Oh

3,4-(OH)2-phenyllactic acid

Figure 2. Rosmarinic acid biosynthesis from phenylalanine and tyrosine. PAL, phenylalanine ammonia lyase; TAT, tyrosine aminotransferase.

based clonal lines for metabolic pathway analysis and for developing gene transfer techniques for subsequent metabolic engineering. This will help provide excellent experimental systems and direction to fill gaps in knowledge of RA biosynthesis using several species used for food and medicinal applications. The basic strategy involves the isolation of genetically uniform, high RA-producing, shoot-based clonal lines from a heterogeneous genetic background. This heterogeneity is found in all species in the family Lamiaceae, and the breeding character is influenced by natural cross-pollination (15). The use of genetically uniform, shoot-based clonal lines envisioned in this article has the following advantages:

1. Shoot clones are genetically more stable than undifferentiated callus cultures.

2. Shoot clones allow the characterization of lightregulated pathways associated with RA synthesis.

3. Shoot clones can easily be targeted for large-scale greenhouse production of elite clonal lines or for incorporating into plant breeding programs to develop superior RA-producing seed varieties.

4. Shoot clones targeted for genetic engineering of metabolic pathways can be easily regenerated to whole plants for incorporation into plant-variety improvement programs.

Role of Proline-Linked Pentose Phosphate Pathway

High RA-producing, shoot-based clonal lines originating from a single heterozygous seed among a heterogeneous bulk-seed population are being selected based on tolerance to the proline analogue, azetidine-2-carboxylate (A-2-C) and a novel Pseudomonas sp. isolated from oregano. This strategy for selection of high RA clonal lines is based on the model that the proline-linked pentose phosphate pathway is critical for driving metabolic flux (erythrose-4-phosphate) toward shikimate and phenylpropanoid pathways (Fig. 3). Any clonal line with a deregulated proline synthesis pathway should have an overexpressed pentose phosphate pathway that allows excess metabolic flux to drive shikimate and phenylpropanoid pathways toward RA synthesis. Such proline-overexpressing clonal lines should be tolerant to A-2-C. If the metabolic flux to RA is overexpressed, it is likely to be stimulated in response to Pseudomonas sp. Therefore, such a clonal line is likely to be tolerant to Pseudomonas sp. Such a clonal line should also have high proline or proline oxidation (proline dehydrogenase) and RA content in response to A-2-C and Pseudomonas sp. In addition, in the presence of A-2-C or Pseudomonas sp., increased activity of key enzymes glucose-6-phosphate dehydrogenase (pentose phosphate pathway), pyrroline-5-carboxylate reductase (proline synthesis pathway), proline dehydrogenase (proline oxidation pathway), 3-deoxy-d-arabino heptulosonate-7-phosphate synthase (shikimate pathway), and phenylalanine ammonia-lyase (phenylpropanoid pathway) should be observed. 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

Pentose phosphate pathway

Glycolysis

Mitochondria Proline» - -

Proline

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