Preliminary results (1,69,72,133,134,148) have provided empirical evidence for a link between proline biosynthesis and oxidation, as well as stimulation of G6PDH. In light mediated sprout studies in pea (Pisum sativum), acetylsalicylic acid in combination with fish protein hydrolysates (a potential source of proline precursors) stimulated phenolic content and guaiacol peroxidase (GPX) activity during early germination with corresponding higher levels of proline and G6PDH activity (149). In parallel light mediated studies in pea, low pH and salicylic acid treatments stimulated increased phenolic content and tissue rigidity. Similarly, there was concomitant stimulation of G6PDH and proline (148). This work supported the hypothesis that pentose-phosphate pathway stimulation may be linked to proline biosynthesis and that modulation of a proton linked redox cycle may also be operating through proline linked pentose-phosphate pathway (148). In dark germinated studies in pea, high cytokinin-containing anise root extracts stimulated phenolic content and antioxidant activity, which correlated with increased proline content but inversely with G6PDH activity (150). In further dark germination studies in mung bean (Vigna radiata), dietary grade microbial polysaccharide treatments stimulated phenolic content and enzyme activity, G6PDH and GPX compared to controls (151), with concomitant stimulation of proline content. In addition, specific elicitors xanthan gum, yeast extract, and yeast glucan stimulated antioxidant activity. In additional studies, oregano phenolic extracts were used as elicitors to stimulate phenolic content during dark germination of mung bean. Again, increased phenolic content corresponded to an increase in activity of G6PDH and GPX and phenolic related antioxidant activity were also stimulated (152). In studies with dark germinated fava bean, support for the hypothesis that stimulation of proline linked pentose-phosphate pathway would stimulate phenolic metabolism under elicitor and stress response was probed (69). In polysaccharide elicitor studies, gellan gum stimulated fava bean total phenolic content ninefold in late stages of germination with a corresponding increase in proline content and GPX activity, although the effect on antioxidant and G6PDH activity was inconclusive (100). In the same fava bean system, UV mediated stimulation of phenolic content in dark germinated fava bean
sprouts indicated a positive correlation to G6PDH and GPX activities with a concomitant increase in proline content (153). It was further confirmed that proline analog, A2C also stimulated phenolic content in fava bean with positive correlation to G6PDH and GPX activities as well as proline content (154). Similar to studies in clonal shoot cultures of thyme (133) and oregano (134), the proline analog mediated studies in fava bean confirmed that proline overexpression was not only possible, but involved stimulating G6PDH and therefore likely diverted the pentose-phosphate pathway toward phenylpropanoid biosynthesis. The late stage stimulation of phenolic content and GPX activity in response to microwave mediated thermal stress in dark germinated fava bean strongly correlated with stimulation of free radical scavenging activity of free phenolics as measured by the quenching of 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical assay and stimulation of superoxide dismutase (SOD) activity (155). Therefore, using elicitor and physical stress in early stages of seed sprouting could be effectively used to stimulate key phenolic phytochemicals for design of functional foods. In designing appropriate phenolic phytochemical extracts diverting carbohydrate and protein metabolic flux through the proposed CCP, proline linked pentose-phosphate pathway (PLPPP) has substantial potential. This CCP link to PLPPP could be the basis for a more effective Systems Biology approach for metabolic control of functional phenolic phyto-chemicals and appropriate design of phytochemicals from different seed sprout systems for different oxidation linked disease targets.
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