extfieldsofstudy: Medicine Biology

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Genome-Wide Identification and Functional Analysis of NADPH Oxidase Family Genes in Wheat During Development and Environmental Stress Responses

As the key producers of reactive oxygen species (ROS), NADPH oxidases (NOXs), also known as respiratory burst oxidase homologs (RBOHs), play crucial roles in various biological processes in plants with considerable evolutionary selection and functional diversity in the entire terrestrial plant kingdom. However, only limited resources are available on the phylogenesis and functions of this gene family in wheat. Here, a total of 46 NOX family genes were identified in the wheat genome, and these NOXs could be classified into three subgroups: typical TaNOXs, TaNOX-likes, and ferric reduction oxidases (TaFROs). Phylogenetic analysis indicated that the typical TaNOXs might originate from TaFROs during evolution, and the TaFROs located on Chr 2 might be the most ancient forms of TaNOXs. TaNOXs are highly expressed in wheat with distinct tissue or organ-specificity and stress-inducible diversity. A large-scale expression and/or coexpression analysis demonstrated that TaNOXs can be divided into four functional groups with different expression patterns under a broad range of environmental stresses. Different TaNOXs are coexpressed with different sets of other genes, which widely participate in several important intracellular processes such as cell wall biosynthesis, defence response, and signal transduction, suggesting their vital but diversity of roles in plant growth regulation and stress responses of wheat.

INTRODUCTION
Reactive oxygen species (ROS) have dual roles in plant growth regulation and environmental stress response. They not only lead to programmed cell death by damaging the cellular components as deleterious factors of oxidative metabolism but are also necessary for plant tolerance to different biotic and abiotic stresses acting as signal molecules Dietz et al., 2016). Although there are various ROS-generating pathways in plants, the plasma membrane NADPH oxidases (NOXs), also named after respiratory burst oxidase homologs (RBOHs), were considered to be the key enzymes for apoplastic ROS production under both normal and stress conditions and play crucial roles in various biological processes in plants.
NOX homologs are universally present in a broad range of organisms including animals, higher plants, and fungi . Until recently, various types of NOXs including the ancestral NOXs, namely ferric reductases (FREs) or ferric reduction oxidases (FROs), were reported in life kingdom with four types. These types were NOXA, NOXB, NOXC, and FRE in fungi (Aguirre et al., 2005). There were also seven types including NOX1-5, DUOX1, and DUOX2 in animals  and one type in plants known as NOX5-Like (Sagi and Fluhr, 2006). Although the first NOX gene OsRbohA (OsNOX2) was characterized as a gp91 phox homolog of mammalian NOX in rice, multiple NOX members were identified in plants such as Arabidopsis thaliana (Sagi and Fluhr, 2006), rice , maize (Potocký et al., 2007), tobacco (Yoshioka et al., 2001), potato (Yoshioka et al., 2003), tomato (Amicucci et al., 1999), Medicago truncatula (Marino et al., 2011), and Phaseolus vulgaris (Nestler et al., 2014), and more than 50 FRO and 77 NOX gene homologs were identified (Chang et al., 2016). All the plant NOX members are NOX5-like homologs except the ancestral NOXs (FROs), which were considered as the isoforms of yeast FREs (Sagi and Fluhr, 2006;. In addition to having their special NADPH_Ox domain and several calcium-binding EF-hand motifs in its N terminus, the NOX5-like homologs in plants also contain membrane-spanning domains, two hemes, NADPH-binding motifs, and FAD-binding motifs just like all the other NOX/DUOX enzymes in animals (Geiszt, 2006). Many plants have multiple NOX members, and each member has its own special function during the growth and development regulation and stress response. In Arabidopsis, there are 10 typical NOX members reported as AtRbohA-J (Sagi and Fluhr, 2006). Among these NOXs, AtRbohD was found to participate in various important processes including abscisic acid-mediated ROS production and stomatal closure (Zhang et al., 2009), lignin assembly (Denness et al., 2011), endosperm development (Penfield et al., 2006) as well as in systemic signal transduction, (Miller et al., 2009), and survival of seedlings under anoxic conditions (Yamauchi et al., 2017). Moreover, it was reported that AtRbohB mainly participates in seed after ripening (Muller et al., 2009), AtRbohC regulates root hair formation and root-hair-cell growth (Takeda et al., 2008), whereas both AtRbohD and AtRbohF not only regulate the immune response (Chaouch et al., 2012) and salt stress tolerance (Xie et al., 2011) but also function in jasmonic acid (JA)-induced gene expression (Maruta et al., 2011). Furthermore, AtRbohE was found to regulate tapetal programmed cell death (PCD) and pollen development (Xie et al., 2011), whereas AtRbohH and AtRbohJ function in pollen tube development (Kaya et al., 2014). In rice, at least nine typical NOX members were identified to be of OsRbohA-I or OsNOX1-9 (Wong et al., 2007;Wang et al., 2013). It was found that OsRbohA (OsNOX2), OsRbohB (OsNOX1), and OsRbohE (OsNOX3) are involved in immune response (Yoshie et al., 2005;Nagano et al., 2016), and OsRbohA (OsNOX2) also plays a crucial role in drought-stress tolerance . Most recently, OsRbohH (OsNOX9) was found to regulate the aerenchyma formation in roots of rice (Yamauchi et al., 2017). In maize, both ZmRbohH (ZmNOX13) and ROOTHAIRLESS5 (ZmNOX7) were found to function in root development (Rajhi et al., 2011;Nestler et al., 2014).
Wheat (Triticum aestivum) is a worldwide staple crop, necessitating a clear deciphering of its developmental characteristics and stress tolerance mechanisms. However, as of today, only two NOXs in wheat have been reported to participate in brown rust infection response (Dmochowska-Boguta et al., 2013), and the functions of wheat NOX family genes and their regulatory mechanisms in both plant growth regulation and environmental stress response are still largely unknown. In the present study, a comprehensive analysis based on bioinformatics approaches and experimental methods was performed to identify the wheat NOX family genes and their functions during the plant development and stress response. The results obtained here will largely broaden our understanding of the roles of NOXs and their regulation in plants, especially in wheat.

Sequence Retrieval and Identification of the NOX Gene Family in Wheat
We retrieved the potential sequences of NOX family members in wheat from IWGSC (http://www.wheatgenome.org/, last accessed May 25, 2017), NCBI (https://www.ncbi.nlm.nih. gov/, last accessed May 25, 2017), and Ensembl Plants (http://plants.ensembl.org/Triticum_aestivum/Info/Index, last accessed May 25, 2017) websites, with Arabidopsis and rice NOX sequences as queries. We identified each NOX member by predicting the conserved domains. For further information, we analyzed some physicochemical parameters, predicted the subcellular localization and the numbers of transmembrane helix, and performed amino acid sequence alignment (see Supplementary Information S1 for detailed procedures).

Chromosomal Location and Exon/Intron Structure Analysis
Using Adobe_Photoshop_CS6 software, a total of 46 candidate genes were mapped to 18 different chromosomes according to the information from scaffolds and Gene ID reported in IWGSC and NCBI websites. The exon/intron logos of individual NOX and FRO genes were obtained from the Gene Structure Display Server (http://gsds.cbi.pku.edu.cn) by aligning the coding or cDNA sequences with their corresponding genomic DNA sequences.

Sequence Alignment and Gene Structure Analysis
The phylogenetic tree of wheat NOX and FRO family members was constructed with MEGA 6.06. The logos of domain organization were obtained from EMBL-EBI (http:// pfam.xfam.org/search#tabview=tab1) or SMART (http:// smart.embl-heidelberg.de/) websites and were amended with Adobe_Photoshop_CS6. The four conserved domain motifs, namely NADPH_Ox, Ferric_reduct, FAD_binding_8, and NAD_binding_6 in each NOX sequence, were generated by MEME suite (http://meme-suite.org/) (see Supplementary Information S1 for detailed procedures).

Phylogenetic Relationships of NOX and FRO Gene Families in Wheat and Seven Other Plant Species
Multiple sequence alignments and the phylogenetic relationship analysis of NOX and FRO gene families from eight plant species were performed using MEGA 6.06. Meanwhile, the non-synonymous (Ka) and synonymous (Ks) in paralogous and/or orthologous gene pairs from four species were also estimated using the bioinformatics software PAMLX 1.2 (see Supplementary Information S1 for detailed procedures).

Subcellular Localization Analysis
The subcellular location of wheat NOXs was examined in rice protoplasts using transient transformation systems with some modifications ; at least three monoclones were sent for sequencing upon gene cloning and vector construction for each examined gene (see Supplementary Information S1 for detailed procedures).

Prediction and Functional Analysis of cis-Regulatory Elements
We selected 2,000-bp genomic DNA sequences upstream of the transcriptional start sites of TaNOXs as the promoter sequences (named TaNOX-pros) to analyze the cis-acting elements using the databases: PlantCARE (http://bioinformatics.psb.ugent.be/ webtools/plantcare/html/) (Lescot et al., 2002) and PLACE (http://www.dna.affrc.go.jp/PLACE/). Then, the activity of the promoters was analyzed by dual luciferase reporter assay Gu et al., 2013). Three or more monoclones were sent for sequencing to identify each promoter sequence and related vector construction. All values were shown as the average of the data collected from five or more replicates (see Supplementary Information S1 for detailed procedures).

