The YABBY Family Transcription Factor AaYABBY5 Directly Targets Cytochrome P450 Monooxygenase (CYP71AV1) and Double-Bond Reductase 2 (DBR2) Involved in Artemisinin Biosynthesis in Artemisia Annua

Abstract

Artemisinin is an effective antimalarial sesquiterpene lactone synthesized in Artemisia annua. Various transcription factors have been previously reported that can influence the biosynthesis of artemisinin; however, the effect of YABBY family transcription factors on artemisinin biosynthesis was unknown. In the present study, we cloned and characterized AaYABBY5: a homolog of MsYABBY5 in Mentha spicata which is involved in modulating the monoterpenes, as a positive regulator of artemisinin biosynthesis in A. annua. AaYABBY5 was found localized to the nucleus, and its expression was found to be induced by exogenous methyl jasmonic acid (MeJA) treatment. In the dual-luciferase reporter assay, it was found that AaYABBY5 significantly increased the activities of promoters of amorpha-4,11-diene synthase (ADS), cytochrome P450 monooxygenase (CYP71AV1), double-bond reductase 2 (DBR2), and aldehyde dehydrogenase 1 (ALDH1) genes. Yeast one hybrid assay showed that AaYABBY5 directly bonds to the promoters of CYP71AV1 and DBR2 genes. Quantitative real-time polymerase chain reaction (qPCR) of AaYABBY5 overexpression and AaYABBY5 antisense plants revealed a significant increase in the expression of ADS, CYP71AV1, DBR2, and ALDH1 in AaYABBY5 overexpression plants and a significant decrease in the expression of these genes in AaYABBY5 antisense A. annua, respectively. Furthermore, the results of high-performance liquid chromatography (HPLC) showed that the artemisinin and its precursor dihydroartemisinic acid were significantly increased in the AaYABBY5 overexpression plants while AaYABBY5 downregulation resulted in a significant decrease in the concentration of artemisinin. Taken together, these results explicitly represent that AaYABBY5 is a positive regulator of artemisinin biosynthesis in A. annua.

Keywords: Artemisia annua; CYP71AV1; DBR2; YABBY family transcription factor; artemisinin; sesquiterpene.

Figure 1

Biosynthetic pathway for artemisinin in Artemisia annua. HMGR, 3-hydroxy-3-methylglutaryl-CoA reductase; DXS, 1-deoxyxylulose 5-phosphate synthase; DXR, 1-deoxyxylulose 5-phosphate reductoisomerase; DMAPP, dimethylallyl diphosphate; IPP, isopentenyl diphosphate; IPPI, isopentenyl diphosphate isomerase; FPS, farnesyl diphosphate synthase; ADS, amorpha-4,11-diene synthase; CYP71AV1, cytochrome P450 monooxygenase; CPR, cytochrome P450 reductase; ADH1, alcohol dehydrogenase I; DBR2, double-bond reductase 2; ALDH1, aldehyde dehydrogenase 1.

Figure 2

Phylogenetic analysis. The phylogenetic tree was constructed by the maximum likelihood method using MEGA 6.06. Bootstrap analysis was performed using 500 replicates. (A) Phylogenetic relationship was found using protein sequences from Artemisia annua YABBYs and MsYABBY5 protein. (B) Phylogenetic analysis was performed using YABBY family proteins from A. annua, Mentha spicata, Arabidopsis thaliana, and various other plant species. AaYABBY5 clustered among YABBY5 members of other plants. The accession number of AaYABBY5 in GenBank is MK675289.

Figure 3

Subcellular localization of AaYABBY5 protein. (A) The sketch map shows the constructs used for finding subcellular localization. (B) Subcellular localization found under confocal laser microscope using leaf cells of Nicotiana benthamiana after infiltration. AaYABBY5 was found localized to the nucleus in N. benthamiana leaf cells infiltrated with Agrobacterium strains containing the coding sequence of AaYABBY5 fused with YFP in the pEG101 vector. (C) Empty vector, i.e., YFP alone signal, was found in both the nucleus and cytoplasm of leaf cells agroinfiltrated with control. Bars represent 100 µm.

