Eicosapentaenoic acid: New insights into an oomycete-driven elicitor to enhance grapevine immunity.

The widespread use of pesticides in agriculture remains a matter of major concern, prompting a critical need for alternative and sustainable practices. To address this, the use of lipid-derived molecules as elicitors to induce defence responses in grapevine plants was accessed. A Plasmopara viticola fatty acid (FA), eicosapentaenoic acid (EPA) naturally present in oomycetes, but absent in plants, was applied by foliar spraying to the leaves of the susceptible grapevine cultivar (Vitis vinifera cv. Trincadeira), while a host lipid derived phytohormone, jasmonic acid (JA) was used as a molecule known to trigger host defence. Their potential as defence triggers was assessed by analysing the expression of a set of genes related to grapevine defence and evaluating the FA modulation upon elicitation. JA prompted grapevine immunity, altering lipid metabolism and up-regulating the expression of several defence genes. EPA also induced a myriad of responses to the levels typically observed in tolerant plants. Its application activated the transcription of defence gene's regulators, pathogen-related genes and genes involved in phytoalexins biosynthesis. Moreover, EPA application resulted in the alteration of the leaf FA profile, likely by impacting biosynthetic, unsaturation and turnover processes. Although both molecules were able to trigger grapevine defence mechanisms, EPA induced a more robust and prolonged response. This finding establishes EPA as a promising elicitor for an effectively managing grapevine downy mildew diseases.

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.

A New Perspective for Vineyard Terroir Identity: Looking for Microbial Indicator Species by Long Read Nanopore Sequencing.

Grapevine is one of the most important fruit crops worldwide, being Portugal one of the top wine producers. It is well established that wine sensory characteristics from a particular region are defined by the physiological responses of the grapevine to its environment and thus, the concept of terroir in viticulture was established. Among all the factors that contribute to terroir definition, soil microorganisms play a major role from nutrient recycling to a drastic influence on plant fitness (growth and protection) and of course wine production. Soil microbiome from four different terroirs in Quinta dos Murças vineyard was analysed through long-read Oxford Nanopore sequencing. We have developed an analytical pipeline that allows the identification of function, ecologies, and indicator species based on long read sequencing data. The Douro vineyard was used as a case study, and we were able to establish microbiome signatures of each terroir.

Grapevine-Associated Lipid Signalling Is Specifically Activated in an Rpv3 Background in Response to an Aggressive P. viticola Pathovar.

Vitis vinifera L. is highly susceptible to the biotrophic pathogen Plasmopara viticola. To control the downy mildew disease, several phytochemicals are applied every season. Recent European Union requirements to reduce the use of chemicals in viticulture have made it crucial to use alternative and more sustainable approaches to control this disease. Our previous studies pinpoint the role of fatty acids and lipid signalling in the establishment of an incompatible interaction between grapevine and P. viticola. To further understand the mechanisms behind lipid involvement in an effective defence response we have analysed the expression of several genes related to lipid metabolism in three grapevine genotypes: Chardonnay (susceptible); Regent (tolerant), harbouring an Rpv3-1 resistance loci; and Sauvignac (resistant) that harbours a pyramid of Rpv12 and Rpv3-1 resistance loci. A highly aggressive P. viticola isolate was used (NW-10/16). Moreover, we have characterised the grapevine phospholipases C and D gene families and monitored fatty acid modulation during infection. Our results indicate that both susceptible and resistant grapevine hosts did not present wide fatty acid or gene expression modulation. The modulation of genes associated with lipid signalling and fatty acids seems to be specific to Regent, which raises the hypothesis of being specifically linked to the Rpv3 loci. In Sauvignac, the Rpv12 may be dominant concerning the defence response, and, thus, this genotype may present the activation of other pathways rather than lipid signalling.

Membrane remodelling and triacylglycerol accumulation in drought stress resistance: The case study of soybean phospholipases A.

Agriculture is facing major constraints with the increase of global warming, being drought a major factor affecting productivity. Soybean (Glycine max) is among the most important food crops due to the high protein and lipid content of its seeds despite being considerably sensitive to drought. Previous knowledge has shown that drought induces a severe modulation in lipid and fatty acid content of leaves, related to alteration of membrane structure by lipolytic enzymes and activation of signalling pathways. In that sense, little is known on lipid modulation and lipolytic enzymes' role in soybean drought stress tolerance. In this work, we present for the first time, soybean leaves lipid content modulation in several drought stress levels, highlighting the involvement of phospholipases A. Moreover, a comprehensive analysis of the phospholipase A superfamily was performed, where 53 coding genes were identified and 7 were selected to gene expression analysis in order to elucidate their role in soybean lipid modulation under water deficit. Proportionally to the drought severity, our results revealed that galactolipids relative abundance and their content in linolenic acid decrease. At the same time an accumulation of neutral lipids, mainly due to triacylglycerol content increase, as well as their content in linolenic acid, is observed. Overall, PLA gene expression regulation and lipid modulation corroborate the hypothesis that phospholipases A may be channelling the plastidial fatty acids into extraplastidial lipids leading to a drought-induced accumulation of triacylglycerol in soybean leaves, a key feature to cope with water stress.

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.

Vitis vinifera 'Pinot noir' leaves as a source of bioactive nutraceutical compounds.

Agricultural by-products are often hidden sources of healthy plant ingredients. The investigation of the nutritional values of these by-products is essential towards sustainable agriculture and improved food systems. In the vine industry, grape leaves are a bulky side product which is strategically removed and treated as waste in the process of wine production. In this work we performed an untargeted metabolomic profiling of the methanol extract of the leaves of Vitis vinifera cultivar 'Pinot noir', analysed their fatty acid content, and estimated their antioxidative capacity, with the purpose of investigating its nutritional and medicinal value. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) analysis identified the presence of numerous compounds which are known to possess diverse nutritional and pharmacological properties, particularly polyphenols and phenolic compounds (e.g. caffeic acid, catechin, kaempferol and quercetin), several phytosterols and fatty acids. Fatty acids were the most represented lipids' secondary class, with the essential alpha-linolenic acid being the most abundant in 'Pinot noir' leaves, with a relative content of 42%. Also, we have found that 'Pinot noir' leaves present a high antioxidant capacity, putting grapevine leaves at the top of the list of foods with the highest antioxidative activity. Our findings scientifically confirmed that 'Pinot noir' leaves have a high content and diversity of biologically active phytochemical compounds which make it of exceptional interest for pharmaceutical and food industries.

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

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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.