Plant Materials, Treatments, Expression Profiles, and Coexpression Network Analysis
Tissue-specific expression profiles, inducible expression profiles of TaNOX genes, and coexpression analysis between TaNOXs and other genes in wheat (Triticum aestivum cv. Chinese Spring) were performed using the online Genevestigator v3 (https:// genevestigator.com/gv/) and/or by qRT-PCR with TaActin (AB181991.1) and TaGAPDH (ABS59297.1) as the internal transcript level controls. The wheat seedlings were harvested from different developmental stages for tissue-specific expression profiles analysis. At the same time, the wheat seedlings treated with abiotic stresses and hormones were also collected for inducible expression profiles and coexpression network analysis. Total RNA was extracted from different samples using RNAiso TM Plus (Takara, Dalian, China) performance, and the subsequent quantitative real-time PCR (qRT-PCR) analysis was referenced to our previous study . The results were presented as heat maps, histogram, and/or also as table lists. All the expression levels represent the mean ± SD of data collected from three independent experiments with each having three or four replicates (see Table S6 and Supplementary Information S1 for detailed procedures).

Identification of NOX Family Genes in Wheat Genome
A hidden markov model (HMM) search was performed to investigate and characterize the NOX gene family in wheat genome, and a total of 46 genes including 36 NOX and 10 FRO candidates were identified (Table S1). These NOX and FRO candidates can be divided into three types: TaNOXs, TaNOX-likes, and TaFROs based on their chromosome localization and domain composition (Table S1, Figures 1, 2). The homologous genes from different subgenomes (A, B, and D) were assigned the same number in gene denomination due to their similarity in gene structure and protein size. The typical NOXs (TaNOXs) have all four conserved domains, namely NADPH_Ox (Pfam accession number PF08414), Ferric_reduct (PF01794), FAD_binding_8 (PF08022), and NAD_binding_6 domain (PF08022). The TaNOX-likes have NADPH_Ox domain but lack one or two other domains, whereas the TaFROs lack NADPH_Ox domain but have the other three domains (Figure 2). Intriguingly, the NOX and FRO family genes in wheat genome are non randomly distributed across the chromosomes. It seems that FRO candidates are mainly distributed on Chr 2 and less on Chr 1; however, all the predicted NOX candidates are mainly distributed on Chr 1, followed by Chr 5, 3, 4, and 6, and none of the NOX members on Chr 2 ( Figure S1). Moreover, the characteristics of gene structure and protein size are quite different between the NOX and FRO members (Figure 1, Table S1). TaNOXs are much bigger than TaFROs, whereas the TaNOX-likes are the smallest.

Gene Structure and Domain Composition of the Wheat NOX Family Proteins
As can be seen in Figure 1, the gene structures are quite diverse between the TaNOXs and TaFROs with different intron numbers for different genes except for the certain homologous genes from different subgenomes (A, B, and D). On the whole, the gene constructions became more and more complicated from TaFROs to TaNOXs for increasing exons and introns. As a special type of NOX, TaNOX-likes are much simple with less exons and introns. According to the phylogenetic tree (Figure 2A), we found that all the homologous genes assigned as the same number in gene denomination were generally clustered into one group earlier than the others, and all the NOX family members could be classified into three big branches: FRO I (including TaFRO3 and (C) Four conserved motifs, namely NADPH_Ox, Ferric_reduct, FAD_binding_8, and NAD_binding_6, from all 46 NOX proteins in wheat. The logos of four conserved domain sequences were obtained from MEME Suite website. The bit score means information content of each position in the amino acid sequence. 4) on the Chr 2, FRO II (including TaFRO1 and 2) on the Chr 1, and NOX (including TaNOXs and TaNOX-likes) on the Chr 1, 3, 4, 5, and 6, respectively.
As shown in Figure 2B, almost all the typical NOXs contain one or two transmembrane regions, one to four calcium-binding EF-hand motifs, and four conserved constructions, namely one or two typical NADPH_Ox domain, one Ferric_reduction domain, one FAD_binding_8 domain, and one NAD_binding_6 domain. Only TaNOX14 does not have an EF-hand motif ( Figure 2B). All TaFROs possess the conserved domains that the typical TaNOXs have but lack the NADPH_Ox domain and the EF-hand motif. In contrast, the TaNOX-likes have only one or two of the three conserved domains similar to TaFROs possession, besides the typical NADPH_Ox and EF-hand motifs. Furthermore, the four functional domains exhibited to be all considerably conserved in construction, and the distribution of amino acid residues in every domain is quite similar but not identical among the NOX members ( Figure 2C). Four conservative amino acid residues Y, G, G, and P presented with Y××G×G××P motif in the 5 ′ end sequence of NAD_binding_6 domain from TaFROs were the same as those from the TaNOXs and TaNOX-likes. There were also other quite conserved residues, for example, the P in FAD_binding_8 domain and the G in Ferric_reduct domain, as indicated in capital letters in Figure 2C. However, the conservatism of amino acid residues in NADPH _Ox domain was lower than the other three conserved domains. Taken together, all the results mentioned above imply a closer evolutionary relationship between TaFROs and TaNOXs (Figure 2).

Subcellular Localization of the Wheat NOX Family Proteins
As shown in Table S1, all the NOXs including TaNOXs, TaNOXlikes, and TaFROs are located in the plasma membrane of cells as predicted by the Plant-mPLoc. Although no typical transmembrane helixes were identified in TaNOX-likes, these proteins were also predicted to be located at the plasma membrane. To verify the subcellular localization of TaNOXs, the full-length cDNA sequences of four genes, TaNOX7-3AS, TaNOX10-5BL, TaFRO4-2BL, and TaNOX-like4, were cloned and analyzed by a transient transformation system. As shown in Figure 3, the proteins encoded by the four genes are all located on the plasma membrane as expected.

Systematic Evolutionary Relationship of NOX Gene Family in Wheat
To comprehensively dissect and characterize the evolutionary relationship of NOX gene family in wheat, a total of 112 NOX and FRO homologs were identified and selected from wheat and other seven plant species for the multiple sequence alignments and phylogenetic relationship analysis (Figure 4; Table S2). The evolutionary relationships between wheat (T. aestivum) and seven other species were presented in Figure 4A, and the numbers of NOX and FRO members were provided in Figure 4B. All the members of NOX and FRO families could be classified into six subgroups I-VI based on the difference of protein topological structure ( Figure 4C). The wheat TaNOXs and TaFROs are distributed in every subgroup but dominantly distributed in subgroup I, whereas, the Arabidopsis AtNOXs (AtRbohs) and AtFROs are mainly distributed in subgroup VI ( Figure 4D). The members within a certain subgroup exhibited a high identical percentage of amino acid sequences as shown in Table S3.
To estimate evolutionary relatedness and divergence time of NOX family genes between wheat (T. aestivum) and its diploid relatives: Aegilops tauschii, Triticum urartu, and Hordeum vulgare, the rates of non synonymous (Ka) and synonymous (Ks) nucleotide substitutions and their ratios (ω = Ka/Ks) of four putative paralogous and 19 orthologous gene pairs of NOXs were calculated using the bioinformatics software pamlX (Table S4). As can be seen, the majority of Ka/Ks ratios (ω) are more than 1 among the paralogous gene pairs, indicating that the NOX family genes have undergone strong positive selection pressure for functional expansion, and the duplication event was estimated to have occurred 3.16630-6.2533 Mya. On the contrary, the values of ω are almost less than 1 among the orthologous gene pairs, suggesting that the NOX family genes have undergone strong negative selection pressure for functional conservation, and the duplication event was estimated to have occurred 0.0060-0.6466 Mya.

Development and Tissue-Specific Expression of NOX Family Genes in Wheat
To clarify the expression profiles of NOX family genes during the development of wheat, a set of microarray data for the gene expression were obtained from Genevestigator v3. To simplify the phraseology in the following experiments, the homologous genes located on different subchromosomes (Chr A, B, and C) are referred to as TaNOXx; for example, TaNOX1-1AL, -1BL, and/or -1DL were all named as TaNOX1. The expression levels of TaNOXs at 10 developmental stages were presented in Figure 5A. As can be seen, different TaNOXs exhibited different expression patterns with some genes dominantly expressed at certain stages. For example, the genes TaNOX2, 7, and 12 are widely expressed in whole developmental stages with the highest in stem elongation and inflorescence emergence stages, whereas TaNOX15 and TaNOX-like4 are mainly expressed in germinating seeds and at booting stage. The expression of TaFROs has more specificity in wheat. For instance, TaFRO1 and TaFRO2 are only expressed at stem elongation stage, whereas TaFRO3 is mainly expressed at young seedling, inflorescence emergence, and milk stages ( Figure 5A).
The tissue-specific expression profiles of TaNOXs were examined by qRT-PCR ( Figure 5B). Seven wheat tissues from different developmental stages were used for the analysis. According to the tissue-expression profiles obtained from Genevestigator v3, seven TaNOX genes, including TaNOX3, 5, 7, 8, 10, 11, and 15, were selected as the candidates to study the tissue specificities of the genes in wheat. The results showed that every gene had its obvious tissue specificities in expression. The expression of TaNOX3 is the highest in leaves at booting stage, whereas the expression of TaNOX5, 10, and 11 is the highest in young leaves at seedling stage, TaNOX7 in young panicle at booting stage, TaNOX8 in young roots at seedling stage, and TaNOX15 in stems at booting stage. All these results indicated that the expression of each NOX gene is unique and displays a strong spatio-temporal and tissue specificity in wheat. The developmental expression pattern analysis of NOX and FRO family genes in 10 developmental stages during wheat growth. All the data were selected from the Ta_mRNASeq_WHEAT_GL-0 database. (B) Expression profiles of seven wheat NOX genes in seven different tissues. The seven tissues are as follows: the young leaf and young root came from 2-week-old seedlings; the leaf, stem, leaf sheath, and young panicle came from the booting stage; the panicle came from the inforescence emergence stage. The developmental expression pattern for each NOX or FRO family gene was performed by the three independent experiments with three replicates for each experiment.
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Analysis of the Promoters of NOX Genes and Their Response to Exogenous Treatments
The promoters of all TaNOX genes were analyzed, and the cis-acting elements existing in the promoters were predicted by the databases of PlantCARE, PLACE, and Neural Network Promoter Prediction. In total, 31 cis-regulatory elements with the prediction score being greater than or equal to 5 remained and were depicted on the corresponding sequences. The elements responsive to environmental stresses and hormones were considered for further analysis (Figure 6, Figure S2). As can be seen, almost all the stress or hormone responsive elements are distributed randomly on the promoter sequences of the TaNOX genes. However, some cis-elements are distributed in clusters in certain promoters, for example, MBS-motif, TGACG-motif, and G-box in the promoters of TaNOX7AS, TaNOX5-3AL and TaNOX5-3BL genes, respectively, implying the essential roles of these elements in the expression of these genes. Interestingly, the numbers and distribution patterns of the cis-elements are also greatly varied among the promoters of homologous genes with the same number in gene denomination although their encoding proteins have an extreme similarity in amino acids sequence and domain construction (Figure 6). This means that the homologous genes might have different regulatory mechanisms in expression, implying their functional divergence during the polyploidization of wheat genome.
To get further insight into the regulation of gene expression of TaNOXs, the promoters of seven genes were cloned, and their activities in response to heat, cold, ABA, and MeJA treatments were determined using a dual luciferase reporter system. As shown in Figure 7, each promoter examined exhibited different levels of biological activities in response to different treatments. The activities of almost all the promoters of seven TaNOX genes could be greatly stimulated by heat (Figure 7B), whereas no marked changes in the activities were observed under cold stress compared with the controls. Only the activity of TaNOX7-3AS promoter was obviously decreased by cold ( Figure 7C). ABA treatment also significantly stimulated the activities of the promoters of the TaNOX genes except for the promoter of TaNOX3-1DL ( Figure 7D). Although the MeJA-responsive cis-elements are widely distributed in the promoters of the seven genes examined here, only the promoters of TaNOX7-3AS, TaNOX12-5BL, and TaNOX13-5AL responded to the exogenous MeJA treatment (Figure 7E). These results indicated that the responses of the TaNOX genes to environmental factors are quite different, and a complicated mechanism might be involved in the control of gene expression in wheat.

