Essential Oils of <em>Thymbra capitata</em> and <em>Thymus hyemalis</em> and Their Uses Based on Their Bioactivity

Essential oils (EO) are volatile compounds produced by the secondary metabolism of aromatic plants. They are complex mixtures whose main components are synthesized by the mevalonic acid and the methyl erythritol phosphate pathways, which lead to the biosynthesis of terpenes, and the shikimic acid pathway, responsible for the biosynthesis of phenylpropanoid compounds. In nature, EOs are stored in the aerial parts of the plant, being of vital importance for their survival due to their antimicrobial properties. In addition, EOs provide protection against herbivores to the aromatic plants and allow them to repel or attract insects because of their strong fragrance, as well as compete with other plants of the same environment. Humans have exploited the properties of their EOs since ancient times, being used as medicinal remedies, among other uses. Currently, aromatic plants are used in pharmaceutical and food industries. One of the most commonly used aromatic plants is thyme. Thyme is a perennial aromatic plant, taxonomically belonging to the genera Thymus and Thymbra, belonging to the family Lamiaceae. These plants are very abundant in the Mediterranean Region. In this review, we focus on the study of the properties and use of EOs of Thymbra capitata (L) Cav. and Thymus hyemalis Lange., whose EOs are rich in phenolic monoterpenes. These compounds are responsible for their antioxidant, anti-inflammatory, anticarcinogenic, antibacterial, antifungal, and antiparasitic properties

Enhanced extracellular production of trans-resveratrol in Vitis vinifera suspension cultured cells by using cyclodextrins and coronatine

In the present work the effect of cyclodextrin and coronatine on both trans-resveratrol production and the expression of stilbene biosynthetic genes in Vitis vinifera L. cv Monastrell suspension cultured cells were evaluated. The results showed the maximum level of trans-resveratrol produced by cells and secreted to the culture medium with 50 mM cyclodextrins and 1 μM coronatine. Since the levels of trans-resveratrol produced in the combined treatment were higher than the sum of the individual treatments, a synergistic effect between both elicitors was assumed. In addition, all the analysed genes were induced by cyclodextrins and/or coronatine. The expression of the phenylalanine ammonia lyase and stilbene synthase genes was greatly enhanced by coronatine although an increase in the amount of trans-resveratrol in the spent medium was not detected. Therefore, despite the fact that trans-resveratrol production is related with the expression of genes involved in the biosynthetic process, other factors may be involved, such as post-transcriptional and post-traductional regulation. The expression maximal levels of cinnamate 4-hydroxylase and 4-coumarate-CoA ligase genes were found with cyclodextrins alone or in combination with coronatine suggesting that the activity of these enzymes could be not only important for the formation of intermediates of trans-R biosynthesis but also for those intermediates involved in the biosynthesis of lignins and/or flavonoids.This work has been supported by the Ministerio de Economía y Competitividad (BIO2011-29856-C02-02 and BIO2014-51861-R)

Caracterización de la producción de resveratrol y análisis de proteínas en cultivos celulares de Vitis vinífera cv Monastrell

En esta memoria de investigación se han utilizado cultivos celulares de Vitis vinifera cv Monastrell para la producción de trans-resveratrol. En este sentido se ha realizado la caracterización del crecimiento de suspensiones celulares de Monastrell en diferentes medios minerales y concentraciones de sacarosa. Asimismo se caracterizó la producción extracelular de trans-resveratrol mediante la utilización de distintos elicitores tales como ciclodextrinas de distinta naturaleza, jasmonato de metilo, luz UV, fosetil de aluminio, ácido salicílico y etefón. Asimismo, se realizó el estudio de la implicación del calcio, del peróxido de hidrógeno, el óxido nítrico así como los eventos de fosforilación y desfosforilación de proteínas en los fenómenos de señalización desencadenados por la elicitación de suspensiones celulares de Monastrell que conducen a la producción de trans-resveratrol. Finalmente, se realizó el análisis de las proteínas extracelulares tanto en condiciones control como elicitadas con distintos compuestos con el fin de determinar las respuestas de defensa inducidas en estas condiciones. In this memory Vitis vinifera cv Monastrell cell cultures were used to produce trans-resveratrol. The growth of Monastrell cell cultures were characterized using different mineral media and sucrose concentrations. Extracellular production of trans-resveratrol was also characterized using different elicitors such as different nature cyclodextrins, methyl jasmonate, UV light, fosetyl aluminum, salicylic acid and ethephon. Moreover it was study the involvement of calcium, hydrogen peroxide, nitric oxide and phosphorylation and dephosphorylation of proteins in the signaling events triggered by elicitation of Monastrell cell cultures leading to trans-resveratrol production. Finally, it was analyzed extracellular proteins in control and elicited cell cultures in the presence of different elicitors to determine defense responses induced in these conditions

