Tanacetum cinerariifolium ‘W99’ plants grown in the flower base of Huazhong Agricultural University, Wuhan, China were used in this study. This cultivar has the advantages of easily rooting cuttings, rapid growth, and high pyrethrins content and, therefore, was suitable for the present experiments. W99 seedlings were subcultured in half-strength Murashige and Skoog medium for 1 month (25°C, 16 h light/8 h dark) prior to transient treatment with MeJA or ABA. The rooted cuttings were sprayed with 5 ml of 2 mM MeJA/ABA solution as a single foliar application. Leaves were sampled in triplicate at 0 (control), 2, 4, 6, 8, 12, and 24 h after treatment, immediately placed in liquid nitrogen, and stored at −80°C until further analysis.

Flower heads and leaves were harvested at seven flowering stages: S1, well-developed closed buds; S2, ray floret limb in vertical position; S3, ray floret limb in horizontal position and disc florets of the outermost whorl open; S4, three whorls of disc florets open; S5, all disc florets open; S6, termed the early overblown condition, the disc floret color is faded but the ray florets remain intact; S7, termed the late overblown condition, the disc florets retain little color and the ray florets are desiccated. Three biological replicates were collected for each sample, which were immediately frozen in liquid nitrogen and stored at −80°C for further analysis.

Our previously established transgenic chrysanthemum (Chrysanthemum × morifolium) harboring TcCHS-promoter-driven GFP and tobacco (Nicotiana tabacum) harboring TcGLIP-promoter-driven GUS were treated with MeJA (Sultana et al., 2015). The plants were sprayed with 5 ml of 300 μM MeJA dissolved in 0.8% ethanol as a single foliar application. Leaves were collected in triplicate at 0 (control) and 12 h after treatment, and were immediately frozen in liquid nitrogen. The GFP expression level was determined by quantitative real-time PCR (qRT-PCR) analysis. The GUS activity was measured as previously described (Luo et al., 2013).

Total RNA was extracted from the collected samples using the standard phenol–chloroform extraction method. The first-strand cDNA was synthesized using the EasyScript^® One-step gDNA Removal and cDNA Synthesis SuperMix Kit (TransGen Biotech, Beijing, China), in accordance with the manufacturer’s instructions, using the total RNA extracts as the template. The TcbZIP60 gene sequence of T. cinerariifolium was identified from the inflorescence transcriptome database determined by our laboratory. The Primer Premier software was used to design specific primers for amplifying the open reading frame (ORF) fragment. The primers are listed in Supplementary Table S1 . The cis-acting elements of associated genes (TcCHS, TcAOC, TcALDH, and TcGLIP) were determined using the PlantCARE database. The cis-regulatory E-box and G-box elements were determined using PlantPAN 3.0 (Chow et al., 2019).

Total RNA was extracted using the Ultrapure RNA Kit (CWBIO, Beijing, China), and the EasyScript One-step gDNA Removal and cDNA Synthesis SuperMix Kit (TransGen Biotech) was used to reverse-transcribe the RNA into cDNA. A qRT-PCR analysis was performed using a LightCycler^® 96 Real-Time PCR System (Roche, Basel, Switzerland) in accordance with the manufacturer’s instructions, with SYBR Premix Ex Taq II (Takara, Kusatsu, Japan) and sequence-specific primers (listed in Supplementary Table S1 ). Glyceraldehyde-3-phosphate dehydrogenase (GADPH) was used as the internal reference gene (Li et al., 2019). The relative expression levels were calculated using the 2^{−ΔΔ C t} method with three biological and three technical replicates (Livak and Schmittgen, 2001). Student’s two-tailed t-test was used to determine statistical significance. Differences at P < 0.05 were considered to be significant and those at P < 0.01 were considered to be highly significant.

The ORF of TcbZIP60 was cloned from T. cinerariifolium cDNA using the sequence-specific primers TcbZIP60-F and TcbZIP60-R ( Supplementary Table S1 ). Arabidopsis sequences homologous to the TcbZIP60 sequence were identified by a Basic Local Alignment Search Tool (BLAST) search against The Arabidopsis Information Resource (TAIR) database, and homologous sequences from other plant species were identified by a BLAST search against the National Center for Biotechnology Information (NCBI) database. A phylogenetic tree was constructed with the maximum likelihood method using MEGA X software with 1000 bootstrap replications (Kumar et al., 2018). The GenBank accession numbers of the genes used in the analysis are listed in Supplementary Tables S2 and S3 .

