Secondary metabolites production elicitors

Plant cell and organ cultures can produce higher metabolite concentrations than found in the corresponding intact plant organs (6, 9). However, plant cells grown in culture may also produce lower quantities of the desired secondary metabolites which are commonly stored intracellularly. The challenges to increase product yield and to enhance the release of secondary metabolites can be met in various ways (7). These include immobilization (9), permeabilization (12, ), the use of precursors (12,13), and the induction of secondary metabolite production via elicitors (14). 

To date, progress achieved clearly demonstrates the potential of cultured plant cells for secondary metabolite production. Use of concurrent immobilization/permeabilization procedures, as well as precursor and elicitor treatments, may open new avenues of increasing product yields and will consequently affect the economic aspects of plant cell culture in a positive manner. However, our understanding of the many biosynthetic pathways of desired secondary metabolites is incomplete and successful industrial scale plant cell culture processes are still limited. Results of research in the area of plant cell culture will increase our understanding of the biosynthesis of plant metabolites, enhance our knowledge of plant-microorganism or plant-plant interactions and can lead to entirely new products or product lines of desirable compounds currently not available to use. Such work can also lead to development of industrial scale production processes for products now produced and recovered by conventional methods. Also, the genetic variety of the 250,000 to 750,000 plant species available remains to be explored. Presently only 5 to 15% of these species have been subject to even... 

Another strategy for the improvement of secondary metabolites production is the addition of elicitors (biotic or abiotic) [68]. 

It is well known that the elicitors are compounds that can induce defense responses on plant cultures deriving in an increase on secondary metabolites production [42]. In addition, it was frequently observed that these compounds were able to release the plant products formed to the medium [42]. 

Secondary product formation is a form of differentiation, induction of the required type of differentiation does lead to secondary product formation, e.g. phytoalexins will readily be formed upon stress conditions (additions of fungal elicitors) and compounds usually found in the roots will also be found in root cultures or hairy-root (cells transformed with Agrobacterium rhizogenes) cultures. In Table 4 some examples are summarized of the influence of differentiation on secondary metabolite production. 

In industry, enhancement of secondary metabolite production is crucial for successful application of plant cell culture technology. Among the manipulative techniques available to promote the productivity of useful secondary metabolites from plant cell cultures, the treatment of plant cells with various elicitors has been one of the best approaches for dramatically increasing the production of useful secondary metabolites. ... 

Sometimes explant has been one of the important factor for secondary metabolites production, which could be economicallly feasible and the protective of the plant (Figure 40.1). This study was done in different hours such as 6,12,24,48,72, and 168 h with different concentrations of chitosan (used by an elicitor). A positive effect on the production was exerted in particular by elicitation of... 

The term elicitor refers to chemicals from various sources, biotic or abiotic, as well as physical factors, that can trigger a response in living organisms resulting in a high accumulation of secondary metabolites. Therefore, elicitors are usefiil tools for improving the production of plant valuable compounds ([36] and references therein). The effectiveness of elicitation as a tool to enhance the production of secondary metabolites depends on a complex interaction between the elicitor and the plant cell. There is evidence that the same elicitor can stimulate secondary metabolism in different cell cultures and that certain plant cultures are responsive to diverse elicitors. Treatment of a particular plant cell culture with different elicitors will result in the accumulation of a particular class of compounds, since these are specific of each plant culture. Although the class of metabolite depends on the plant species, the kinetics of induction or accumulation levels can vary with different elicitors. 

Zhao J, Davis LC, Verpoorte R. 2005. Elicitor signal transduction leading to production of plant secondary metabolites. Biotechnol Adv 23 283-333. 

What can be done to achieve this necessary improvement Most plant cell products are secondary metabolites, which means that plant suspension culture systems involve a growth phase and a production phase. In any commercial system, one will probably want to separate physically these two phases. Unfortunately, one has to add a growth hormone such as 2-4-D for a successful growth phase, and these hormones must be completely removed before elicitors are added to the production phase to induce the secondary product. Optimizing this two-stage system is an engineering challenge. 

DiCosmo and Misawa (14) suggested the immense potential of plant cell culture- elicitor (inducer) interactions to the large scale production of secondary metabolites with the induction of shikonin formation by agar in Lithospermum erythrorhizon cell suspension cultures (39) this is so far one of the most successful examples of elicitor effects. Some reports on the induction of enzymes of plant... 

To increase production and facilitate isolation, plant cells have been immobilised on various matrices such as polyurethane foam and calcium alginate gel beads,24 while elicitation (i.e. the induction of a defence response) is generally critical for the production of secondary metabolites. The rationale for the use of elicitors is that plants produce secondary metabolites as part of a defence response to stress, either biotic (pathogen infection) or abiotic (ultraviolet, toxic heavy metals and rare earth ions). Jasmonic acid plays a crucial role in plant stress responses and, along with fungal polysaccharides and heavy metals, is the most widely employed elicitor in plant tissue cultures.30... 

We can distinguish between secondary metabolites that are already present prior to an attack or wounding, so-called constitutive compounds, and others that are induced by these processes and made de novo. Inducing agents, which have been termed elicitors by phytopathologists, can be cell wall fragments of microbes, the plant itself, or many other chemical constituents (4,17,22-24). The induced compounds are called phytoalexins, which is merely a functional term, since these compounds often do not differ in structure from constitutive natural products. In another way this term is misleading, since it implies that the induced compound is only active in plant-microbe interactions, whereas in reality it often has multiple functions that include antimicrobial and antiherbivoral properties (see below). 

Phytoalexins are an Important component of the plant disease defense reaction called the hypersensitive response. Successful pathogens have evolved methods for dealing with plant phytoalexins. Including suppressors of their production, detoxification of the phytoalexins and In some cases avoiding elaboration of substances, called elicitors, that would otherwise Initiate the defense reaction. Elicitors obtained from pathogens are of considerable utility for study of various aspects of plant biology because of their interaction with the products of plant disease resistance genes. Substantial information has been obtained on how elicitors are perceived by plant cells and how they function, but much remains to be done. Finally, elicitors may prove of value for the economic production of exotic plant secondary metabolites and as specific herbicides. 

With a few exceptions, the characteristic problem of cultivation of plant explants in in vitro cultures is a low production of secondary metabolites by these cultures. One of the methods which can achieve an increase in the production of natural substances in in vitro cultures, is elicitation of cell cultures with biotic elicitors. For example, hairy root cultures of Cassia obtusifolia L. clones transformed with Agrobacterium rhizogenes strain 9402 were established to investigate anthraquinone production. It was found that changes of the elements in the culture medium and the addition of rare earth element Eu3+ can greatly influence the contents of free anthraquinones in the hairy root [320],... 

Elicitors are probably the best-studied exogenous signals in plants. They are connected with microbial infections and induce plant defense responses. Among others, the production of certain secondary metabolites (phytoalexins) is induced by biotic or abiotic elicitors (e.g., see reviews in refs. 326-328). 

For many plants, methyl jasmonate is a key elicitor, which initiates a series of response mechanisms to herbivores, like the synthesis of protease inhibitors and the increased formation of secondary metabolites (cf section 3.3- Jasmonoids). The effects were already well-known for tomato and tobacco plants, but also for Catharanthus and Cinchona seedlings and for soya beans. In the case of yew cells, this additive enabled to increase the paclitaxel production from 3 to 117 mg, and later to 295 mg per litre of fermentation broth. 

---