Cap ‘n’ Collar C and Aryl Hydrocarbon Receptor Nuclear Translocator Facilitate the Expression of Glutathione S-Transferases Conferring Adaptation to Tannic Acid and Quercetin in Micromelalopha troglodyta (Graeser) (Lepidoptera: Notodontidae)

Micromelalopha troglodyta (Graeser) (Lepidoptera: Notodontidae) is a notorious pest of poplar. Coevolution with poplars rich in plant secondary metabolites prompts M. troglodyta to expand effective detoxification mechanisms against toxic plant secondary metabolites. Although glutathione S-transferases (GSTs) play an important role in xenobiotic detoxification in M. troglodyta, it is unclear how GSTs act in response to toxic secondary metabolites in poplar. In this study, five GST gene core promoters were accurately identified by a 5’ loss luciferase reporter assay, and the core promoters were significantly induced by two plant secondary metabolites in vitro. Two transcription factors, cap ‘n’ collar C (CncC) and aryl hydrocarbon receptor nuclear translocator (ARNT), were cloned in M. troglodyta. MtCncC and MtARNT clustered well with other insect CncCs and ARNTs, respectively. In addition, MtCncC and MtARNT could bind the MtGSTt1 promoter and strongly improve transcriptional activity, respectively. However, MtCncC and MtARNT had no regulatory function on the MtGSTz1 promoter. Our findings revealed the molecular mechanisms of the transcription factors MtCncC and MtARNT in regulating the GST genes of M. troglodyta. These results provide useful information for the control of M. troglodyta.


Introduction
Plants are the main food source for animals, while insects serve as important consumers of plants. They constantly fight against each other. From a plant perspective, many plants produce defensive toxins or inhibitors to repel insects, including secondary metabolites such as isoflavones, furanocoumarins, terpenoids, alkaloids and cyanogenic glycosides [1]. From an insect perspective, insects are protected from plant secondary metabolites mainly through increased physiological tolerance, metabolic capacity of the detoxification system, or behavioral avoidance [2]. As insects attempt to increase new host plant species, these mechanisms will continue to evolve [3].
Micromelalopha troglodyta (Graeser), which is mainly found in China, is an important leaf-feeding pest of poplar trees and can be widely spread causing heavy losses to the forestry industry [4,5]. Poplar secondary metabolites such as tannic acid and quercetin, as toxic natural products, have a toxic effect on M. troglodyta [6,7]. It is well known that the powerful detoxification metabolism mechanism of insects is an important way to overcome plant chemicals [8]. There are several enzymes involved in metabolizing heterologous substances and converting them into less toxic hydrophilic compounds. Major enzyme

Induction Effect of Tannic Acid and Quercetin on the Core Region of Two Promoters
Due to the high transcriptional activity of MtGSTt1 (−574/+340) and MtGSTz1 (−350/+521) promoters, we used them as examples to explore the promoter response to tannic acid and quercetin (Figures 2 and 3). The promoter activity of MtGSTt1 (−574/+340) was significantly induced by tannic acid at a low concentration (0.56 mg/L), while it was inhibited by tannic acid at 2.8, 14 and 70 mg/L ( Figure 2A). The MtGSTt1 (−574/+340) promoter showed an increasing and then decreasing trend under quercetin stress, and the highest promoter activity was observed when the quercetin concentration was 7 mg/L ( Figure 2B). These data showed that the promoter activity of the core region of MtGSTt1 (−574/+340) could be induced by tannic acid and quercetin. For MtGSTz1 (−350/+521) promoter, the promoter activity was strongly induced by 2.8 mg/L tannic acid ( Figure 3A), while it was repressed at 70 mg/L tannic acid ( Figure 3A). We also observed that the promoter activity of MtGSTz1 (−350/+521) was notably increased when Sf9 cells were treated with quercetin at 0.28, 1.4 and 7 mg/L and sharply repressed at 35 mg/L ( Figure 3B). These data suggested that tannic acid and quercetin could influence the core region of MtGSTz1 promoter.  Each value is presented as the mean ± SD of three replicates, and different lowercase letters show significant differences (P < 0.05).

Cloning and Phylogenetic Analysis of MtCncC
To explore the regulatory mode of promoters by transcription factors. The CncC gene of M. troglodyta was obtained from the transcriptome database and confirmed by cloning Each value is presented as the mean ± SD of three replicates, and different lowercase letters show significant differences (p < 0.05).

