Ana Belén Sabater-Jara

A rational approach to improving the biotechnological production of taxanes in plant cell cultures of Taxus spp.

Taxol is a complex diterpene alkaloid scarcely produced in nature and with a high anticancer activity. Biotechnological systems for taxol production based on cell cultures of Taxus spp. have been developed, but the growing commercial demand for taxol and its precursors requires the optimization of these procedures. In order to increase the biotechnological production of taxol and related taxanes in Taxus spp. cell cultures, it is necessary not only to take an empirical approach that strives to optimize in-put factors (cell line selection, culture conditions, elicitation, up-scaling, etc.) and out-put factors (growth, production, yields, etc.), but also to carry out molecular biological studies. The latter can provide valuable insight into how the enhancement of taxane biosynthesis and accumulation affects metabolic profiles and gene expression in Taxus spp. cell cultures. Several rational approaches have focused on studying the transcriptomic profiles of key genes in the taxol biosynthetic pathway in Taxus spp. cell cultures treated with elicitors such as methyl jasmonate, coronatine and cyclodextrins in relation with the taxane pattern, production and excretion to the culture medium. These studies have provided new insights into the taxol biosynthetic pathway and its regulation. Additionally, identifying genes with low levels of expression even in the presence of elicitors, together with metabolomics studies, has shed light on the limiting steps in taxol biosynthesis and could help define suitable metabolic targets for engineering with the main aim of obtaining highly productive Taxus cultured cells. In this review, we have summarized the latest endeavors to enhance the molecular understanding of the action mechanism of elicitors in Taxus spp. cell cultures. Developments in the ongoing search for new and more effective elicitation treatments and the application of metabolic engineering to design new transgenic cell lines of Taxus with an improved capacity for taxane production are described.

Induction of sesquiterpenes, phytoesterols and extracellular pathogenesis-related proteins in elicited cell cultures of Capsicum annuum

Capsicum annuum suspension cell cultures were used to evaluate the effect of cyclodextrins and methyl jasmonate as elicitors of defence responses. The induced defence responses included the accumulation of sesquiterpenes and phytosterols and the activation of pathogenesis-related proteins, leading to reinforcement and modification of the cell wall architecture during elicitation and protection cells against biotic stress. The results showed that the addition of both cyclodextrins and methyl jasmonate induced the biosynthesis of two sesquiterpenes, aromadendrene and solavetivone. This response was clearly synergistic since the increase in the levels of these compounds was much greater in the presence of both elicitors than when they were used separately. The biosynthesis of phytosterols was also induced in the combined treatment, as the result of an additive effect. Likewise, the exogenous application of methyl jasmonate induced the accumulation of pathogenesis-related proteins. The analysis of the extracellular proteome showed the presence of amino acid sequences homologous to PR1 and 4, NtPRp27-like proteins and class I chitinases, peroxidases and the hydrolytic enzymes LEXYL1 and 2, arabinosidases, pectinases, nectarin IV and leucin-rich repeat protein, which suggests that methyl jasmonate plays a role in mediating defence-related gene product expression in C. annuum. Apart from these methyl jamonate-induced proteins, other PR proteins were found in both the control and elicited cell cultures of C. annuum. These included class IV chitinases, beta-1,3-glucanases, thaumatin-like proteins and peroxidases, suggesting that their expression is mainly constitutive since they are involved in growth, development and defence processes.

Enhancement of phytosterols, taraxasterol and induction of extracellular pathogenesis-related proteins in cell cultures of Solanum lycopersicum cv Micro-Tom elicited with cyclodextrins and methyl jasmonate.

Suspension-cultured cells of Solanum lycopersicum cv Micro-Tom were used to evaluate the effect of methyl jasmonate and cyclodextrins, separately or in combination, on the induction of defense responses. An extracellular accumulation of two sterols (isofucosterol and β-sitosterol) and taraxasterol, a common tomato fruit cuticular triterpene, were observed. Their levels were higher in Micro-Tom tomato suspension cultured cells elicited with cyclodextrins than in control and methyl jasmonate-treated cells. Also, their accumulation profiles during the cell growth phase were markedly different. The most striking feature in response to cyclodextrin treatments was the observed enhancement of taraxasterol accumulation. Likewise, the exogenous application of methyl jasmonate and cyclodextrins induced the accumulation of pathogenesis-related proteins. Analysis of the extracellular proteome showed the presence of amino acid sequences homologous to pathogenesis-related 1 and 5 proteins, a cationic peroxidase and a biotic cell death-associated protein, which suggests that methyl jasmonate and cyclodextrins could play a role in mediating defense-related gene product expression in S. lycopersicum cv Micro-Tom.

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 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.

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.

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.

Biotechnological approaches to enhance the biosynthesis of ginkgolides and bilobalide in Ginkgo biloba

Ginkgo biloba is one of the oldest living tree species and its extracts or powdered leaves are one of the best selling herbal preparations. The main bioactive constituents are flavonoids and the terpene trilactones, ginkgolides and bilobalide, which are responsible for their pharmacological activity. However, there are many difficulties for ginkgo leaves supply and the chemical synthesis is far from of being applicable for commercial-scale production. G. biloba cell cultures have arisen as a useful alternative source of pharmacologically active terpene trilactones. This review sheds light on the chemistry and biosynthesis of terpene trilactones with the aim of increasing the production of these high value compounds by biotechnological approaches. Different biotechnological strategies to improve ginkgolides and bilobalide production will be discussed, including screening and selection of in vitro ginkgo cultures, cell differentiation levels of these cultures, optimization of culture conditions, feeding and elicitation strategies. Special attention will be paid in developing new methodologies to enhance ginkgo cell biomass and provide high amounts of these bioactive terpene trilactones using large-scale cell cultures.

