Research ArticlePLANT SCIENCES

Jasmonate promotes artemisinin biosynthesis by activating the TCP14-ORA complex in Artemisia annua

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Science Advances  14 Nov 2018:
Vol. 4, no. 11, eaas9357
DOI: 10.1126/sciadv.aas9357
  • Fig. 1 AaTCP14 protein interacts with AaORA.

    (A) Y2H analysis of AaTCP14 interaction with AaORA. Yeast cells transformed with different combinations of constructs containing AaTCP14 fused with the DNA binding domain (BD-AaTCP14), AaORA fused with the activation domain (AD-AaORA), the BD alone, and the AD alone were grown on two different selective media, SD/-Trp/-Leu/-His (TDO) and SD/-Trp/-Leu/-His/-Ade (QDO), and the control medium SD/-Trp/-Leu (DDO). Pictures were taken after 4 days of incubation at 30°C. Y2H assays were repeated three times, and representative results are shown. (B) In vitro pulldown assays of AaTCP14 and AaORA recombinant proteins. His-AaTCP14 proteins were pulled down with GST-AaORA and further detected on Western blots probed with anti-His antibody. Experiments were carried out three times, and representative results are shown. (C) Bimolecular fluorescence complementation (BiFC) analysis of the interaction between AaTCP14 and AaORA in N. benthamiana cells. AaTCP14 was fused to the N-terminal fragment of yellow fluorescent protein (AaTCP14-nYFP), and AaORA was fused to the C-terminal fragment of YFP (AaORA-cYFP). Colocalization of reconstituted YFP and nuclei was determined by 4′,6-diamidino-2-phenylindole (DAPI) staining. Three independent transfection experiments were performed. Scale bars, 20 μm. (D) Co-IP studies of AaTCP14 and AaORA complex formation in N. benthamiana leaves. Total protein extracts from N. benthamiana leaves infiltrated with constructs harboring AaTCP14-GFP and AaORA-Flag were immunoprecipitated with anti-Flag antibody. The coimmunoprecipitated proteins were detected by anti-GFP antibody. Experiments were repeated three times and similar results were obtained.

  • Fig. 2 Expression pattern and subcellular localization of AaTCP14.

    (A) Relative expression levels of AaTCP14 and AaORA in leaves at different positions. The expression levels of AaTCP14 and AaORA in leaf 1 were set as 1. Actin was used as an internal control. The data represent the means ± SD of three replicates from three independent A. annua plants. (B) An image of a 3-month-old A. annua plant labeled with the leaves numbered as in (A). (C) Relative expression levels of AaTCP14 in roots, stems, flowers, shoots, buds, leaves, and trichomes were measured by qRT-PCR. The expression level of AaTCP14 in roots was set as 1. Actin was used as an internal control. The data represent the means ± SD of three replicates from three independent A. annua plants. (D) GUS expression (blue staining) in A. annua plants transformed with the 1391Z-GUS empty vector (control plants) and 1391Z-proTCP14-GUS. (a and d) Leaves of control plants. (b and e) Leaves of 1391Z-proTCP14-GUS plants. (c and f) Stems of 1391Z-proTCP14-GUS plants. Scale bars, 200 μm (a to c) and 50 μm (d to f). (E) Relative expression levels of AaTCP14 and AaORA in plants treated with MeJA (100 μM) over 24 hours. Actin was used as an internal control. The data represent the means ± SD of three replicates from three independent experiments. (F) Subcellular localization of AaTCP14. Colocalization of AaTCP14-YFP and nuclei was determined by DAPI staining. YFP was used as a negative control. Three independent transfection experiments were performed. Scale bars, 20 μm.

  • Fig. 3 Analysis of AaTCP14 transgenic plants.

    (A and D) Expression levels of AaTCP14 in different A. annua AaTCP14 overexpression (A) and antisense lines (D), plants transformed with the empty vector, and WT plants. Actin was used as the internal standard. (B and E) Expression levels of ADS, CYP71AV1, DBR2, and ALDH1 in different A. annua AaTCP14 overexpression (B) and antisense lines (E), plants transformed with the empty vector, and WT. Actin was used as the internal control. (C and F) HPLC analysis of artemisinin (AN) in the leaves of different A. annua AaTCP14 overexpression (C) and antisense lines (F), plants transformed with the empty vector, and WT. All data represent the means ± SD of three replicates from three cutting propagations. *P < 0.05, **P < 0.01, Student’s t test.

  • Fig. 4 AaTCP14 is a transcriptional activator of DBR2 and ALDH1.