Inducible Expression Profiles Range of TaNOXs Under a Wide Range of Environmental Stresses and Hormone Treatments
To further study the expression characteristics of wheat NOX family genes under suboptimal conditions, we carried out a comprehensive analysis using both the wheat microarray data in Genevestigator v3 and qRT-PCR experiment (Figure 8, Figure S3). As can be seen in Figure 8A, many NOX genes in wheat can respond to a number of environmental stresses. Different inducible expression patterns could be seen in different NOX genes responding to different biotic stresses such as B. graminis, F. graminearum, P. graminis, P. striliformis, and X. translucens and to different abiotic stresses such as cold, heat, drought, and salt. According to the expression patterns, the TaNOX family genes can be simply classified into four groups: Group I including TaNOX1, 2, 6, 7, 8, 12, 13, and 15 and TaNOX-like1 and 4 was upregulated by cold but downregulated by almost all the other environmental treatments; Group II including TaFRO1 and TaNOX4 had no obvious responses to all the environmental treatments; Group III including TaNOX5, TaFRO3, and 4 was upregulated by P. Graminis attack but downregulated by all abiotic treatments and B. graminis and F. graminearum attacks; and Group IV including TaNOX3, 9, 10, 11, 14, and TaFRO2 was upregulated by B. graminis and F. graminearum attacks but downregulated by P. graminis attack ( Figure 8A).
The different responsive profiles of the NOX gene members to different abiotic stresses were also observed by qRT-PCR experiment ( Figure 8B, Figure S3B). Here, only more than twofold difference in transcript levels was considered to be a true difference for the genes under treatments. Under cold (4 • C) stress, most TaNOX genes were downregulated in both shoots and roots at 12 and 24 h time points of the treatment, whereas TaNOX7 was upregulated in roots at 12 h time point. Similarly, most of the genes were downregulated by salt stress (200 mM NaCl) in both shoots and roots at 12 h and/or 24 h time point, whereas TaNOX5 was upregulated in roots at 12 h time point. However, heat stress (40 • C) markedly upregulated the transcript levels of TaNOX3, 8, 11, and 15 genes but downregulated TaNOX2, 5, 7, 10, and 12 genes in shoots. In contrast, except for TaNOX12 and 13, almost all the other genes examined were downregulated by heat in roots ( Figure S3B). All the examined TaNOXs could be upregulated by the oxidative stress (30 µM MV) in roots. On the contrary, almost all TaNOX genes had no obvious changes by osmotic stress (20% PEG6000) in both roots and shoots except TaNOX13 was upregulated in shoots. Furthermore, except TaNOX7, which showed no changes, and TaNOX12, which was upregulated, all the other genes were downregulated by ABA treatment (100 µM) in shoots, whereas except TaNOX5, which was greatly upregulated, and TaNOX12 and 13, which were downregulated, all the others showed no obvious changes in roots. With the treatment of MeJA (100 µM), the majority of TaNOXs had no significant changes, but TaNOX12 in shoots and TaNOX5, 7, and 10 in roots were exhibited upregulation. For SA treatment (500 µM), the genes of TaNOXs exhibited more complicated expression profiles, and no distinct consistency in expression patterns was observed. Simply, it can be seen that TaNOX3 and 5 genes were upregulated by SA in shoot, whereas TaNOX8 and 10 were upregulated in roots.
Although not all the NOX family genes were examined, the results obtained here suggested that each NOX member in wheat has its unique inducible expression profiles and thus has its own functional specificity in the plant stress responses.

Coexpression Relationships Between the Genes of TaNOXs and Others
To further explore the biological functions of TaNOX genes, we generated the coexpression patterns of TaNOXs with others using the wheat microarray data in the database of Ta_mRNASeq_WHEAT_GL-0 from Genevestigator v3 ( Figures  S4-S6). Eight genes were selected for the analysis according to their inducible expression profiles. Interestingly, several genes showed obvious coexpression relationships with certain NOX genes either at 10 different developmental stages ( Figure  S4) or in 22 different tissues ( Figure S5). These coexpressed genes are mainly involved in the substance transport, cell wall synthesis, redox reaction, phosphorylation, and/or gene expression regulation (Table S5). For instance, TaNOX2 and 3 showed positive correlation with the genes encoding two S-locus lectin protein kinase family proteins; TaNOX3, 7, and TaFRO3 are coexpressed with the genes encoding four leucinerich repeat protein kinase family proteins. The eight NOX genes also exhibited distinct coexpression with other genes under a lot of biotic and abiotic stress conditions ( Figure S6). These coexpression genes are widely involved in stress response, signal transduction, cell wall formation, protein degradation, substance transport, and transcriptional regulation (Table S5). For example, TaNOX2 is highly coexpressed with a gene encoding S-adenosyl-L-methionine-dependent methyltransferase and a gene encoding ubiquitin-specific protease family C19-related protein, whereas TaNOX3 is highly coexpressed with the genes encoding two brassinosteroid-Insensitive 1 precursor kinases and a gene encoding glutathione S-transferase tau 7.
To better understand the coexpression relationships of the TaNOXs with other genes under abiotic stresses, we chose some genes as targets for qRT-PCR. According to the results of tissue and inducible expression profiles, five TaNOX genes and six stress conditions were selected for the experiment. Here, a more than two fold difference in transcript levels was considered to be a true difference for the genes as well. As shown in Figure 9, most of the genes selected exhibited significant coexpression patterns with the TaNOXs under a certain stress condition. Only a few genes had an opposite response with the TaNOX gene under some special stress. As can be seen, TaNOX2 exhibited an opposite response with its predicted coexpression genes only under salt stress. For TaNOX3, except under drought and MeJA treatments, it seems that only one predicted gene was not obviously coexpressed with it under four other stress conditions. The distinct coexpression relationships between TaNOX7 and the predicted genes could be observed under heat, natural drying, and ABA treatments. By contrast, TaNOX8 exhibited positive coexpression with the predicted genes under all the six stress treatments. Except drought and salt stresses, TaNOX12 also showed obvious coexpression with its predicted genes examined under other four treatments. These results indicated that the coexpression relationships of TaNOXs with the predicted genes might be stress dependent, implying the specific but complicated functions of NOX genes in wheat development and/or stress responses.

