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

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>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

Position of putative <i>cis</i>-acting regulatory elements known to be involved in responsiveness towards external factors in the four cloned sesquiterpene synthase promoters.

<p>Elements marked above the promoters are located to the (+)-strain and the elements marked under the promoters are located to the (–)-strain.</p

Some features of the cloned sesquiterpene promoters.

*<p>The position of the TSS (+1) is given as bases upstream of the ATG start codon.</p>**<p>The position of the TATA- and CAAT-boxes are given relative to the TSS.</p

Nucleotide sequence of primers used. Restriction sites are underlined; F = forward; R = reverse.

<p>Nucleotide sequence of primers used. Restriction sites are underlined; F  =  forward; R  =  reverse.</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

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

Additional file 1 of Using machine learning to determine age over 16 based on development of third molar and periodontal ligament of second molar

Additional file 1: Supplementary Table S1. List of the tuned hyperparameters for each Machine Learning algorithm. For each hyperparameter, the values inside square brackets were explored by Grid Search. Supplementary Table S2. Parameter estimates for logistic model for I3M