    (A) Schematic diagrams of the effector (pCambia1300-AaTCP14-GFP) and reporter (35S: REN-ADS/CYP/DBR2/ALDH1pro:LUC) plasmids used in dual-LUC assays. CYP, CYP71AV1; REN, Renilla luciferase; LUC, firefly luciferase. (B) Dual-LUC assay in N. benthamiana cells using the constructs shown in (A). The GFP effector was used as a negative control, and the LUC/REN ratios of GFP were set as 1. Three independent transfection experiments were performed. The data represent the means ± SD of three replicates from three independent experiments. *P < 0.05, **P < 0.01, Student’s t test. (C and D) Schematic diagrams of the DBR2 and ALDH1 promoters. The positions of potential TBS DNA binding sites (D1 and D2 and A1, A2, and A3) are shown as black and white triangles and are numbered on the basis of their distance from the translational start site (ATG), which is set as +1. (E and F) Y1H assays showing that AaTCP14 binds to the TBS motifs of DBR2 and ALDH1. Three tandem repeats of each motif were used as baits. Yeast cells coexpressing pB42AD, pB42AD-AaTCP14, and the DNA motifs from the DBR2 and ALDH1 promoters were grown on selective medium, SD/-Trp/-Ura, containing X-gal (20 mg/liter), and pictures were taken after 4 days of incubation at 30°C. Blue plaques indicate protein-DNA interactions. The Y1H assays were repeated three times, and representative results are shown. (G and H) The sequences of WT and mutated probes used for EMSAs. Class I TCP binding motifs are shown in bold, and the mutated nucleotides are indicated in red. (I and J) EMSAs showing that AaTCP14 binds to the D2q motif from DBR2 and the A1q motif from ALDH1. Unlabeled D2q and A1q were used as cold competitors, and two labeled mutated D2q and A1q probes were tested as negative controls. 10×, 20×, and 40× indicate the fold excess of cold competitors relative to that of the labeled probe. His-TF protein was used as a negative control.

  • Fig. 5 AaTCP14 and AaORA synergistically promote artemisinin biosynthesis, and AaORA is partly dependent on AaTCP14.

    (A) A schematic representation of the constructs used in dual-LUC assays. (B and C) Activation of the DBR2 (B) and ALDH1 (C) promoters by AaORA and AaTCP14 proteins in the presence or absence of MeJA in N. benthamiana leaves. The GFP effector in the mock treatment served as a negative control, and the LUC/REN ratios of GFP were set as 1. Three independent transfection experiments were performed. The reporter strain harboring DBR2pro:LUC or ALDH1pro:LUC was mixed with the effector strains harboring 35Spro:AaTCP14 and 35Spro:AaORA at a ratio of 1:1:1. The data represent the means ± SD of three replicates from three independent experiments. *P < 0.05, **P < 0.01, Student’s t test. (D and E) Expression levels of AaTCP14 and AaORA (D) and DBR2 and ALDH1 (E) in different A. annua plants including AaTCP14-AaORA co-overexpression (AaTCP14-AaORA), AaTCP14 overexpression lines, and plants transformed with the empty vector. Actin was used as the internal standard. WT plants served as controls. The data represent the means ± SD of three replicates from three cutting propagations. **P < 0.01, Student’s t test. (F) HPLC analysis of artemisinin (AN) in the leaves of different A. annua plants including AaTCP14-AaORA and AaTCP14 overexpression lines, plants transformed with the empty vector, and the WT control. The data represent the means ± SD of three replicates from three cutting propagations. *P < 0.05, **P < 0.01, Student’s t test. (G and H) Expression levels of AaTCP14 and AaORA (G) and DBR2 and ALDH1 (H) in different A. annua plants including the AaTCP14 antisense–AaORA overexpression (ANTCP14-AaORA), AaORA overexpression lines, and plants transformed with the empty vector. Actin was used as the internal standard. WT plants served as controls. The data represent the means ± SD from three replicates from three cutting propagations. *P < 0.05, **P < 0.01, Student’s t test. (I) HPLC analysis of artemisinin (AN) in the leaves of different A. annua plants including the ANTCP14-AaORA, AaORA overexpression lines, plants transformed with the empty vector, and the WT control. The data represent the means ± SD of three replicates from three cutting propagations. *P < 0.05, **P < 0.01, Student’s t test. (J and K) Dual-LUC experiments showing the activation of the DBR2 (J) and ALDH1 (K) promoters by AaORA in A. annua protoplasts from WT and AaTCP14 antisense (ANTCP14) lines. GFP was used as a negative control, and the LUC/REN ratios of GFP in WT A. annua were set as 1. Three independent transfection experiments were performed. The data represent the means ± SD of three independent experiments. Student’s t test, **P < 0.01.

  • Fig. 6 AaJAZ8 interacts with both AaTCP14 and AaORA.

