Transcriptional reprogramming in sound-treated Micro-Tom plants inoculated with Pseudomonas syringae pv. tomato DC3000.

Sound vibrations (SV) are known to influence molecular and physiological processes that can improve crop performance and yield. In this study, the effects of three audible frequencies (100, 500 and 1000 Hz) at constant amplitude (90 dB) on tomato Micro-Tom physiological responses were evaluated 1 and 3 days post-treatment. Moreover, the potential use of SV treatment as priming agent for improved Micro-Tom resistance to Pseudomonas syringae pv. tomato DC3000 was tested by microarray. Results showed that the SV-induced physiological changes were frequency- and time-dependent, with the largest changes registered at 1000 Hz at day 3. SV treatments tended to alter the foliar content of photosynthetic pigments, soluble proteins, sugars, phenolic composition, and the enzymatic activity of polyphenol oxidase, peroxidase, superoxide dismutase and catalase. Microarray data revealed that 1000 Hz treatment is effective in eliciting transcriptional reprogramming in tomato plants grown under normal conditions, but particularly after the infection with Pst DC3000. Broadly, in plants challenged with Pst DC3000, the 1000 Hz pretreatment provoked the up-regulation of unique differentially expressed genes (DEGs) involved in cell wall reinforcement, phenylpropanoid pathway and defensive proteins. In addition, in those plants, DEGs associated with enhancing plant basal immunity, such as proteinase inhibitors, pathogenesis-related proteins, and carbonic anhydrase 3, were notably up-regulated in comparison with non-SV pretreated, infected plants. These findings provide new insights into the modulation of Pst DC3000-tomato interaction by sound and open up prospects for further development of strategies for plant disease management through the reinforcement of defense mechanisms in Micro-Tom plants.

Biotechnological production of ß-carotene using plant in vitro cultures.

MAIN CONCLUSION: ß-carotene is biologically active compound widely distributed in plants. The use of plant in vitro cultures and genetic engineering is a promising strategy for its sustainable production. ß-carotene is an orange carotenoid often found in leaves as well as in fruits, flowers, and roots. A member of the tetraterpene family, this 40-carbon isoprenoid has a conjugated double-bond structure, which is responsible for some of its most remarkable properties. In plants, ß-carotene functions as an antenna pigment and antioxidant, providing protection against photooxidative damage caused by strong UV-B light. In humans, ß-carotene acts as a precursor of vitamin A, prevents skin damage by solar radiation, and protects against several types of cancer such as oral, colon and prostate. Due to its wide spectrum of applications, the global market for ß-carotene is expanding, and the demand can no longer be met by extraction from plant raw materials. Considerable research has been dedicated to finding more efficient production alternatives based on biotechnological systems. This review provides a detailed overview of the strategies used to increase the production of ß-carotene in plant in vitro cultures, with particular focus on culture conditions, precursor feeding and elicitation, and the application of metabolic engineering.

Phenylpropanoids in Silybum marianum cultures treated with cyclodextrins coated with magnetic nanoparticles.

The glucose oligosaccharide-derived cyclodextrins (CDs) are used for improving bioactive compound production in plant cell cultures because, in addition to their elicitation activity, CDs promote product removal from cells. However, despite these advantages, the industrial application of CDs is hampered by their high market price. A strategy to overcome this constraint was recently tested, in which reusable CD polymers coated with magnetic Fe3O4 nanoparticles were harnessed in Vitis vinifera cell cultures to produce t-resveratrol (t-R). In this study, we applied hydroxypropyl-ß-CDs (HPCD) and HPCDs coated with magnetic nanoparticles (HPCD-EPI-MN) in methyl jasmonate (MJ)-treated transgenic Silybum marianum cultures ectopically expressing either a stilbene synthase gene (STS) or a chalcone synthase gene (CHS), and compared their effects on the yields of t-R and naringenin (Ng), respectively. HPCD-EPI-MN at 15 g/L stimulated the accumulation of metabolites in the culture medium of the corresponding transgenic cell lines, with up to 4 mg/L of t-R and 3 mg/L of Ng released after 3 days. Similar amounts were produced in cultures treated with HPCD. Concentrations higher than 15 g/L of HPCD-EPI-MN and prolonged incubation periods negatively affected cell growth and viability in both transgenic cell lines. Reutilization of HPCD-EPI-MN was possible in three elicitation cycles (72 h each), after which the polymer retained 25-30% of its initial efficiency, indicating good stability and reusability. Due to their capacity to adsorb metabolites and their recyclability, the application of magnetic CD polymers may reduce the costs of establishing efficient secondary metabolite production systems on a commercial scale. KEY POINTS: • Long-term transgenic S. marianum suspensions stably produce transgene products • t-R and Ng accumulated extracellularly in cultures elicited with HPCD and HPCD-EPI-MN • The recyclability of HPCD-EPI-MN for metabolite production was proven.

