Diverse Terpenoids and Their Associated Antifungal Properties from Roots of Different Cultivars of Chrysanthemum Morifolium Ramat.

Roots provide anchorage and enable the absorption of water and micronutrients from the soil for plants. Besides these essential functions, roots are increasingly being recognized as an important organ for the production of diverse secondary metabolites. The goal of this study was to investigate the chemical composition and function of terpenoid secondary metabolites in roots of different cultivars of the popular ornamental plant Chrysanthemum morifolium Ramat. Although C. morifolium is known for rich production of secondary metabolites in its flower heads and leaves, the diversity of secondary metabolites in roots remains poorly characterized. In this study, 12 cultivars of C. morifolium were selected for comparative analysis. From their roots, a total of 20 terpenoids were detected, including four monoterpenes, 15 sesquiterpenes, and one diterpene. The cultivar 'She Yang Hong Xin Ju' exhibited the highest concentration of total terpenoids at approximately 730 µg·g-1 fresh weight. Most cultivars contained sesquiterpenes as the predominant terpenoids. Of them, (E)-ß-farnesene was detected in all cultivars. Based on their terpenoid composition, the 12 cultivars were planed into four groups. To gain insights into the function of root secondary metabolites, we performed bioassays to assess their effects on growth of three species of pathogenic fungi: Fusarium oxysporum, Magnaporthe oryzae, and Verticillium dahliae. Significant variability in antifungal activity of the root extracts among different cultivars were observed. The cultivar 'Xiao Huang Ju' was the only cultivar that had significant inhibitory effects on all three species of fungi. Our study reveals the diversity of terpenoids in roots of C. morifolium and their function as a chemical defense against fungi.

Characterization of Composition and Antifungal Properties of Leaf Secondary Metabolites from Thirteen Cultivars of Chrysanthemum morifolium Ramat.

Chrysanthemum morifolium Ramat is an ornamental plant of worldwide cultivation. Like many other species in the family Asteraceae, C. morifolium is a rich producer of secondary metabolites. There are two objectives in this study: (I) to determine and compare the diversity of apolar secondary metabolites among different cultivars of C. morifolium and (II) to compare their properties as antifungal agents. To attain these objectives, we selected 13 cultivars of C. morifolium that are commonly used for making chrysanthemum tea as experimental materials. Leaves at the same developmental stage were collected from respective mature plants and subjected to organic extraction. The extracts were analyzed using gas chromatography-mass spectrometry. A total of 37 apolar secondary metabolites including 26 terpenoids were detected from the 13 cultivars. These 13 cultivars can be largely divided into three chemotypes based on chemical principal components analysis. Next, the extracts from the 13 cultivars were examined in in vitro assays for their antifungal properties against three species of pathogenic fungi: Fusarium oxysporum, Magnaporthe oryzae, and Verticillium dahliae. Significant variability in antifungal activity of the leaf extracts among different cultivars was observed. The 13 cultivars can be divided into four groups based on their antifungal activities, which could be partly correlated to the contents of terpenoids. In short, this study reveals large variations in chemical composition, particularly of terpenoids, of leaf secondary metabolites among different cultivars of C. morifolium and their different abilities in functioning as antifungal agents.

The occurrence and formation of monoterpenes in herbivore-damaged poplar roots.

Volatiles are often released upon herbivory as plant defense compounds. While the formation of volatiles above-ground has been intensively studied, little is known about herbivore-induced root volatiles. Here, we show that cockchafer larvae-damaged roots of Populus trichocarpa and P. nigra release a mixture of monoterpenes, including (-)-α-pinene, (-)-camphene, (-)-ß-pinene, p-cymene, and 1,8-cineole. Three terpene synthases, PtTPS16 and PtTPS21 from P. trichocarpa and PnTPS4 from P. nigra, could be identified and characterized in vitro. PnTPS4 was found to produce 1,8-cineole as sole product. PtTPS16 and PtTPS21, although highly similar to each other, showed different product specificities and produced γ-terpinene and a mixture of (-)-camphene, (-)-α-pinene, (-)-ß-pinene, and (-)-limonene, respectively. Four active site residues were found to determine the different product specificities of the two enzymes. The expression profiles of PtTPS16, PtTPS21, and PnTPS4 in undamaged and herbivore-damaged poplar roots generally matched the emission pattern of monoterpenes, indicating that monoterpene emission in roots is mainly determined at the gene transcript level. Bioassays with Phytophtora cactorum (Oomycetes) revealed inhibitory effects of vapor-phase 1,8-cineole and (-)-ß-pinene on the growth of this important plant pathogen. Thus herbivore-induced volatile monoterpenes may have a role in defense against pathogens that cause secondary infections after root wounding.

