Genomic and transcriptomic insights into cytochrome P450 monooxygenase genes involved in nicosulfuron tolerance in maize(Zea mays L.)

LIU Xiao-min, XU Xian, LI Bing-hua, YAO Xiao-xia, ZHANG Huan-huan, WANG Gui-qi, HAN Yu-jun

1 Institute of Cereal and Oil Crops, Hebei Academy of Agriculture and Forestry Sciences, Shijiazhuang 050035, P.R.China

2 Xingtai Academy of Agriculture Sciences, Xingtai 054000, P.R.China

3 College of Agriculture, Northeast Agricultural University, Harbin 150030, P.R.China

Abstract Postemergence application of nicosulfuron for weed control in maize fields can cause great damage to certain maize inbred lines and hybrids. Two maize genotypes, tolerant inbred (HBR) and sensitive inbred (HBS), were found to significantly differ in their phenotypic responses to nicosulfuron, with the EC50 (50% effective concentration) values differed statistically(763.6 and 5.9 g a.i. ha–1, respectively). Pre-treatment with malathion, a known cytochrome P450 inhibitor, increased nicosulfuron injury in both HBR and HBS. Our results support the hypothesis that nicosulfuron selectivity in maize is associated with cytochrome P450 metabolism. Further analysis of the maize genome resulted in the identification of 314 full length cytochrome P450 monooxygenase (CYP) genes. These genes were classified into 2 types, 10 clans and 44 families. The CYP71 clan was represented by all A-type genes (168) belonging to 17 families. Nine clans possessed 27 families containing 146 non-A-type genes. The consensus sequences of the heme-binding regions of A-type and non-A-type CYP proteins are ‘PFGXGRRXCPG’ and ‘FXXGPRXCXG’, respectively. Illumina transcriptome sequence results showed that there were 53 differentially expressed CYP genes on the basis of high variation in expression between HBS and HBR,nicosulfuron-treated and untreated samples. These genes may contribute to nicosulfuron tolerance in maize. A hierarchical clustering analysis obtained four main clusters named C1 to C4 in which 4, 15, 21, and 13 CYP genes were found in each respective cluster. The expression patterns of some CYP genes were confirmed by RT-qPCR analysis. The research will improve our understanding of the function of maize cytochrome P450 in herbicide metabolism.

Keywords: cytochrome P450, Zea mays L., malathion, nicosulfuron, herbicide metabolism

1. Introduction

Cytochrome P450 monooxygenases (CYP) are one of the largest family of enzymes, exhibiting an intricate and versatile metabolic function in most organisms (Urlacher and Marco 2012). CYP genes are detected in bacteria,plants, animals and humans, and account for about 1%of the protein-coding genes in plants (Nielsen and M?ller 2005; Nelson and Werckreichhar 2011). More CYP genes have been found to exist in plants compared with other organisms because an estimated 200 000 metabolites are produced by plants (Renaultet al.2014). For example,245 and 343 CYP genes were identified inArabidopsisandOryza sativa, while less than 100 CYP genes exist in animal genomes (Rasool and Rozi 2015). CYP genes are named and classified according to their sequence identity and phylogeny. The CYP51, CYP71-99, and CYP701-CYP999 families in plants were traditionally grouped into A-type and non-A-type (Kahn and Francis 2000; Nelson 2009). More than 50% of A-type CYP genes encoding plant-specific enzymes are involved in the synthesis of secondary products in plants, while non-A-type CYP genes encode enzymes that function in signaling molecules and synthesis of compounds like hormones, sterols and oxygenated fatty acids (Schuler and Werckreichhart 2003).

