The lotus variety “Qiuxing” was rhizome propagated in pots of 40 cm diameter and 40 cm height in late April. All pots were set outdoor on a flat ground at the Wuhan Botanical Garden of the Chinese Academy of Sciences (Wuhan, Hubei province, China). Routine water and fertilization management were applied to all the plants during the growth season. Nicotiana benthamiana plants were cultivated in the growth room under controlled conditions: day/night temperature, 24/22°C; day/night length, 16/8 h; light intensity, 250 μmol m⁻²s⁻¹; and relative humidity, 60%.

For JA treatment, methyl jasmonate (MeJA, 100 μM) was exogenously applied on lotus leaves at developmental stage 3 (S3) (Deng et al., 2016) and then wrapped with transparent plastic bags. A final concentration of 100 μM MeJA was prepared by first dissolving 11.2 μL MeJA (4.4 M) in 488.8 μL pure ethanol to make a 100 mM (1000× ) MeJA stock solution, which was then diluted with deionized water to the desired concentration. Leaf samples were collected at 0, 3, 6, and 24 h time intervals after MeJA treatment. For a tissue-specific expression analysis, eight lotus organ samples, including, root, rhizome, leaf, petal, embryo, seedpod, petiole, and stamen, as well as leaves at seven developmental stages (S1–S7) were harvested in July. All the samples were frozen immediately in liquid nitrogen and then stored at −80°C until use. The BIA alkaloids in the lotus were extracted and quantified as previously described (Deng et al., 2016).

The protein sequences of all WRKYs previously reported to be involved in secondary metabolism were obtained from the National Center for Biotechnology Information (NCBI) GenBank. The genomic and coding sequences of AtWRKY70 were retrieved from the TAIR database (https://www.arabidopsis.org/). The sequences of lotus genes were PCR amplified from the lotus variety “Qiuxing” and have been deposited in the GenBank with the following accession numbers: NnWRKY70a, OL469000; NnWRKY70b, OL469001; NnWRKY53b, OL468999; and NnJAZ1, OL469002. The full-length protein sequences were aligned with MUSCLE (Edgar, 2004), followed by a phylogenetic analysis in MEGA7.0 (Kumar et al., 2016) with a neighbor-joining method and 1,000 bootstrap replicates. The tree was finally viewed and modified with FigTree V1.4.2.

Total RNA was extracted using the RNAprep Pure Plant Kit (Tiangen Biotec, Beijing, China), and cDNA was synthesized with the cDNA Synthesis SuperMix (TransGen, Beijing, China). The quantitative real-time PCR was carried out using SYBR^® Premix Ex Taq™ II (Takara, Dalian, China) on a StepOnePlus^TM Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The 2^−ΔΔCt method was used to calculate the relative gene expression (Pfaffl, 2001), and the NnACTIN gene was used as an internal reference for the normalization of gene expression levels (Gu et al., 2013). The primers used for real-time PCR are listed in Supplementary Table S1.

The full-length coding sequences of NnWRKY70a and NnWRKY70b without the stop codon were amplified and inserted into the pMDC83 vector to generate CaMV 35S:NnWRKY70a-GFP and 35S:NnWRKY70b-GFP constructs. The empty pMDC83-GFP vector was used as a control. The recombinant constructs were transformed into the Agrobacterium GV3101 strain. Agro-infiltration was conducted on fully expanded leaves of 5–6-weeks-old N. benthamiana plants. The subcellular localization of fluorescent protein fusions was checked 2 days after the inoculation. A 5 μg/mL DAPI nuclear marker was infiltrated into the same area of agro-infiltration 30 min before sampling. The images were taken with the Zeiss Confocal Fluorescence Microscope (LSM710 Meta, Carl Zeiss) as previously reported (Li et al., 2019). The primers used for cloning subcellular localization vectors are listed in Supplementary Table S1.