Transfecting Taxus � media Protoplasts to Study Transcription Factors BIS2 and TSAR2 as Activators of Taxane-Related Genes.

Taxane diterpenes are secondary metabolites with an important pharmacological role in the treatment of cancer. Taxus spp. biofactories have been used for taxane production, but the lack of knowledge about the taxane biosynthetic pathway and its molecular regulation hinders their optimal function. The difficulties in introducing foreign genes in Taxus spp. genomes hinder the study of the molecular mechanisms involved in taxane production, and a new approach is required to overcome them. In this study, a reliable, simple and fast method to obtain Taxus � media protoplasts was developed, allowing their manipulation in downstream assays for the study of physiological changes in Taxus spp. cells. Using this method, Taxus protoplasts were transiently transfected for the first time, corroborating their suitability for transfection assays and the study of specific physiological responses. The two assayed transcription factors (BIS2 and TSAR2) had a positive effect on the expression of several taxane-related genes, suggesting their potential use for the improvement of taxane yields. Furthermore, the results indicate that the developed method is suitable for obtaining T. � media protoplasts for transfection with the aim of unraveling regulatory mechanisms in taxane production.

Effect of diflufenican on total carotenoid and phytoene production in carrot suspension-cultured cells.

MAIN CONCLUSION: Diflufenican increased 493-fold the level of phytoene. Diflufenican-induced inhibition of phytoene desaturase gene expression in carrot cells resulted in an increased production of phytoene. This work analyzes the effect of diflufenican, an inhibitor of phytoene desaturase, on the gene expression profiles of the biosynthetic pathway of carotenoids related with the production of these compounds in carrot cell cultures. The results showed that the presence of 10 µM diflufenican in the culture medium increased phytoene levels, which was 493-fold higher than in control cells after 7 days of treatment but did not alter cell growth in carrot cell cultures. The maximal production of phytoene was reached with 10 µM diflufenican after 7 days of incubation in the presence of light and with 30 g/L sucrose in the culture medium. Moreover, diflufenican decreased the expression of phytoene synthase and phytoene desaturase genes at all the times studied. This diflufenican-induced inhibition of phytoene desaturase gene expression in carrot cell cultures resulted in an increased production of phytoene. Our results provide new insights into the action of diflufenican in carrot cell cultures, which could represent an alternative more sustainable and environmentally friendly system to produce phytoene than those currently used.

Effect of terbinafine on the biosynthetic pathway of isoprenoid compounds in carrot suspension cultured cells.

