Gene expression of enzymes related to biosynthesis of antioxidative flavonoids in Siamese neem tree leaves

Siamese neem tree (Azadirachta indica A. Juss. var. siamensis Valeton) is a medicinal plant of which the young leaves and flowers are popularly consumed as a bitter tonic vegetable in Thailand [1]. The study of flavonoid biosynthesis gene expression by reverse transcription-polymerase chain reaction (RT-PCR) and electrophoresis showed that Siamese neem tree leaves accumulate mRNAs corresponding to plant F3′H, FLS, DFR and F3′5′H genes. The nucleotide and deduced amino acid sequences share higher similarities to the sequences identified in fruits and leaves than those identified in flowers, which confirmed the identification of the enzyme isoforms specifically accumulated in the leaves of Siamese neem. The leaf extract of Siamese neem tree exhibited higher antioxidant activity than extracts from the fruits and seeds. Antioxidant activity and total flavonoid content determination of the leaf extract of Siamese neem tree from different developmental stages showed that all leaf extracts exhibit high antioxidant activity and total flavonoid content. The expression of FLS, DFR and F3′5′H genes in the leaves is also higher than in other parts of Siamese neem tree. This could imply that FLS, DFR and F3′5′H gene expression are related to total flavonoid content and antioxidant activity of Siamese neem tree leaves. The expression of flavonoid biosynthesis genes in Siamese neem tree, especially for FLS, DFR and F3′5′H is strong in the early stages of leaf development such as in top shoot, first young leaves, top part and basal part of young leaves while the expression is lower in mature leaves. Comparing among different Siamese neem tree tissues, the expression levels of FLS, DFR and F3′5′H are clearly higher in leaves than other tissues of Siamese neem tree while F3′H expression level is equally detected in all tested tissues. The results may implicate roles of FLS, and F3′5′H contributing to the production of flavonoids related to antioxidant activity. Flavonols/dihydroflavonols may contribute to the activity. There has been no report about nucleotide or amino acid sequences of flavonoid biosynthesis related enzymes and also no report about F3′H, FLS, DFR and F3′5′H gene expression in Siamese neem tree and other Azadirachta plants before. Therefore, this study could be beneficial for further biotechnological work about flavonoid biosynthesis and antioxidant activity of Siamese neem tree. This also confirms that the early developmental stages or young leaves of Siamese neem tree exhibit higher antioxidant activity, total flavonoid content and flavonoid biosynthesis related enzyme expression than the mature leaves or other plant parts supporting the ethnomedical uses of the young leaves of Siamese neem tree as bitter tonic vegetables.

Acknowledgements: The Royal Golden Jubilee PhD. Program, Thailand Research Fund.

References: [1] Clayton T. et al. (1996) Medicinal plants in Thailand volume 1. Amarin Printing. Bangkok.