Figure 1.
Enzymes in Artemisia annua utilizing farnesyl diphosphate as substrate.
ADS: amorpha-4,11-diene synthase; CPS: β-caryophyllene synthase; ECS: epi-cedrol synthase; FS: β-farnesene synthase; GAS: germacrene A synthase; GDS: germacrene D synthase; SQS: squalene synthase; PPO: diphosphate moiety
Table 1.
Nucleotide sequence of primers used. Restriction sites are underlined; F = forward; R = reverse.
Table 2.
Some features of the cloned sesquiterpene promoters.
Table 3.
Number of putative cis-acting regulatory elements in sesquiterpene synthase promoters from Artemisia annua.
Figure 2.
Position of putative cis-acting regulatory elements known to be involved in responsiveness towards external factors in the four cloned sesquiterpene synthase promoters.
Elements marked above the promoters are located to the (+)-strain and the elements marked under the promoters are located to the (–)-strain.
Figure 3.
Southern blot of genomic DNA isolated from wild-type (lane 1-2) and transgenic (lane 3-10) plants using a digoxigenin-labeled NPTII probe.
Lane 1: wild-type plant digested with HindIII; lane 2: wild-type plant digested with EcoRI; lane 3: transgenic plant carrying the pECS-GUS fusion digested with HindIII: lane 4: transgenic plant carrying the pECS-GUS fusion digested with EcoRI; lane 5: transgenic plant carrying the pFS-GUS fusion digested with HindIII: lane 6: transgenic plant carrying the pFS-GUS fusion digested with EcoRI; lane 7: transgenic plant carrying the pCPS-GUS fusion digested with HindIII: lane 8: transgenic plant carrying the pCPS-GUS fusion digested with EcoRI; lane 9: transgenic plant carrying the pADS-GUS fusion digested with HindIII: lane 10: transgenic plant carrying the pFS-GUS fusion digested with EcoRI; lane 11: positive control (882 bp fragment carrying the NPTII gene). Sample size: 10–15 µg/lane.
Figure 4.
GUS expression controlled by the CPS promoter in transgenic plants of Artemisia annua.
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.
Figure 5.
GUS expression controlled by the ECS promoter in transgenic plants of Artemisia annua.
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.
Figure 6.
GUS expression controlled by the FS promoter in transgenic plants of Artemisia annua.
A: young leaf; B: close-up of young leaf; C: leaf; D: old leaf; E: root.
Figure 7.
Relative expression of the wild-type pCPS (A), pECS (B) and pFS (C) in different tissues of Artemisia annua.
All activities are relative to the activity of the β-actin promoter, which was set to 1.0.
Figure 8.
Wounding of leaves of transgenic Artemisia annua carrying the pCPS::GUS fusion.
A: unwounded; B: immediately after wounding; C: 1h; D: 2h; E: 4h; F: 8h; G: 12h; H: 24h; I: 48h.
Figure 9.
Wounding of leaves of transgenic Artemisia annua carrying the pECS::GUS fusion.
A: unwounded; B: immediately after wounding; C: 1h; D: 2h; E: 4h; F: 8h; G: 12h; H: 24h; I: 48h.
Figure 10.
Relative expression of the wild-type pADS (A), pCPS (B), pECS (C), and pFS (D) in leaves after wounding.
The β-actin promoter activity was set to 1.0.
Figure 11.
Relative expression of the wild-type pADS (A), pCPS (B), pECS (C) and pFS (D) in leaves after spraying MeJA.
The β-actin promoter activity was set to 1.0.
Figure 12.
Relative levels of GAS, FS, ECS, CPS and ADS transcripts in flower buds (A) and leaf primordia (B) compared to the β-actin transcript level in A. annua.
The relative amount of β-actin transcripts was set to 1.0.