Wheat NOXs Are Diverse in Members and Structures With a Complicated Evolution History
As is well-known, common wheat (T. aestivum) is a hexaploid species with three closely related subgenomes (A, B, and D). In this study, a total of 46 NOX family genes, which encode 15 TaNOXs, 4 TaNOX-likes, and 4 TaFROs, were identified according to the sequence analysis and domain composition (Table S1). Phylogenetic analysis showed that the homologous genes from different subgenomes for a certain NOX protein always clustered together first, as expected (Figure 2). Interestingly, not every protein has three homologous genes distributed on the subchromosomes A, B, and D. For example, TaNOX3 includes two homologous genes (TaNOX3-1BL and -1DL) but TaNOX4 has only one (TaNOX4-1DL) (Figure 1). This means that their homologs on a certain subchromosome might be lost during the long-term evolution and natural selection. In fact, many previous studies have shown that in an allopolyploid species, the phenomenon of genovariation including gene rearrangement, structural variation, DNA sequence loss or amplification, and transposon activation occurred frequently during the polyploidization of genome (Jackson and Chen, 2010).
As suggested earlier, gene duplication, fusion, and/or exon shuffling are commonly occurred in eukaryotes for the biological diversity and functional divergence of certain gene family during evolution in plants (Morgante et al., 2005;Van de Peer et al., 2009;Kaessmann, 2010;Magadum et al., 2013). This is also involved in the expansion of NOX gene family in plants (Chang et al., 2016). The typical NOXs of wheat exhibited high similarity in the sequences of the conserved domains but obvious diversity in gene structure and protein size (Table S1, Figures 1, 2). TaFROs also showed very high similarity in sequences of the conserved domains compared with those of TaNOXs although there are still some differences in same conserved amino acids (Figure 2), implying an evolutionary relationship between TaFROs and TaNOXs. In fact, it was suggested that FRO proteins are the ancestral forms of NOXs in both animals and plants (Chang et al., 2016). Interestingly, although the structures of TaNOXs and TaFROs are conserved, they have undergone a diverse structural evolution. It can be seen that the numbers and positions of intron and exon of the genes became more and more complicated from TaFROs to TaNOXs (Figure 1), exhibiting a structural diversity of NOX family genes in wheat, and the evolutionary mechanisms such as gene duplication, gene fusion, and exon shuffling might be also involved in the evolution of wheat NOX gene family. Moreover, although TaFROs are the ancient forms of TaNOXs, the TaFROs genes located on Chr 1 and Chr 2 seem to be somewhat different in evolution and function. The TaFROs located on Chr 1 (TaFRO1 and TaFRO2) and Chr 2 (TaFRO3 and TaFRO4) could not be clustered into the same subgroup within the phylogenetic tree. The TaFROs located on Chr 1 were clustered into the small subgroup and were closer to the TaNOXs and TaNOX-likes than the TaFROs located on Chr 2 (Figure 2A), indicating their differential but complicated evolution of the two types of TaFROs. The TaFROs encoded by the genes located on Chr 2 exhibited different numbers and arrangement of transmembrane regions and different sizes of the three conserved domains compared with those located on Chr 1 (Figure 2), showing their diversity in protein structure and probably also in function. Moreover, it seems that the arrangement and similarity of Ferric_reduct domains and transmembrane regions of the TaFROs encoded by the genes located on Chr 1 are more similar to those of the typical TaNOXs, further suggesting that the TaFROs encoded by the genes located on Chr 1 perhaps are closer to TaNOXs in genetics than those on Chr 2 and the TaFROs on Chr 2 might be the more ancient forms of TaNOXs in wheat.
As reported earlier, typical NOXs only exist in terrestrial plants evolved from the ancient FROs by gaining a NADPH_Ox domain (Chang et al., 2016). The NADPH_Ox domain endowed NOX with a new function of producing ROS as a defence mechanism. Although the FROs in lower organisms are responsible for both ROS production and iron uptake (Aguirre et al., 2005), a number of increasing literatures have shown that in higher plants, NOX-mediated ROS production is mainly responsible for the stress tolerance and development regulation (Kaya et al., 2014;Gupta et al., 2017;Yamauchi et al., 2017), whereas FROs are mainly responsible for iron uptake and iron homeostasis (Sperotto et al., 2010). Furthermore, TaNOXs perhaps will become more complicated in construction; for example, TaNOX3 possessing two NADPH_Ox domains, and this is different from the others that just possesses one NADPH_Ox domain in animo acid sequences ( Figure 2B). Further study is needed to check whether it has some biological significance.
The four TaNOX-likes are very special because they have the NADPH_Ox domain but lack one or two other conserved domains that the typical TaNOXs have (Figure 2), and although FIGURE 9 | Expression profile analysis of coexpression genes with TaNOXs by qRT-PCR under different abiotic stress treatments on the shoot of wheat seedlings. The 7-day-old wheat seedlings treated with soil drought for 2 days (A), 42 • C for 24 h (B), 200 mM NaCl for 6 h (C), nature drying for 30 min (D), 10 µM ABA for 6 h (E), and 10 µM/L MeJA for 6 h (F), respectively, were used as the materials for RNA extraction. Data are mean values from at least five replicates. Abbreviations and locus names of the coexpression genes used here are available in Table S5. no typical transmembrane helixes were identified in these proteins, they are also located in the plasma membrane (Figure 3). The analysis for phylogenetic relationships showed that TaNOX-like1 and 3 are much closer to TaNOX4, whereas  TaNOX-like2 is much closer to TaNOX3 and TaNOX-like4 is much closer to TaNOX15 (Figures 3, 4), indicating that they might derive from these three typical TaNOXs, respectively, probably by DNA sequence loss during evolution. As no NOXlike proteins were identified in other plant species and no experiments have been done on them, their biological functions are unclear. All this information suggest that wheat NOXs underwent a complicated evolutionary history during the gene expansion and functional divergence.

Wheat NOXs Exhibit a Great Specificity in Expression and Play Vital Roles in Both the Plant Growth Regulation and Stress Response
Specific expression is a common characteristic of the genes of a certain protein family in plants, which often reflects the cross-talk and/or difference in functions of the family members. Many studies have shown that different NOXs have different functions both in animals and in plants Kaye et al., 2011). In Arabidopsis, 10 NOXs were found, and these NOXs participate in different biological processes and/or stress responses with obvious tissue specificity in the gene expression (Maruta et al., 2011;Chaouch et al., 2012;Kaya et al., 2014;Xie et al., 2014). However, in wheat, although it was reported that NOXs could ameliorate the nickel-induced oxidative stress (Hao et al., 2014), to date, only two genes have been reported functioning in brown rust infection (Dmochowska-Boguta et al., 2013). In the present study, both the results obtained from database research and qRT-PCR analysis indicated that TaNOXs widely function in the plant growth regulation and stress response (Figures 5-8, Table 1). For example, TaNOX2 is expressed in a whole plant with dominantly existing in stem elongation and inflorescence emergence stages, and its transcripts were greatly affected by a set of biotic and abiotic stresses, implying its vital roles in plant growth regulation and also stress responses. TaNOX2 is more similar to Arabidopsis AtRbohD and AtRbohF and rice OsNOX2 (OsRbohA) in phylogenetics (Figure 4). AtRbohD and AtRbohF are all expressed throughout the plant (Sagi and Fluhr, 2006) and are essential not only for the hypersensitive response but also for the ABA-, JA-, and ethylene-induced signaling pathways (Suhita et al., 2004;Desikan et al., 2006;Zhang et al., 2009;Maruta et al., 2011), disease resistance (Chaouch et al., 2012), salt stress tolerance (Xie et al., 2011), and endosperm development (Penfield et al., 2006). OsNOX2 was also found to be expressed in the whole plant and plays crucial roles in both growth regulation and drought stress tolerance . TaNOX8 and TaNOX12 are also much close to AtRbohD, AtRbohF, and OsNOX2 (Figure 4), and as expected, the two wheat genes exhibit very similar expression profiles with TaNOX2, although they are somewhat different under a certain stress condition, implying their functional identity or coordination. In contrast, TaNOX3 showed marked tissue and/or development specific expression (Figure 5, Table 1) with a great response for many different treatments (Figures 7B, 8, Table 1), suggesting somewhat specificity in functions. TaNOX3 is much closer to Arabidopsis AtRbohB, which was found to be specifically expressed in root and elongation zone and to be involved in seed after ripening (Sagi and Fluhr, 2006;Muller et al., 2009). Similar to TaNOX3, the TaNOXs including TaNOX4, 9, 10, and 11 are also much closer to AtRbohB in phylogenetics, but these TaNOXs exhibit varied tissue and/or development expression profiles as well as the inducible expression (Figures 5, 7, 8, Table 1), implying somewhat divergence in functions among these NOX proteins. TaNOX15 also shows notable tissue-and developmentspecific expression with more transcripts in stems at booting stage ( Figure 5, Table 1), but the transcripts were less induced by different treatments (Figure 8, Table 1). TaNOX15 has high identity in amino acid sequence with Arabdopsis AtRbohC, which was reported participating in root-hair-tip growth and specifically expressed in roots (Sagi and Fluhr, 2006;Takeda et al., 2008), which means that it has different functions in wheat as AtRbohC has in Arabidopsis.
However, TaNOX7, 8, 13, and 14 exhibit much closeness to rice OsNOX1 (OsRbohB) and OsNOX9 (OsRbohH) (Figure 4), and all of them are sensitive to biotic stresses and ABA treatments (Figures 7B, 8, Table 1), implying their possible functions in plant immunity. It was reported that OsNOX1 (OsRbohB) involves in immune response (Nagano et al., 2016), whereas OsNOX9 (OsRbohH) regulates the ethyleneinduced aerenchyma formation in roots (Yamauchi et al., 2017). Furthermore, TaNOX1 and TaNOX5 are much closer to OsNOX3, which was also found to play crucial roles in plant immune response (Yoshie et al., 2005;Nagano et al., 2016). TaNOX1 and TaNOX5 having similar functions require further study. Interestingly, due to the inducible expression profiles under a set of biotic and abiotic stresses, all the TaNOXs could be divided into four subgroups (Figure 8), implying their functional divergence between the groups but coordination within the same group. However, as discussed above, different TaNOXs exhibit strong spatio-temporal and tissue-specific expressions, demonstrating an obvious cross-talk and/or divergence in both biological roles and evolutionary relationship of the NOX family genes in wheat.

