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The high effectiveness of cyclic oligosaccharides like cyclodextrins in the production of trans-resveratrol in Vitis vinifera cell cultures is enhanced in the presence of methyl jasmonate. In order to dissect the basis of the interactions among the elicitation responses triggered by these two compounds, a transcriptional analysis of grapevine cell cultures treated with cyclodextrins and methyl jasmonate separately or in combination was carried out. The results showed that the activation of genes encoding enzymes from phenylpropanoid and stilbene biosynthesis induced by cyclodextrins alone was partially enhanced in the presence of methyl jasmonate, which correlated with their effects on trans-resveratrol production. In addition, protein translation and cell cycle regulation were more highly repressed in cells treated with cyclodextrins than in those treated with methyl jasmonate, and this response was enhanced in the combined treatment. Ethylene signalling was activated by all treatments, while jasmonate signalling and salicylic acid conjugation were activated only in the presence of methyl jasmonate and cyclodextrins, respectively. Moreover, the combined treatment resulted in a crosstalk between the signalling cascades activated by cyclodextrins and methyl jasmonate, which, in turn, provoked the activation of additional regulatory pathways involving the up-regulation of MYB15, NAC and WRKY transcription factors, protein kinases and calcium signal transducers. All these results suggest that both elicitors cause an activation of the secondary metabolism in detriment of basic cell processes like the primary metabolism or cell division. Crosstalk between cyclodextrins and methyl jasmonate-induced signalling provokes an intensification of these responses resulting in a greater trans-resveratrol production.

Elicitation with methyl jasmonate (MeJA) or/and cyclodextrin (CD) strongly induced silymarin (Sm) accumulation in suspensions of Silybum marianum, with most of Sm isomers being detected in the culture medium. This induction provides a model platform to characterize the regulation of flavonolignan accumulation and release in response to elicitors and, with this aim, changes in the S. marianum cell proteome were investigated. The DIGE technique was used to detect statistically significant changes in the cell's proteome. A total number of 1269 unique spots were detected, 67 of which were de-regulated upon elicitation. Nineteen spots were identified by nLC-MS/MS database search analysis. Identified proteins belong to a few categories, including metabolism, stress and defense responses and transport processes. The most abundant group was represented by pathogenesis-related (PR) proteins and heat shock proteins. Two proteins related to transport process were identified and both were upregulated by elicitation. One was identified as Ras-related protein Rab11C of the Rab family of small ATPase superfamily. A second protein was identified as an ABC transporter. Some of the identified proteins are discussed with respect to their putative role in the extracellular flavonolignan accumulation in S. marianum cultures., Biological Significance: Most approaches to increase secondary metabolite yields using plant cell cultures have been focused on the optimization of its biosynthesis. The study of other post biosynthetic events, like chemical or enzymatic modifications, transport, storage/secretion and catabolism/degradation are also biotechnologically relevant. Secretion is of particular interest since if cell cultures are to be used routinely for the commercial production, they must release the targeted metabolites into the extracellular medium. Elicitor-induced silymarin accumulation and release in S. marianum cell cultures provide a responsive model system to profile both alterations in proteins related to monolignol/flavonoid biosynthesis and to identify potential systems involved in secretion of secondary metabolites. The proteomic approach undertaken in this work has permitted identify some of the events occurring in elicited S. marianum cell cultures. One attainment of this study is that a vesicular transport mechanism could be involved in the release of this class of secondary metabolites to the extracellular compartment. This finding forms a baseline for future research on a non-sequenced medicinal plant S. marianum at molecular level., (Copyright © 2013 Elsevier B.V. All rights reserved.)

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 Ca(2+) in mediating elicitor-induced trans-R production in grapevine SCC. The Ca(2+) elevation was promoted by an uptake of Ca(2+) from the extracellular medium, and by Ca(2+) 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 H(2)O(2) 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., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

We analyze, for the first time, the early signal transduction pathways triggered by methyl jasmonate (MJ) and cyclodextrins (CDs) in tobacco (Nicotiana tabacum) cell cultures, paying particular attention to changes in cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)), the production of hydrogen peroxide (H(2)O(2)) and nitric oxide (NO), and late events like the induction of capsidiol. Our data indicate that MJ and CDs trigger a [Ca(2+)](cyt) rise promoted by Ca(2+) influx through Ca(2+)-permeable channels. The joint presence of MJ and CDs provokes a first increase in [Ca(2+)](cyt) similar to that observed in MJ-treated cells, followed by a second peak similar to that found in the presence of CDs alone. Moreover, oxidative burst induced by MJ is more pronounced when tobacco cells are incubated with CDs alone or in combination with MJ. The presence of both elicitors provokes H(2)O(2) production similar to that found in CD-treated cells, and a sustained response similar to that found in MJ-treated cells. In all treatments, H(2)O(2) production is dependent on Ca(2+) influx and protein phosphorylation events. Similarly, the joint action of both elicitors provokes NO accumulation, although to a lesser extent that in MJ-treated cells because CDs alone do not trigger this accumulation. This NO production is dependent on Ca(2+) influx but independent of both H(2)O(2) production and staurosporine-sensitive phosphorylation events. Taken as a whole, these results suggest the existence of different intracellular signaling pathways for both elicitors. Likewise, CDs might act by regulating the signaling pathway triggered by MJ since, in the presence of both compounds, CDs neutralize the strong oxidative and nitrosative bursts triggered by MJ and therefore, they regulate both H(2)O(2) and NO levels., (Copyright © 2011. Published by Elsevier Masson SAS.)

In this work, the effect of different inducing factors on trans-resveratrol extracellular production in Monastrell grapevine suspension cultured cells is evaluated. A detailed analysis provides the optimal concentrations of cyclodextrins, methyljasmonate and UV irradiation dosage, optimal cell density, elicitation time and sucrose content in the culture media. The results indicate that trans-resveratrol production decreases as the initial cell density increases for a constant elicitor concentration in Monastrell suspension cultured cells treated with cyclodextrins individually or in combination with methyljasmonate; the decrease observed in cell cultures elicited with cyclodextrins alone is far more drastic than those observed in the combined treatment. trans-Resveratrol extracellular production observed by the joint use of cyclodextrins and methyljasmonate (1,447.8 ± 60.4 μmol trans-resveratrol g(-1) dry weight) is lower when these chemical compounds are combined with UV light short exposure (669.9 ± 45.2 μmol trans-resveratrol g(-1) dry weight). Likewise, trans-resveratrol production is dependent on levels of sucrose in the elicitation medium with the maximal levels observed with 20 g l(-1) sucrose and the joint action of cyclodextrins and 100 μM methyljasmonate. The sucrose concentration did not seem to limit the process although it affects significantly the specific productivity since the lowest sucrose concentration is 10 g l(-1), the highest productivity is reached (100.7 ± 5.8 μmol trans-resveratrol g(-1) dry weight g(-1) sucrose) using cyclodextrins and 25 μM methyljasmonate.