Metabolism in Plant tissue cultures and its industrial applications in the era of genetic manipulations.
Metabolism in Plant tissue cultures and its industrial applications in the era of genetic manipulations. Humans have been manipulating the genetic makeup of agricultural plants (and animals) for millennia. Recently, the pace of change has increased markedly as researchers apply molecular tools to modify a wide range of agronomic characteristics and to develop and market transgenic crops that generate specific novel products (ap Rees, 1995). Among many others, these include vaccines and other pharmaceuticals, plastics, and proteins that may render certain plants effective tools for environmental decontamination (John and Keller, 1996; Raskin, 1996; Arntzen, 1997; Hezari and Croteau, 1997). Related techniques can be adapted to manipulate endogenous biochemical pathways. Here, the objective is to generate transgenic crops in which the range, scope, or nature of a plant’s existing natural products is modified to provide beneficial commercial, agronomic, and/or post-harvest processing characteristics (Kinney, 1998). Such plants may, for example, accumulate unusual edible or industrial oils (Broun and Somerville, 1997; Zou et al., 1997), lignins with different subunit compositions that interfere less extensively with paper production (Campbell and Sederoff, 1996), starches with different milling qualities, and so on. In theory, any biochemical pathway can be subjected to this “metabolic engineering.” In practice, however, efforts to modify the biosynthesis of a variety of plant primary and secondary metabolites have been confounded by the very metabolic flexibility that researchers are trying to exploit. Although these studies may uncover novel information about a pathway and its regulation that will inform subsequent attempts to manipulate it, such difficulties must be overcome if a genetically engineered crop is to become commercially viable. There are four major variables that appear to affect the outcome of metabolic engineering experiments. First, it is not always possible to predict the effects that directed perturbations (i.e., changing the level or activity of a single biosynthetic enzyme) may have on the entire pathway—unidentified endogenous feedback and/or feedforward controls may constrain metabolic pathways in manners that are poorly defined. Second, manipulating primary metabolic pathways can have pleiotropic (and of-ten detrimental) effects on plant growth and development. Third, no matter how well a transgenic plant is characterized in an experimental setting, crop plants in the field will be subjected to environmental perturbations that may adversely affect accumulation of the engineered product. Fourth, because many applications of genetic engineering demand that the engineered products get to the right place in the plant at the right time, it is critical to ensure that these products are appropriately targeted within and/or between cells in the transgenic plant. These variables can be addressed by adapting metabolic engineering strategies in a number of ways. Using cultured plant cells as metabolic factories instead of whole plants can circumvent problems associated with inappropriate targeting and with the vagaries in metabolite accumulation that can occur in field-grown material. Growing cell cultures in defined media may also bypass a number of other potential problems, such as tissue-specific or developmental regulation of some (or all) of the genes in a pathway. Clearly, the more that is known about the metabolic pathway in question, the better the chances that metabolic engineering will succeed. However, detailed understanding of the biochemical changes that take place at each step and of the enzymes that catalyze the reactions may not always be sufficient. It is also critical to determine how the activities of these enzymes are regulated, either at the level of gene expression or by their substrates and products through feedback and/or feedforward control. In fact, regulatory genes are becoming valuable tools for metabolic engineering. Combined genetic and biochemical studies over the past 20 years suggest that different transcription factors operating in a single pathway may affect the expression of distinct (but sometimes overlapping) suites of “downstream” genes. These distinct suites of genes may encode enzymes that participate in individual branches of a complex pathway (see e.g., Grotewold et al., 1994; Sablowski et al., 1994; Tamagnone et al., 1998). Thus, by manipulating the expression of a single transcription factor, it is theoretically possible to affect the expression of several coordinately regulated biosynthetic enzymes. Furthermore, work in a number of systems suggests that many transcriptional regulators are capable of faithfully recognizing their homologous target genes in heterologous species. For example, Tamagnone et al. (1998) have recently shown that two transcriptional regulators that repress lignin biosynthesis in Antirrhinum also affect lignin synthesis in transgenic tobacco.