Effect of UV Light on Secondary Metabolite Biosynthesis in Plant Cell Cultures Elicited with Cyclodextrins and Methyl Jasmonate

All the chemical reactions that occur in the cells of a living organism are called metabolism. By these reactions, a large number of organic compounds, including sugars, amino acids, fatty acids, nucleotides and their polymers derived, that is, polysaccharides, proteins, lipids, RNA, DNA, etc ... are produced. These processes are essential and common to all organisms and are known as primary metabolism and related compounds are known as primary metabolites. In the case of plants, in addition to the primary metabolic pathways, other metabolic pathways are activated under certain situations and the compounds produced are called secondary metabolites. The role of secondary metabolites may seem irrelevant, but the truth is that the plant spends a great deal of energy in their synthesis and they have remained in the plant kingdom up to date. This is because that wide variety and high diversity of secondary metabolites have apparently evolved as a means for plants to interact with the environment and for the development of resistance against both abiotic and biotic stress. In fact, secondary metabolites are useful to protect plants against herbivores (insects and vertebrates), mammals, bacteria, fungi, viruses and even other competing plants. In addition, some plants use secondary metabolites to attract pollinators and seed dispersers, as signals for communication between plants and symbiotic microorganisms or for protection against UV light and other physical stress (Wink 2003, 2008). Daucus carota L. (Umbelliferae) is a biennial herb, whose fruits (common name: wild carrot fruits) have been used in traditional Chinese medicine for the treatment of ancylostomiasis, dropsy, chronic kidney disease and bladder afflictions (Pant & Manandhar, 2007) due to a wide range of reported pharmacological effects, including antibacterial (Rossi et al., 2007), antifungal (Tavares et al., 2008), antihelminthic, hepatoprotective (Bishayee et al., 1995) and cytotoxic activities (Yang et al., 2008; Fu et al., 2009). Carrot roots contain a variety of carotenoids and anthocyanins that are responsible for the typical colour of the root. In addition, this vegetable also produces phenolic compounds such as scopoletin, p-hydroxy benzoic acid and the isocoumarin, 6-methoxymellein, all major components of the phytoalexin complex (Mercier et al., 2000). These compounds are induced in carrot by fungal infection, heavy metals or UV light (Marinelli et al., 1994), and therefore they are involved in plant defence responses. Recently, Sabater-Jara et al., (2008) have described the production of sterols in different cell cultures including D. carota.

Induction of extracellular defense-related proteins in suspension cultured-cells of Daucus carota elicited with cyclodextrins and methyl jasmonate.

Suspension cultured-cells (SCC) of Daucus carota were used to evaluate the effect of methyl jasmonate and cyclodextrins, separately or in combination, on the induction of defense responses, particularly the accumulation of pathogenesis-related proteins. A comparative study of the extracellular proteome (secretome) between control and elicited carrot SCC pointed to the presence of amino acid sequences homologous to glycoproteins which have inhibitory activity against the cell-wall-degrading enzymes secreted by pathogens and/or are induced when carrot cells are exposed to a pathogen elicitor. Other amino acid sequences were homologous to Leucine-Rich Repeat domain-containing proteins, which play an essential role in defense against pathogens, as well as in the recognition of microorganisms, making them important players in the innate immunity of this plant. Also, some tryptic peptides were shown to be homologous to a thaumatin-like protein, showing high specificity to abiotic stress and to different reticuline oxidase-like proteins that displayed high levels of antifungal activity, suggesting that methyl jasmonate and cyclodextrins could play a role in mediating defense-related gene product expression in SCC of D. carota. Apart from these elicitor-inducible proteins, we observed the presence of PR-proteins in both control and elicited carrot SCC, suggesting that their expression is mainly constitutive. These PR-proteins are putative class IV chitinases, which also have inhibitory activity against pathogen growth and the class III peroxidases that participate in response to environmental stress (e.g. pathogen attack and oxidative), meaning that they are involved in defense responses triggered by both biotic and abiotic factors.

Kinetic characterization of a basic peroxidase from garlic (Allium sativum L.) cloves.

Peroxidases catalyze the reduction of H(2)O(2) by taking electrons from a variety of compounds from the secondary metabolism including flavonoids and lignin precursors. This work describes the purification and kinetic characterization of a basic peroxidase from garlic cloves using quercetin and p-coumaric acid, flavonoid and phenolic compounds found in garlic cloves. The high catalytic efficiency shown by this basic peroxidase in the oxidation of quercetin at acidic pH suggests good adaptation of this enzyme, involved in quercetin catabolism in the acidic physiological pH conditions of the vacuoles, where it is presumably located. Likewise, garlic peroxidase showed similar oxidation rates for hydroxycinnamyl (p-coumaric) and sinapyl-type structures, which suggests its involvement in the cross-coupling reactions that occur in the cell wall during lignification. On the other hand, the high affinity of this enzyme for H(2)O(2) would be in accordance with the oxidation of both flavonoid and phenolic compounds to regulate H(2)O(2) levels in tissues/organelles, where this peroxidase is expressed.