Plant cell permeabilization

In the interdisciplinary field of biophysics and biotechnology, the bioeffects of electric field have received considerable interest for both fundamental studies on these interaction mechanisms and potential application. However, the effects of pulsed electric field (PEF) on secondary metabolism in plant cell cultures and fermentation processes have been unknown. Therefore, it would be very interesting to find out whether PEF could be used as a new tool for stimulating secondary metabolism in plant cell cultures for potential application to the value-added plant-specific secondary metabolite production. Furthermore, if the PEF permeabilization and elicitation are discovered in a cell culture system, the combination of... 

Brearley, C.A. Hanke, D.E. Metabolic relations of inositol 3,4,5,6-tetrakis-phosphate revealed by cell permeabilization. Identification of inositol 3,4,5, 6-tetrakisphosphate 1-kinase and inositol 3,4,5,6-tetrakisphosphate phosphatase activities in mesophyll cells. Plant Physiol., 122, 1209-1216 (2000)... 

Integrated bioprocesses can be used to enhance the production of valuable metabolites from plant cell cultures. The in situ removal of product during cell cultivation facilitates the rapid recovery of volatile and unstable phytochemicals, avoids problems of cell toxicity and end-product inhibition, and enhances product secretion. In situ extraction, in situ adsorption, the utilization of cyclodextrin, and the application of aqueous two-phase systems have been proposed for the integration of cell growth and product recovery in a bioreactor. The simultaneous combination of elicitation, immobilization, permeabilization, and in situ recovery can promote this method of plant cell culture as a feasible method to produce various natural products including proteins. 

Plant cell cultures represent a potentially rich source of secondary metabolites of commercial importance and have been shown to produce them in higher concentrations than the related intact plants. However, plant cell cultures often produce metabolites in lower concentrations than desired and commonly store them intracellularly. These limitations can be overcome by product yield enhancement procedures, including immobilization of cultured cells, and permeabilization, or ideally using a combined immobilization/ permeabilization process with retained plant cell viability. Complex coacervate capsules consisting of chitosan and alginate or carrageenan proved to be effective biomaterials for entrapment, controlled permeabilization of cells and to allow control of capsule membrane diffusivity. 

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). 

Secondary metabolites produced by plant cell culture are commonly accumulated in the cells. With few exceptions such as Capsicum frutescens, Thalictrum minus (9) and Vanilla planifolia (Knorr, D. and Romagnoli, L., Univ. of Delaware, unpublished data) cultures, which release valuable compounds such as capsaicin, berberine and vanillin, respectively, into the medium, procedures to induce product release are required to develop continuous production processes. Reported permeabilization methods include treatment with dimethylsulfoxide (DMSO), isopropanol, toluene, phenethyl alcohol or chloroform (9, 28). But as Fontanel and Tabata (9) pointed out, such treatments with organic solvents are severe and other methods of permeabilization need to be developed. 

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... 

Products formed in plant cells are usually intracellular storage products and must therefore be released from the cells if the product is to be continuously collected in the medium. This is especially important in immobilized cell systems. There are several environmental factors that will allow for release of products such as modifications in the media pH (35) and addition of solvents that permeabilize the cells Q, 36). It has also been demonstrated that operation in two-phase reactors, where the products are extracted into the nonaqueous phase, can be effective. Both liquid-liquid (37) and liquid-solid (38) systems have been used. The continuous release of products in this manner would also favor their continuous production since the storage capacity of the cells would not become limiting. 

When considering economically feasible production of secondary metabolites by employing in vitro cultures, it is worth determining whether the products can be released into the medium and collected without destroying the biomass. Attempts are being made to develop methods for the permeabilization of plant cells for release of intracellularly stored products. The cells should remain viable after the treatment to be... 

Unfortunately the majority of secondary metabolites produced by plant cells are accumulated in the vacuole or elsewhere in the cell. It is therefore necessary to devise methods for selective permeabilization and controlled release. Dimethylsulfoxide (DMSO) treatment allowed alkaloid release from Cinchona ledgeriana cells... 

ISPR applications have also been reported with raising interest for plant and animal cell technology. Table 4 does not give a complete summary on the ISPR activities with these two types of cells, but it gives a good overview for their main applications. Plant cells are mostly used for the production of secondary metabolites. Besides the technique of permeabilization of the cell wall... 

Arabinogalactans are secondary metabolites isolated from immobilized callus cultures of Tinospora cardifolia. These plant cells were immobilized in calcium chloride, sodium alginate with chitosan solution, and the cells could be permeabilized with polymeric neutral resin were used for trouble-free secondary metabolites production. The research findings show that the resin and chitosan additions increased the production of arabinogalactan 10 fold compared with controls (D Souza 2005). 

The two most often used methods for DNA delivery into protoplasts are electroporation and treatment with polyethylene glycol (Paszkowski et al., 1984). The observation that short electric pulses of high field strength transiently permeabilize cell membranes led to the development of electroporation-mediated gene transfer techniques for mammalian cells (Neumann et al., 1982) and plant cell protoplasts (Fromm et al., 1985). Electroporation is now the preferred technique for direct DNA transfer to plant protoplasts. 

During MEF treatment, the eleetrie field may cause changes in the permeability of plant cell membranes at temperatures below those at which thermal permeabilization occurs (Personius Sharp, 1938). Diffusion is enhanced, electrical conductivity changes are more linear during heating, and moisture migrates more easily out of the tissue. (Lima et al., 2001 Schreier et al., 1993). Electropermeabilization is a mechanism that can accormt for these... 

More complex staining methods using selective and fluorescent dyes have been used by Phoon et al. (2008), in combination with microscopic analysis. The measurement of cell membrane permeabilization of single cells or protoplasts, using fluorescent dyes, is also possible by means of flow cytometry (Shapiro, 2003), and this technique is gaining increasing interest for the characterization of plant cell microstructures. 

The treatment involves the application of very short electric pulses (1-100 ps) at electric field intensities in the range of 0.1-1 kVcm for the reversible permeabilization by means of stress induction in plant cells, 0.5-3 kVcm for the irreversible permeabilization of plant and animal tissues, and 15—40kVcm for the irreversible permeabilization of microbial cells. The aforementioned field intensities lead to the formation of a critical transmembrane potential, which is regarded as being the precondition for cell membrane breakdown and electroporation (Tsong, 1996). 

Brodelius P, Nilsson K (1983) Permeabilization of immobilized plant cells, resulting in release of intracellular stored products with preserved cell viability. Eur J Appl Microbiol Biotechnol 17 275-280... 

Defensins Mammals, birds, Invertebrates, plants, fungi Membrane permeabilization, macromolecular synthesis inhibition Cell proliferation/differentiation chemotaxis induction of gene expression adaptive immune polarization in vivo protection cytokine/chemokine induction CCR6, TLRs -1, -2 and -4... 

The use of saponin, a plant glycoside, for permeabilization was introduced by Endo and coworkers (1977). Saponin removes the surface membrane without impairment of the functions of SR. The plasma membrane, unlike that of triton skinned smooth muscle (Spedding, 1983), appears fairly intact in electron microscopic pictures of cross sections of permeabilized cells. However, when patches of isolated membranes were viewed face on in homogenates of permeabilized cells, numerous 70- to 80-A holes were visible (Kargacin and Lay, 1987). 

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