Seeds of C. roseus cv. Rose Red were germinated in a greenhouse at 28°C, 16/8 h photoperiod on MS plates. After 4 weeks, seedlings with two to three pairs of true leaves were transferred to soil. Samples were collected from 1-month-old plants which were treated with MeJA for 0, 1 or 4 h. The MeJA experiment was with three replicates. Three plants were combined from each replicate time sample. MeJA treatment consisted of spraying 100 μM MeJA then placing plants under a clear plastic dome sealed with tape. Leaves of the plants were harvested and then frozen in liquid nitrogen.

Total RNA extracted from each material were subjected to cDNA library preparation and sequenced at Novogene Bioinformatics Institute (China) on Illumina HiSeq^TM 2000 sequencer (Illumina Inc., United States). Clean reads were aligned based on the reference genome (Medicinal Plant Genomics Resource, MPGR) (Kellner et al., 2015), transcript abundant estimation and expression level in fragment per kilobase exon per million mapped fragment (FPKM) were performed as described by Chen et al. (2017).

Full-length amino acid sequences of candidate transcription factor as well as the AP2/ERFs which are potentially involved in secondary metabolite biosynthesis were aligned by ClustalW program. The neighbor-joining phylogeny was generated using MEGA 6 with bootstrap analysis of 1,000 replicates.

Seeds of C. roseus cv. Rose Red were germinated in a greenhouse at 28°C, 16/8 h photoperiod. Four weeks later, plants with two to three pairs of true leaves were used for VIGS. The genomic DNA of C. roseus cv. Rose Red was used as template to amplify CR1 with specific primers (Table 1). The PCR-generated fragment was then introduced into the terminal vector pTRV2 by BamH I and Xho I digestion. Catharanthus phytoene desaturase (PDS) involved in chlorophyll biosynthesis produces visible photo bleaching and serves as a useful marker for determining the best time to collect samples for testing. The purified plasmid DNA pTRV2-CR1, pTRV2 (negative control), pTRV2-PDS were respectively transformed into Agrobacterium tumefaciens strain GV3101 by electroporation. Three kinds of strains and GV3101 harboring pTRV1 were cultured overnight at 28°C in 300 ml Luria-Bertani medium containing 10 Mm MES, 20 μM acetosyringone and 50 μg/mL kanamycin. These bacterial pellets were collected and then resuspended in 5 ml infiltration buffer (10 mM MES, 200 μM acetosyringone and 1 mM MgCl₂), and further incubated at 28°C for 3 h with shaking. The suspension of strain harboring pTRV1 was mixed with pTRV2-CR1, pTRV2 or pTRV2-PDS in equal volume just before the infection. The leaves of the seedlings were injected by a needle less syringe to fill the whole leaf with the bacteria, and each seedling was injected with 2–4 leaves. After injection, the plants were cultured in a constant temperature incubator. The PDS phenotype was observed 3 weeks after inoculation of leaves, and the other two sample plants were harvested at this stage. After recording the fresh weights of harvested materials, the samples were frozen in liquid nitrogen, one member of a leaf pair was used for RNA extraction, while the other was used for metabolite analysis.

Total RNAs of CR1-silenced plants and control were extracted and reverse-transcribed as described previously (Wang et al., 2015), and qRT-PCR was performed according to the same reference. The primers of CR1, CR2 and CR3 as well as 7 genes whose expression levels were detected in our work are shown in Table 1. Relative expression levels were determined using 2^-ΔΔC_T method (Livak and Schmittgen, 2001). All qRT-PCR analyses were performed with three independent biological replicates.

The selected samples were quickly pulverized in liquid nitrogen. Precisely weighed 0.3 g powder, mixed with 2 mL methanol, then subjected to extraction using the ultrasonic for 20 min, the supernatant was obtained after centrifugation. Repeatedly extracted with methanol for three times, then combined supernatant extract and compressed to dry, diluted to 2 mL with methanol, finally, filtrated with 0.45 μm microporous membrane before metabolite analyses by MS. The three standards catharanthine, vindoline and vinblastine were configured as a reserve solution of 10 ng/L with methanol, stored at -20°C, diluted to 10 ng/mL working fluid at the same time for testing purposes. ESI/MS/MS was used to detect the catharanthine, vindoline and vinblastine; using time-of-flight mass spectrometry (TOF-MS) to detect the serpentine.

