Additional file 2: of Biodegradation of caffeine by whole cells of tea-derived fungi Aspergillus sydowii, Aspergillus niger and optimization for caffeine degradation

Figure S1. Colony characteristics of strain No. 5 on culture medium. Figure S2. Conidia structure of strain No.5 under optical microscope. Figure S3. Colony characteristics of strain No.1 on culture medium. Figure S4. Conidia structure of strain No.1 under optical microscope. (DOCX 3679 kb

Additional file 3: of Biodegradation of caffeine by whole cells of tea-derived fungi Aspergillus sydowii, Aspergillus niger and optimization for caffeine degradation

Figure S5. ITS sequences data of the target strains. (DOCX 16 kb

Studies on the Expression of Sesquiterpene Synthases Using Promoter-β-Glucuronidase Fusions in Transgenic <i>Artemisia annua</i> L

<div><p>In order to better understand the influence of sesquiterpene synthases on artemisinin yield in <i>Artemisia annua</i>, the expression of some sesquiterpene synthases has been studied using transgenic plants expressing promoter-GUS fusions. The cloned promoter sequences were 923, 1182 and 1510 bp for β-caryophyllene (CPS), <i>epi</i>-cedrol (ECS) and β-farnesene (FS) synthase, respectively. Prediction of <i>cis</i>-acting regulatory elements showed that the promoters are involved in complex regulation of expression. Transgenic <i>A. annua</i> plants carrying promoter-GUS fusions were studied to elucidate the expression pattern of the three sesquiterpene synthases and compared to the previously studied promoter of amorpha-4,11-diene synthase (ADS), a key enzyme of artemisinin biosynthesis. The CPS and ECS promoters were active in T-shaped trichomes of leaves and stems, basal bracts of flower buds and also in some florets cells but not in glandular secretory trichome while FS promoter activity was only observed in leaf cells and trichomes of transgenic shoots. ADS, CPS, ECS and FS transcripts were induced by wounding in a time depended manner. The four sesquiterpene synthases may be involved in responsiveness of <i>A. annua</i> to herbivory. Methyl jasmonate treatment triggered activation of the promoters of all four sesquiterpene synthases in a time depended manner. Southern blot result showed that the <i>GUS</i> gene was inserted into genomic DNA of transgenic lines as a single copy or two copies. The relative amounts of CPS and ECS as well as germacrene A synthase (GAS) transcripts are much lower than that of ADS transcript. Consequently, down-regulation of the expression of the <i>CPS</i>, <i>ECS</i> or <i>GAS</i> gene may not improve artemsinin yield. However, blocking the expression of <i>FS</i> may have effects on artemisinin production.</p></div

Relative expression of the wild-type <i>pADS</i> (A), <i>pCPS</i> (B), <i>pECS</i> (C), and <i>pFS</i> (D) in leaves after wounding.

<p>The β-actin promoter activity was set to 1.0.</p

GUS expression controlled by the <i>FS</i> promoter in transgenic plants of <i>Artemisia annua</i>.

<p>A: young leaf; B: close-up of young leaf; C: leaf; D: old leaf; E: root.</p

Wounding of leaves of transgenic <i>Artemisia annua</i> carrying the <i>pCPS::GUS</i> fusion.

<p>A: unwounded; B: immediately after wounding; C: 1h; D: 2h; E: 4h; F: 8h; G: 12h; H: 24h; I: 48h.</p

Wounding of leaves of transgenic <i>Artemisia annua</i> carrying the <i>pECS::GUS</i> fusion.

<p>A: unwounded; B: immediately after wounding; C: 1h; D: 2h; E: 4h; F: 8h; G: 12h; H: 24h; I: 48h.</p

Enzymes in <i>Artemisia annua</i> utilizing farnesyl diphosphate as substrate.

<p>ADS: amorpha-4,11-diene synthase; CPS: β-caryophyllene synthase; ECS: <i>epi</i>-cedrol synthase; FS: β-farnesene synthase; GAS: germacrene A synthase; GDS: germacrene D synthase; SQS: squalene synthase; PPO: diphosphate moiety</p

GUS expression controlled by the <i>ECS</i> promoter in transgenic plants of <i>Artemisia annua</i>.

<p>A: leaf primordia; B: lower leaf; C: leaf at bottom at early vegetative stage; D: leaf primordia; E: leaf primordia; F: leaf at upper node; G: close-up of panel F; H: leaf at lower node; I: leaf at bottom at late vegetative stage; K: stem; L: stem ; M: flower buds; N: flower buds; O: flower at early flower stage; P: florets; Q: florets; R: flower at late flower stage; S: hermaphroditic floret; T: pistillate floret; U: root.</p

GUS expression controlled by the <i>CPS</i> promoter in transgenic plants of <i>Artemisia annua</i>.

<p>A: leaf primordia; B: lower leaf; C: leaf at bottom at early vegetative stage; D: leaf primordia; E: leaf at upper node; F: close-up of panel E; G: leaf at upper node; H: leaf at lower node at late vegetative stage; I: leaf at lower node at late vegetative stage; K: stem; L: stem; M: flower buds; N: flowers at early flowering stage; O: floret; P: flowers at late flowering stage; Q: florets; R: pollen; S: flower bracts; T: roots.</p