Coexpression Analysis Reveals the Wide Functions but Complicated Regulatory Mechanisms of the TaNOXs in Wheat
Acting as the key producer of ROS, NOXs play vital roles in plant growth and development including root and root hair elongation (Foreman et al., 2003), endosperm development (Penfield et al., 2006), cellular vesicle cycle (Hao et al., 2014), seed after ripening (Muller et al., 2009), pollen tube growth, lipid microdomain polarization (Potocký et al., 2007;Hao et al., 2014;Nagano et al., 2016), plant-rhizobial symbiosis (Marino et al., 2011(Marino et al., , 2012, and also stress responses as reviewed previously (Chang et al., 2016). The functional diversity was also observed in the wheat NOX gene family as discussed above. The results from our thorough coexpression analysis showed that different TaNOXs have different sets of coexpression genes against development stages, tissues, or stress conditions (Figure 9, Figures S4-S6, Table S5), implying the specific but complicated functions of NOX proteins in wheat development regulation and/or stress responses.
Corresponding to the developmental stages and tissue specificity, the coexpression genes with TaNOXs are mainly related to enzymatic glycosyl transfer, energy metabolism, substrate transport, cell wall synthesis, redox reaction, phosphorylation, and/or gene expression regulation ( Figures  S4, S5, Table S5), suggesting their diverse and vital function in wheat development. For example, TaNOX2 is highly coexpressed with a kinase (casein kinase 1.5) and two cellulose synthases (CESA3 and CESA5) in all the tissues examined, implying that its function may be tightly related to the cell wall synthesis and protein phosphorylation. Casein kinase 1 (CK1) plays a crucial role in regulating growth and development via phosphorylating various substrates throughout the eukaryote kingdom. In Arabidopsis, two CK1s were found to regulate blue light signaling by phosphorylating cryptochrome 2 (Tan et al., 2013). CESA3 and CESA5 are two important proteins functioning in plant cell wall formation (Griffiths et al., 2015) and also in fiber cellulose production . Several RLK family genes and a phytosulfokin receptor 1 gene were found to have significant coexpression with TaNOX3 (Table  S5), demonstrating that TaNOX3 might participate in a series of cellular signaling pathways. As reported earlier, RLKs play crucial roles in plant growth, development, and defence response by perceiving and transmitting extracellular or intracellular signals . As the receptor of phytosulfokine (PSK), the phytosulfokin receptor 1 functions in cell growth, pollen tube guidance, and cell differentiation (Sauter, 2015). Several heavy metal transport-related proteins and DHHC-type zinc finger family proteins are highly coexpressed with TaNOX7 (Table S5), implying its functions in substrate transport and protein anchoring to the membrane. DHHC-type zinc finger family proteins were found to play crucial roles in protein palmitoylation for cell membrane anchoring (Mitchell et al., 2006). Besides being coexpressed with two CESA1-cellulose synthases, TaNOX8 also has high coexpression with a SCAMP family protein and two kinesin motor family proteins, indicating that it might not only participate in cell wall biosynthesis but also function in intracellular substance transport. SCAMP family proteins acting as carriers are responsible for the protein transport functioning to the cell surface in post-Golgi recycling pathways (Fernandez-Chacon et al., 2000), and kinesin family proteins unidirectionally transport various cargos including membranous organelles, protein complexes, and mRNA (Li et al., 2012). TaNOX12, however, is highly coexpressed with several glycosyl transfer-related proteins, demonstrating their roles in enzymatic glycosyl transfer. Moreover, two riboflavin synthase-like superfamily proteins have high coexpression with TaNOX12. Riboflavin synthases are responsible for the biosynthesis of vitamin B 2 , which is vital for the cellular redox balance (Fischer and Bacher, 2008). TaNOX13 is coexpressed with three CESA proteins and one CK1 protein, suggesting its functions in the cell wall formation and protein phosphorylation. It should be noticed that TaNOX12 has obvious coexpression with TaNOX2 and TaNOX8 at different development stages and in different tissues (Table S4), showing somewhat cross-talk between the function of different TaNOXs. All these results indicate the distinct but crossing functions of the TaNOXs in wheat growth and development.
The coexpression patterns of TaNOXs with other genes are greatly affected by different stresses, and different sets of genes were found to be highly coexpressed with the TaNOXs under stress conditions (Figure 9, Figure S6, Table S5), showing the vital roles of TaNOXs with complicated regulatory mechanisms during their functioning in stress tolerance. The genes that are highly coexpressed with TaNOXs under stress conditions are widely involved in stress signal transduction, cell wall formation, protein degradation, transcriptional regulations, and so on. Among the eight TaNOXs examined here, four genes including TaNOX2, 3, 8, and 13 have distinct coexpression with the genes encoding several protein degradation-related proteins including one ubiquitin-specific protease family C19-related protein, two ARM repeat superfamily proteins, one RING/U-box superfamily protein, and five COP1-interacting protein-related proteins ( Figure S6, Table S5). All these coexpressed proteins function as E3 ubiquitin ligase or involve in ubiquitination of proteins (Zeng et al., 2008;Acosta et al., 2012;Xu et al., 2014), implying these TaNOXs might participate in protein degradation in wheat under stress conditions. Furthermore, a set of signal transduction-related genes were found to be highly coexpressed with TaNOXs, especially under stress conditions. At first, it was found that the genes encoding five protein kinase superfamily proteins are highly coexpressed with the TaNOXs, including TaNOX2, 7, and 13 (Table S5). These protein kinases, including an LRR family protein and two CC-NBS-LRR proteins, belong to receptor-like protein kinase (RLK) family, often functioning in signal transduction in plant cells, especially in the pathogen detection and defense response . Moreover, it was observed that TaNOX3 is highly coexpressed with the genes encoding two BRASSINOSTEROID INSENSITIVE1 (BRI1) precursor kinases, and TaNOX7 is coexpressed with three PLC-like phosphodiesterases superfamily proteins (Figure 9, Figure S6, Table S5). BRI1 is a key component in brassinosteroid (BR) perception and signal transduction and has a broad impact on plant growth and development (Oh et al., 2012). Phospholipase C (PLC) is a class of membrane-associated enzymes playing a crucial role in signal transduction pathways (Singh et al., 2015). Our results obtained here have given much cues for the functions of TaNOXs and the possible working mechanism in stress tolerance but needs further investigation.
The coexpression of TaNOXs with several CESA proteins is another interesting event, especially under stress conditions. A total of nine CESA cellulose synthases were found to be highly coexpressed with the TaNOXs including TaNOX2, 8, and 13 (Table S5), indicating that these TaNOXs have important roles in cell wall formation both under normal and stress conditions. CESA cellulose synthases are the key enzymes for cellulose synthesis during the cell wall formation (Endler and Persson, 2011). It was also proposed that ROS, such as superoxide anion radical and hydrogen peroxide, function in cell wall loosing and stiffing, and in these processes, the NOXmediated ROS production plays an important role (Karkonen and Kuchitsu, 2015). Furthermore, the ROS production also plays a key role in the process of cell wall remodeling under abiotic stresses (Tenhaken, 2015). All of these results suggest that some complicated regulatory mechanisms might be involved in the developmental regulation and stress response of TaNOXs in wheat.
Several other stress response-related genes were also found to be highly coexpressed with certain TaNOXs, further demonstrating the wide functions but complicated regulatory mechanisms of the TaNOXs during the plant development and stress responses. For instance, TaNOX3 is coexpressed with glutathione S-transferase tau 7, and TaNOX13 has coexpression with NAD kinase 1 (NADK1). Glutathione S-transferase (GST) is a multifunctional ROS scavenging enzyme that is involved in a variety of cellular processes. In Arabidopsis, a homolog of glutathione S-transferase tau 7 was found to be related to detoxification, secondary metabolism, and stress responses to Botrytis cinerea or Pseudomonas syringae (Fode et al., 2008). NADKs are the enzymes found in both prokaryotes and eukaryotes, generating pyridine nucleotide NADP(H) from substrates ATP and NAD(H). NADP(H) can be used as the electron donor to NOXs for generating ROS in plants (Turner et al., 2004). In Arabidopsis, NADK1 was found to be induced by environmental stresses and participated in plant pathogen interaction (Berrin et al., 2005). Moreover, the TaNOXs including TaNOX7, 12, and 13 also exhibit high coexpression with the genes encoding two O-fucosyltransferase family proteins, one starch synthase 2, and two melibiase family proteins, respectively, which all play important roles in glycometabolism. All these results suggest that the TaNOXs themselves or with their coexpression genes, synergistically or antagonistically, display pleiotropic functions in a wide range of processes of substance and energy metabolism, detoxification, substance transport, development regulation, and defence response, again implying the specific but complicated functions of the NOX genes in the wheat development and/or stress responses.
Taken together, wheat has multiple members of NOXs with diverse but vital functions in plant growth and development regulation and stress responses. Every member of TaNOXs has its specific expression pattern and function. Meanwhile, the functions of each wheat NOX and its regulatory mechanism in development regulation and defence response are still unclear. The interactions among TaNOXs and other genes are still barely reported in detail. The results obtained have provided a valuable foundation for further exploring the functions of NOX genes in wheat and in other plants as well.

AUTHOR CONTRIBUTIONS
K-MC proposed concept. C-HH and P-QW collected data. C-HH, X-YW, and BY made the abiotic stress treatments.
C-HH, X-YW, L-BY, T-TM, P-PZ, XW, W-TL, W-QL, and L-SM contributed for coexpression data. K-MC and C-HH wrote the manuscript.

FUNDING
This work was supported by the National Natural Science Foundation of China (grant nos. 31770204 and 31270299) and the Program for New Century Excellent Talents in University of China (NCET-11-0440).


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Insulin and 20-hydroxyecdysone oppose each other in the regulation of phosphoinositide-dependent kinase-1 expression during insect pupation

Insulin promotes larval growth of insects by stimulating the synthesis of the steroid hormone 20-hydroxyecdysone (20E), which induces pupation and apoptosis. However, the mechanism underlying the coordinate regulation of insect pupation and apoptosis by these two functionally opposing hormones is still unclear. Here, using the lepidopteran insect and serious agricultural pest Helicoverpa armigera (cotton bollworm) as a model, we report that phosphoinositide-dependent kinase-1 (PDK1) and forkhead box O (FoxO) play key roles in these processes. We found that the transcript levels of the PDK1 gene are increased during the larval feeding stages. Moreover, PDK1 expression was increased by insulin, but repressed by 20E. dsRNA-mediated PDK1 knockdown in the H. armigera larvae delayed pupation and resulted in small pupae and also decreased Akt/protein kinase B expression and increased FoxO expression. Furthermore, the PDK1 knockdown blocked midgut remodeling and decreased 20E levels in the larvae. Of note, injecting larvae with 20E overcame the effect of the PDK1 knockdown and restored midgut remodeling. FoxO overexpression in an H. armigera epidermal cell line (HaEpi) did not induce apoptosis, but promoted autophagy and repressed cell proliferation. These results reveal cross-talk between insulin and 20E and that both hormones oppose each other's activities in the regulation of insect pupation and apoptosis by controlling PDK1 expression and, in turn, FoxO expression. We conclude that sufficiently high 20E levels are a key factor for inducing apoptosis during insect pupation.