KEY MESSAGE: Terbinafine induced a significant increase of squalene production. Terbinafine increased the expression levels of squalene synthase. Cyclodextrins did not work as elicitors due to the gene expression levels obtained. Plant sterols are essential components of membrane lipids, which contributing to their fluidity and permeability. Besides their cholesterol-lowering properties, they also have anti-inflammatory, antidiabetic and anticancer activities. Squalene, which is phytosterol precursor, is widely used in medicine, foods and cosmetics due to its anti-tumor, antioxidant and anti-aging activities. Nowadays, vegetable oils constitute the main sources of phytosterols and squalene, but their isolation and purification involve complex extraction protocols and high costs. In this work, Daucus carota cell cultures were used to evaluate the effect of cyclodextrins and terbinafine on the production and accumulation of squalene and phytosterols as well as the expression levels of squalene synthase and cycloartenol synthase genes. D. carota cell cultures were able to produce high levels of extracellular being phytosterols in the presence of cyclodextrins (12 mg/L), these compounds able to increase both the secretion and accumulation of phytosterols in the culture medium. Moreover, terbinafine induced a significant increase in intracellular squalene production, as seen after 168 h of treatment (497.0 ± 23.5 µg g dry weight-1) while its extracellular production only increased in the presence of cyclodextrins.The analysis of sqs and cas gene expression revealed that cyclodextrins did not induce genes encoding enzymes involved in the phytosterol biosynthetic pathway since the expression levels of sqs and cas genes in cyclodextrin-treated cells were lower than in control cells. The results, therefore, suggest that cyclodextrins were only able to release phytosterols from the cells to the extracellular medium, thus contributing to their acumulation. To sum up, D. carota cell cultures treated with cyclodextrins or terbinafine were able to produce high levels of phytosterols and squalene, respectively, and, therefore, these suspension-cultured cells of carrot constitute an alternative biotechnological system, which is at the same time more sustainable, economic and ecological for the production of these bioactive compounds.

Biosynthesis and bioactivity of glucosinolates and their production in plant in vitro cultures.

MAIN CONCLUSION: Glucosinolates are biologically active compounds which are involved in plant defense reaction. The use of plant in vitro cultures and genetic engineering is a promising strategy for their sustainable production. Glucosinolates are a class of secondary metabolites found mainly in Brassicaceae, which contain nitrogen and sulfur in their structures. Glucosinolates are divided into three groups depending on the amino acid from which they are biosynthesized. Aliphatic glucosinolates are generally derived from leucine, valine, methionine, isoleucine and alanine while indole and aromatic glucosinolates are derived from tryptophan and phenylalanine or tyrosine, respectively. These compounds are hydrolyzed by the enzyme myrosinase when plants are stressed by biotic and abiotic factors, obtaining different degradation products. Glucosinolates and their hydrolysis products play an important role in plant defense responses against different types of stresses. In addition, these compounds have beneficial effect on human health because they are strong antioxidants and they have potent cardiovascular, antidiabetic, antimicrobial and antitumoral activities. Due to all the properties described above, the demand for glucosinolates and their hydrolysis products has enormously increased, and therefore, new strategies that allow the production of these compounds to be improved are needed. The use of plant in vitro cultures is emerging as a biotechnological strategy to obtain glucosinolates and their derivatives. This work is focused on the biosynthesis of glucosinolates and the bioactivity of these compounds in plants. In addition, a detailed study on the strategies used to increase the production of several glucosinolates, in particular those synthesized in Brassicaceae, using in vitro plant cultures has been made. Special attention has been paid for increasing the production of glucosinolates and their derivatives using metabolic engineering.

Combinatorial biosynthesis of sapogenins and saponins in Saccharomyces cerevisiae using a C-16α hydroxylase from Bupleurum falcatum.

The saikosaponins comprise oleanane- and ursane-type triterpene saponins that are abundantly present in the roots of the genus Bupleurum widely used in Asian traditional medicine. Here we identified a gene, designated CYP716Y1, encoding a cytochrome P450 monooxygenase from Bupleurum falcatum that catalyzes the C-16α hydroxylation of oleanane- and ursane-type triterpenes. Exploiting this hitherto unavailable enzymatic activity, we launched a combinatorial synthetic biology program in which we combined CYP716Y1 with oxidosqualene cyclase, P450, and glycosyltransferase genes available from other plant species and reconstituted the synthesis of monoglycosylated saponins in yeast. Additionally, we established a culturing strategy in which applying methylated ß-cyclodextrin to the culture medium allows the sequestration of heterologous nonvolatile hydrophobic terpenes, such as triterpene sapogenins, from engineered yeast cells into the growth medium, thereby greatly enhancing productivity. Together, our findings provide a sound base for the development of a synthetic biology platform for the production of bioactive triterpene sapo(ge)nins.

