Plants of Artemisia annua produce artemisinin, a sesquiterpene lactone widely used in malaria treatment. Amorpha-4,11-diene synthase (ADS), a sesquiterpene synthase, and CYP71AV1, a P450 monooxygenase, are two key enzymes of the artemisinin biosynthesis pathway. Accumulation of artemisinin can be induced by the phytohormone jasmonate (JA). Here, we report the characterization of two JA-responsive AP2 family transcription factors--AaERF1 and AaERF2--from A. annua L. Both genes were highly expressed in inflorescences and strongly induced by JA. Yeast one-hybrid and electrophoretic mobility shift assay (EMSA) showed that they were able to bind to the CRTDREHVCBF2 (CBF2) and RAV1AAT (RAA) motifs present in both ADS and CYP71AV1 promoters. Transient expression of either AaERF1 or AaERF2 in tobacco induced the promoter activities of ADS or CYP71AV1, and the transgenic A. annua plants overexpressing either transcription factor showed elevated transcript levels of both ADS and CYP71AV1, resulting in increased accumulation of artemisinin and artemisinic acid. By contrast, the contents of these two metabolites were reduced in the RNAi transgenic lines in which expression of AaERF1 or AaERF2 was suppressed. These results demonstrate that AaERF1 and AaERF2 are two positive regulators of artemisinin biosynthesis and are of great value in genetic engineering of artemisinin production.

Plant metabolites vary at different stages of their life cycle. Although it is well documented that environmental factors stimulate biosynthesis of secondary metabolites, the regulation by endogenous developmental cues remains poorly understood. The microRNA156 (miR156)-targeted squamosa promoter binding protein-like (SPL) factors function as a major age cue in regulating developmental phase transition and flowering. We show here that the miR156-targeted SPL transcription factor plays an important role in the spatiotemporal regulation of sesquiterpene biosynthesis. In Arabidopsis thaliana, the miR156-SPL module regulates the formation of (E)-β-caryophyllene in the flowering stage through modulating expression of the sesquiterpene synthase gene TPS21. We demonstrated that SPL9 directly binds to TPS21 promoter and activates its expression. In the perennial fragrant herb Pogostemon cablin, the accumulation of patchouli oil, largely composed of sesquiterpenes dominated by (-)-patchoulol, is also age-regulated, and the SPL promotes biosynthesis of sesquiterpenes in elder plants by upregulating patchoulol synthase (PatPTS) gene expression. As miR156-SPLs are highly conserved in plants, our finding not only uncovers a molecular link between developmental timing and sesquiterpene production but also suggests a new strategy to engineer plants for accelerated growth with enhanced production of terpenoids.

Cotton fibres are unicellular seed trichomes. Our previous study suggested that the cotton R2R3 MYB transcript factor GaMYB2 is a functional homologue of the Arabidopsis trichome regulator GLABRA1 (GL1). Here, the GaMYB2 promoter activity is reported in cotton (Gossypium hirsutum), tobacco (Nicotiana tabacum), and Arabidopsis plants. A 2062 bp promoter of GaMYB2 was isolated from G. arboreum, and fused to a b-glucuronidase (GUS) reporter gene. In cotton, the GaMYB2 promoter exhibited activities in developing fibre cells and trichomes of other aerial organs, including leaves, stems and bracts. In Arabidopsis the promoter was specific to trichomes. Different from Arabidopsis and cotton that have unicellular nonglandular simple trichomes, tobacco plants contain more than one type of trichome, including multicellular simple and glandular secreting trichomes (GSTs). Interestingly, in tobacco plants the GaMYB2 promoter directed GUS expression exclusively in glandular cells of GSTs. A series of 5#-deletions revealed that a 360 bp fragment upstream to the translation initiation codon was sufficient to drive gene expression. A putative ciselement of the T/G-box was located at -233 to -214; a yeast one-hybrid assay showed that Arabidopsis bHLH protein GLABRA3 (GL3), also a trichome regulator, and GhDEL65, a GL3-like cotton protein, had high binding activities to the T/G-box motif. Overexpression of GL3 or GhDEL65 enhanced the GaMYB2 promoter activity in transgenic Arabidopsis plants. A comparison of GaMYB2 promoter specificities in trichomes of different plant species with different types of trichomes provides a tool for further dissection of plant trichome structure and development.

