O

COO-

Phenolic radical

Figure 1.1 Model for the role of proline linked pentose-phosphate pathway in phenolic synthesis and its utilization for screening high phenolic clonal lines of single seed genetic origin.

Phenolic radical

Figure 1.1 Model for the role of proline linked pentose-phosphate pathway in phenolic synthesis and its utilization for screening high phenolic clonal lines of single seed genetic origin.

e e stimulated somatic embryogenesis in anise, which correlated with increased phenolic content (135). It was also established that during Pseudomonas mediated stimulation of total phenolic and rosmarinic acid (RA), proline content was stimulated in oregano clonal shoot cultures (136). Therefore, it was proposed that NADPH demand for proline synthesis during response to microbial interaction and proline analog treatment (1,69), may reduce the cytosolic NADPH/NADP+ ratio, which should activate G6PDH (137,138). Therefore, deregulation of the pentose-phosphate pathway by proline analog and microbial induced proline synthesis may provide the excess erythrose-4-phosphate (E4P) for shikimate and, therefore, the phenylpropanoid pathways (69). At the same time, proline and P5C could serve as superior reducing equivalents (RE), alternative to NADH (from Krebs/TCA cycle) to support increased oxidative phosphorylation (ATP synthesis) in the mitochondria during the stress response (69,127,128).

The proline analog, azetidine-2-carboxylate (A2C), is an inhibitor of proline dehydrogenase (139). It is also known to inhibit differentiation in Leydig cells of rat fetal testis, which can be overcome by exogenous proline addition (140). Another analog, hydroxy-proline, is a competitive inhibitor of proline for incorporation into proteins. According to the model of Shetty (1,69), either analog, at low levels, should deregulate proline synthesis from feedback inhibition and stimulate proline synthesis (1,69). This would then allow the proline linked pentose-phosphate pathway to be activated for NADPH synthesis, and concomitantly drive metabolic flux toward E4P for biosynthesis of shikimate and phenylpro-panoid metabolites, including RA. Proline could also serve as a RE for ATP synthesis via mitochondrial membrane associated proline dehydrogenase (129,69).

Using this rationale for the mode of action of proline analogs and links to the pentose-phosphate pathway, high RA-producing, shoot based clonal lines originating from a single heterozygous seed among a heterogeneous bulk seed population of lavender, spearmint, and thyme have been screened and isolated based on tolerance to the proline analog, A2C, and a novel Pseudomonas sp. isolated from oregano (Figure 1.2; 141-143). This strategy for screening and selection of high RA clonal lines is also based on the model that proline linked pentose phosphate pathway is critical for driving metabolic flux (i.e., E4P) toward shikimate and phenylpropanoid pathways (Figure 1.1). Any clonal line with a deregulated proline synthesis pathway should have an overexpressed pentose phosphate pathway which allows excess metabolic flux to drive shikimate and phenylpropanoid pathway toward total phenolic and RA synthesis (69). Similarly, such proline overexpressing clonal lines should be more tolerant to proline analog, A-2-C. If the metabolic flux to RA is overexpressed, it is likely to be stimulated in response to Pseudomonas sp (69). Therefore,

Independent heterozygous seeds (# 1, 2, 3 representing different clonal origin)

2 Sensitive

3 Sensitive

Each plate represents clonal shoots of single heterozygous seed (clonal line) origin (1,2,3 represent clonal lines from different heterozygous seeds)

Challenge:

Pseudomonas or proline analog

3 Sensitive

Figure 1.2 Isolation of clonal lines of single seed origin based bacterial or proline analog tolerance. Clonal lines can be used for investigations on Rosmarinic acid inducibility and endogenous antioxidant enzyme response in various clonal lines under various nutritional conditions. Further, high phenolic clonal lines can be used for development of ingredient profiles of single seed clonal origin.

such a clonal line is equally likely to be tolerant to Pseudomonas sp. Further, such a clonal line should also exhibit high proline oxidation and RA content in response to A2C and Pseudomonas sp. The rationale for this model is based on the role of the pentose-phosphate pathway in driving ribose-5-phosphate toward purine metabolism in cancer cells (127), differentiating animal tissues (140), and plant tissues (144). The hypothesis of this model is that the same metabolic flux from over expression of proline linked pentose-phosphate pathway regulates the interconversion of ribose-5-phosphate to E4P driving shikimate pathway (69). Shikimate pathway flux is critical for both auxin and phenylpropanoid biosynthesis, including RA (69). This hypothesis has been strengthened by preliminary results in which RA biosynthesis in several oregano clonal lines was significantly stimulated by exogenous addition of proline analog (e.g., A2C) and ornithine (133,134). The same clonal lines are also tolerant to Pseudomonas sp. and respond to the bacterium by increasing RA and proline biosynthesis (136,145). High RA-producing clonal lines selected by approaches based on this model (141-143,145-147) are being targeted for preliminary characterization of the key enzymes in phenolic synthesis and large scale production of such phenolics using tissue culture generated clonal lines in large scale field production systems. This strategy for investigation and stimulation of phenolic biosynthesis in clonal plant systems using PLPPP as the CCP can be the foundation for designing dietary phenolic phytochemicals from cross-pollinating, heterogeneous species for functional foods (1,69).

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