is essential in the establishment of the incompatible interaction between grapevine and Plasmopara viticola. In the first hours after pathogen challenge, jasmonic acid (JA) accumulation, activation of its biosynthetic pathway and an accumulation of its precursor, the polyunsaturated α-linolenic acid (C18:3), were observed in the leaves of the tolerant genotype, Regent. This work was aimed at a better comprehension of the desaturation processes occurring after inoculation. We characterised, for the first time in Vitis vinifera, the gene family of the FA desaturases and evaluated their involvement in Regent response to Plasmopara viticola. Upon pathogen challenge, an up-regulation of the expression of plastidial FA desaturases genes was observed, resulting in a higher content of polyunsaturated fatty acids (PUFAs) of chloroplast lipids. This study highlights FA desaturases as key players in membrane remodelling and signalling in grapevine defence towards biotrophic pathogens.

Fatty acid modulation and desaturase gene expression are differentially triggered in grapevine incompatible interaction with biotrophs and necrotrophs.

Grapevine (Vitis vinifera L.) is prone to fungal and oomycete diseases. Downy and powdery mildews and grey mold, are caused by Plasmopara viticola, Erisiphe necator and Botrytis cinerea, respectively. P. viticola and E. necator are obligatory biotrophs whereas B. cinerea is a necrotroph. In tolerant grapevine cultivars, plant-pathogen interaction induces defence responses, including metabolite and protein accumulation and hypersensitive reaction. Lipid and lipid-derived molecules may have a key role in the activation of defence mechanisms. Previous results suggest that V. vinifera cv Regent tolerance to P. viticola may be mediated in the first hours post inoculation by fatty acid (FA) associated signalling. In the present study we characterized FA modulation in V. vinifera cv Regent leaves upon inoculation with P. viticola, E. necator and B. cinerea and correlated FA modulation with the expression profiles of genes encoding the FA desaturases FAD6 and FAD8. In all the interactions, a progressive desaturation of stearic acid to α-linolenic acid, precursor of jasmonic acid, occurred, which was observed for a longer period against B. cinerea. Our results provide evidence of a distinct FA meditated signalling pattern in grapevine interaction with biotrophs and necrotrophs. While the interaction with the biotrophs may trigger a higher synthesis of polyunsaturated FA (PUFA) at early time-points with a tendency to return to basal levels, the interaction with B. cinerea may trigger a later and more durable induction of PUFA synthesis. In all interactions, membrane fluidity modulation occurred, which may be crucial to maintain cellular function during infection.

Speaking the language of lipids: the cross-talk between plants and pathogens in defence and disease.

Lipids and fatty acids play crucial roles in plant immunity, which have been highlighted over the past few decades. An increasing number of studies have shown that these molecules are pivotal in the interactions between plants and their diverse pathogens. The roles played by plant lipids fit in a wide spectrum ranging from the first physical barrier encountered by the pathogens, the cuticle, to the signalling pathways that trigger different immune responses and expression of defence-related genes, mediated by several lipid molecules. Moreover, lipids have been arising as candidate biomarkers of resistance or susceptibility to different pathogens. Studies on the apoplast and extracellular vesicles have been highlighting the possible role of lipids in the intercellular communication and the establishment of systemic acquired resistance during plant-pathogen interactions. From the pathogen perspective, lipid metabolism and specific lipid molecules play pivotal roles in the pathogen's life cycle completion, being crucial during recognition by the plant and evasion from the host immune system, therefore potentiating infection. Studies conducted in the last years have contributed to a better understanding of the language of lipids during the cross-talk between plants and pathogens. However, it is essential to continue exploring the knowledge brought up to light by transcriptomics and proteomics studies towards the elucidation of lipid signalling processes during defence and disease. In this review, we present an updated overview on lipids associated to plant-pathogen interactions, exploiting their roles from the two sides of this battle.

An apoplastic fluid extraction method for the characterization of grapevine leaves proteome and metabolome from a single sample.