The TcbZIP60 coding sequence was amplified by PCR using sequence-specific primers ( Supplementary Table S1 ). The gene was cloned using the ClonExpress^® II One Step Cloning Kit (Vazyme Biotech, Nanjing, China). The full-length sequence of TcbZIP60 (without the stop codon) was spliced into the pSuper-1300 GFP vector. The recombinant product was transferred into Escherichia coli strain DH5α, then sequenced, and the correct plasmid was extracted and constructed. The resultant plasmid was introduced into Agrobacterium tumefaciens strain GV3101, and then co-infiltrated into Nicotiana benthamiana leaves together with the RFP-NLS plasmid (a nuclear marker). The empty vector was used as a control. After 72 h of weak light exposure, fluorescence signals were observed with a confocal laser scanning microscope (TCS-SP8, Leica, Wetzlar, Germany).

The full-length cDNA of TcbZIP60 was cloned into the pGADT7 vector used for homologous recombination, and the promoter sequences of TcCHS and TcAOC were cloned separately into the pHis2.1 vector. The resultant pGADT7:TcbZIP60 plasmid was co-transformed into yeast strain Y187, together with pHis2.1:TcCHS or pHis2.1:TcAOC, using the Super Yeast Transformation Kit (Coolaber, Beijing, China). The transformed yeast cells were selected on DDO (SD/−Leu/−Trp) medium and interreacted with TDO (SD/−Leu/−Trp/−His) medium at 30°C for 3 days.

To generate reporter constructs, and in accordance with the ORF sequence of the cloned TcbZIP60 gene and the map of the plant expression vector, the downstream sequences of the gene promoter region (for TcCHS and TcAOC) were cloned into the linearized pGreenII0800-LUC vector. To generate effector constructs, the coding sequence of TcbZIP60 was cloned into the linearized pGreenII62-SK vector under the control of the Cauliflower mosaic virus (CaMV) 35S promoter. The resultant vectors were transiently co-expressed in N. benthamiana leaves. Luminescence was detected using the LB 985 Nightshade system (Berthold, Bad Wildbad, Germany). Introduction of the pGreen-62-SK empty vector and the pGreen-62-SK/pGreenII-0800-LUC : TcCHS/TcAOC constructs served as negative controls. Three biological replicates per treatment were measured.

The CDS of TcbZIP60 was ligated into pGreenII62-SK vector to generate an effector plasmid, while TcCHS/TcAOC were fused into the vector pGreenII0800-LUC to produce the reporter plasmids. The effector, each of the two reporter constructs were co-transformed into A. tumefaciens GV3101, respectively. Transient expression assay in N. benthamiana leaves was as described previously (Geng and Liu, 2018). The activities of firefly luciferase (LUC) and Renilla luciferase (REN) were measured using the Dual-Luciferase^® Reporter Assay System (Promega, WI, USA). The promoter activity was expressed as the ratio of LUC to REN ( Supplementary Figure S1 ).

The ORF without the TcbZIP60 terminator codon was used to generate the pET6×HN-C vector protein, which was fused into the N-terminal frame of 6×His, and the vector pET6×HN-C-TcbZIP60 was then transformed into E. coli strain Rosetta (DE3). For the induced recombinant protein, 0.5 mM IPTG was used, and the cultures were incubated at 18°C for 16 h. Then, Ni²⁺–nitrilotriacetic acid was used to purify the recombinant proteins. For the electrophoretic mobility shift assay (EMSA), the promoter fragments of TcCHS and TcAOC containing E-box or G-box cis-regulatory elements labeled with fluorescein amidite (FAM) as probes, the same but unlabeled DNA fragments, and cis-element mutant DNA fragments were used as competitors in the assay. After performing the EMSA assays, FAM-labeled DNA was detected from the chemiluminescent signal.