A new regulatory mechanism controlling carotenogenesis in the fungus Mucor circinelloides as a target to generate ß-carotene over-producing strains by genetic engineering.

BACKGROUND: Carotenoids are natural pigments with antioxidant properties that have important functions in human physiology and must be supplied through the diet. They also have important industrial applications as food colourants, animal feed additives and nutraceuticals. Some of them, such as ß-carotene, are produced on an industrial scale with the use of microorganisms, including fungi. The mucoral Blakeslea trispora is used by the industry to produce ß-carotene, although optimisation of production by molecular genetic engineering is unfeasible. However, the phylogenetically closely related Mucor circinelloides, which is also able to accumulate ß-carotene, possesses a vast collection of genetic tools with which to manipulate its genome. RESULTS: This work combines classical forward and modern reverse genetic techniques to deepen the regulation of carotenoid synthesis and generate candidate strains for biotechnological production of ß-carotene. Mutagenesis followed by screening for mutants with altered colour in the dark and/or in light led to the isolation of 26 mutants that, together with eight previously isolated mutants, have been analysed in this work. Although most of the mutants harboured mutations in known structural and regulatory carotenogenic genes, eight of them lacked mutations in those genes. Whole-genome sequencing of six of these strains revealed the presence of many mutations throughout their genomes, which makes identification of the mutation that produced the phenotype difficult. However, deletion of the crgA gene, a well-known repressor of carotenoid biosynthesis in M. circinelloides, in two mutants (MU206 and MU218) with high levels of ß-carotene resulted in a further increase in ß-carotene content to differing extents with respect to the crgA single-null strain; in particular, one strain derived from MU218 was able to accumulate up to 4 mg/g of ß-carotene. The additive effect of crgA deletion and the mutations present in MU218 suggests the existence of a previously unknown regulatory mechanism that represses carotenoid biosynthesis independently and in parallel to crgA. CONCLUSIONS: The use of a mucoral model such as M. circinelloides can allow the identification of the regulatory mechanisms that control carotenoid biosynthesis, which can then be manipulated to generate tailored strains of biotechnological interest. Mutants in the repressor crgA and in the newly identified regulatory mechanism generated in this work accumulate high levels of ß-carotene and are candidates for further improvements in biotechnological ß-carotene production.

Indole alkaloids from Catharanthus roseus: bioproduction and their effect on human health.

Catharanthus roseus is a medicinal plant belonging to the family Apocynaceae which produces terpenoid indole alkaloids (TIAs) of high medicinal importance. Indeed, a number of activities like antidiabetic, bactericide and antihypertensive are linked to C. roseus. Nevertheless, the high added value of this plant is based on its enormous pharmaceutical interest, producing more than 130 TIAs, some of which exhibit strong pharmacological activities. The most striking biological activity investigated has been the antitumour effect of dimeric alkaloids such as anhydrovinblastine, vinblastine and vincristine which are already in pre-, clinical or in use. The great pharmacological importance of these indole alkaloids, contrasts with the small amounts of them found in this plant, making their extraction a very expensive process. To overcome this problem, researches have looked for alternative sources and strategies to produce them in higher amounts. In this sense, intensive research on the biosynthesis of TIAs and the regulation of their pathways has been developed with the aim to increase by biotechnological approaches, the production of these high added value compounds. This review is focused on the different strategies which improve TIA production, and in the analysis of the beneficial effects that these compounds exert on human health.

Higher Plant-Derived Biostimulants: Mechanisms of Action and Their Role in Mitigating Plant Abiotic Stress.