Cloning and Phylogenetic Analysis of MtCncC
To explore the regulatory mode of promoters by transcription factors. The CncC gene of M. troglodyta was obtained from the transcriptome database and confirmed by cloning and resequencing. MtCncC is a 1671 bp open reading frame (ORF) sequence and encodes 556 AA residues. The amino acid sequence of MtCncC is listed in Supplemental Material S1. Twenty-six representative insect CncC amino acid sequences were selected for constructing the phylogenetic tree using the maximum likelihood (ML) method of MEGA X based on the multiple alignment built with Clustal W. The phylogenetic analysis showed that CncCs from different species were classified into six order clusters, and MtCncC was clearly classified into Lepidoptera subclusters, which suggested that MtCncC had a high identity with other lepidopteran insects CncCs ( Figure 4).

Cloning and Phylogenetic Analysis of MtARNT
Another transcription factor ARNT was also identified from the transcriptome database and confirmed by cloning and resequencing. MtARNT is a 1440 bp ORF sequence and encodes 479 AA residues. The amino acid sequence of MtARNT is listed in Supplemental Material S1. The phylogenetic tree of MtARNT was built according to the above method. Phylogenetic analysis showed that MtARNT was grouped into Lepidoptera subclusters and had a high similarity with ARNTs from other insects ( Figure 5).

Cloning and Phylogenetic Analysis of MtARNT
Another transcription factor ARNT was also identified from the transcriptome database and confirmed by cloning and resequencing. MtARNT is a 1440 bp ORF sequence and encodes 479 AA residues. The amino acid sequence of MtARNT is listed in Supplemental Material S1. The phylogenetic tree of MtARNT was built according to the above method. Phylogenetic analysis showed that MtARNT was grouped into Lepidoptera subclusters and had a high similarity with ARNTs from other insects ( Figure 5).

Discussion
GSTs are broadly distributed and important detoxifying enzymes in aerobic organisms that catalyze glutathione (GSH) binding to endogenous and exogenous compounds and excrete them outside cells, thus reducing their damage to the cells [29,30]. In insects, GSTs are a family of multifunctional enzymes involved in the detoxification of toxic compounds, including plant secondary metabolites [4,31,32]. There are six cytoplasmic GST gene families, including epsilon, omega, delta, theta, sigma, and zeta [33]. In a previous study, we demonstrated that all five GST genes of M. troglodyta could be significantly induced by tannic acid, which belong to the omega, delta, theta, sigma, and zeta families. Subsequently, we cloned the 5' flanking promoter sequences of these five GST genes and found that they could be induced by tannic acid [4].
The promoter is a very important regulatory element in gene transcription that determines the pattern and intensity of gene expression [34]. Many inducible promoters have been identified from insects, plants and pathogens to explore the in-depth mechanisms of their regulation [35][36][37]. The promoter of Drosophila heat shock protein (Hsp70) could enhance the expression of Hsp70 more than 200-fold after heat stimulation treatment [36]. Adding a stress-inducible promoter before the DREB1A gene in plants could enhance the drought, high salt and low temperature resistance of transgenic plants [37]. Bombyx mori nucleopolyhedrovirus (BmNPV)-inducible promoters were applied for gene therapy [38]. Currently, the promoters of GST have been reported in a few insects. In Spodoptera litura, the GST promoter acted as an important element for upregulating the expression of GST and improved S. litura tolerance to insecticides [22]. It was reported that the promoters of S. exigua GSTs were coregulated by two transcription factors, which enhanced the resistance of insects to xenobiotic stress [27]. Although we previously obtained five MtGST promoters, their regulatory mechanisms for MtGST genes are not clear. In the present study, we further identified the core regions of the five MtGST promoters in vitro by a 5' loss fragment assay. The activity of the core regions of MtGSTt1 (−574/+340) and Each value is presented as the mean ± SD of three replicates, and lowercase letters show significant differences (p < 0.05).