The promoter regions spanning about 1.5–2 kb of NnNCS1, NnTYDC1, NnCYP80G, and Nn7OMT genes were PCR amplified and inserted into the dual-luciferase (LUC) reporter gene expression vector pGreen 0800-LUC to form NnNCS1pro::LUC, NnTYDC1pro::LUC, NnCYP80Gpro::LUC, and Nn7OMTpro::LUC constructs. The complete coding sequence (CDS) of NnWRKY70a and NnWRKY70b genes were cloned into the pSAK277 vector under the 35S promoter to form the TF expression vectors. Agrobacterium transformation, agro-infiltration, and the measurements of Firefly luciferase (F-Luc) and Renilla luciferase (R-Luc) activities were performed as previously described (Deng et al., 2018). The primers used in the dual-luciferase reporter assay are listed in Supplementary Table S1.

The yeast one-hybrid assay was conducted according to the instructions of Matchmaker^® Gold Yeast One-Hybrid Library Screening System User Manual (Clonteck, USA). The promoter sequences of approximately 1.5-kb upstream of the BIA pathway gene start codon were PCR amplified and cloned into pAbAi vectors to generate bait-reporter yeast strains, while whole coding sequences of NnWRKY70a and NnWRKY70b were cloned into the pGADT7 vector to generate pGADT7-TF constructs. The minimal inhibitory concentration of Aureobasidin A (100 ng/mL) was determined with different bait-reporter yeast strains transformed with empty pGADT7 AD vectors. To evaluate whether NnWRKY70a and NnWRKY70b interact with BIA pathway gene promoters, pGADT7-TF plasmids were transformed into Y1HGold bait strains carrying different promoters and cultured on SD/-Leu containing 100 ng/mL AbA. Y1HGold bait strains transformed with empty pGADT7 were set as negative controls, while Y1HGold (p53-AbAi) transformed with pGADT7-p53 was set as a positive control. To check the specific binding of NnWRKY70a and NnWRKY70b to the W-box cis-elements, the promoter mutants were generated with overlap extension PCR to remove all W-box cis-elements.

For the yeast two-hybrid library screening, a lotus “Mate & Plate” library was constructed in Y187 yeast strain according to the “Make Your own Mate & Plate Library System User Manual” (Clonteck, USA). cDNA for the library was generated from a tissue mixture including leaves, petals, and embryos of the lotus variety “China Antique”. The library screening and yeast two-hybrid assays (Y2H) were conducted as described in the “Matchmaker Gold Yeast Two-Hybrid System User Manual” (Clonteck, USA). Truncated NnWRKY70b coding sequence harboring the WRKY domain was PCR amplified and cloned into the pGBKT7 (BD) vector as a bait to avoid autoactivation, while the full-length CDS of NnWRKY70a, NnWRKY70b, NnWRKY53b, and NnJAZ1 were cloned into the pGADT7(AD) vector as a prey. Combinations of the different prey vectors and BD-NnWRKY70b bait were co-transformed into the Y2H Gold yeast strain. Meanwhile, the combinations of BD-53 + AD-T and BD-Lam + AD-T were co-transformed as positive and negative controls, respectively. The yeast cells were cultured on DDO (SD/-Leu/-Trp) selective medium and on TDO (SD/-Leu/-Trp/-His) and QDO (SD/-Leu/-Trp/-His/-Ade) media for interaction assays. The primers used for cloning Y1H and Y2H vectors are listed in Supplementary Table S1.

Transient overexpression of NnWRKY70s was conducted in the lotus petals of the “Qiuxing” variety 2 days before blooming. Agrobacterium GV3101 strain carrying the pMDC83:NnWRKY70a and pMDC83:NnWRKY70b vectors were infiltrated on the lower side of petals at OD600 = 0.5, using a 1 mL needleless syringe. The lotus petals were sampled 2 days after infiltration for subsequent gene expression analysis and determination of alkaloid content.