Modern agriculture is being challenged by deteriorating edaphoclimatic conditions and increasing anthropogenic pressure. This necessitates the development of innovative crop production systems that can sustainably meet the demands of a growing world population while minimizing the environmental impact. The use of plant biostimulants is gaining ground as a safe and ecologically sound approach to improving crop yields. In this review, biostimulants obtained from different higher plant sources are presented under the term higher plant-derived biostimulants (hPDBs). Their mechanisms of action regulate physiological processes in plants from germination to fructification, conditioned by responses induced in plant mineral nutrition and primary metabolism, specialized metabolism, photosynthetic processes, oxidative metabolism, and signaling-related processes. The aim of this review is to collect and unify the abundant information dispersed in the literature on the effects of these biostimulants, focusing on crops subjected to abiotic stress conditions and the underlying mechanisms of action.

Synergistic and cytotoxic action of indole alkaloids produced from elicited cell cultures of Catharanthus roseus.

CONTEXT: Catharanthus roseus (L.) G. Don (Apocynaceae) is a medicinal plant that produces more than 130 alkaloids, with special attention given to the production of the anti-hypertensive monomeric indole alkaloids, serpentine and ajmalicine, and the antitumor dimeric alkaloids, vinblastine and vincristine. OBJECTIVE: This study evaluated the cytotoxic activity of the indole alkaloid-enriched bioactive extract obtained from suspension cultured-cells of C. roseus elicited with methyl jasmonate (MJ) and cyclodextrins (CDs) in three cell lines: JURKAT E.6 human lymphocytic leukemia, THP-1 human monocytic leukemia and BL 1395 non-tumor human B-cell line. MATERIALS AND METHODS: An indole alkaloid-enriched bioactive extract was obtained from C. roseus cell cultures elicited with MJ and CDs. The indole alkaloids were identified using an HPLC-diode array system coupled to a time-of-flight mass spectrometer using electrospray ionization (ESI) source. The cytotoxic assays were made using the colorimetric assay 2, 3-bis (2-methoxy-4-nitro-5-sulfophenyl)-S-[(phenylamino)carbonyl]-2 tetrazolium hydroxide (XTT). RESULTS: Four indole alkaloids were identified (catharanthine, ajmalicine, tabersonine and lochnericine) but only catharanthine and ajmalicine were quantified. The concentration of the indole alkaloid-enriched bioactive extract that inhibited cell growth by 50% was 211 and 210 ng/mL for the JURKAT E.6 and THP-1 cell lines, respectively. DISCUSSION AND CONCLUSION: The results confirm that the powerful antitumor activity of this indole alkaloid-enriched bioactive extract is not due to the effect of a single compound but depends on the synergistic action of the four compounds identified.

Biotechnological Approach to Increase Oxyresveratrol Production in Mulberry In Vitro Plants under Elicitation.

Morus alba L. is used for a range of therapeutic purposes in Asian traditional medicine, and its extracts are reported to be effective against lipidemia, diabetes, and obesity, as well as being hepatoprotective and tyrosinase-inhibitory. They are also included in cosmetic products as anti-aging and skin-whitening agents. Stilbenes, the major bioactive compounds found in M. alba, have received renewed attention recently because of their putative activity against COVID-19. In this study M. alba plants were established in vitro, and the effect of elicitation on plant growth and stilbene accumulation, specifically oxyresveratrol and trans-resveratrol, was investigated. Different concentrations of the elicitors including methyl jasmonate and cyclodextrins were applied, and stilbene levels were determined in leaves, roots, and the culture medium. Elicitation of the M. alba plants with 5 mM cyclodextrins, alone or in combination with 10 µM methyl jasmonate, significantly increased the total phenolic content in the culture medium and leaves after 7 days of treatment. The higher total phenolic content in the roots of control plants and those treated only with methyl jasmonate indicated that cyclodextrins promoted metabolite release to the culture medium. Notably, the cyclodextrin-treated plants with the highest levels of oxy- and trans-resveratrol also had the highest total phenolic content and antioxidant capacity. These results indicate that elicited M. alba in vitro plants constitute a promising alternative source of bioactive stilbenes to supply pharmaceutical and cosmeceutical industries.

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

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.