Discussion
GSTs are broadly distributed and important detoxifying enzymes in aerobic organisms that catalyze glutathione (GSH) binding to endogenous and exogenous compounds and excrete them outside cells, thus reducing their damage to the cells [29,30]. In insects, GSTs are a family of multifunctional enzymes involved in the detoxification of toxic compounds, including plant secondary metabolites [4,31,32]. There are six cytoplasmic GST gene families, including epsilon, omega, delta, theta, sigma, and zeta [33]. In a previous study, we demonstrated that all five GST genes of M. troglodyta could be significantly induced by tannic acid, which belong to the omega, delta, theta, sigma, and zeta families. Subsequently, we cloned the 5' flanking promoter sequences of these five GST genes and found that they could be induced by tannic acid [4].
The promoter is a very important regulatory element in gene transcription that determines the pattern and intensity of gene expression [34]. Many inducible promoters have been identified from insects, plants and pathogens to explore the in-depth mechanisms of their regulation [35][36][37]. The promoter of Drosophila heat shock protein (Hsp70) could enhance the expression of Hsp70 more than 200-fold after heat stimulation treatment [36]. Adding a stress-inducible promoter before the DREB1A gene in plants could enhance the drought, high salt and low temperature resistance of transgenic plants [37]. Bombyx mori nucleopolyhedrovirus (BmNPV)-inducible promoters were applied for gene therapy [38]. Currently, the promoters of GST have been reported in a few insects. In S. litura, the GST promoter acted as an important element for upregulating the expression of GST and improved S. litura tolerance to insecticides [22]. It was reported that the promoters of S. exigua GSTs were coregulated by two transcription factors, which enhanced the resistance of insects to xenobiotic stress [27]. Although we previously obtained five MtGST promoters, their regulatory mechanisms for MtGST genes are not clear. In the present study, we further identified the core regions of the five MtGST promoters in vitro by a 5' loss fragment assay. The activity of the core regions of MtGSTt1 (−574/+340) and MtGSTz1 (−350/+521) promoters were also well induced by low concentrations of tannic acid and quercetin, which suggested that the core sequences of MtGST promoters have significant activity in response to plant secondary metabolites. However, we also found that higher concentrations of tannic acid and quercetin would decrease the activity of luciferase despite that they were not toxic to Sf9 cells. Based on a previous study, when human cells were exposed to xenobiotic stress, low concentrations of xenobiotics induced the promoter activity of pituitary adenylate cyclase-activating polypeptide (PACAP) receptor 1 (PAC1-R) while high concentrations of xenobiotics inhibited PAC1-R promoter activity [39]. Thus, we hypothesized that the relationship between GST promoter activity and plant secondary metabolites (tannic acid and quercetin) also presented a dose-dependent way. Once the concentrations of plant secondary metabolites exceeded the range causing induction, they might inhibit the activity of promoters. These findings provided useful information for understanding the mechanism of GST transcriptional regulation in M. troglodyta.
The transcription factor Nrf2, which is a member of the basic leucine-zipper family, is an oxygen-sensitive transcription factor and a vital physiological stress response mechanism in organisms [40]. Under oxidative stress, Nrf2 can translocate into the nucleus to bind to antioxidant response elements (AREs) and heterodimerize with MafK to regulate the expression of detoxification genes [41]. The mutation of Nrf2 in mice makes it more sensitive to xenobiotic stress [42]. Nrf2 can recognize specific DNA sequences in the presence of nuclear factor-erythroid 2 [43]. CncC in insects is homologous to Nrf2 and is an important transcription factor for regulating detoxification genes. In silkworm, both CncC and detoxification genes (including GSTs and P450s) regulated by CncC were upregulated after phoxim treatment [44]. The CncC-mediated detoxification pathway was associated with oxidative stress in Drosophila, and it was found that CncC could upregulate GSTd expression to enhance the ability to resist oxidative stress [45]. Nrf2 was able to regulate the detoxification enzyme gene CYP6A2 and increase resistance to DTT in Drosophila [46]. In Tribolium castaneum, the transcription factors CncC and Maf could regulate the expression of the CYP6BQ gene and increase resistance to deltamethrin [47]. Based on the results of phylogenetic analysis in this study, MtCncC was highly similar to CncCs from other insects. Therefore, we speculated that MtCncC is relatively conserved and has similar characteristics to other CncCs. In our study, by cotransfecting constructs containing MtCncC sequences and MtGST promoter sequences, we observed a significant induction of the MtGSTt1 (−574/+340) promoter by MtCncC, which suggests that MtCncC acts as a transcription factor responsible for the activity of the MtGSTt1 (−574/+340) promoter.
ARNT is also a regulatory element of xenobiotic stress response genes and a member of the bHLH-PAS transcription factor superfamily [25]. AhR is another bHLH-PAS protein family member that is a ligand-activated transcription factor [48]. In vertebrates, AhR has two isoforms, AhR1 and AhR2. AhR1 is found in all vertebrates, while AhR2 is present in some vertebrates [49]. AhR and ARNT can form heterodimers to bind enhancer DNA sequences and activate antioxidant and xenobiotic metabolic genes such as GSTs and P450s [26,50]. In mammals, some GSTs were regulated by AhR/ARNT [51,52]. In insects, AhR/ARNT was associated with the regulation of Aphis gossypii Glover CYP450 to improve its tolerance to spirotetramat [53]. In M. persicae, AhR/ARNT could upregulate the expression levels of CYP450 to confer resistance to pesticides [54]. NlARNT could bind to the CarE7 promoter and strongly induce transcriptional activity to enhance resistance to xenogenic stress in Nilaparvata lugens [55]. In this study, the phylogenetic relationship of MtARNT was closely related to that of other insect ARNTs. We hypothesized that MtARNT is highly similar to other ARNTs and has similar functions to other ARNTs. In this study, by cotransfecting constructs containing MtARNT sequences and MtGST promoter sequences, the MtGSTt1 (−574/+340) promoter was significantly induced by MtARNT, which suggests that MtARNT acts as an important cis-regulatory element responsible for the transcriptional activity of MtGSTt1 (−574/+340). CncC and ARNT coordinately regulated the expression of GST in S. exigua [27]. In mammals, the interaction between AhR and Nrf2 may be achieved through multiple mechanisms, including Nrf2 as a target gene of AhR, indirect activation of Nrf2 via CYP1A1-generated reactive oxygen species, and direct cross-interaction of AhR/XRE and Nrf2/ARE signaling [56]. According to our results, MtCncC and MtARNT did not coregulate the MtGST promoters and even appeared to reduce the transcriptional activity of the promoters. Thus, we speculated that MtCncC and MtARNT regulate the GST genes of M. troglodyta in a complex process.
In summary, this study identified the core regions of the five MtGST promoters and demonstrated their involvement in the response to tannic acid and quercetin stress. Furthermore, we identified two important transcription factors MtCncC and MtARNT involved in the regulation of the GST gene promoter. These results suggested that transcription factors regulate the expression of GSTs conferring resistance to plant secondary metabolites in M. troglodyta, and provided useful information for a better understanding of the regulatory mechanism between transcription factors and GSTs in M. troglodyta. Future studies will need to examine the mechanism of posttranscriptional regulation of GSTs in M. troglodyta.