The insulin/insulin-like growth factor (IGF) 3 signaling (IIS) pathway is a conserved pathway in animals (1). Mammalian insulin is produced in the islets of Langerhans in the pancreas, and insect IGFs are produced in the brain and various tissues. Although the production organs for mammalian insulin and insect IGFs are different, the structures of insulin and IGFs are similar and consist of two peptide chains, namely the A and B chains (2). Bombyxin is the first insulin-like peptide identified in Bombyx mori (3). In insects, up to 38 insulin-like peptides have been identified in each species (4). The function of the IIS pathway is to promote growth (5,6). In mammals, the IIS pathway regulates the metabolism of carbohydrates, fats, and proteins by promoting the absorption of glucose from the blood into the liver and fat and skeletal muscle cells (7). In Drosophila, the IIS pathway promotes cell growth and proliferation (5) (8). The effect of the IIS on metabolism of insects is little known.
Phosphoinositide-dependent kinase-1 (PDK1) is a central mediator in various pathways, including the insulin pathway, by phosphorylating many AGC kinases, such as protein kinase B (Akt/PKB), p70 ribosomal S6 kinase, p90 ribosomal S6 kinase, protein kinase N, and all protein kinase Cs (9). In the insulin pathway, PDK1 phosphorylates Akt/PKB (10), which then phosphorylates the transcription factor forkhead box O (FoxO). The phosphorylated FoxO is located in the cytoplasm and loses its transcription activity in the nucleus (11,12). Thus, insulin can promote cell proliferation and repress apoptosis (13). Repression of the IIS pathway influences the morphology of Drosophila (14). Repression of insulin/TOR signaling in Drosophila suppresses larval growth and delays pupation time (15). Overexpression of PDK1 in Drosophila increases cell and organ size (9,14). However, there is limited information on the involvement and mechanism of PDK1 during pupation in lepidopteran insects.
In insects, pupation is promoted by 20-hydroxyecdysone (20E), which is a steroid hormone that promotes insect molting and metamorphosis (16). 20E combines with its nuclear receptor EcRB1 to form the EcRB1-USP1 heterodimeric transcription complex (17,18), which regulates the expression of genes in the 20E pathway, including hormone receptor 3 (HR3) (19) and broad (Br) (20), that initiate metamorphosis and midgut remodeling. Midgut remodeling is controlled by 20E, and it includes imaginal midgut formation after the proliferation of regenerative cells and larval midgut programmed cell death (PCD). During larval midgut PCD, the color of the larval midgut changes to red, and it is finally degraded through PCD (21)(22)(23).
In B. mori, autophagy and apoptosis successively occur during larval midgut PCD (24). Insulin and 20E cross-talk to regulate insect development. Insulin promotes 20E production by promoting insect growth to critical body weight, and the prothoracic glands (PGs) grow larger to produce more 20E for metamorphosis (25,26). In Drosophila melanogaster, overexpression of FoxO in the PGs delays ecdysone biosynthesis and critical weight (27). Human insulin can induce Akt phosphorylation in Helicoverpa armigera, thereby inducing FoxO phosphorylation and cytoplasm localization. In contrast, 20E represses Akt phosphorylation, thereby repressing FoxO phosphorylation and inducing FoxO nuclear localization (28). Because PDK is a key protein kinase in the insulin pathway, we hypothesized that PDK is involved in the cross-talk between insulin and 20E.
Using H. armigera, the cotton bollworm, as a model, we identified PDK1 as a key factor for the coordinated regulation of insect midgut remodeling and pupation by insulin and 20E. Our results showed that PDK1 plays key roles in insect pupation by participating in the insulin pathway. Insulin and 20E cross-talk by counteractively regulating PDK1 mRNA levels to regulate insect pupation.

Insulin promoted PDK1 expression and 20E repressed PDK1 expression
To examine the involvement of PDK1 in insect development, the expression profile of PDK1 was examined (Fig. 1). The qRT-PCR results showed that PDK1 was expressed in the epidermis, fat body, and midgut. The transcript levels of PDK1 increased during the feeding stages of the sixth instar at 6 h to sixth instar at 24 h (6th-6h to 6th-24h) in the fat body, epidermis, and midgut and then decreased from the sixth instar at 48 h and were low during the metamorphic stages (6th-72h to 6th-120h) (Fig. 1A), indicating that PDK1 transcript levels are correlated with feeding.
HaEpi, a cell line established in our laboratory from the fifth instar larval epidermis (29), was used to examine the regulation of PDK1 mRNA levels by insulin and 20E. After insulin stimulation for 3 h, PDK1 transcripts were up-regulated in a dose-de-  (3-24 h). DMSO was used as the solvent control. F-I, hormonal regulation of the expression of PDK1 in the larval midgut. All of the experiments were performed in triplicate, and statistical analysis was conducted using Student's t test. Bars, mean Ϯ S.D. The asterisks indicate the significant differences when compared with PBS or DMSO: *, p Ͻ 0.05; **, p Ͻ 0.01.

Insulin and 20E cross-talk to regulate insect pupation
pendent manner from 2 to 5 g/ml (Fig. 1B). By stimulation with 2 g/ml insulin, PDK1 transcripts were up-regulated from 1 to 3 h gradually, and they then recovered to the base levels (Fig. 1C). These results suggested that insulin increases the mRNA levels of PDK1 in a dose-dependent manner in a short period.
Because the 20E titer is higher at metamorphic molting in lepidopteran insects (16), HaEpi cells were treated with exogenous 20E to examine the effects of regulation of 20E on PDK1 expression. We found that 5 M 20E decreased PDK1 transcript levels significantly in 6 h (Fig. 1D), and 5 M 20E consistently decreased PDK1 transcript levels significantly from 3 to 24 h (Fig. 1E). The in vivo results of the larval midgut also confirmed that insulin promoted PDK1 expression and 20E repressed PDK1 expression (Fig. 1, F-I). These results suggested that 20E consistently decreases the expression of PDK1 in a dose-dependent manner.

PDK1 determined pupation time and pupa weight
To study the functions of PDK1 during H. armigera growth and pupation, PDK1 was knocked down by injecting dsRNA against PDK1 into sixth-instar 6-h larvae. The larvae formed small pupae ( Fig. 2A). Statistical analysis showed no differences in the pupation rate after injection with dsPDK1 or dsGFP, with 75% survival and 25% death (Fig. 2B); however, the dsPDK1 injection prolonged pupal duration and delayed the initiation time of pupation for 34 h on average (recorded from sixth instar 0 h) (Fig. 2C). Moreover, the pupal weight decreased to an average of 0.16 g, compared with 0.32 g of the dsGFP injection control (Fig. 2D). These results suggested that PDK1 is critical for pupation.
To understand how PDK1 knockdown delayed pupation, the downstream genes in the insulin pathway were detected. The qRT-PCR assay showed that the expression of Akt decreased and that of FoxO increased after knockdown of PDK1 in the midgut (Fig. 2E). Therefore, PDK1 affects the transcript levels of Akt and FoxO.

Knockdown of PDK1 in the larvae repressed midgut remodeling and decreased 20E titer
To examine the effects of PDK1 knockdown on midgut remodeling, we dissected the larvae 90 h after dsRNA injection of sixth-instar 6 h larvae (6th-6h larvae). All midguts of the dsGFP-injected larvae appeared in red color (23), which implies that the midgut was entering PCD (30); in contrast, all midguts of the dsPDK1-injected larvae appeared yellow, indicating that the midgut did not enter PCD (Fig. 3A). Hematoxylin-eosin   (HE) staining showed that the larval midgut separated from the imaginal midgut after dsGFP injection, indicating the occurrence of midgut remodeling. In contrast, the imaginal midgut did not form after dsPDK1 injection (Fig. 3B), confirming that PDK1 knockdown repressed midgut remodeling.

Insulin and 20E cross-talk to regulate insect pupation
The detailed structure of the midgut was observed using transmission EM (TEM) after PDK1 knockdown. The number of typical autophagosomes, which are two-membraned and contain degenerating cytoplasmic organelles or cytosol (31), increased in the midgut after PDK1 knockdown in the midgut when compared with the dsGFP control (Fig. 3, C and D), suggesting that PDK1 knockdown arrested the midgut at the autophagic stage.
Because 20E is the key factor for promotion of midgut remodeling, the 20E titer was suspected to be insufficient on the basis of decreased body weight after PDK1 knockdown. Therefore, we determined the 20E titer in the whole larvae 90 h after injection with dsRNA and found that the 20E titer had decreased significantly after injection with dsPDK1 (Fig. 3E). These results suggested that knockdown of PDK1 decreases the 20E titer, thereby repressing midgut remodeling.