BiFC assay was conducted with a 2-in-1 system based on the splitting enhanced yellow fluorescent protein (EYFP) as previously described (Grefen and Blatt, 2012). Coding DNA sequences of two independent genes were gateway cloned into two different expression cassettes on the pBiFCt-2in1-CN vector. To construct the NnWRKY70b:nYFP fusion protein expression cassette, a termination code of NnWRKY70b was dropped out. Agrobacterium tumefaciens strain GV3101 carrying different constructs was infiltrated in N. benthamiana leaves. Yellow and red fluorescence was observed with Confocal Fluorescence Microscope as described in the subcellular localization assays.

Previously, two independent studies have identified NnWRKY70a (Nnu_24385) and NnWRKY70b (Nnu_12194) as key candidate regulators of the lotus BIA biosynthesis based on a positive correlation between their expression and BIA accumulation in lotus (Deng et al., 2018; Meelaph et al., 2018). To evaluate the possible roles of these two WRKYs in regulating the lotus BIA biosynthesis, we first analyzed their nucleotide and amino acid sequence characteristics. NnWRKY70a was found to be located on the lotus chromosome 5, which contains a 981 nucleotide (nt) open reading frame (ORF), encoding a protein with a calculated molecular weight of 36.5 kDa. NnWRKY70b was located on chromosome 2, which has a 990 nt ORF, encoding a 37.06 kDa protein. Pairwise sequence alignment showed that the two NnWRKY70s shared high-sequence identity, with nucleotide and amino acid sequence similarity of 70.6 and 74.4%, respectively.

A phylogenetic analysis was conducted using two NnWRKY70s and other previously characterized WRKYs involved in the regulation of plant secondary metabolism (Supplementary Table S2). Two NnWRKYs clustered together with group III members (Figure 2A). NnWRKY70s showed the highest sequence similarity with the AtWRKY70, followed by CrWRKY1 and OpWRKY1. Both CrWRKY1 and OpWRKY1 are group III WRKYs involved in regulating MIA biosynthesis in Catharanthus roseus and Ophiorrhiza pumila, respectively (Suttipanta et al., 2011; Xu et al., 2020). Similar to the other group III WRKYs, both NnWRKY70s contained a core WRKYGQK heptapeptide and a conserved CX₇CX₂₃HXC zinc-finger motif in their WRKY domains (Figure 2B). Therefore, NnWRKY70a and NnWRKY70b were both typical group III WRKY proteins similar to AtWRKY70.

Next, we analyzed the spatial expression patterns of NnWRKY70 genes in the lotus variety “Qiuxing” by quantitative real-time PCR. The two WRKY70s showed quite distinct expression patterns (Figure 3A). Overall, NnWRKY70b was predominantly expressed in the lotus root, followed by the rhizome and leaf. In contrast, NnWRKY70a had the highest transcript levels in the lotus leaf, where mono-BIA are primarily accumulated.

Their expression profiling was also evaluated in seven lotus leaf developmental stages (Figure 3B). Two lotus NnWRKY70 genes showed quite similar expression patterns, with both consistently increasing throughout the tested developmental stages. This expression pattern followed well with our previously reported BIA accumulation pattern in lotus leaves (Deng et al., 2016). In addition, we carried out a subcellular localization assay by fusing the coding regions of NnWRKY70s with a GFP reporter (Figure 3C). The GFP fluorescence of both NnWRKY70a:GFP and NnWRKY70b:GFP fused proteins co-localized with the DAPI-stained nucleus, demonstrating an obvious nuclear localization of both NnWRKY70a and NnWRKY70b proteins.

JA is known to trigger most biosynthetic pathways leading to secondary metabolites by activating the JA-responsive TFs (Wasternack and Strnad, 2019; Yamada et al., 2020). To verify whether NnWRKY70s are JA responsive, we checked their expression in the MeJA-treated samples, wherein the lotus leaves were treated with 100 μM MeJA at the developmental stage 4 (Li et al., 2019). MeJA significantly induced both NnWRKY70 genes, with their expression increasing approximately 5-fold at 24 h after treatment (Figure 4A). This was consistent with the increased BIA content in the lotus leaves, a 15–30% increase between 3 and 6 h, and an approximately 50% increase at 24 h after the MeJA treatment (Li et al., 2019).