Cytotoxic effect of natural trans-resveratrol obtained from elicited Vitis vinifera cell cultures on three cancer cell lines.

trans-Resveratrol (trans-R) has been reported to be a potential cancer chemopreventive agent. Although its cytotoxic activity against different cancer cell lines has been tested, its effect on human acute leukemia cell lines has scarcely been investigated, and only a few in vitro studies were performed using human breast epithelial cell lines. Due to its potential value for human health, demand for trans-R has rapidly increased, and new biotechnological strategies to obtain it from natural edible sources have been developed. Thus, grapevine cell cultures represent a reliable system of trans-R production since they biosynthesize trans-R constitutively or in response to elicitation. In addition, there are no studies deepen on the inhibitory effect of trans-R, produced by elicited grapevine cell cultures, on growth of human tumor cell lines. In this work, the effect of trans-R extracted from the culture medium, after elicitation of grapevine cell cultures, was tested on two human acute lymphocytic and monocytic leukemia cell lines, and one human breast cancer cell line. The effect of trans-R on cell proliferation was not only dose- and time-dependent but also cell type-dependent, as seen from the different degrees of susceptibility of cancer cell lines tested. As regards the effect of trans-R on cell cycle distribution, low trans-R concentrations increased cells in the S phase whereas a high trans-R concentration increased G0/G1 phase in all cell lines. Perturbation of the cell cycle at low trans-R concentrations did not correlate with the induction of cell death, whereas a high trans-R concentration, cell proliferation decreased as a result of increasing apoptosis in the three cell lines. In leukemia cells, trans-R up-regulated the expression of caspase-3 while trans-R-induced apoptosis in breast cells occur through a caspase-3-independent mechanism mediated by a down-regulation of Bcl-2.

Differential Response of Phenol Metabolism Associated with Antioxidative Network in Elicited Grapevine Suspension Cultured Cells under Saline Conditions.

Highly productive trans-resveratrol (t-R) grapevine suspension cultured cells (SCC) and two effective elicitors, methyl jasmonate (MJ) and randomly methylated ß-cyclodextrins (CDs), were used to analyze the extent to which salt treatments alter the production of bioactive phenolic compounds. The expression/activity profile of the enzymes involved in phenol metabolism and antioxidant networks were also studied. A marked extracellular accumulation of phenolic compounds, especially t-R, was found in SCC elicited with CDs and/or MJ under saline conditions. However, the treatments with MJ alone and all those combined with salt favored the intracellular accumulation of catechin and ferulic acid. The salt-induced accumulation of phenolics was correlated with the higher total antioxidant capacity values found in cells, suggesting that cellular redox homeostasis under saline conditions was largely maintained by increasing phenolic compound production. These higher levels of phenolics found in elicited cells under saline conditions fit well with the highest activity of phenylalanine ammonia-lyase. Moreover, antioxidant enzyme activities were boosted in treatments with MJ and/or in those combined with salt and decreased in those treated with CDs. These results suggest a differential response of the antioxidative network to the presence of elicitors under saline conditions.

Cyclodextrins Increase Triterpene Production in Solanum lycopersicum Cell Cultures by Activating Biosynthetic Genes.

In this work, Solanum lycopersicum cv. Micro-Tom suspension-cultured cells were used to analyze the effect of different elicitors including ß-cyclodextrins (CD), methyl jasmonate (MJ), ß-glucan (Glu) and 3-hexenol (Hex) separately and the combined treatments of CD + MJ, CD + glu and CD + Hex on triterpene compound production after 24, 72 and 96 h. Moreover, we studied the changes induced by elicitors in the expression of key biosynthetic genes to elucidate the regulation of the triterpene biosynthetic pathway. The relative abundance of the triterpene compounds identified in the extracellular medium after elicitation (squalene, fucosterol, avenasterol, ß-sitosterol, cycloartenol and taraxasterol) was determined by gas chromatography coupled to mass spectrometry, and the expression level of genes in treated-cells was analyzed by real-time quantitative polymerase chain reaction (qRT-PCR). Results showed that, in CD-treated cells (CD, CD + MJ, CD + Glu, CD + Hex), specialized metabolites were accumulated mainly in the extracellular medium after 72 h of elicitation. Moreover, qRT-PCR analysis revealed that the highest triterpene levels in CD-treated cells (CD, CD + MJ, CD + Glu, CD + Hex) were highly correlated with the expression of cycloartenol synthase, 3-hydroxy-3-methylglutaryl-CoA reductase and squalene epoxidase genes at 24 h of treatment, whereas the expression of sterol methyltransferase was increased at 72 h. According to our findings, CD acts as a true elicitor of triterpene biosynthesis and can promote the release of bioactive compounds from the tomato cells into the extracellular medium. The results obtained provide new insights into the regulation of the triterpene metabolic pathway, which might be useful for implementing metabolic engineering techniques in tomato.