The full-length TcbZIP60 coding sequence was cloned into the HindIII-linearized pGreenII62-SK vector downstream of the CaMV 35S promoter using gene-specific primers ( Supplementary Table S1 ). The experimental method followed a previously described procedure (Jung et al., 2015). The pGreenII62-SK : TcbZIP60 vector was co-transformed together with the helper plasmid pSoup19 into A. tumefaciens strain GV3101. The pSoup19-transformed Agrobacterium cells were inoculated into YEB liquid medium containing 100 mg/l kanamycin and cultured on a rotating shaker at 28°C for 12 h. The supernatant was removed and resuspended in MES (containing 100 μM acetosyringone) to attain the final optical density (OD₆₀₀ = 0.6). Leaves of T. cinerariifolium were placed in a 500 ml beaker containing a suspension of A. tumefaciens, and the beaker was then placed in a vacuum chamber at 0.23 ATM for 5 min. The soaked leaves were then dried with filter paper and stored in a petri dish lined with moist filter paper in the base. After 4 days of culture, leaves were sampled for qRT-PCR and high-performance liquid chromatography (HPLC) analyses. To determine the effect of transient expression, transient overexpression of GUS in T. cinerariifolium leaves was verified by staining with X-Gluc reagent ( Supplementary Figure S2 ).

The Tobacco rattle virus (TRV)-based vectors pTRV1 and pTRV2 were used for the VIGS assay ( Supplementary Table S1 ). The pTRV2:TcbZIP60 and pTRV1 plasmids were transformed into chemically active A. tumefaciens strain GV3101 cells using a liquid nitrogen freeze–thaw method. The positive transformant cells were inoculated into YEB liquid medium containing 100 mg/l kanamycin, incubated overnight on a shaker at 28°C, then reactivated in an infiltrating buffer (pH = 5.6) containing 10 mM MgCl₂, 10 mM MES, and 100 µM acetoeugenone, and adjusted to OD₆₀₀ = 0.6. After standing for 3 h, Agrobacterium cells containing the pTRV2 plasmid and pTRV1 plasmid were mixed (1:1, v/v), and the bacterial solution was injected through the adaxial epidermis of T. cinerariifolium leaves using a needle-free syringe. The leaves were then incubated in a culture room in the dark for 3 days and under light for 11 days. The pTRV1 and pTRV2 vectors were used as the control group. The method followed a previously described procedure (Senthil-Kumar and Mysore, 2014). The VIGS assay was conducted with three biological replicates. The empty vectors were used as controls. After 14 days, the samples were subjected to qRT-PCR and HPLC analyses.

Transiently transformed leaf samples were dried for 48 h in an oven at 50°C to constant dry weight. The dried leaves were ground to a fine powder, then 100 mg powder was placed in a screw-capped glass tube, and dissolved in 600 μl n-hexane. After vortex-oscillation for 30 s, the sample was extracted in an ultrasonic water bath for a further 10 min, followed by vortex-oscillation for 30 s. The extracted samples were filtered with a 0.22 μm filter and analyzed by HPLC. The pyrethrins content was determined using a Waters HPLC system equipped with a photodiode array detector as described previously (Hu et al., 2018). Three biological replicates were analyzed for each sample, and commercial pyrethrum extract (Sigma-Aldrich, St. Louis, MO, USA) was used as the standard. Pyrethrins standard solution mother liquor was obtained by absorbing 3.0 μl pyrethrins standard and dissolving it in a small amount of n-hexane, and finally the volume was standardized to 1.00 ml. The mother liquor of 200, 100, 50, 25, 12.5, and 6.25 μl was standardized to 250 μl with methanol to obtain the pyrethrins standard sample solutions with concentrations of 1.15, 0.575, 0.288, 0.144, 0.072, and 0.036 mg/ml, respectively. After absorbing 20 μl of each standard sample solution, the samples were analyzed by HPLC and a standard curve for the pyrethrins standard samples was generated with Microsoft Excel.

All experiments were repeated using at least three biological replicates. The data were analyzed with one-way ANOVA and Student’s t-test using SPSS 18 software. Significant differences were determined with Student’s t-test, with p < 0.05 considered to be statistically significant.

Pyrethrins are derived from two independent pathways: the terpenoid biosynthesis pathway and the JA biosynthesis pathway ( Figure 1A ). Most pyrethrins biosynthesis enzymes have been identified to date, including those encoded by the key regulatory genes CHS, AOC, GLIP, and ALDH (Kikuta et al., 2012; Xu et al., 2018a). Therefore, we first analyzed the promoter elements of these key genes involved in pyrethrins synthesis using the PlantCARE and PlantPAN3.0 databases ( Figure 1B ), revealing several hormone-responsive elements, including those responding to MeJA, ABA, and salicylic acid (SA). This analysis indicated that MeJA treatment might play a role in pyrethrins synthesis.