Next, we screened the promoter regions of the BIA biosynthetic genes, including NnTYDC1, NnNCS1, NnCYP80G, and Nn7OMT, for potential binding sites of WRKY TFs. Conserved W-box (TTGACT) was found in the 2 kb promoter regions of NnTYDC1, NnCYP80G, and Nn7OMT, but not of the NnNCS1 gene. To further confirm whether NnWRKY70a and NnWRKY70b could activate the three BIA gene promoters, a dual-luciferase assay was conducted. Transient overexpression of NnWRKY70a significantly enhanced the pNnTYDC1-driven firefly luciferase transcription but had no effect on the other three promoters (Figure 4B). In contrast, transient overexpression of NnWRKY70b was able to activate three promoters, including pNnTYDC1, pNnCYP80G, and pNn7OMT2 (Figure 4C).

To further test whether NnWRKY70a and NnWRKY70b bind directly to these promoters, we conducted the yeast one-hybrid assays for putative BIA structural gene promoters and their corresponding mutants (with W-box removed). NnWRKY70b was bound to pNnTYDC1 and pNnCYP719A and weakly bound to pNnCYP80G and pNn7OMT (Figure 4D). In contrast, NnWRKY70a was bound obviously to the NnCYP80G and NnCYP719A promoters, while weakly bound to the NnTYDC1 and Nn7OMT promoters. However, neither NnWRKY70b nor NnWRKY70a bound to the NnNCS1 promoter. Markedly, when the W-boxes were removed from these promoters, none of them were bound by NnWRKY70a or NnWRKY70b, indicating a specific binding of NnWRKY70a or NnWRKY70b to the W-box cis-elements in the BIA biosynthetic gene promoters. Taken together, these results suggest that both NnWRKY70a and NnWRKY70b can bind and activate the BIA pathway gene promoters.

To determine in planta whether NnWRKY70a and NnWRKY70b activate the BIA biosynthesis in lotus, an Agrobacterium-mediated transient overexpression assay was conducted. Given that the lotus leaves were almost unable to be infiltrated, we performed agroinfiltration in the lotus petals in the “Qiuxing” variety, which is known to accumulate moderate levels of BIAs (Deng et al., 2016, 2022). As a result, a significant 4.2-fold increase in the transcript levels of NnWRKY70a was observed in the petals infiltrated with Agrobacterium carrying an overexpression vector (Figure 5A). Similarly, an approximately 2-fold increase in the expression of NnWRKY70b was observed in the NnWRKY70b overexpressing petals (Figure 5B). Interestingly, the overexpression of either NnWRKY70a or NnWRKY70b significantly promoted the transcription of the other members (Figures 5A,B), indicating a possible inter-activation ability between the two NnWRKY70s.

Moreover, the overexpression of the two NnWRKY70s enhanced the BIA contents in the lotus petals (Figures 5A,B). Petals overexpressing NnWRKY70a had approximately 60% increase in the total BIA content, whereas those overexpressing NnWRKY70b exhibited an even higher increase of about 126%. Consistently, increased BIA contents in petals overexpressing NnWRKY70s were accompanied by significant elevation of BIA pathway genes. Of the six tested BIA biosynthetic genes, three, including NnCYP80G, NnCNMT, and Nn7OMT, were markedly enhanced in the petals overexpressing NnWRKY70a, with approximately 8.1-, 4.4-, and 11.4-fold increase in the expression, respectively (Figure 5A). In contrast, a more dramatic increase in the gene expression was observed in the petals overexpressing NnWRKY70b, with NnTYDC1, NnCYP80G, NnCNMT, Nn7OMT, and NnCYP719A genes exhibiting approximately 151.4-, 2.2-, 3.9-, 9.7-, and 309.3-fold increase in their expression, respectively (Figure 5B). This further revealed that NnWRKY70b overexpression could induce a more dramatic increase in the BIA accumulation.