The organ-specific expression of terpene synthase genes contributes to the terpene hydrocarbon composition of chamomile essential oils.

BACKGROUND: The essential oil of chamomile, one of the oldest and agronomically most important medicinal plant species in Europe, has significant antiphlogistic, spasmolytic and antimicrobial activities. It is rich in chamazulene, a pharmaceutically active compound spontaneously formed during steam distillation from the sesquiterpene lactone matricine. Chamomile oil also contains sesquiterpene alcohols and hydrocarbons which are produced by the action of terpene synthases (TPS), the key enzymes in constructing terpene carbon skeletons. RESULTS: Here, we present the identification and characterization of five TPS enzymes contributing to terpene biosynthesis in chamomile (Matricaria recutita). Four of these enzymes were exclusively expressed in above-ground organs and produced the common terpene hydrocarbons (-)-(E)-ß-caryophyllene (MrTPS1), (+)-germacrene A (MrTPS3), (E)-ß-ocimene (MrTPS4) and (-)-germacrene D (MrTPS5). A fifth TPS, the multiproduct enzyme MrTPS2, was mainly expressed in roots and formed several Asteraceae-specific tricyclic sesquiterpenes with (-)-α-isocomene being the major product. The TPS transcript accumulation patterns in different organs of chamomile were consistent with the abundance of the corresponding TPS products isolated from these organs suggesting that the spatial regulation of TPS gene expression qualitatively contribute to terpene composition. CONCLUSIONS: The terpene synthases characterized in this study are involved in the organ-specific formation of essential oils in chamomile. While the products of MrTPS1, MrTPS2, MrTPS4 and MrTPS5 accumulate in the oils without further chemical alterations, (+)-germacrene A produced by MrTPS3 accumulates only in trace amounts, indicating that it is converted into another compound like matricine. Thus, MrTPS3, but also the other TPS genes, are good markers for further breeding of chamomile cultivars rich in pharmaceutically active essential oils.

Herbivore-induced SABATH methyltransferases of maize that methylate anthranilic acid using s-adenosyl-L-methionine.

Volatile methyl esters are common constituents of plant volatiles with important functions in plant defense. To study the biosynthesis of these compounds, especially methyl anthranilate and methyl salicylate, we identified a group of methyltransferases that are members of the SABATH enzyme family in maize (Zea mays). In vitro biochemical characterization after bacterial expression revealed three S-adenosyl-L-methionine-dependent methyltransferases with high specificity for anthranilic acid as a substrate. Of these three proteins, Anthranilic Acid Methyltransferase1 (AAMT1) appears to be responsible for most of the S-adenosyl-L-methionine-dependent methyltransferase activity and methyl anthranilate formation observed in maize after herbivore damage. The enzymes may also be involved in the formation of low amounts of methyl salicylate, which are emitted from herbivore-damaged maize. Homology-based structural modeling combined with site-directed mutagenesis identified two amino acid residues, designated tyrosine-246 and glutamine-167 in AAMT1, which are responsible for the high specificity of AAMTs toward anthranilic acid. These residues are conserved in each of the three main clades of the SABATH family, indicating that the carboxyl methyltransferases are functionally separated by these clades. In maize, this gene family has diversified especially toward benzenoid carboxyl methyltransferases that accept anthranilic acid and benzoic acid.