Using our previously established transgenic platform for CHSpro-green fluorescent protein (GFP) and GLIPpro-b-glucuronidase (GUS) reporter genes (Sultana et al., 2015), we evaluated the MeJA-induced upregulation on the CHS and GLIP promoters individually. After treatment with 300 μM MeJA, the specific GFP expression level was measured by real-time polymerase chain reaction (PCR) and GUS activity was measured. Consequently, the GFP gene driven by the CHS promoter and the GUS gene driven by the GLIP promoter were both significantly activated at 12 h after MeJA treatment ( Figure 1C ), confirming that the CHS and GLIP promoters are MeJA-inducible, which will ultimately lead to enhanced pyrethrins production.

Indeed, in our previous study, we found that the pyrethrins content increased for a short time under high-concentration (2 mM) treatment of MeJA (Zeng et al., 2022). However, treatment of such a high concentration of MeJA leads to a dramatic decline in the pyrethrins content after 6 h due to disturbed homeostasis or metabolites feedback. Thus, in the present study, we treated T. cinerariifolium plants with a reduced concentration of MeJA of 300 μM. As expected, T. cinerariifolium plants showed a higher yield of pyrethrins after MeJA treatment, including all six constituents. The pyrethrins content increased by 2-fold to 0.99 mg/g per fresh weight at 12 h after MeJA treatment, and this accumulation level was maintained for 4 days ( Figure 1D ). These results strongly suggested that pyrethrins biosynthesis genes are MeJA-inducible and that the optimal working concentration of MeJA treatment could maintain a high pyrethrins yield for several days.

Considering that many E-box and G-box elements that bind to members of the bZIP TF family were identified in the promoter regions of pyrethrins biosynthesis genes, we searched for genes encoding putative bZIP TFs in the transcriptome data of T. cinerariifolium. Since we found that MeJA treatment can activate the CHS and GLIP promoter-driven reporter genes and improve pyrethrins production, we selected Unigene27160, which showed the highest expression level induced by MeJA, as the candidate bZIP TF ( Figure 2A ).

To identify the Unigene27160 gene in the T. cinerariifolium genome, the sequence was input as a query object in The Arabidopsis Information Resource (TAIR). In the phylogenetic comparison between Unigene27160 and members of the Arabidopsis bZIP TF family, Unigene27160 clustered with Arabidopsis AtbZIP60 and AtHY5 in the same evolutionary branch with high homology, suggesting that these genes exhibit similar functions ( Figure 2B , blue box). Unigene27160 showed the closest relationship with AtbZIP60 ( Figure 2C ). Moreover, 10 bZIP60 proteins from different plant species were selected for further comparative sequence analysis. These proteins clustered into five branches, represented by boxes with different colors in Figure 2D . Unigene27160 clustered with NabZIP60, NbbZIP60, and AtbZIP60, and contained a conserved bZIP domain similar to that of other bZIP proteins ( Figure 2E ). Thus, Unigene27160 was named TcbZIP60, and its open reading frame region was cloned for further analysis.

Since TcbZIP60 expression was most strongly induced by MeJA, we confirmed this result by real-time PCR and further investigated whether other hormones could also induce the expression of TcbZIP60. As expected, treatment with both MeJA and ABA strongly induced TcbZIP60 expression, and high-level expression was maintained for 24 h ( Figures 3A, B ). To confirm that TcbZIP60 is involved in pyrethrins biosynthesis, the relative expression levels of TcbZIP60 and other pyrethrins biosynthesis genes were analyzed at different flowering stages. The expression profiles were similar, with high expression detected in the first two stages ( Figure 3C ). Based on these results, we speculated that the TF TcbZIP60 regulates pyrethrins synthesis through interaction with the pyrethrins synthase gene.

To determine the subcellular localization of TcbZIP60, the coding sequence of TcbZIP60 was fused into the GFP framework under control of the CaMV35S promoter. When TcbZIP60-GFP was instantaneously expressed in Nicotiana benthamiana leaves, strong and specific fluorescence was observed in the nucleus ( Figure 3D ). In addition, the co-localization of the GFP signal and red fluorescent protein (as a nuclear localization signal) resulted in a fused yellow fluorescence signal ( Figure 3E ). These results suggested that TcbZIP60 is a nuclear-localized protein, further supporting its potential role as a TF.