It was known that lotus petals mainly accumulate five types of BIAs, nuciferine, N-nornuciferine, O-nornuciferine, roemerine, and anonaine, with nuciferine as a predominant one (Deng et al., 2016). Of the six tested BIA pathway enzymes, NnTYDC1, NnNCS1, NnCYP80G, and NnCNMT are located upstream and are involved in the biosynthesis of all the five BIAs in lotus petals (Figure 1). In contrast, Nn7OMT is especially involved in the production of nuciferine, N-, and O-nornuciferine, whereas NnCYP719A mainly catalyzes the production of roemerine and anonaine. An HPLC analysis detected three BIA peaks corresponding to N-nornuciferine (peak 1), O-nornuciferine (peak 2), and nuciferine (peak 4) in the uninfiltrated “Qiuxing” petals (Figure 5C). The overexpression of NnWRKY70s significantly increased the accumulation of all the three detected BIAs (Supplementary Figure S1). Interestingly, the two additional peaks, representing anonaine (peak 3) and roemerine (peak 5), respectively, were evidently detected (Figure 5C; Supplementary Figures S1A,B). This is obviously due to the significant elevation of NnCYP719A expression in the lotus petals. Taken together, our results demonstrate that both NnWRKY70a and NnWRKY70b are involved in the BIA biosynthesis in planta.

As shown above, NnWRKY70a and NnWRKY70b might interregulate the expression of each other. Previous studies also showed that the group II and III WRKY members in Arabidopsis often function by forming homodimers or heterodimers with other WRKY TFs (Xu et al., 2006; Besseau et al., 2012). To investigate the possible interaction between NnWRKY70s and other proteins in regulating the lotus BIA biosynthesis, we performed a yeast two-hybrid cDNA library screen assay. NnWRKY70b was selected as a bait due to its significant ability to regulate the BIA biosynthesis. The bait construct harboring full-length NnWRKY70b displayed a strong autoactivation activity. Thus, truncated NnWRKY70b bait was used to screen the library. In this bait, the WRKY and zinc-finger domains were kept, while its activation domain was dropped out (Figure 6A). As a result, a total of 32 proteins potentially interacting with NnWRKY70b were identified (Supplementary Table S3). Of these interactions, NnWRKY70b vs. NnWRKY70a, NnWRKY70b vs. NnWRKY53b, and NnWRKY70b vs. NnJAZ1 were proved to be genuine during the subsequent yeast two-hybrid assays.

The truncated NnWRKY70b bait could not interact with NnWRKY70b itself but positively interacted with NnWRKY70a, and another group III type protein NnWRKY53b (Figure 6B). NnWRKY70b also interacted with NnJAZ1, a lotus JAZ repressor protein. Both NnWRKY53b and NnJAZ1 were also JA responsive and displayed similar expression patterns as NnWRKY70a and NnWRKY70b under the JA treatment (Figures 6C,D). These interactions were also confirmed in vivo by the BiFC assays. The EYFP fluorescence was detected in the living N. benthamiana epidermal cells infiltrated with pBiFCt-2in1 vectors carrying coding sequence of NnWRKY70b and NnWRKY70a, NnWRKY70b and NnWRKY53b, and NnWRKY70b and NnJAZ1 (Figure 7).

Next, we evaluated whether NnWRKY53b interacts with NnJAZ1 and the capacity of NnWRKY53b to activate BIA pathway gene promoters. The yeast two-hybrid assay showed that NnWRKY53b did not physically interact with NnJAZ1 (Supplementary Figure S2). A transient trans-activation assay in tobacco indicated that NnWRKY53b alone can activate pNnTYDC1 (Supplementary Figure S3). A combination of NnWRKY70b and NnWRKY53b, however, did not show enhanced activation abilities on BIA gene promoters, in compared to either of the singletons. These results suggest NnWRKY70b may regulate the lotus BIA biosynthesis through the JA-signaling pathway. The interaction between NnWRKY70b and NnWRKY53b, however, did not enhance the BIA biosynthesis.