Changes in the secretome of Vitis vinifera cv. Monastrell cell cultures treated with cyclodextrins and methyl jasmonate

Elicitors induce defense responses that resemble those triggered by pathogen attack, including the synthesis of phytoalexins and pathogen-related proteins, which are accumulated in the extracellular space. In this work we analyze the changes in the secretome of Vitis vinifera cv. Monastrell cell cultures. This refers to the secreted proteome obtained from cell suspension cultures, in response to treatment with cyclodextrins and methyl jasmonate, separately or in combination using label-free quantitative approaches. Of the proteins found, thirty-three did not show significant differences in response to the different treatments carried out, indicating that these proteins were expressed in a constitutive way in both control and elicited grapevine cell cultures. These proteins included pathogenesis-related proteins 4 and 5, class III peroxidases, NtPRp-27, chitinases and class IV endochitinases, among others. Moreover, eleven proteins were differentially expressed in the presence of cyclodextrins and/or methyl jasmonate: three different peroxidases, two pathogenesis related protein 1, LysM domain-containing GPI-anchored protein 1, glycerophosphoryl diester phosphodiesterase, reticulin oxidase, heparanase, β-1,3-glucanase and xyloglucan endotransglycosylase. Treatments with cyclodextrins reinforced the defensive arsenal and induced the accumulation of peroxidase V and xyloglucan endotransglycosylase. However, elicitation with methyl jasmonate decreased the levels of several proteins such as pathogenesis related protein 1, LysM domain-containing GPI-anchored protein 1, cationic peroxidase, and glycerophosphoryl diester phosphodiesterase, but increased the levels of new gene products such as heparanase, β-1,3 glucanase, reticulin oxidase, and peroxidase IV, all of which could be used as potential biomarkers in the grapevine defense responses.This work has been supported by the Ministerio de Economía y Competitividad (No. BIO2017-82374-R) and Fundación Séneca - Agencia de Ciencia y Tecnología de la Región de Murcia (No. 19876/GERM/15). University of Alicante’s lab is a member of Proteored, PRB3 and is supported by grant PT17/0019, of the PE I+D+i 2013-2016, funded by ISCIII and ERDF

Induction of trans-resveratrol and extracellular pathogenesis-related proteins in elicited suspension cultured cells of Vitis vinifera cv Monastrell

Suspension-cultured cells of Vitis vinifera cv Monastrell were used to investigate the effects of methyljasmonate, ethylene and salicylic acid separately or in combination with cyclodextrins on both trans-resveratrol production and the induction of defense responses. The results showed that the addition of methyljasmonate or ethylene to suspension-cultured cells jointly treated with cyclodextrins and salicylic acid provoked a decrease of trans-resveratrol levels suggesting that salicylic acid has a negative and antagonistic effect with methyljasmonate or ethylene on trans-resveratrol production. Likewise, the exogenous application of these compounds induced the accumulation of pathogenesis-related proteins. Analysis of the extracellular proteome showed the presence of amino acid sequences homologous to an specific β-1,3-glucanase, class III peroxidases and a β-1,4-mannanase, which suggests that these signal molecules could play a role in mediating defense-related gene product expression in V. vinifera cv Monastrell. Apart from these inducible proteins, other proteins were found in both the control and elicited cell cultures of V. vinifera. These included class IV chitinase, polygalacturonase inhibitor protein and reticuline oxidase-like protein, suggesting that their expression is constitutive being involved in the modification of the cell wall architecture during cell culture growth and in the prevention of pathogen attack.L. Almagro, AB, Sabater-Jara and F. Fernández-Pérez held a grant from the Ministerio de Ciencia e Innovación. This study was supported by the Fundación Séneca, Agencia de Ciencia y Tecnología de la Región de Murcia en el marco de II PCTRM 2007-10 (08799/PI/08) and by MICINN-FEDER (BIO2008-2941 and BIO2011-29856-C02-02)