The analysis of complex biological systems keeps challenging researchers. The main goal of systems biology is to decipher interactions within cells, by integrating datasets from large scale analytical approaches including transcriptomics, proteomics and metabolomics and more specialized 'OMICS' such as epigenomics and lipidomics. Studying different cellular compartments allows a broader understanding of cell dynamics. Plant apoplast, the cellular compartment external to the plasma membrane including the cell wall, is particularly demanding to analyze. Despite our knowledge on apoplast involvement on several processes from cell growth to stress responses, its dynamics is still poorly known due to the lack of efficient extraction processes adequate to each plant system. Analyzing woody plants such as grapevine raises even more challenges. Grapevine is among the most important fruit crops worldwide and a wider characterization of its apoplast is essential for a deeper understanding of its physiology and cellular mechanisms. Here, we describe, for the first time, a vacuum-infiltration-centrifugation method that allows a simultaneous extraction of grapevine apoplastic proteins and metabolites from leaves on a single sample, compatible with high-throughput mass spectrometry analyses. The extracted apoplast from two grapevine cultivars, Vitis vinifera cv 'Trincadeira' and 'Regent', was directly used for proteomics and metabolomics analysis. The proteome was analyzed by nanoLC-MS/MS and more than 700 common proteins were identified, with highly diverse biological functions. The metabolome profile through FT-ICR-MS allowed the identification of 514 unique putative compounds revealing a broad spectrum of molecular classes.

Author Correction: The interplay between membrane lipids and phospholipase A family members in grapevine resistance against Plasmopara viticola.

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Preliminary diversity assessment of an undervalued tropical bean (Lablab purpureus (L.) Sweet) through fatty acid profiling.

Several large-scale metabolic profiling studies have been directed to prospect crops with a major focus on yield-related traits and, ultimately, with the definition of specific markers for plant selection in breeding programs. However, some of these technologies are expensive, time-consuming and not easily feasible for a quick approach. Fatty acid profiling was described as reliable biomarkers and as a chemotaxonomic tool allowing to study not only the diversity in germplasm collections but also to discriminate their geographic origin. We have used fatty acids profiling for a preliminary assessment of Lablab purpureus (L.) Sweet (hyacinth bean) diversity and landraces discrimination. Hyacinth bean displays an enormous variability of agro-morphological traits, probably linked to the multi-purpose uses in different regions, i.e. as pulse, or as food with nutraceutical potential (Africa and Asia), forage (Africa and Australia) and ornamental (Europe and USA). Only two forage cultivars are widely marketed, cv. Rongai and cv. Highworth, with several landraces remaining to be addressed in terms of diversity. We show that fatty acids profiling was able to distinguish landraces, which display shared fatty acids with cultivars from the center of hyacinth bean diversity origin (East Africa). We propose that fatty acid profiling is a tool that may be used not only for nutritional value assessment but also as a chemodiversity tool in crop research.

The interplay between membrane lipids and phospholipase A family members in grapevine resistance against Plasmopara viticola.

Grapevine downy mildew, caused by the biotrophic oomycete Plasmopara viticola, is one of the most important diseases in modern viticulture. The search for sustainable disease control measure is of extreme importance, thus becoming imperative to fully characterize the mechanisms leading to an incompatible interaction. We have previously shown that lipid signalling events play an important role in grapevine's response to this pathogen, namely through changes in linolenic acid content, lipid peroxidation and jasmonic acid synthesis. Here, we have characterized the modulation of lipid metabolism in leaves from two V. vinifera cultivars (resistant and susceptible to P. viticola) in the first hours after pathogen inoculation. Prior to pathogen inoculation both genotypes present an inherently different fatty acid composition that is highly modulated in the resistant genotype after pathogen challenge. Such changes involve modulation of phospholipase A activity suggesting that the source of lipids mobilized upon pathogen infection are the chloroplast membranes. This work thus provides original evidence on the involvement of lipid signalling and phospholipases in grapevine immune responses to pathogen infection. The results are discussed considering the implications on the plant's physiological status and the use of discriminating lipid/fatty acids pattern in future selection procedures of cultivars.