Aging affects artemisinin synthesis in Artemisia annua.

Artemisinin (ART) is the most effective component in malaria treatment, however, the extremely low content restricts its clinical application. Therefore, it is urgent to increase the yield of ART. ART gradually accumulates with aging, small RNA (sRNA) and transcriptome analysis were applied on the leaves of 2-week-old (2 w) and 3-month-old (3 m) A. annua respectively. Among all the annotated sRNAs, 125 were upregulated and 128 downregulated in the 3 m sample compared to the 2 w one. Whereas 2183 genes were upregulated and 2156 downregulated. Notably, the level of miR156 and several annotated miRNAs gradually decreased while SPLs increased. In addition, the genes on ART biosynthesis pathway were significantly upregulated including ADS, CYP71AV1, ADH1, DBR2 and ALDH1, and so were the positive transcription factors like AaERF1, AaORA and AaWRKY1 indicating that age influences the ART biosynthesis by activating the expression of the synthesizing genes as well as positive transcription factors. This study contributes to reveal the regulatory effects of age on ART biosynthesis both in sRNA and transcription levels.

Two new secolignans from the roots of Urtica fissa.

Two new secolignans, 3,4-trans-3-hydroxymethyl-4-[bis(4-hydroxy-3- methoxyphenyl)methyl]butyrolactone (1) and 3,4-trans-3-hydroxymethyl-4- [bis(3,4-dimethoxyphenyl)methyl]butyrolactone (2) have been isolated from the roots of Urtica fissa E.Pritz. Their structures were determined on the basis of spectroscopic methods, especially 1H NMR, 13C NMR, 2D NMR, and HR-ESI-MS. The inhibitory effects on N1 and N2, two subtypes of neuraminidases (NAs), of these two compounds were assayed.

Progressive regulation of sesquiterpene biosynthesis in Arabidopsis and Patchouli (Pogostemon cablin) by the miR156-targeted SPL transcription factors.

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)-ß-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.

Progressive Regulation of Sesquiterpene Biosynthesis in Arabidopsis and Patchouli (Pogostemon cablin) by the miR156-Targeted SPL Transcription Factors.

Plant metabolites vary at different stages of 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)-tageted 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)-ß-caryophyllene in 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 up-regulating 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 plant for accelerated growth with enhanced production of terpenoids.

The jasmonate-responsive AP2/ERF transcription factors AaERF1 and AaERF2 positively regulate artemisinin biosynthesis in Artemisia annua L.

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.

Promoter of a cotton fibre MYB gene functional in trichomes of Arabidopsis and glandular trichomes of tobacco.

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 beta-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 non-glandular 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 cis-element 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.