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.

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.

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.

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.

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.

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.

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.

A Novel Hydroxylation Step in the Taxane Biosynthetic Pathway: A New Approach to Paclitaxel Production by Synthetic Biology.

Engineered plant cell lines have the potential to achieve enhanced metabolite production rates, providing a high-yielding source of compounds of interest. Improving the production of taxanes, pharmacologically valuable secondary metabolites of Taxus spp., is hindered by an incomplete knowledge of the taxane biosynthetic pathway. Of the five unknown steps, three are thought to involve cytochrome P450-like hydroxylases. In the current work, after an in-depth in silico characterization of four candidate enzymes proposed in a previous cDNA-AFLP assay, TB506 was selected as a candidate for the hydroxylation of the taxane side chain. A docking assay indicated TB506 is active after the attachment of the side chain based on its affinity to the ligand 3'N-dehydroxydebenzoyltaxol. Finally, the involvement of TB506 in the last hydroxylation step of the paclitaxel biosynthetic pathway was confirmed by functional assays. The identification of this hydroxylase will contribute to the development of alternative sustainable paclitaxel production systems using synthetic biology techniques.

A Smart Strategy to Improve t-Resveratrol Production in Grapevine Cells Treated with Cyclodextrin Polymers Coated with Magnetic Nanoparticles.

One of the most successfully procedures used to increase the production of t-resveratrol in Vitis vinifera suspension-cultured cells is the application of cyclodextrins (CDs) and methyl jasmonate (MJ) as elicitors. In particular, ß-CDs are characterized by their chemical structure which makes them special, not only by acting as elicitors, but also because they are compounds capable of trapping high added-value hydrophobic molecules such as t-resveratrol. However, the use of ß-CDs as elicitors increases the production costs of this compound, making their industrial exploitation economically unfeasible. Therefore, the development of ß-CDs recovery strategies is necessary to provide a viable solution to their industrial use. In this work, carboxymethylated and hydroxypropylated ß-CDs have been used to form polymers using epichlorohydrin (EPI) as a cross-linking agent. The polymers were coated to Fe3O4 nanoparticles and were jointly used with MJ to elicit V. vinifera suspension-cultured cells. Once elicitation experiments were finished, a magnet easily allowed the recovery of polymers, and t-resveratrol was extracted from them by using ethyl acetate. The results indicated that the production of t-resveratrol in the presence of free carboxymethyl-ß-CDs was much lower than that found in the presence of carboxymethyl-ß-cyclodextrins-EPI polymer coated magnetic nanoparticles. In addition, the maximal levels of t-resveratrol were found at 168 h of elicitation in the presence of 15 g/L hydroxypropyl-ß-CDs polymer coated magnetic nanoparticles and MJ, and non-t-resveratrol was found in the extracellular medium, indicating that all the t-resveratrol produced by the cells and secreted into the culture medium was trapped by the polymer and extracted from it. This work also showed that polymers can be regenerated and reused during three cycles of continuous elicitation since the induction and adsorption capacity of hydroxypropyl-ß-CDs polymer-coated magnetic nanoparticles after these cycles of elicitation remained high, allowing high concentrations of t-resveratrol to be obtained.

Non-enzymatic screen-printed sensor based on PtNPs@polyazure A for the real-time tracking of the H2O2 secreted from living plant cells.

Monitoring of hydrogen peroxide (H2O2) in living cells has high significance for understanding its functions. We herein report an enzymeless H2O2 sensor consisting of a previously activated screen-printed carbon electrode modified with Pt nanoparticles electrogenerated on a supporting conductive layer of polyazure A-dodecyl sulfate. This electrode was used to investigate the dynamic process of H2O2 release from living grapevine cells under different (a)biotic stresses. The modified surfaces were characterized by FESEM/EDX, EIS and cyclic voltammetry. Sensor analytical performance was studied in a cell culture medium under aerobic conditions, as required for cell survival. In relation to the synergistic effect between the metal nanoparticles and the conjugated polymer, this electrode showed good stability, excellent analytical performance combined with a rapid response (<2s) and limit of detection of 24.9 nM in the culture medium. The modified electrodes could fulfill the real-time measurement requirement of H2O2 release from living plant cells to the extracellular medium operating continuously, even in experiments lasting more than 12 h. Methyl jasmonate, L-methionine, clopyralid and the fungus Botrytis cinerea were the eliciting agents chosen to induce oxidative stress in the plant cells. This work demonstrates the huge potential of this sensor for the real-time tracking of the H2O2 released from living cells under different physiological conditions.

Enhanced bioactive compound production in broccoli cells due to coronatine and methyl jasmonate is linked to antioxidative metabolism.