Enzymes of the cytochrome P450 superfamily are known to function in herbicide resistance and bioremediation of agrochemical and environmental chemical residues, and are responsible for the Phase I herbicide metabolism (Siminszky 2006; Ohkawa and Hideyuki 2015). Barrett (1995)demonstrated that cytochrome P450 monooxygenases were involved in the detoxification of herbicides in corn and other crops. In recent years, identifying the function of CYP genes associated with non-target-site herbicide resistance mechanisms in weeds and crops has become a major research topic (Powles and Qin 2010). CYP genes involved in herbicide metabolism were identified and used to optimize new active compounds (e.g., adding a methylenedioxo or acetylenic function) to improve control of herbicide resistance in weeds (Werckreichhartet al.2000).CYP71A10,CYP71AH11,CYP71C6v1,CYP71R4,CYP76B1,CYP81B1, andCYP81B2in different plants have been isolated and found to be involved in metabolism of the phenylurea herbicide chlorotoluron (Cabellohurtadoet al.1998; Robineauet al.1998; Siminszkyet al.1999;Werckreichhartet al.2000; O’Keefeet al.2002). Recently,a novel rice cytochrome P450 gene,CYP72A31, was found to confer tolerance to acetolactate synthase-inhibiting herbicides such as bispyribac sodium (Saikaet al.2014).

Maize (Zea maysL.) is one of the most economically important crops in the world. In the last couple of decades,researchers have documented that some corn hybrids and inbreds were severely injured or killed after post-emergence application of several herbicides, including nicosulfuron,mesotrione, primisulfuron and rimsulfuron (Patakyet al.2006; Meyeret al.2010). Metabolism of different herbicides in maize may involve a three-phase process with the coordinated action of several types of enzymes. Four gene families participate in herbicide detoxification metabolism and they include P450s, GSTs, ABC transporters and glycosyltransferases. Unfortunately, knowledge of genes conferring herbicide metabolic resistance is still limited.Cytochrome P450 enzymes are primarily responsible for the detoxification of some corn herbicides. Mutations of a CYP gene, such as thensf1orben1on the short arm of chromosome 5, can induce sensitivity to multiple P450-metabolized herbicides (Williamset al.2006).Because the maize genome has been sequenced, we now have a valuable resource for genomic analysis of this important gene family (Schnableet al.2009). In this study, we investigated the effects of a cytochrome P450 inhibitor, malathion (an organo-phosphate insecticide),on nicosulfuron injury to the growth of maize. We have also identified different P450 genes from the maize genome, and analyzed the co-expression of P450 genes in nicosulfuron-tolerant and -sensitive inbred lines of maize using Illumina sequencing data. Our objective was to establish a centralized resource of P450 genes in maize for the benefit of future functional studies on herbicide metabolism with these genes.

2. Materials and methods

2.1. Herbicide and malathion treatments

The tolerant inbred (HBR) and sensitive inbred (HBS) lines of maize were obtained from the Institute of Cereal and Oil Crops, Hebei Academy of Agriculture and Forestry Sciences,China. Seeds of HBR and HBS lines were sown in pots(three plants per pot) and grown in a greenhouse with 70 to 80% relative humidity, at (25±2)°C and 14-h/10-h light/dark cycle. Both genotypes were used to examine the function of cytochrome P450 in plant response to nicosulfuron and malathion. Different genotypes of maize plants were treated with 1 000 g a.i. ha?1of malathion when maize plants were at the V3 stage (the three-leaf stage) and left for 1 h in a ventilated room before being treated with nicosulfuron.We carried out three experimental treatments: a control,herbicide, and malathion followed by herbicide treatment.The nicosulfuron application rates assayed were as follows:60, 120, 240 g a.i. ha–1for HBR, and 15, 30, 45 g a.i. ha–1for HBS. Each rate was also assayed in plantlets treated with 1 000 g a.i. ha–1of malathion. The experiment was conducted under a completely randomized design with three replicates. The aboveground biomass from each pot was harvested and weighed after 14 d of treatment. Plant response to herbicides was evaluated as a ratio of fresh biomass relative to that of the untreated control.

2.2. ldentification and motif analysis of CYP genes

The CYP genes of maize were identified as described below.First,Z. maysgenome sequences from the phytozome v11.0 database (http://www.phytozome.jgi.doe.gov/) were queried with a CYP search in the ontologies option. Second, the CYPs were confirmed by the presence of a CYP motif using the ExPASy-PROSITE tool (http://prosite.expasy.org/). All the full-length CYP proteins were named according to the standard CYP nomenclature using sequences from a wellannotated CYP database as reference sequences (Nelsonet al.2004). In particular, 40, 55 and 97% sequence identities were used as cutoffs for family, subfamily and allelic variants, respectively. Lastly, the names of the CYP proteins were assigned by Prof. David Nelson (University of Tennessee, USA).