Redox post-translational modifications and their interplay in plant abiotic stress tolerance.

The impact of climate change entails a progressive and inexorable modification of the Earth's climate and events such as salinity, drought, extreme temperatures, high luminous intensity and ultraviolet radiation tend to be more numerous and prolonged in time. Plants face their exposure to these abiotic stresses or their combination through multiple physiological, metabolic and molecular mechanisms, to achieve the long-awaited acclimatization to these extreme conditions, and to thereby increase their survival rate. In recent decades, the increase in the intensity and duration of these climatological events have intensified research into the mechanisms behind plant tolerance to them, with great advances in this field. Among these mechanisms, the overproduction of molecular reactive species stands out, mainly reactive oxygen, nitrogen and sulfur species. These molecules have a dual activity, as they participate in signaling processes under physiological conditions, but, under stress conditions, their production increases, interacting with each other and modifying and-or damaging the main cellular components: lipids, carbohydrates, nucleic acids and proteins. The latter have amino acids in their sequence that are susceptible to post-translational modifications, both reversible and irreversible, through the different reactive species generated by abiotic stresses (redox-based PTMs). Some research suggests that this process does not occur randomly, but that the modification of critical residues in enzymes modulates their biological activity, being able to enhance or inhibit complete metabolic pathways in the process of acclimatization and tolerance to the exposure to the different abiotic stresses. Given the importance of these PTMs-based regulation mechanisms in the acclimatization processes of plants, the present review gathers the knowledge generated in recent years on this subject, delving into the PTMs of the redox-regulated enzymes of plant metabolism, and those that participate in the main stress-related pathways, such as oxidative metabolism, primary metabolism, cell signaling events, and photosynthetic metabolism. The aim is to unify the existing information thus far obtained to shed light on possible fields of future research in the search for the resilience of plants to climate change.

Improved biotechnological production of paclitaxel in Taxus media cell cultures by the combined action of coronatine and calix[8]arenes.

Paclitaxel (PTX), a widely used anticancer agent, is found in the inner bark of several Taxus species, although at such low levels that its extraction is ecologically unsustainable. Biotechnological platforms based on Taxus sp. cell cultures offer an eco-friendlier approach to PTX production, with yields that can be improved by elicitation. However, the also limited excretion of target compounds from the producer cells to the medium hampers their extraction and purification. In this context, we studied the effect of treating T. media cell cultures with the elicitor coronatine (COR) and calix[8]arenes (CAL), nanoparticles that can host lipophilic compounds within their macrocyclic scaffold. The highest taxane production (103.5 mg.L-1), achieved after treatment with COR (1 µM) and CAL (10 mg.L-1), was 15-fold greater than in the control, and PTX represented 82% of the total taxanes analyzed. Expression levels of the flux-limiting PTX biosynthetic genes, BAPT and DBTNBT, increased after the addition of COR, confirming its elicitor action, but not CAL. The CAL treatment significantly enhanced taxane excretion, especially when production levels were increased by COR; 98% of the total taxanes were found in the culture medium after COR + CAL treatment. By forming complexes with PTX, the nanoparticles facilitated its excretion to the medium, and by protecting cells from PTX toxicity, its intra-and extra-cellular degradation may have been avoided. The addition of COR and CAL to T. media cell cultures is therefore a bio-sustainable and economically viable system to improve the yield of this important anticancer compound.