Insect Rearing and Cell Culture
M. troglodyta larvae were gathered from poplar (Populus × euramericana 'Nanlin 895') trees in Nanjing, Jiangsu Province, China. The larvae were fed fresh poplar leaves with a photoperiod of 16 h:8 h (light: dark), a temperature of 26 ± 1 • C and a relative humidity of 70-80%. Third-instar larvae were used for subsequent experiments. Sf9 cells were routinely maintained with SF-900 II serum-free medium (Invitrogen, Carlsbad, CA, USA) with 10% fetal bovine serum (HyClone, Logan, UT, USA), 50 mg/mL streptomycin and 50 mg/mL penicillin (Invitrogen) at 28 • C. Sf9 cells were cultured for 3 days and then used for transfection experiments.

Cloning and Sequencing 5' Loss Fragments of GST Promoters
First, the transcription factor-binding sites for all full-length MtGST promoter sequences were predicted on the website http://alggen.lsi.upc.es (accessed on 10 November 2022) to avoid disrupting the integrity of the binding sites when the promoters of different fragments were cloned. The 5' loss fragments of each MtGST promoter were amplified from the full-length sequences of the MtGST promoters using TaKaRa Ex Premier™ DNA Polymerase (Takara, Dalian, Liaoning, China). All primers were designed by Primer 5 software and were listed in Table 1. Each forward primer sequence and reverse primer sequence were added with Nhe I and Xho I restriction enzyme cleavage sites, respectively. Each 5' loss fragment of MtGST promoter was ligated to a TA clone vector pMD-19T (Takara, Dalian, Liaoning, China), and the correct clone product was obtained by sequencing. The PGL4.10-Basic vector and the pMD-19T with the 5' loss fragment were digested with Nhe I and Xho I. Then, the 5' loss fragment of MtGST promoter was ligated to the PGL4.10-Basic vector using T4 DNA ligase (Takara, Dalian, Liaoning, China), and the ligation product was transformed into E. coli cells. The plasmid DNA was purified from E. coli cells for subsequent cell transfection experiments.