    (A) Y2H assays to detect the pairwise interactions between AaJAZ8, AaTCP14, and AaORA. AaORA and AaJAZ8 were fused with the activation domain (AD), and AaTCP14 and AaJAZ8 were fused with the binding domain (BD). Yeast cells harboring bait and prey plasmids were grown on two types of selective media, QDO and TDO, and the control medium, DDO. Pictures were taken after 4 days of incubation at 30°C. The Y2H assays were repeated three times, and representative results are shown. (B) BiFC analysis to detect the pairwise interactions between AaTCP14, AaORA, and AaJAZ8. AaTCP14 and AaORA were fused to the N-terminal fragment of YFP (TCP14-nYFP and ORA-nYFP), and AaJAZ8 was fused to the C-terminal fragment of YFP (JAZ8-cYFP). Colocalization of reconstituted YFP and nuclei was determined by DAPI staining. Three independent transfection experiments were performed. Scale bars, 20 μm. (C) Colocalization of AaTCP14 and AaJAZ8 and of AaORA and AaJAZ8 to the same NBs in N. benthamiana cells. N. benthamiana leaves were infiltrated with A. tumefaciens strains harboring different combinations of CFP-TCP14, CFP-ORA, and YFP-JAZ8 fusion protein constructs, and CFP-TCP14 and YFP, CFP-ORA and YFP, and CFP and YFP-JAZ8 were cotransformed as negative controls. Pictures were taken after 60 to 72 hours of incubation at 23°C. Three independent transfection experiments were performed. Scale bars, 5 μm. (D) LUC complementation assay to detect the pairwise interactions between AaTCP14, AaORA, and AaJAZ8. AaJAZ8 was fused to the C-terminal fragment of LUC (Cluc-JAZ8), and AaTCP14 and AaORA were fused to the N-terminal fragment of LUC (TCP14-Nluc and ORA-Nluc). LUC activity of Cluc-JAZ8 and Nluc was set to 1. Three independent transfection experiments were performed. The data represent the means ± SD of three independent experiments. **P < 0.01, Student’s t test. (E) Co-IP analysis of TCP14-GFP, ORA-Flag, and JAZ8-Flag in N. benthamiana leaves. Total protein extracts from N. benthamiana leaves infiltrated with A. tumefaciens harboring TCP14-GFP, ORA-Flag, and JAZ8-Flag fusion protein constructs were immunoprecipitated with anti-GFP antibody. The coimmunoprecipitated proteins were detected by anti-Flag antibody. Similar results were obtained in three independent experiments. (F) Colocalization of TCP14-nYFP, ORA-cYFP, and CFP-JAZ8 in the same NBs in N. benthamiana leaves. Colocalization of TCP14-nYFP, ORA-cYFP, and CFP was used as the negative control. After A. tumefaciens infiltration, pictures were taken after 60 to 72 hours of incubation at 23°C. Three independent transfection experiments were performed. Scale bars, 5 μm. IB, immunoblotting.

  • Fig. 7 AaJAZ8 interferes with the interaction between AaORA and AaTCP14 and attenuates the transcriptional activation activity of AaTCP14 and AaORA.