Figure 4

Dual-luciferase (LUC) reporter assay. (A) The sketch map shows the constructs for dual-LUC assay. The coding sequence of AaYABBY5 was fused to the yellow fluorescent protein (YFP) present in the pEG104 vector to make the effector construct. Empty vectors were used as negative controls. Promoters of ADS, CYP71AV1, DBR2, and ALDH1 were fused with the N-terminal of the LUC gene for the preparation of reporter constructs. (B–E) Dual-LUC analysis was performed by transient infiltration of Nicotiana benthamiana leaves with equal concentrations of Agrobacterium GV3101 cells transformed with effector and reporter constructs, respectively. The values were obtained as a ratio of the activity of firefly luciferase and renilla luciferase. A significant increase in the activities of ADS, CYP71AV1, DBR2, and ALDH1 promoters was found in the presence of the AaYABBY5 effector. Data represent values obtained as mean ± SD of four independent infiltrations. Error bars represent standard deviation for n = 4. Statistical significance was performed using Student’s t-test with paired and two-tailed distribution methods. **P < 0.01.

Figure 5

Yeast one hybrid (Y1H) assay. Y1H assay was performed using the open reading frame (ORF) sequence of AaYABBY5 as prey and promoters of ADS, CYP71AV1, DBR2, and ALDH1 as bait. The GenBank accession numbers of pADS, pCYP71AV1, pDBR2, and pALDH1 are DQ448294, FJ870128, KC118523.1, and KC118525.1, respectively. AaYABBY5 directly bound to full-length CYP71AV1 and DBR2 promoters in yeast cells transformed with pB42AD-AaYABBY5 and plac-Z-CYP71AV1/plac-Z-DBR2, as shown by the appearance of blue colonies, whereas no binding was observed in the case of ADS and ALDH1 promoters. Yeast cells transformed with empty pB42AD in combination with placZ-ADS/placZ-CYP71AV1/placZ-DBR2/plac-Z-ALDH1 and empty vectors pB42AD + placZ were used as negative controls.

Figure 6

Expression and induction pattern of AaYABBY5. (A) Relative expression of AaYABBY5 was analyzed from different tissues of 5-month-old Artemisia annua plants. β-Actin was used as an internal standard. Values represent the mean ± SD of three replicates. Error bars represent the standard deviation for n = 3. (B) AaYABBY5 expression, in response to 100-µM MeJA, was analyzed in 4-week-old A. annua leaves. Water with 1% DMSO solution was used as mock solution. β-Actin was used as an internal standard. Values represent the mean ± SD of three experimental replicates. Error bars represent standard deviation for n = 3. Statistical significance was determined by Student’s t-test with paired and two-tailed distribution methods. Asterisks indicate the difference between treated and nontreated plants. *P < 0.05.

Figure 7

Relative expression analysis of AaYABBY5, ADS, CYP71AV1, DBR2, and ALDH1 in AaYABBY5 overexpression and AaYABBY5 antisense Artemisia annua. (A) Expression of AaYABBY5, ADS, CYP71AV1, DBR2, and ALDH1 in selected AaYABBY5 overexpression A. annua. β-Actin was used as an internal control of each gene. Values represent mean ± SD of three experimental replicates. Error bars represent standard deviation for n = 3. Statistical significance was determined by Student’s t-test with paired and two tailed distribution methods. Asterisks indicate the difference between AaYABBY5 overexpression and control plants. **P < 0.01. (B) Expression of AaYABBY5, ADS, CYP71AV1, DBR2, and ALDH1 in selected AaYABBY5 antisense plants of A. annua. β-Actin was used as an internal control of each gene. Values represent mean ± SD of three experimental replicates. Error bars represent standard deviation for n = 3. Statistical significance was determined by Student’s t-test with paired and two-tailed distribution methods. Asterisks indicate the difference between AaYABBY5 antisense and control plants. **P < 0.01.

Figure 8

High-performance liquid chromatography (HPLC) analysis of metabolites in AaYABBY5 overexpression and antisense plants. The concentration of (A) artemisinin, (B) dihydroartemisinic acid, and (C) artemisinic acid in AaYABBY5 overexpression and antisense plants of Artemisia annua. Values of all metabolites represent mean ± SD of three independent experiments. Error bars represent the standard deviation for n = 3. Statistical significance was determined by Student’s t-test with paired and two-tailed distribution methods. Asterisks indicate the difference between transgenic and control plants. **P < 0.01.