2.3. Expression analysis of CYP genes using RNASeq data

For the analysis of CYP gene expressions, HBS and HBR maize plants were grown under the greenhouse conditions(27°C/24°C day/night and 16-h/8-h light/dark cycles) with three plants per pot. Two weeks later, the sensitive and tolerant maize seedlings were treated with 60 g a.i. ha–1nicosulfuron. After 24 h of spraying, the nicosulfurontreated and untreated leaves of each HBS and HBR maize plant were harvested. Solexa sequencing libraries for four samples, a sample from each of the untreated and 24 h treated HBS and HBR plants, were utilized to quantify the transcriptomic expression of CYP genes in maize. The expression levels of CYP genes were calculated using the Trinity Program (version 2.0.2, released 22nd January 2015)and represented as numbers of fragments per kilobase per million fragments mapped (FPKM). The genes having a FPKM≤1 were considered as not expressed. The FPKM was added to 1, log transformed and centered with the mean of the expression levels of the four samples and subjected to hierarchical analysis using JMP (version 10, SAS, North Carolina).

2.4. Quantitative RT-PCR analysis

Gene-specific primers were designed using the NCBI Primer-Blast Tool (http://www.ncbi.nlm.nih.gov/tools/primerblast/). The list of genes and primers used for amplification are shown in Table 1. Maize β-actin gene was used as an internal control for gene expression studies. cDNA synthesis and RT-qPCR were performed as described here. The cDNAs were amplified by RT-qPCR in a final volume of 20 μL containing 1 μL cDNA, 10 μL 2× qPCR Master Mix, and 10 μmol of each primer. Amplification was standardized in a 7500 Real-time Fast Thermal Cycler (Applied Biosystems,Foster City, CA, USA) using the following conditions: 50°C for 20 s, 95°C for 10 min followed by 45 cycles of 3 min at 94°C, 15 s at 94°C, 15 s at 58°C and 20 s at 72°C. The PCR products for each primer set were subjected to a melting curve analysis to verify the presence of primer dimers or non-specific amplicons. The temperatures of the melting curve analysis ranged from 60 to 95°C with a stepwise increase in temperature by 1%.

3. Results

3.1. Effect of malathion on nicosulfuron tolerance in maize

Acetolactate synthase (ALS) is responsible for target-site tolerance or sensitivity to sulfonylurea herbicide. Thus,ALS activity was estimatedin vitroin the crude enzyme extracts of maize leaves exposed to different concentrations of nicosulfuron. The results revealed that there were no differences in enzyme sensitivity between HBR and HBS.We also sequenced the full-length genomic DNA sequences of the two maizeALSgenesAHAS108andAHAS109;however, we did not observe any sequence polymorphism within the conserved region ofALSbetween the two genes(data not shown). The results indicated that tolerance to nicosulfuron observed in the study is not controlled by a target site but by a non-target site.

To determine the involvement of cytochrome P450 detoxification, responses of HBR and HBS to nicosulfuron were recorded for a period of 14 d with or without malathion.Statistical analysis showed that nicosulfuron treatment without malathion had a significant main effect of reducing plant biomass of HBS, but the reduction was not significantly different for HBR. However, following pretreatment withmalathion, the application of nicosulfuron caused severe injury to both the tolerant and sensitive genotypes (Fig. 1).The results support the hypothesis that nicosulfuron selectivity in maize is associated with cytochrome P450 metabolism.

Table 1 Forward and reverse primers used for the real-time RT-PCR

3.2. Characterization of CYP proteins in maize

Analysis ofZ. maysgenomes showed that there were 314 cytochrome P450 genes which belonged to 2 types, 10 clans and 44 families (Tables 2 and 3). The CYP71 clan contained 168 A-type CYP genes which belonged to 17 families. A total of 146 non-A-type CYP genes were distributed in nine other clans. Those nine clans formed two groups: singlefamily clans (CYP51, CYP74, CYP97, CYP710, CYP711,and CYP727) and multi-family clans (CYP72, CYP85, and CYP86). Single family clans appear to be ancient because of the presence of orthologs found in green algae, and they have a unique and highly conserved function (Nelson 2006). The CYP72 clan (56 genes) is classified into seven families. The CYP85 clan (29 genes) contains 10 families.The CYP86 clan (40 genes) contains four families, including CYP86, CYP94, CYP96 and CYP704 (Table 3). CYP71 is the largest A-type family, with 57 members, while CYP72 is the largest non-A type family, with 19 members.