The binding sites of bZIP TFs were predicted in the promoter sequence of the pyrethrins synthesis genes ( Figure 1B ). In T. cinerariifolium, similar expression profiles were observed between TcbZIP60 and pyrethrins synthesis genes during different flowering stages ( Figure 3C ). Therefore, we further speculated that TcbZIP60 can regulate the expression of these genes by directly binding to the corresponding promoters. To test this hypothesis, we performed a yeast monohybrid (Y1H) experiment. The promoters of pyrethrins synthesis genes were cloned and inserted into the pHis2.1 vector to generate reporter genes. TcbZIP60 was fused to the GAL4 activation domain to generate the effector construct pGADT7-TcbZIP60 ( Figure 4A ).

In the interaction experiments with several pyrethrins synthesis gene promoters, yeast clones were observed on solid synthesized Leu, Trp, and His (SD/-Leu-Trp-His) media only when pGADT7-TcbZIP60 was transferred into yeast cells expressing pHis2.1-TcCHS and pHis2.1-TcAOC, but not in those expressing pHis2.1. This suggested that TcbZIP60 binds directly to the promoters of TcCHS and TcAOC ( Figure 4B ). In addition, we hypothesized that TcbZIP60 directly binds to the motifs on the promoters of the two genes, which was further verified by electrophoretic mobility shift assays (EMSAs).

We predicted that TcbZIP60 binds to the potential E-box and G-box sites on the two promoters, and designed the corresponding mutation sites ( Figure 4C ). The migration bands were detected in the presence of the purified and concentrated TcbZIP60 protein and the labeled probe of the E-box containing the TcCHS promoter or the G-box containing the TcAOC promoter ( Figure 4D ). When 50 times the concentration of the cold probe (unlabeled probe) was added, the intensity of the migration band decreased. When the mutant probe (unlabeled probe) was added, the intensity of the migration band recovered.

To further verify the interaction between TcbZIP60 and the TcCHS or TcAOC promoter in plants, we performed a transient dual-luciferase (dual-LUC) assay with a reporter structure (TcCHS/TcAOC Pro : LUC) and effector structure ( Figure 4E ). Strong LUC activity was observed in tobacco leaves co-transformed by TcCHS/TcAOC Pro : LUC and 35S:TcbZIP60. However, almost no fluorescence signal was detected in tobacco leaves co-transformed with TcCHS/TcAOC Pro : LUC and an empty carrier ( Figure 4F ). These results suggested that the TF TcbZIP60 directly activates promoters of the TcCHS and TcAOC genes and regulates pyrethrins biosynthesis in T. cinerariifolium.

Previous studies demonstrated that bZIP TFs in A. annua bind to gene promoters and regulate artemisinin biosynthesis (Hao et al., 2019; Lv et al., 2019). Similarly, we confirmed that TcbZIP60 can directly bind to the promoters of the pyrethrins biosynthesis genes TcCHS and TcAOC. Therefore, to further verify the role of TcbZIP60 in regulating pyrethrins biosynthesis, we constructed TcbZIP60-overexpressing plants (TcbZIP60-OE) and TcbZI60-suppressed plants (pTRV2-TcbZIP60) in T. cinerariifolium. Transient overexpression and transient RNA interference-mediated silencing of TcbZIP60 were performed in the leaves of T. cinerariifolium, and the expression of the pyrethrins biosynthesis genes was detected by reverse transcription-quantitative PCR after transformation. Compared with wild-type (Mock) plants of pGreenII 62-SK, the transcription level of TcbZIP60 in TcbZIP60-OE plants significantly increased by 49.65 times, and the expression levels of the pyrethrins synthesis genes TcCHS and TcAOC also increased by 1.76 times and 1.88 times, respectively. The other two pyrethrins biosynthesis genes, TcALDH and TcGLIP, were also upregulated with TcbZIP60 overexpression ( Figure 5A ). By contrast, in the TcbZIP60-silenced plants, the transcription level of TcbZIP60 decreased by 0.8-fold ( Figure 5B ) and the expression levels of four pyrethrins synthesis genes also decreased ( Figure 5C ).

Subsequently, we measured the contents of pyrethrins in the transient overexpression and RNA interference plants. We used the pyrethrins standard to compare and identify the six main components of pyrethrins in the sample as shown by the arrow and the black line in Figures 5E, F , whereas the red and green lines represent the HPLC results of the experimental group and the control group, respectively. Compared with the pGreenII 62-SK Mock plants in the blank control, the total amount of pyrethrins in the overexpression plants significantly increased by 1.239 times ( Figures 5B, E ) and decreased by 0.273 times in the TcbZIP60 interference plants ( Figures 5D, F ). These results showed that TcZIP60 can positively regulate pyrethrins accumulation by activating pyrethrins biosynthesis genes.