RESUMEN
Dioscorea zingiberensis is a perennial medicinal herb rich in a variety of pharmaceutical steroidal saponins. Squalene epoxidase (SE) is the key enzyme in the biosynthesis pathways of triterpenoids and sterols, and catalyzes the epoxidation of squalene in coordination with NADPH-cytochrome P450 reductase (CPR). In this study, we cloned DzSE and DzCPR gene sequences from D. zingiberensis leaves, encoding proteins with 514 and 692 amino acids, respectively. Recombinant proteins were successfully expressed in vitro, and enzymatic analysis indicated that, when SE and CPR were incubated with the substrates squalene and NADPH, 2,3-oxidosqualene was formed as the product. Subcellular localization revealed that both the DzSE and DzCPR proteins are localized to the endoplasmic reticulum. The changes in transcription of DzSE and DzCPR were similar in several tissues. DzSE expression was enhanced in a time-dependent manner after methyl jasmonate (MeJA) treatments, while DzCPR expression was not inducible.
Asunto(s)
Dioscorea/enzimología , NADPH-Ferrihemoproteína Reductasa/metabolismo , NADP/metabolismo , Proteínas de Plantas/metabolismo , Escualeno-Monooxigenasa/metabolismo , Escualeno/metabolismo , Acetatos/metabolismo , Ciclopentanos/metabolismo , Dioscorea/genética , Dioscorea/metabolismo , Regulación de la Expresión Génica de las Plantas , NADPH-Ferrihemoproteína Reductasa/genética , Oxilipinas/metabolismo , Proteínas de Plantas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Escualeno/análogos & derivados , Escualeno-Monooxigenasa/genéticaRESUMEN
Rhizomes of the perennial herb Paris polyphylla have been used in traditional Chinese medicine for hundreds of years. Agricultural production of the rhizomes requires 7-10 years, which is too long to meet the demand of the medicinal industry. Therefore, studies on improving the yield of the herb and shortening the culturing period are imperative. The present work aimed to investigate the effect of sodium bisulfite (NaHSO (3)) on rhizome yield and quality, as well as some related metabolic features of P. polyphylla. The rhizome yield was improved by NaHSO (3) treatment in long-term experiments conducted during 2006 and 2007, with 2 mM NaHSO (3) giving the highest yield. HPLC analysis revealed that NaHSO (3) treatment increased the total saponin content (49 %), including three pennogenin glycosides and two diosgenin glycosides. In a short-term experiment, NaHSO (3) treatment resulted in an enhanced net photosynthetic rate (Pn) for about 4 days without significant changes in the chlorophyll or carotenoid content. The total soluble sugars and sucrose contents in the leaves also significantly increased after 2 mM NaHSO (3) treatment, whereas the starch content changed only slightly. The activities of the enzymes involved in ammonium assimilation (glutamine synthetase [GS] and glutamate dehydrogenase [GDH]) were not significantly influenced. In a long-term experiment, chlorophylls and carotenoids were not significantly affected, and neither was the starch content in leaves, but the total soluble sugars and sucrose contents in leaves increased significantly. The NaHSO (3) treatment significantly increased GS and GDH activities. These results indicate that NaHSO (3) treatment improved the rhizome yield in P. polyphylla, not only through enhancement of Pn but also by improving carbohydrate accumulation and ammonium assimilation. The increased saponin content after NaHSO (3) treatment was indicative of high rhizome quality.