Analysis of CYP genes revealed that there were more CYP81, CYP89, CYP92, CYP99, CYP72, CYP709, CYP714 and CYP734 genes in maize than those in soybean,Arabidopsisand rice. CYP99 and CYP729 are present in maize and rice, but are absent in soybean andArabidopsis,whereas CYP82, CYP83, CYP712, CYP716, CYP718,and CYP720 are absent in maize and rice, but are present in soybean andArabidopsis. The CYP705, CYP702 and CYP708 families are absent in maize, soybean and rice,but present inArabidopsis. CYP92, CYP728, CYP733 and CYP727 are present in maize, soybean and rice, but absent inArabidopsis. We did not find any P450 families that are unique to maize.

3.3. Analysis of conserved motifs in the CYP proteins

The cysteine (Cys) heme-iron ligand signature motif, the PERF motif, the conserved EXXR motif, and the conserved threonine are the four distinct characteristics for all CYP protein sequences (Nelsonet al.2006), and the Phe-X-XGly-X-Arg-X-Cys-X-Gly motif near the C-terminus is the most conserved sequence among P450s. Hydrophobic aromatic residue phenylalanine (Phe), invariant hydrophobic Cys and simple small amino acid glycine (Gly) are the conserved motif signature (Saxenaet al.2013). In the study, analysis of motifs revealed varied heme signatures between A-type and non-A-type CYPs. Almost all of the A-type CYPs showed the consensus sequences of the heme-binding regions‘PFGXGRRXCPG’, while almost all of the non-A-type CYPs had the signature sequence ‘FXXGPRXCXG’ (Fig. 2). The consensus sequences of these motifs are similar to those that have previously been described (Duhouxet al.2015).Analysis of the presence of a CYP motif using the ExPASy-PROSITE tool also showed that there were no CYP motif sequences in some CYP proteins, which was also found in the common bean (Kumaret al.2015).

3.4. P450 gene expression profiling using lllumina transcriptome analysis

Fig. 1 Responses of maize to different dosages of nicosulfuron with or without a malathion pretreatment. A, the tolerant inbred(HBR). B, the sensitive inbred (HBS). Plant fresh weight was recorded at 14 d after treatment. Bars represent the means±SE of three replicates. Different letters above error bars indicate significant differences according to Duncan’s test (P＜0.05).

Table 2 Distribution of A-type P450 families in maize, soybean,Arabidopsis and rice

We utilized Illumina transcriptome sequencing technology to analyze the expression of all 314 maize P450 genes.These data sets were generated from total RNAs isolated from leaves of the four samples, including nicosulfurontreated and untreated HBS and HBR samples. The CYP genes with FPKM≤1 in all the four samples were considered as not expressed and were removed. As shown in Fig. 3,a total of 53 differentially expressed genes (DEGs) were selected on the basis of high variation in expression among the four samples (log2Ratio≥1 and the false discovery rate(FDR) significance score＜0.01). To study the co-expression patterns of CYP genes, we performed hierarchical clustering of the expression profiles of these 53 full-length CYP genes, and obtained four main clusters including C1 to C4.There were four highly expressed genes in the untreated nicosulfuron-sensitive HBS maize in cluster C1, includingCYP709E9,CYP79A61,CYP81N5andCYP71C57. The clusters C2 and C4 possessed theCYPgenes that were highly expressed in the nicosulfuron-treated HBS and HBR plants, which included 15 and 13 genes, respectively. The cluster C3 was composed of 21 of the highest expressing CYP genes in the nicosulfuron-tolerant maize HBR, which includedCYP709D2,CYP75B87,CYP714C11,CYP71K15,CYP73A122,CYP90A22,CYP728B1,CYP92A92,CYP51H12,CYP99A5,CYP92A1,CYP72A124,CYP71C4,CYP81A9,CYP90D10,CYP71C2,CYP72A5,CYP714B10,CYP71C3v2,CYP74A39andCYP97A16.