Early signaling events in grapevine cells elicited with cyclodextrins and methyl jasmonate

The use of cyclic oligosaccharides like cyclodextrins (CDs), alone or combined with methyl jasmonate (MJ), as elicitors has proved very effective in stimulating the production of trans-resveratrol (trans-R) in Vitis vinifera suspension-cultured cells (SCC). Since elicitors can be used to increase trans-R production, understanding the molecular mechanisms involved would improve the management of grapevine cells as factories of this compound. The results obtained in this study provide evidence for a role of Ca2+ in mediating elicitor-induced trans-R production in grapevine SCC. The Ca2+ elevation was promoted by an uptake of Ca2+ from the extracellular medium, and by Ca2+ mobilization from intracellular organelles. Moreover, protein phosphorylation/dephosphorylation events seem to be involved in the signal transduction pathways triggered by CDs separately or in combination with MJ since trans-R production is dependent on both, the phosphorylation status of several proteins through mitogen-activated kinase pathway and the activity of tyrosine phosphatases. Our results also suggest that H2O2 and NO participated in the production of trans-R triggered by both elicitors in grapevine SCC. Finally, a fast alkalinization of the extracellular medium is induced in the presence of CDs and/or MJ.L. Almagro, AB. Sabater-Jara and F. Fernández-Pérez have grants from the MICINN. This study was supported by the Fundación Séneca, Agencia de Ciencia y Tecnología de la Región de Murcia en el marco de II PCTRM 2007e10 (08799/PI/08) and MICINN-FEDER (BIO2008-2941 and BIO2011-29856-C02-02)

Methyl jasmonate induces extracellular pathogenesis-related proteins in cell cultures of Capsicum chinense

Suspension cultured cells of Capsicum chinense secrete proteins to the culture medium in both control conditions and under methyl jasmonate treatment. The exogenous application of methyl jasmonate induced the accumulation of putative pathogenesis-related proteins, class I chitinase, leucin-rich repeat protein, NtPRp27-like protein and pectinesterase which were also found in suspension cultured cells of C. annuum elicited with methyl jasmonate. However, a germin-like protein, which has never been described in methyl jasmonate-elicited C. chinense suspension cultured cells, was found. The different effects described as being the result of exogenous application of signalling molecules like methyl jasmonate on the expression of germin-like protein suggest that germin-like proteins may play a variety of roles in protecting plants against pathogen attacks and different stresses. Further studies will be necessary to characterize the differential expression of these pathogenesis-related proteins and to throw light on the complexity of their regulation

Modulation of <i>trans</i>-resveratrol biosynthesis pathway encoding enzymes gene expression by CD (cyclodextrins) and/or MJ (methyl jasmonate).

<p>Enzyme abbreviations: 4CL, 4-coumarate CoA ligase; ACC, acetyl-CoA carboxylase; C4H, cinnamate-4-hydroxylase; CHI, chalcone isomerase; CM, chorismate mutase; CS, chorismate synthase; DAHP synthase, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase; DHD, dehydroquinate dehydratase; DHQS, 3-dehydroquinate synthase; PAL, phenylalanine ammonia lyase; PDT, prephenate dehydratase; PEPC, phosphoenolpyruvate carboxylase; SDH, shikimate dehydrogenase; SK, shikimate kinase; STS, stilbene synthase.</p

Mapman visualization of the significant genes in the ‘Phenylpropanoid metabolism’ pathway.

<p>A, MJ (methyl jasmonate); B, CD (cyclodextrins); C, CDMJ (cyclodextrins and methyl jasmonate). Resultant transcripts were considered after the average of significant redundant probesets expression values. Significant probesets according to a 5% FDR and <i>P</i>-value <0.05 for model variable in the corresponding control versus treatment 24 h series maSigPro comparison. Expression changes in the treatment normalized to these in the control are shown. Red, treatment up-regulated transcripts; blue, treatment down-regulated transcripts; grey, transcripts not significant in the treatment.</p