Elicited broccoli suspension-cultured cells (SCC) provide a useful system for obtaining bioactive compounds, including glucosinolates (GS) and phenolic compounds (PCs). In this work, coronatine (Cor) and methyl jasmonate (MJ) were used to increase the bioactive compound production in broccoli SCC. Although the use of Cor and MJ in secondary metabolite production has already been described, information concerning how elicitors affect cell metabolism is scarce. It has been suggested that Cor and MJ trigger defence reactions affecting the antioxidative metabolism. In the current study, the concentration of 0.5 µM Cor was the most effective treatment for increasing both the total antioxidant capacity (measured as ferulic acid equivalents) and glucosinolate content in broccoli SCC. The elicited broccoli SCC also showed higher polyphenol oxidase activity than the control cells. Elicitation altered the antioxidative metabolism of broccoli SCC, which displayed biochemical changes in antioxidant enzymes, a decrease in the glutathione redox state and an increase in lipid peroxidation levels. Furthermore, we studied the effect of elicitation on the protein profile and observed an induction of defence-related proteins. All of these findings suggest that elicitation not only increases bioactive compound production, but it also leads to mild oxidative stress in broccoli SCC that could be an important factor triggering the production of these compounds.

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.

Production and localization of hydrogen peroxide and nitric oxide in grapevine cells elicited with cyclodextrins and methyl jasmonate.

The use of methyl jasmonate, alone or in combination with cyclic oligosaccharides such as cyclodextrins, has proved to be a successful strategy for increasing the production of trans-resveratrol in Vitis vinifera cell cultures. However, understanding the intracellular signalling pathways involved in its production would improve the management of grapevine cells as biofactories of this high-value natural product. The results obtained herein confirm the involvement of hydrogen peroxide and nitric oxide in cyclodextrins and methyl jasmonate-induced trans-resveratrol production in grapevine cell cultures. In fact, methyl jasmonate led to maximal intracellular levels of hydrogen peroxide and nitric oxide after 24 h of treatment, but extracellular hydrogen peroxide was only detected in the culture medium when grapevine cells were treated with cyclodextrins. The results derived from the cytochemical detection of H2O2 in elicited grapevine cell cultures also suggested that the combined treatment with cyclodextrins and methyl jasmonate not only increased the production of H2O2 but also released cell wall fragments with electron-dense deposits. Moreover, nitric oxide was localized in all the cellular compartments, particularly in the nucleus and cytoplasmic organelles, whereas hydrogen peroxide was mainly found in cytoplasmic areas close to the cell wall, and in the nucleoplasm.

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.

Perfluorodecalins and Hexenol as Inducers of Secondary Metabolism in Taxus media and Vitis vinifera Cell Cultures.

Plant cell cultures constitute a potentially efficient and sustainable tool for the production of high added-value bioactive compounds. However, due to the inherent restrictions in the expression of secondary metabolism, to date the yields obtained have generally been low. Plant cell culture elicitation can boost production, sometimes leading to dramatic improvements in yield, as well as providing insight into the target biosynthetic pathways and the regulation of the genes involved. Among the secondary compounds successfully being produced in biotechnological platforms are taxanes and trans-resveratrol (t-R). In the current study, perfluorodecalins (PFDs) and hexenol (Hex) were tested for the first time with Taxus media and Vitis vinifera cell cultures to explore their effect on plant cell growth and secondary metabolite production, either alone or combined with other elicitors already established as highly effective, such as methyl jasmonate (MeJa), coronatine (Coro) or randomly methylated ß-cyclodextrins (ß-CDs). The total taxane content at the peak of production in T. media cell cultures treated with PFDs together with Coro plus ß-CDs was 3.3-fold higher than in the control, whereas the t-R production in MeJa and ß-CD-treated V. vinifera cell cultures increased 552.6-fold compared to the extremely low-yielding control. Hex was ineffective as an elicitor in V. vinifera cell cultures, and in T. media cell suspensions it blocked the taxol production but induced a clear enhancement of baccatin III. Regarding biosynthetic gene expression, a strong positive relationship was observed between the transcript level of targeted genes and taxol production in the T. media cell cultures, but not with t-R production in the elicited V. vinifera cell cultures.

Cyclodextrins increase phytosterol and tocopherol levels in suspension cultured cells obtained from mung beans and safflower.

In this work, suspension-cultured cells of mung beans and safflower were used in order to analyze the effect of methyl jasmonate and/or cyclodextrins, on bioactive compound production such as phytosterols and tocopherols. The results indicated that mung bean suspension-cultured cells produced higher amount of total phytosterols and tocopherols. In particular, mung bean suspension-cultured cells produced almost 220-fold higher levels of tocopherols than safflower suspension-cultured cells in the best conditions. However, while cyclodextrins were able to enhance extracellular production of phytosterols, in the case of tocopherols, they only increased their intracellular accumulation. Our results showed that mung bean cells could be used as a highly efficient system for the production of phytosterols and tocopherols which have a wide range of biological activities. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1662-1665, 2017.

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.