Complementation of 20E rescued the effect of PDK1 knockdown
To prove that knockdown of PDK1 repressed apoptosis because of the low titer of 20E, 20E (500 ng/larva) was injected into sixth-instar 6-h larvae after injection with dsPDK1 for 48 h. When compared with the DMSO control, the larval midgut appeared red (23, 32) (Fig. 4A), and the larval midgut separated from the imaginal midgut ( Fig. 4B), which indicated that complementation of 20E recovered midgut remodeling when PDK1 was knocked down.
PDK1 was knocked down in HaEpi to confirm that it caused autophagy, as observed in the midgut. The autophagy-related protein, microtubule-associated protein 1 light chain 3 (LC3, one isoform of ATG8) (33), was overexpressed as a fusion protein with the cell-penetrating TAT peptide and red fluorescent protein (RFP) (His-TAT-RFP-LC3-His) to detect autophagosomes in HaEpi (34). The TAT peptide helped the fusion protein His-TAT-RFP-LC3-His to enter the cells (35), RFP showed red fluorescence, and LC3 protein indicated the puncta of autophagosomes by its location in the autophagosome membrane (31). His-TAT-RFP-LC3-His indicated that LC3 formed puncta after PDK1 was knocked down in dsPDK1 ϩ DMSOtreated cells, but 5 M 20E blocked the formation of the LC3 puncta significantly in dsPDK1 ϩ 20E-treated cells (Fig. 4, C and D).
In contrast, caspase-3 activity was detected in few dsPDK1 ϩ DMSO-treated cells, but caspase-3 activity was detected in 85% of dsPDK1 ϩ 20E-treated cells (Fig. 4, E and F). The efficiency of interference of PDK1 in HaEpi was significant (Fig.  4G). These results suggested that PDK1 interference indeed induces autophagy but could not induce apoptosis, and 20E is the key factor for inducing apoptosis.

Overexpression of FoxO induced autophagy but not apoptosis
We observed that PDK1 knockdown caused an increase in FoxO mRNA levels in the larval midgut; however, the midgut was arrested at the autophagic stage, which implied that FoxO could induce autophagy but not apoptosis without sufficient 20E. To confirm that FoxO induced autophagy, we detected the transformation of LC3-I to LC3-II (phosphatidylethanolamine form) by using Western blotting, which indicates autophagy (36). FoxO-GFP was confirmed to be overexpressed in the HaEpi cells (Fig. 5A). Overexpression of FoxO-GFP increased the LC3-II level, whereas the addition of 5 M 20E for 72 h decreased the LC3-II level significantly (Fig. 5, B and C).
RFP-LC3 was overexpressed in HaEpi to indicate the autophagosomes to further confirm that overexpression of FoxO induces autophagy. RFP-LC3 is a fluorescent protein that can reveal autophagosomes (34,37,38). The number of autophagosomes increased after overexpression of FoxO-GFP-His; however, the addition of 20E for 72 h decreased the autophagosomes (Fig. 5, D and E). The negative control by the RFP tag had fewer autophagosomes (Fig. 5F). These results confirmed that FoxO promotes autophagy, but treatment with 5 M 20E for 72 h caused the disappearance of autophagosomes.

Insulin and 20E cross-talk to regulate insect pupation
FoxO-His was overexpressed in HaEpi ( Fig. 6A) to evaluate the role of FoxO in apoptosis. Caspase-3 activity was detected in 18% of His tag overexpression cells after the addition of 20E, with DMSO treatment as a solvent control, suggesting 20Einduced apoptosis. Similarly, caspase-3 activity was detected in 18% cells in FoxO-His-overexpressing cells after the addition of 20E (Fig. 6, B and C). Flow cytometry analysis further confirmed that His tag overexpression and FoxO-His overexpression induced 3-4% cell apoptosis, but 20E supplementation induced 18% cell apoptosis (Fig. 6, D and E). These results confirmed that overexpression of FoxO alone cannot induce apoptosis.

Overexpression of FoxO repressed cell proliferation
PDK1 knockdown in the larvae produced small pupae and repressed the formation of imaginal midgut cells, which was accompanied by an increase in FoxO mRNA levels. Thus, FoxO was hypothesized to be involved in the repression of cell proliferation. To verify the assumption, cell proliferation was detected by 5-ethynyl-2Ј-deoxyuridine (EdU) staining after overexpression of FoxO-GFP by using the pIEx-FoxO-GFP-His plasmid in HaEpi. The results showed that the proliferative sig-nal EdU was detected in the nucleus of GFP-overexpressing cells when compared with the 20E-treated cells (5 M for 72 h), suggesting that the GFP-overexpressing cells proliferate normally and 20E represses cell proliferation. However, a very low EdU signal was detected in the FoxO-GFP-overexpressing cells, regardless of DMSO treatment or 5 M 20E treatment (Fig. 7, A and B); this suggested that FoxO overexpression results in an effect similar to that after 20E treatment to repress cell proliferation.

PDK1 knockdown inhibits insulin-induced Akt and FoxO phosphorylation
To verify the function of PDK1 in the insulin pathway in H. armigera, we analyzed the involvement of PDK1 in insulininduced Akt and FoxO phosphorylation in HaEpi by overexpression of Akt fused with RFP-His and FoxO fused with GFP-His in the cells, respectively. Western blotting showed insulin-induced phosphorylation of both Akt and FoxO. However, knockdown of PDK1 blocked insulin-induced Akt and FoxO phosphorylation significantly (Fig. 8, A (a) and B (b)). These results confirmed that insulin, via PDK1, induced Akt and FoxO phosphorylation in H. armigera. Moreover, insulin

Insulin and 20E cross-talk to regulate insect pupation
induced FoxO translocation to the cytosol from the nucleus by GFP co-fluorescence indication. However, knockdown of PDK1 blocked insulin-induced FoxO translocation to the cytosol (Fig. 8C). These results confirmed that PDK1 is involved in the insulin pathway in H. armigera.

Discussion
IGF intensifies the cell proliferation and inhibition of cell apoptosis via the PI3K/Akt signaling pathway (39), whereas the steroid hormone 20E promotes apoptosis during insect metamorphosis (40). It is intriguing how these functionally counteractive hormones regulate insect pupation and apoptosis coordinately. Our study showed that PDK1 plays a critical role in the cross-talk between insulin and the steroid hormone 20E. PDK1 is necessary for 20E synthesis. PDK1 functions as a repressor of FoxO. 20E represses PDK1 expression to release FoxO expression for autophagy, and 20E finally promotes apoptosis.

PDK1 is necessary for 20E synthesis to regulate insect metamorphosis and apoptosis
Repression of insulin and reduced ecdysone levels have similar effects on larval pupation (41). Insulin promotes insect body growth and PG growth, thereby promoting 20E synthesis (25). The 20E titer is lower than 0.5 g/ml during the feeding stage, but it increases to about 5 g/ml in the hemolymph during the metamorphic stage of Antheraea mylitta (42). In this study, we found that repression of the insulin pathway via knockdown of PDK1 in the larvae resulted in a decrease in the 20E titer from 1.5 to 0.1 g/g, suggesting that insulin via PDK1 regulates the increase in 20E levels.
Larval midgut PCD has been proven to be apoptosismediated during metamorphosis in the lepidopteran insects Bombyx mori (43) and H. armigera (30). Autophagy was observed before apoptosis in the midgut of B. mori (44,45). In H. armigera, a high concentration of 20E promotes apoptosis, but a low concentration of 20E induces autophagy; autophagy is switched to apoptosis when a high concentration of 20E triggers Ca 2ϩ influx (34). We found that the decreased 20E titer after PDK1 knockdown could not trigger midgut remodeling and pupation. By complementation of 20E in the larvae, midgut remodeling was recovered, confirming that 20E is the critical factor that promotes midgut remodeling. Our study confirmed that the insulin and 20E interplay for regulating metamorphosis is a common mechanism in insects.

PDK1 plays a significant role in repressing FoxO mRNA levels
FoxO is a transcription factor that regulates diverse processes, such as energy homeostasis, apoptosis, and cellular dif-

Insulin and 20E cross-talk to regulate insect pupation
ferentiation, and life span. FoxO is phosphorylated by Akt and arrested in the cytoplasm to lose its transcriptional function under insulin regulation (13). The phosphorylation of Akt and FoxO in H. armigera is induced by insulin and repressed by 20E in HaEpi, and the nonphosphorylated FoxO is localized in the nucleus (28). However, little is known about the transcriptional regulation of FoxO, compared with its post-translational modifications (46). Previous studies have reported that 20E represses FoxO phosphorylation and promotes FoxO nuclear translocation in Drosophila (41) and Bombyx (47). In H. armigera, 20E via its nuclear receptor EcRB1 promotes FoxO expression (28). In this study, we found that PDK1 knockdown caused an increase in FoxO mRNA, which suggests that PDK1 is a repressor of FoxO. PDK1 plays a key role in the insulin pathway by regulating Akt phosphorylation (10); thus, FoxO transcription is possibly repressed by the insulin pathway.
FoxO regulates the life span of Drosophila (48,49). We found that PDK1 knockdown increased FoxO mRNA levels, elongated the larval stage, and delayed pupation time, suggesting that FoxO plays a role in the elongated larval life span. Further analyses of the HaEpi cells showed that the overexpressed FoxO induced autophagy. This phenomenon is correlated to the finding that FoxO promotes autophagy-related gene expression in animals (50). However, overexpressed FoxO did not induce apoptosis. In addition, we found that, although PDK1 knockdown decreased Akt expression and increased FoxO expression, midgut PCD was not induced when 20E was insufficient. Therefore, apoptosis is controlled by 20E in a FoxO-independent manner, which is inconsistent with the finding that FoxO induces apoptosis in humans (51, 52). This may be because 20E is necessary for inducing high cellular Ca 2ϩ levels for apoptosis (53) and the transformation of autophagy to apoptosis in insects (34).