To confirm the accuracy of data from the Illumina sequencing, we carried out RT-qPCR analyses on a setof selected CYP genes. Total RNAs were isolated from the nicosufuron-treated and untreated maize leaves. The expression levels of the above-mentionedCYP81A9,CYP71C3v2,CYP72A5,CYP92A1,CYP71C2andCYP71C4were measured. All six genes showed the same expression trends in the Solexa-sequencing analysis.Compared to HBS, the expression of these six genes were more highly expressed in HBR, andCYP81A9was the most differentially expressed gene with significance between HBR and HBS. The CYP genes were up-regulated after nicosulfruon treatment (Fig. 4).

Table 3 Distribution of non-A-type P450 families in maize,soybean, Arabidopsis and rice

Fig. 2 Conserved heme-binding motifs identified from maize cytochrome P450 monooxygenases (CYPs). A, A-type. B, non-A-type.

4. Discussion

Encoded by multi-gene families, cytochrome P450 monooxygenases (CYPs) and glutathione transferases (GSTs)are crucial enzymes in herbicide detoxification in plants.The CYPs can catalyze oxidation reactions, and GSTs can conjugate electrophilic xenobiotics with glutathione.However, the relationship between CytP450 and GST gene expression levels in response to herbicides remains unknown, and more work is needed to elucidate their roles in herbicide resistance.

The basis for selectivity of sulfonylurea herbicides in conventional maize is mainly due to the fact that it can metabolize herbicides and convert them into nontoxic substances to maize. However, organophosphate insecticides can interfere with sulfonylurea metabolism.Thus sulfonylurea herbicides can harm conventional maize when applied to a plant treated with organophosphate insecticide (Tanet al.2005). In this study, we found significantly greater herbicide injury for both tolerant and sensitive maize inbreds when nicosulfuron was applied following treatment with malathion. This undesirable interaction revealed that the P450-mediated herbicide metabolism results inin vivodetoxification of nicosulfuron in maize. Our result is congruent with previous reports regarding other sweet maize genotypes. Previous genetic analyses revealed that sensitivity of maize to multiple P450-metabolized herbicides was regulated by a single dominant gene,nsf1/ben1, located on the short arm of chromosome 5(Nordbyet al.2008; Williamset al.2008). Williamset al.(2008) used a map-based cloning approach to sequence the dominant allelensf1from the nicosulfuron-tolerant inbred B73 and nicosulfuron-sensitive inbred W703 lines, and found that W703a contained a 392-base pair insertion in thensf1sequence relative to B73 (Williamset al.2006).

Analysis of maize genomes showed that there were 314 cytochrome P450 genes, which accounted for about 0.96% of the predicted protein-encoding genes. The result is congruent with other findings of CYPs representing 0.57 to 1.07% of protein coding genes in various plant species,such as soybean, rice,Arabidopsis, flax and castor (Nelsonet al.2004; Guttikondaet al.2010; Babuet al.2013; Kumaret al.2014). In the present study, all clans were found in maize except CYP746, which was only found in moss and has no known function (Nelson and Werckreichhart 2011).Families CYP99 and CYP723 were only found in monocots.In our results, we found that there were eight and two genes respectively in CYP99 family in maize and rice, which were absent in soybean andArabidopsis. However, we found no gene in CYP723 family in maize, while there were two genes in CYP723 family in rice.