    (A) Y3H assays of the influence of JAZ8 on TCP14-ORA interaction. Left, schematic representation of the bait and prey constructs used in Y3H assays. ORA was fused with the activation domain (AD), and TCP14 was fused with the binding domain (BD). PMet25 is an inducible promoter that drives the expression of the bridge protein, JAZ8. Right, yeast cells harboring bait and prey plasmids were grown on two types of selective media, SD/-Trp/-Leu/-Met (SD/-T/-L/-M) and SD/-Trp/-Leu/-His/-Ade/-Met (SD/-T/-L/-H/-A/-M), and pictures were taken after 4 days of incubation at 30°C. The different dilutions, 100, 10−1, 10−2, and 10−3, are shown at the top of the figure. The Y3H assays were repeated three times, and representative results are shown. (B) β-Gal activities of yeast in (A) were measured in the presence or absence of JAZ8. The promoter driving JAZ8 expression was suppressed by methionine. The data represent the means ± SD of three replicates from three independent experiments. Student’s t test, **P < 0.01. The β-gal activity assays were repeated three times, and similar results were obtained. (C) LUC complementation assay showing that JAZ8 inhibits TCP14-ORA interaction. The LUC activities of Cluc-ORA, TCP14-Nluc, and Cluc-Flag were set to 1. The data represent the means ± SD of three replicates from three independent experiments. **P < 0.01, Student’s t test. The bottom panel shows a Western blot of proteins isolated from N. benthamiana cells. JAZ8-Flag and Cluc-Flag fusion proteins were detected using anti-Flag antibody. Three independent transfection experiments were performed. (D) Y2H analysis showing the interactions between ORA, JAZ8, and full-length and truncated versions of TCP14. Left, schematic representations of the truncated TCP14 proteins used in this experiment. Numbers indicate the amino acid (a.a.) positions of the truncated TCP14 variants. The location of the TCP domain is indicated by a yellow box. Right, transformed yeast cells were grown on the selective medium, QDO, and the control medium, DDO, and pictures were taken after 4 days of incubation at 30°C. (E) Dual-LUC experiment showing that MeJA treatment partially recovers the activation of the DBR2 promoter by AaTCP14 and AaORA in the presence of AaJAZ8. The LUC/REN ratio of GFP in the mock treatment was set as 1. Three independent transfection experiments were performed. The data represent the means ± SD of three replicates from three independent experiments. *P < 0.05, **P < 0.01, Student’s t test. (F) A working model depicting how JA signaling regulates the DBR2 promoter via interactions between TCP14-ORA and JAZ8. (a) In the absence of JA, JAZ8 interacts with both TCP14 and ORA and disrupts the TCP14-ORA complex. This attenuates the transcriptional activation activity of the TCP14-ORA complex at the DBR2 promoter, decreasing artemisinin biosynthesis. In addition, JAZ8 interacts with MYC2 and may suppress MYC2-mediated artemisinin biosynthesis. (b) In the presence of JA, JAZ8 is recognized by COI1 (the JA receptor) and degraded by the 26S proteasome. In the absence of JAZ8, the ORA and TCP14 proteins form a TCP14-ORA complex and synergistically activate the DBR2 promoter, enhancing artemisinin biosynthesis. In addition, the degradation of JAZ8 releases MYC2, and MYC2 directly or indirectly activates the expression of its target gene GSW1 and the downstream gene ORA to enhance JA-regulated artemisinin biosynthesis. Solid arrow, direct regulation. Broken line arrow, hypothetical direct links. T-bars, negative interactions. 26S, 26S proteasome.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/11/eaas9357/DC1

    Fig. S1. A schematic diagram of the artemisinin biosynthetic pathway and its regulation in A. annua, and AaORA activates the ADS, CYP71AV1, DBR2, and ALDH1 promoters.

    Fig. S2. Y2H assay showing the regions of AaORA with autoactivation activity.

    Fig. S3. Alignment of the protein sequences of AaTCP14 and 29 related proteins.

    Fig. S4. Phylogenetic analysis of TCP14 proteins from A. annua and other plants.

    Fig. S5. Relative expression levels of transcription factors positively regulating artemisinin biosynthesis and JA biosynthetic genes in AaTCP14 transgenic plants.

    Fig. S6. Characterization of A. annua transgenic plants.

    Fig. S7. Neither AaORA nor AaJAZ8 affects the ability of AaTCP14 to bind DNA.

    Fig. S8. The expression patterns of AaJAZ8, MeJA-induced AaJAZ8 degradation, and analysis of artemisinin biosynthesis in A. annua plants overexpressing AaJAZ8 or AaJAZ8Δjas.

    Fig. S9. AaTCP14 and AaORA interact with AaJAZ proteins and mapping of the domains involved in the interaction between AaJAZ8, AaORA, and AaTCP14 using Y2H assays.

    Fig. S10. Artemisinin content in AaTCP14 transgenic plants under MeJA treatment.

    Table S1. List of primers used in this study.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. A schematic diagram of the artemisinin biosynthetic pathway and its regulation in A. annua, and AaORA activates the ADS, CYP71AV1, DBR2, and ALDH1 promoters.
    • Fig. S2. Y2H assay showing the regions of AaORA with autoactivation activity.
    • Fig. S3. Alignment of the protein sequences of AaTCP14 and 29 related proteins.
    • Fig. S4. Phylogenetic analysis of TCP14 proteins from A. annua and other plants.
    • Fig. S5. Relative expression levels of transcription factors positively regulating artemisinin biosynthesis and JA biosynthetic genes in AaTCP14 transgenic plants.
    • Fig. S6. Characterization of A. annua transgenic plants.
    • Fig. S7. Neither AaORA nor AaJAZ8 affects the ability of AaTCP14 to bind DNA.
    • Fig. S8. The expression patterns of AaJAZ8, MeJA-induced AaJAZ8 degradation, and analysis of artemisinin biosynthesis in A. annua plants overexpressing AaJAZ8 or AaJAZ8Δjas.
    • Fig. S9. AaTCP14 and AaORA interact with AaJAZ proteins and mapping of the domains involved in the interaction between AaJAZ8, AaORA, and AaTCP14 using Y2H assays.
    • Fig. S10. Artemisinin content in AaTCP14 transgenic plants under MeJA treatment.
    • Table S1. List of primers used in this study.

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