Three new cucurbitane-type triterpenoid saponins, 23-O-beta-D-allopyranosyl-5beta,19-epoxycucurbita-6,24-diene-3beta,22(S),23(S)-triol-3-O-beta-D-glucopyranoside (1), 23-O-beta-D-allopyranosyl-5beta,19-epoxycucurbita-6,24-diene-3beta,22(S),23(S)-triol-3-O-beta-D-allopyranoside (2), and 23-O-beta-D-allopyranosyl-5beta,19-epoxycucurbita-6,24-diene-3beta,19(R), 22(S),23(S)-tetraol-3-O-beta-D-allopyranoside (3), named momordicoside M, N, and O, respectively, along with one known saponin momordicoside L (4), were isolated from the fresh fruits of Momordica charantia. The structures of these saponins were elucidated on the basis of chemical properties and spectral data.

730000, People's Republic of China ABSTRACT.-Further phytochemical investigation of the roots of Ligularia sagitta and L. veitchiana afforded, in addition to several known compounds, six new eremophilane derivatives [1][2][3][4][5][6]. Their structures were elucidated by means of nmr spectroscopy.Some 27 Ligularia species have long been used as folk remedies due to their antibiotic, antiphlogistic, and antitumor activities (1). Eremophilane sesquiterpenes and pyrrolizidine alkaloids are the most widespread secondary metabolites in this genus (2,3). However, the isolation of a novel norditerpene with a previously unreported carbon skeleton from Ligularia sagitta (Maxim.) Mattf. ex Rehder & Kobuski (Compositae) (4) has increased interest in the phytochemistry of this species. In a reinvestigation on the roots of Ligularia sagitta, we have now isolated eight chemical components, of which four are novel (1-4). A further species, Ligularia veitchiana (Hemsl.) Greenm. (Compositae), from which several eremophilanolides have been isolated (5-7), has now yielded two additional previously unreported eremophilane derivatives (compounds 5 and 6). We report herein the isolation and structural characterization of novel compounds 1-6. RESULTS AND DISCUSSIONA petroleum ether-Et20-MeOH (1:1:1) extract of the powdered roots of Ligularia sagitta was subjected to cc on Si gel and then fractionated, as described in the Experimental section, to yield sagittolactone [8] (4), ß-sitosterol, ß-sitosterol ß-Dglucopyranoside [7], petasin [91 (8), isopetasin [10] (9), and four new compounds, 1-4. From a petroleum ether-Et20-Me2C0 (1:1:1) extract of Ligularia veitchiana, two minor eremophilane derivatives [5 and 6] were obtained by prep. tic. Compounds 1 and 2 were obtained as an epimeric mixture (ca. 1:1 by 'Hand 13Cnmr spectroscopy). The molecular formula, C17H2204, was indicated by its eims (m/z 290[M]+) and elemental analysis. In addition to acetoxy signals at 21.15 (CH3) and 169.71 (CO), the 13C-nmr spectrum of 1 and 2 showed fifteen signals. The 'H-nmr spectra of 1 and 2 showed three methyl signals, the position and splitting patterns of which suggested these were eremophilane sesquiterpenes. Furthermore, two olefinic hydrogen singlets appeared at 6.37 (H-9) and 6.79 or 6.81 (H-6 of 1 and 2). These signals, in conjunction with the 13C-nmr resonances at 153.39/154.61 (CH), 135.38/135.24 (C), 184.84 (C), 127.94 (CH), and 160.46 (C), strongly suggested the presence of a 6(7 ),9( 10)-dien-8-oxo moiety. This concept was supported by the ,ß, ',ß'-unsaturated ketone absorption band at 1664 cm 1 in the ir spectrum. In addition to the molecular ion peak at m/z 290, the eims exhibited a significant [M-1]+ peak at m/z 289.Thus, the 'H-nmr signal at 9-66 (1H, s) and the ir absorption band at 1739 cm 1 were consistent with the presence of an aldehyde group. The location of this group at C-l 1 was deduced from the downfield methine proton at 3.72 and 3.67 attributed to -11, as well as from the chemical shift of the methyl doublet (H-13) at 1.27 (Ta...

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Radix hedysari is not only a restorative food but also a famous Traditional Chinese Medicine. In this study, a simple, reliable, and reproducible high-performance liquid chromatography-ultraviolet method was developed for the first time to determine the true contents of five major flavoniods, naringenin-7-O-alpha-L-rhamanopyranosyl-(1-->2)-beta-d-glucopyranoside, ononin, formononetin, medicarpin, and an unstable flavonoid malonate (formononetin-7-O-beta-D-glucoside-6' '-O-malonate, FGM), and the stability of FGM was also investigated. Flavoniods were selected as chemical markers because they have appeared as one of the major bioactive compound groups in Radix hedysari. The stability results of FGM at different pH showed that it remained relatively stable in acidic aqueous methanol of pH 3. The HPLC assay with an improved sample preparation scheme can be readily utilized as a suitable "chemical" quality control method for Radix hedysari.