20E via repressing PDK1 mRNA levels antagonizes the insulin pathway
Insulin and 20E had an antagonistic effect on the regulation of glycogen mobilization and showed a similar effect on carbo-  for the regulation of pupation by insulin and 20E via counteractive regulation of PDK1 expression. 1, insulin up-regulates PDK1 expression, which induces Akt phosphorylation. Akt induces FoxO phosphorylation and cytosol localization to allow cell proliferation and high titer of 20E production. 2, high 20E titer represses PDK1 mRNA levels by an unknown negative feedback mechanism and therefore represses Akt and FoxO phosphorylation, resulting in FoxO nuclear localization. 3, FoxO in the nucleus induces autophagy and represses cell proliferation. 4, 20E promotes autophagy transformed to apoptosis in the midgut during metamorphosis by increasing cellular calcium (34,53,66). High 20E titer functions as a switch between growth and metamorphosis. Akt-P, phosphorylated Akt. FoxO-P, phosphorylated FoxO. PCD, programmed cell death. *, p Ͻ 0.05; **, p Ͻ 0.01.

Insulin and 20E cross-talk to regulate insect pupation
hydrate reserves in B. mori (54). A lower 20E concentration could not repress insulin function (30), and a higher 20E concentration is critical for antagonizing insulin function in H. armigera (55). The ecdysone counteracts the growth-promoting action of insulin by mediating nuclear localization of dFOXO in Drosophila (41). 20E promotes FoxO expression and represses FoxO phosphorylation by up-regulating PTEN (phosphatase and tensin homolog) expression, which represses Akt and FoxO phosphorylation in H. armigera (28). Here, we showed that 20E counteracts the insulin pathway by repressing PDK1 expression in a dose-dependent manner. 20E repressed the insulin pathway via a negative feedback mechanism when it was increased to a critical titer by insulin. This is consistent with the finding that increased 20E levels negatively repress insulin function (56). However, the mechanism that 20E represses PDK1 expression needs further study in future work.

Conclusions
PDK1 was highly expressed during the larval feeding stage to promote cell proliferation and larval growth under insulin regulation. 20E titer was increased along with larval growth and therefore repressed PDK1 expression in a dose-dependent manner. PDK1 repressed FoxO expression. FoxO induced autophagy but not apoptosis under insufficient 20E titer conditions. A sufficient 20E titer is the key factor for triggering metamorphosis and midgut apoptosis (Fig. 8D).

Experimental animals
H. armigera specimens were raised on an artificial diet, according to a previous study (57), in our laboratory. The larvae were maintained at 27°C with 60 -70% relative humidity and conditions of 14-h light and 10-h dark.

HaEpi culture
HaEpi was established in our laboratory from the fifth-instar larval epidermis (29). The cell line has been used to study the hormonal pathways of 20E, JH, and insulin by RNAi or overexpression (28). The cells were cultured in cell culture flasks with a monolayer at the bottom and maintained at 27°C. In every tissue culture flask, 4 ml of Grace's medium supplemented with 10% heat-inactivated FBS was added, and subculture was performed once a week.

RNAi in the larvae and HaEpi cell line
dsRNA was prepared using PDK1 cDNA as the template (1719 bp) with the MEGAscript TM RNAi kit (Ambion, Austin, TX) and primers (PDK1RNAiF and PDK1RNAiR sequences in Table 1). A single long dsRNA was used for H. armigera and produced good results in our previous study (58,59) and other lepidopteran insects (60). Thus, a long dsRNA was used (870 bp from bp 352 to 1222) for the experiments. The long dsRNA was cleaved into multiple siRNAs in the cells by intracellular RNase to target multiple sites of the gene (61,62). We checked the RNAi ratio by using the primers PDK1-RTF and PDK1-RTR (Table 1). For the RNAi experiment using the larvae, dsRNA was diluted to 400 ng/l with PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 ), and 5 l was injected into the hemocoel of the sixth-instar 6-h larvae. The control larvae were injected with 2 g of dsGFP. In every experiment, 30 larvae were injected with dsRNA, and the injection was repeated twice over a 24-h interval. HaEpi was cultured at 27°C in Grace's medium supplemented with 10% FBS to almost 80% density. Then the cells were incubated in 2 g of dsRNA and 10 l of Lipofectamine in 1 ml of Grace's medium supplemented with 10% FBS after 6 h.

Hormone treatment for the larvae and HaEpi cell line
The stock solution for 20E (10 mg/ml, about 20 mM in DMSO) was diluted 100-fold with PBS (10 mM Na 2 HPO 4 , 1.8

Insulin and 20E cross-talk to regulate insect pupation
mM KH 2 PO 4 , 140 mM NaCl, and 2.7 mM KCl, pH 7.4) and injected into sixth-instar 6-h larvae (100 -500 ng/larva). The stock solution for human insulin (10 mg/ml) was diluted 10-fold with PBS and injected into sixth-instar 6-h (6th-6h) larvae (0.5-5 g/larva). HaEpi was cultured to ϳ80% density, and 20E or stock solution of human insulin (10 mg/ml in PBS) was added at various concentrations or intervals. The control larvae or HaEpi cells were treated with the same amount of DMSO as the solvent control.

Detection of 20E levels
The larvae were frozen in liquid nitrogen for 5 min and homogenized sufficiently in 1 ml of 80% methyl alcohol and centrifuged at 12,000 ϫ g and 4°C for 15 min. The supernatant was air-dried. Then the 20-hydroxyecdysone enzyme immunoassay kit (Bertin Pharma, Montigny-le-Bretonneux, France) was used to measure the 20E titer, according to the manufacturer's instructions.

qRT-PCR
Total RNA was acquired from the larvae using TransZol reagent (Transgen, Beijing, China). Total RNA was transcribed into first-strand cDNA as the midgut template for qRT-PCR. Then 10 l of the reaction mixture, containing 5 l of SsoFast EvaGreen Supermix (Bio-Rad), 2 l of 1 mol/liter forward primer, 2 l of 1 mol/liter reverse primer, and 1 l of cDNA (1:6 dilution), was used. ␤-Actin was used for quality control (59). The data were analyzed using the formula, r ϭ 2 Ϫ(⌬Ct sample Ϫ ⌬Ct control) , where R is the relative transcriptional level, ⌬Ct sample is the difference between the Ct of the gene and average ␤-actin in the experimental sample, and ⌬Ct control is the difference between the Ct of the gene and average ␤-actin in the control sample (63).

Hematoxylin-eosin (HE) staining and TEM
The waxing and dewaxing of the samples and HE staining were performed as described previously (30). The section was washed with running water for 1 min and stained with Scott's liquid for 1 min. Then the section was washed with hydrochloric acid ethanol differentiation medium (70% ethanol in 1% hydrochloric acid) for 20 s, stained with Scott's liquid for 1 min, incubated with 0.5% water-soluble eosin dye solution for 30 s, and then washed with running water. Finally, the sections were sealed with 80% glycerin and observed using an Olympus BX51 fluorescence microscope (Tokyo, Japan). TEM was performed by the Servicebio Company (Wuhan, China) after the larvae were treated in the various experiments.

Overexpression of proteins in the HaEpi cells
FoxO-GFP-His, FoxO-His, GFP-His, or His tag proteins constructed in our laboratory (28) were overexpressed with 4 g of pIEx-4 plasmid (Novagen) and 10 l of QuickShuttle-enhanced transfection reagent (Biodragon Immunotechnologies Co., Ltd., Beijing, China) for 48 h in 1 ml of Grace's medium, according to the manufacturer's instructions and our previous study (34).

Western blotting
The cell homogenates were separated using 12.5% SDS-PAGE. Equal amounts of each sample (50 g) was subjected to SDS-PAGE. The proteins were transferred to the nitrocellulose membrane (0.45 m) by electrotransfer. The membrane was blocked with the blocking buffer (2% nonfat milk in PBS) for 1 h at room temperature. The membrane was then incubated with the primary antibody (diluted 1:5000 in the blocking buffer) against GFP or His tags (Zhong Shan Jin Qiao, Beijing, China) overnight at 4°C and with the secondary antibody for alkaline phosphatase (Zhong Shan Jin Qiao) conjugated with horse antimouse IgG diluted to 1:10,000 in the blocking buffer. The protein signal was visualized using 45 l of nitro blue tetrazolium (75 mg/ml) and 35 l of 5-bromo-4-chloro-3-indolylphosphate (50 mg/ml; Sigma) in 10 ml of TBS in the dark at room temperature. The protein bands on the membrane were analyzed using Quantity One software (Bio-Rad).

Detection of apoptosis
An aspartic acid proteinase called caspase-3 is a highly conserved protein that can form active caspase-3 during apoptosis (65). Thus, caspase-3 activity can be used to indicate apoptosis. The NucView TM caspase-3 assay kit (catalog no. 30029, Biotium, Fremont, CA) was used to detect caspase-3 activity in the HaEpi cells, according to the manufacturer's instructions. Annexin V and propidium iodide (PI) were used to detect apoptosis by using the Annexin V-FITC apoptosis detection kit (GK3603, Genview) and flow cytometry (Amnis). Annexin V-FITC stained the earlier apoptotic cells by binding to the membrane phosphatidylserine, and PI indicated the later apoptotic cells and dead cells by entering the cells.

Detection of cell proliferation
The EdU kit (Ribobio, Guangzhou, China) was used to detect cell proliferation levels, according to the manufacturer's protocol. FoxO-GFP and GFP tags were overexpressed by the pIEx-4-FoxO-GFP-His and pIEx-4-GFP-His plasmids, respectively, in the HaEpi cells for 24 h. Then the cells were treated with 5 M 20E for 72 h. The same volume of DMSO was used as the control.

Statistical analysis
Student's t test was used for statistical analysis of the paired data. The bars represent mean Ϯ S.D. values of three independent biological experiments. Statistical significance was determined using two-tailed paired Student's t test. Differences were considered statistically significant at p Ͻ 0.05 (*) and p Ͻ 0.01 Insulin and 20E cross-talk to regulate insect pupation (**). GraphPad Prism 5 (version 5.01) was used to produce the figures.