Studies on the expression of sesquiterpene synthases using promoter-β-glucuronidase fusions in transgenic Artemisia annua L

Abstract

In order to better understand the influence of sesquiterpene synthases on artemisinin yield in Artemisia annua, 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), epi-cedrol (ECS) and β-farnesene (FS) synthase, respectively. Prediction of cis-acting regulatory elements showed that the promoters are involved in complex regulation of expression. Transgenic A. annua 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 A. annua 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 GUS 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 CPS, ECS or GAS gene may not improve artemsinin yield. However, blocking the expression of FS may have effects on artemisinin production.

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

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.