No abstract

Flavonoids represent a large and important group of plant natural products that are ubiquitous in the plant kingdom. Epidemiological studies have shown the health benefits of a diet high in flavonoids. However, the dietary intake of flavonoids in most western populations is limited, creating a need to find alternative food sources for these polyphenolic secondary metabolites. The domestication of many of our cultivated food crops has resulted in alterations in the biosynthetic pathways of many essential micronutrients and vitamins through inadvertent counterselection against nutritional traits in favor of agronomic ones. Flavonoids are nearly absent from fruits of cultivated tomato (Lycopersicon esculentum Mill.), a major vegetable in human diets. Previous attempts to restore the flavonoid pathway in tomato fruits have been limited to transgenic strategies, suggesting that the problem was intractable through traditional methods. Here, we describe for the first time a nontransgenic metabolic engineering approach to developing a high flavonoid tomato using a wild tomato species (Lycopersicon pennelliiv. puberulum) and demonstrate the opportunities for restoring functional pathways using the genetic resources of wild species, resulting in production of healthier foods.

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We present evidence that the activity of the mannitolcatabolizing enzyme mannitol dehydrogenase (MTD) is repressed by sugars in cultured celery (Apium graveolens L.) cells. Furthermore, this sugar repression appears to be mediated by hexokinases (HKs) in a manner comparable to the reported sugar repression of photosynthetic genes. Glucose (Glc)-grown cell cultures expressed little MTD activity during active growth, but underwent a marked increase in MTD activity, protein, and RNA upon Clc starvation. Replenishment of Glc in the medium resulted in decreased MTD activity, protein, and RNA within 12 h. Addition of mannoheptulose, a competitive inhibitor of HK, derepressed MTD activity in Glc-grown cultures. In contrast, the addition of the sugar analog 2-deoxyglucose, which is phosphorylated by HK but not further metabolized, repressed MTD activity in mannitol-grown cultures. Collectively, these data suggest that HK and sugar phosphorylation are involved in signaling MTD repression. In vivo repression of MTD activity by galactose (Cal), which is not a substrate of HK, appeared to be an exception to this hypothesis. Further analyses, however, showed that the products of Cal catabolism, Glc and fructose, rather than Cal itself, were correlated with MTD repression.

We previously presented evidence that the hexose‐regulated repression of the mannitol catabolic enzyme mannitol dehydrogenase (MTD) in celery (Apium graveolens L.) may be mediated by hexokinase (EC 2.7.1.1) (HK) [Prata et al. (1997) Plant Physiol 114: 307–314]. To see if differential regulation of HK forms might be involved in the sugar‐regulated repression of MTD we characterized two forms of HK with respect to their expression in various plant organs as well as in celery suspension cell cultures. We found that the vast majority of HK activity was membrane‐associated, whereas fructokinase (EC 2.7.1.4) was found largely in the soluble cell fraction. Gel filtration chromatography further revealed the differential expression of two molecular size classes of HK. One HK (HK‐L) chromatographed at 68 kDa, a typical size for a plant HK, while the second (HK‐H) chromatographed at 280 kDa. This unique 280 kDa HK was shown to be composed of a 50 kDa HK protein, possibly complexed with other, as yet unidentified, components. The HK‐L was present in all cells and organs analyzed, and thus may be a likely candidate for mediation of sugar repression. In contrast, the presence of the HK‐H complex was specific to certain organs and cells grown under certain conditions. Our analyses here showed no correlation between the presence of the HK‐H and MTD repression or derepression in celery cells. Instead, the HK‐H complex was present exclusively in rapidly growing organs and cells, but not in non‐growing celery storage tissues or in carbon‐depleted celery suspension‐cultured cells. Furthermore, the HK‐H complex was present when Glc in the growth media was replaced with 2‐deoxy Glc, a HK substrate that does not provide energy for growth and metabolism. These results imply that the HK‐H complex may have a potentially unique role in the metabolism of rapidly growing celery cells, in particular, in hexose phosphorylation. We also found that mitochondria prepared from Glc‐grown celery suspension‐cultured cells contained substantial HK activity, and that oxygen uptake of these mitochondria was stimulated by Glc. These results are consistent with the hypothesis that mitochondrial localization of celery HK may play a role in rapid recycling of adenylate.