MeJA is identified as an important signaling molecule in TIA biosynthetic pathway in many plants (De Geyter et al., 2012). In order to excavate candidate transcription factors that may be involved in the regulation of TIA biosynthesis, we collected the leaves of 1-month-old C. roseus with MeJA treatment and extracted their RNA to generate the transcriptome sequencing data using Illumina platform. Subsequently, the expression profiles of different transcription factor families in C. roseus after MeJA treatment were analyzed. The expression profile of AP2/ERF family indicated that the expression levels of many AP2/ERF genes were changed obviously at 1-h and 4-h after MeJA treatment (their annotated gene names are shown in Supplementary Table S1). Three candidate AP2/ERF genes with different representative response to MeJA were selected for further studies (Figure 1). The expression level of Unigene9835_ALL (here designated as CR1) gradually decreased along the treatment. The expression level of Unigene2253_ALL (here designated as CR2) gradually increased after MeJA treatment. The transcript of Unigene1189_All (here designated as CR3) was up-regulated dramatically at 1-h and 4-h after MeJA treatment, while the expression level at 4-h was slightly lower than at 1-h. The expression pattern suggested that CR3 is sharply induced by MeJA in a short time, and its expression level might gradually restore to the initial level. Above genes show different responses to MeJA treatment, and may be involved in the regulation of TIAs synthesis in C. roseus.

In order to determine the accuracy of the RNA-seq data, the expression levels of three candidate genes at different time point after MeJA treatment were confirmed by qRT-PCR. Figure 2 shows that after MeJA treatment, the expression level of CR1 decreased gradually with time; CR2 showed gradually increased transcript along MeJA treatment; the expression level of CR3 was up-regulated rapidly and reached a peak at 2-h, then gradually declined. According to the results, expression patterns of three candidate genes under MeJA treatment were consistent with the RNA-seq data. Hence, the above candidate genes were subjected to further study.

A phylogenetic tree was constructed based on the protein sequences of the three candidate transcription factors (for their sequences, see Supplementary Table S2), together with the previously reported AP2/ERF transcription factors which are potentially involved in secondary metabolite biosynthesis, to analyze their evolutionary relationships (Figure 3). CR1 was classified to the same group with CR2, CR3 and OpERF2. Besides, CR1 showed the closest relationship with OpERF2 which was identified recently to be involved in the regulation of specialized metabolism in Ophiorrhiza pumila (Udomsom et al., 2016), while CR2 and CR3 form the same cluster. The divergent clusters as well as the opposite response to MeJA between CR1 and CR2 or CR3 suggest the different molecular functions between CR1 and the other two. ORCA2 and ORCA3 involved in MIA biosynthesis in C. roseus; NbERF1, NtERF189 and NtORC1 regulating nicotine alkaloids biosynthesis; AaERF1, AaERF2 and AaORA regulating terpenoid biosynthesis in Artemisia annua, all of them showed more distant relationship with three candidate transcription factors than OpERF2 (Van der Fits and Memelink, 2001; Rushton et al., 2008; Shoji et al., 2010; Todd et al., 2010; De Boer et al., 2011; Yu et al., 2012; Lu et al., 2013; Thagun et al., 2016). Since CR1 showed a close phylogenetic relationship with other secondary metabolite-regulating AP2/ERFs, which suggested that CR1 may be a potential regulator in TIA biosynthesis, we selected CR1 for further functional characterization.

Virus-induced gene silencing is a technique used for functional analysis of genes, which can be effectively applied in plants with long growth cycle and low conversion efficiency. The utility of this approach has been successful in exploring new genes that were involved in iridoid biosynthesis (Geu-Flores et al., 2012) and MIA biosynthesis (Liscombe and O’Connor, 2011) in C. roseus. In order to identify the function of CR1, we selected VIGS technology with the pTRV vector system (Dinesh-Kumar et al., 2003; Burch-Smith et al., 2004; Velásquez et al., 2009).

Transcript analysis of CR1 monitored by qRT-PCR revealed that the transcript level decreased by approximately 78% in CR1-silenced plants compared with the negative control (Figure 4). Subsequently, expression analysis of 7 genes (G10H, SLS, TDC, STR, SGD, DAT and PRX) which are representative in the TIAs synthetic pathway (Figure 5) in CR1-silenced plants was carried out. Their GenBank accession numbers are as follows: KF561461.1, L10081.1, X67662.1, X53602.1, EU072423.1, AF053307.1 and AM236087.1. The results indicated that silencing CR1 in C. roseus could result in obvious changes in the expression levels of these genes. The expression levels of G10H, SLS, TDC, STR, SGD, DAT and PRX showed significant up-regulation (Figure 6). According to the previous results which suggest that the expression level of CR1 is down-regulated in response to MeJA treatment, it can be inferred that CR1 may be a new negative regulatory transcription factor in the pathway of TIA biosynthesis.

ESI/MS/MS was used to detect the catharanthine and vindoline levels; time-of-flight mass spectrometry (TOF-MS) was applied to detect the serpentine. The accumulations of catharathine and vindoline in the leaves of CR1-silenced plants were 0.191 nmol/L and 0.153 nmol/L, compared to 0.198 nmol/L and 0.046 nmol/L in negative control plants (Figure 7). According to the results, silencing CR1 could increase the accumulation of vindoline, but not catharanthine. Based on the peak area of serpentine detected by TOF-MS (Figure 8), we found that the accumulation of serpentine in CR1-silenced samples was higher than that in negative control. These results are consistent with the changes of the expression levels of related genes in CR1-silenced plants, which confirmed the inference that CR1 could regulate the synthesis of TIAs with negatively regulatory function in C. roseus.