SUMMARYThe developmental regulation and organ-specific expression of shikimate dehydrogenase was studied in seedlings of Capsicum annuum L. during their early development. The results obtained suggest that there is both a developmental regulation and an organ-specific expression of shikimate dehydrogenase in seedlings of C. annuum, seen mainly in the cotyledons. For example, shikimate dehydrogenase isoenzyme 3 was differentially expressed in cotyledons, but not at all in roots or hypocotyls. When the amounts of shikimate dehydrogenase were compared with the end-products of the shikimate pathway, chlorogenic acid and lignins, it was observed that the fall in shikimate dehydrogenase activity in cotyledons coincided with a decrease in the concentrations of phenolics during primary leaf development. However, this did not seem to be the case with the hypocotyls, where the fall in phenolics was not related to the amount of shikimate dehydrogenase but rather to an increase in lignin deposition, supporting the view that chlorogenic acid might act as a precursor of the aromatic moiety of cinnamoyl alcohols during cell wall ligniflcation.
Real-time PCR was used to detect and quantify Verticillium dahliae and to assess the susceptibility of four Capsicum annuum cultivars (Luesia, Padrón, SCM331 and PI201234) and the Capsicum chinense cv. C118 to this pathogen. The symptoms which developed after infection included stunting and yellowing, and were more acute in the cv. SCM331, which also suffered defoliation in later stages of the disease and in C118, which suffered severe stunting. Quantification of the pathogen DNA in roots 23 and 34 days post-inoculation (dpi) revealed that there were significantly higher amounts of Verticillium dahliae DNA in C118 than in the other cultivars, followed by SCM331, Padrón and PI201234. The lowest amounts of fungal DNA in roots were found in Luesia. In hypocotyls, the highest amounts of fungal DNA were found in SCM331, while Luesia, Padrón and PI201234 had much lower amounts, and C118 had intermediate levels. When a compatible versus an incompatible system was studied, using the near-isogenic tomato lines LA3030 (susceptible) and LA3038 (resistant to V. dahliae), we were able to detect fungal DNA in both lines. As expected, the fungus/plant DNA ratio was lower in LA3038 than in LA3030 and it decreased with time in LA3038. The amount of Verticillium dahliae DNA in the roots of LA3030 remained constant between days 23 and 34 post-inoculation, but increased 10-fold in collars. Finally, when real-time PCR was applied as a diagnostic method to samples from pepper plants, soil and water collected from farms in northwest Spain, we were able to detect V. dahliae DNA in these samples even when symptoms of the disease were not evident.
Oxidation of dihydrocapsaicin (S-methyl-N-vanillyl-6-nonanamide) by peroxidase (EC 1.11.1.7) from Capsicum annuum var. annuum fruits yielded one absorbent oxidation product with eX6* = 4.7 l@ M-' cm-'. Dependence of oxidation rate on dihydrocapsaicin and HzOz concentrations revealed Michaelis-Menten type kinetics with inhibition at high substrate concentrations and optimal pH near 6.0. Dihydrocapsaicin was oxidized by pepper peroxidase, and participation of peroxidase in capsaicinoid metabolism of pepper fruits should be taken into account.
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