Promoter Activity Analysis by Luciferase Reporter Assays
Sf9 cells (2.0 × 10 6 /well) were cultured in a 24-well culture plate, and each 5' loss fragment promoter plasmid (700 ng/well) and pRL-TK (interference renilla luciferase reporter plasmid, Promega, Madison, WI, USA) (70 ng/well) were cotransfected using 2 µL/well Cellfectin II reagent (Invitrogen) in accordance with our previous method (Tang et al., 2020). After 48 h, Sf9 cells were harvested and lysed in 1 × passive lysis buffer (Promega), and the renilla and firefly luciferase activities were measured using the Dual-Luciferase ® Reporter Assay System kit (Promega) on an FLx800 TM fluorescence microplate reader (BioTek, Winooski, VT, USA). The promoter activity was calculated by normalizing the relative activity of firefly luciferase with that of renilla luciferase. Three replicates were performed for each treatment independently.
Tannic acid was initially solubilized in a small volume of acetone and then diluted in sterilized water to 70, 14, 2.8 and 0.56 mg/L, and sterilized water was used as a control. Quercetin was serially diluted in acetone to 35, 7, 1.4 and 0.28 mg/L, and acetone was used as a control. The concentrations of tannic acid and quercetin were determined according to previous study [4,57]. Sf9 cells (2.0 × 10 6 /well) were cultured in a 24-well culture plate, and then each 5' loss fragment promoter plasmid (700 ng/well) and pRL-TK (interference plasmid) (70 ng/well) were cotransfected using 2 µL/well Cellfectin II reagent (Invitrogen). At 5 h posttransfection, we changed the transfection solution to cell culture medium containing 10 µL of tannic acid or quercetin with serum and double antibiotics. After 48 h, we measured luciferase activity using a Dual-Luciferase ® Reporter Assay System kit on a microplate reader. The luciferase activity was calculated according to the above method.

Cloning the Sequences of MtCncC and MtARNT Genes
Total RNA was extracted from third-instar larvae using TRIzol Reagent (Takara, Dalian, Liaoning, China) according to the protocol. The quality and integrity of RNA were examined by a NanoDrop spectrophotometer and agarose gel electrophoresis, respectively. M. troglodyta RNA was reverse transcribed using the PrimeScript TM 1st Strand cDNA Synthesis Kit (Takara, Dalian, Liaoning, China), and the cDNA was used for cloning MtCncC and MtARNT. The primers for cloning MtCncC and MtARNT were designed according to the transcriptome database and were listed in Table 1. Polymerase chain reaction (PCR) was performed using Premix Ex Taq™ (Takara, Dalian, Liaoning, China). The PCR program was as follows: 98 • C for 3 min; 35 cycles of 98 • C for 10 s, approximately 60 • C for 30 s and 72 • C for 90 s; an extension cycle of 72 • C for 5 min. The MtCncC and MtARNT DNA were ligated to the pMD-19T clone vector. The constructs were transformed into E. coli cells and sequenced by Sangon Biotech (Shanghai) Co., Ltd. The amino acid (AA) sequences of MtCncC and MtARNT were deduced from the NCBI Open Reading Frame (ORF) finder (https://www.ncbi.nlm.nih.gov/orffinder/, accessed on 10 November 2022).

Phylogenetic Analysis of MtCncC and MtARNT
A phylogenetic tree was constructed to investigate the relationship between MtCncC and other insect CncC, and we picked Cap 'n' Collar as a keyword to query the nonredundant database (https://www.ncbi.nlm.nih.gov/ (accessed on 10 November 2022)). Multiple AA sequence alignment analysis was carried out using MEGA X (version 10.1) and Clustal X software (version 2.1). The phylogenetic tree was inferred by the maximum likelihood (ML) method in MEGA X with 1000 bootstrap replicates. The phylogenetic tree of MtARNT was inferred using the same methods. Using two primer pairs pAC-V5-CncC and pAC-V5-ARNT (Table 1), the MtCncC and MtARNT were amplified, respectively. Then MtCncC and MtARNT were cloned into pAC-V5 (Invitrogen). Sf9 cells (2.0 × 10 6 /well) were cultured in a 24-well culture plate, and 350 ng of the promoter plasmid and 350 ng of the pAC-V5, pAC-V5-MtARNT, pAC-V5-MtCncC or pAC-V5-MtARNT and pAC-V5-MtCncC expression plasmids were cotransfected using 2 µL/well Cellfectin II reagent (Invitrogen). After 48 h induction, Sf9 cells were harvested and lysed to measure the renilla and firefly luciferase activities.

Statistical Analysis
ANOVA of the data collected from these experiments was performed using InStat software (GraphPad, San Diego, CA, USA). The significant differences of all two samples were evaluated using Student's t test (two-tailed unpaired t test). The statistical significance of multisample comparisons was assessed with one-way ANOVA followed by Tukey's multiple comparisons. A value of p < 0.05 was considered significantly different.