Cytochrome P450 enzymes that metabolize herbicides in plants play an important role in herbicide selectivity and resistance. Most herbicides, for example, prosulfuron,diclofop, chlortoluron and atrazine, can be converted into several metabolites by P450s by catalyzing ring-methyl hydroxylation or di-N-demethylation (H?feret al.2014;Tanet al.2015). Many genes in CYP71, CYP73, CYP76 and CYP81 subfamilies belonging to the CYP7l clan are involved in metabolizing phenylurea and sulfonylurea herbicides (Ohkawa and Inui 2015). Expression of theCYP71A10gene in soybean,CYP71A11in tobacco andCYP71C6V1in wheat have corresponded with high tolerance of these plants to chlortoluron and triasulfuron(Siminszkyet al.1999; Yamadaet al.2000; Xianget al.2006).CYP76B1isolated fromHelianthus tuberosuswas found to be responsible for chlortoluron resistance (Batardet al.1998). TheCYP81A6in rice andCYP81A12andCYP81A21inEchinochloa phyllopogonconferred resistance to bensulfuron-methyl (Panet al.2006; Iwakamiet al.2014).In our study, we identified 21 CYP genes differentially transcribed among the nicosulfuron-tolerant maize inbred HBR and nicosulfuron-sensitive maize inbred HBS. The expression levels ofCYP81A9,CYP71C3v2,CYP72A5,CYP92A1,CYP71C2andCYP71C4were measuredviaRT-qPCR analysis. TheCYP81A9gene is likely involved in nicosulfuron detoxification in maize (Liet al.2013; Liuet al.2015). Persanset al.(2001) reported that the herbicides safener naphthalic anhydride (NA) and triasulfuron induced expression of maize cytochromeCYP71C1,CYP71C3,CYP72A5andCYP92A1. Furthermore, they found thatCYP71C3v2was involved in triasulfuron tolerance (Persanset al.2001). Further studies should focus on these genes to better understand the pathways of herbicide metabolism in maize.

Fig. 3 Clustering analyses of the 53 full-length cytochrome P450 monooxygenase (CYP) genes based on their expression profiles obtained from the RNA-Seq experiment. The expression levels represented by FPKM (fragments per kilobase per million fragments mapped) values were log transformed and mean-centered. Hierarchical clustering was conducted using JMP software. The heat map reflects the relative expression levels of genes in the four treatments: HBS-CK (untreated sensitive maize), HBS-TR (nicosulfuron-treated sensitive maize), HBR-CK (untreated tolerant maize) and HBR-TR (nicosulfuron-treated tolerant maize). The GenBank accession numbers for the genes are as follows: CYP709E9 (ONL98408), CYP79A61 (AQL04081),CYP81N5 (ONM01607), CYP71C57 (ONM16205), CYP73A6 (AQK99675), CYP94D49 (AQK99675), CYP72A354 (ONM40051),CYP72A351P (AQK96819), CYP89E15P (DAA53516), CYP71Y10 (AQK99675), CYP87A43 (ONM14827), CYP87A42 (AQK45446),CYP71AK4 (AQK78475), CYP71C62 (AQK78475), CYP76H18 (ONM34976), CYP71AF7 (AQL04702), CYP94C71 (AQK86258),CYP72A349 (AQK96828), CYP728C14 (AQK48952), CYP709D2 (AEN14328), CYP75B87(AQK72244), CYP714C11 (ONL97681),CYP71K15 (AQK69153), CYP73A122 (AQK84974), CYP90A22 (NM19847), CYP728B1 (ONM24075), CYP92A92 (ONM22199),CYP51H12 (ONM39954), CYP99A5 (ONL98899), CYP92A1 (ONM22197), CYP72A124 (ONM40043), CYP71C4 (AQK48644),CYP81A9 (ONM34976), CYP90D10 (AQK84718), CYP71C2 (AQK48630), CYP72A5 (AAL66770), CYP714B10 (ONM26218),CYP71C3v2 (AAL66769), CYP74A39 (AQK62615), CYP97A16 (AQK76218), CYP707A5 (AQK73979), CYP703A12 (AQK41827),CYP74A11 (AQL08337), CYP98A7 (ONM37166), CYP71X20 (AQK69143), CYP76M15 (DAA47388), CYP92A93 (ONM55293),CYP96D3 (AQK84634), CYP94E4 (AQK85542), CYP78A55 (ONL93396), CYP72A28v2 (AAM77718), CYP71Z17 (AQK72671),and CYP71W11 (AFW71000).

Fig. 4 RT-qPCR confirmation of maize cytochrome P450 monooxygenase (CYP) genes highly expressed in nicosulfuron-tolerant maize as revealed by the analysis of Solexa transcriptome datasets. HBS, the sensitive inbred; HBR, the tolerant inbred; CK, control check; NS, nicosulfuron. β-actin was used as internal control genes. Means and standard errors from three biological replicates are shown. Bars with the same lowercase letters within a treatment indicate non-significant differences at P＜0.05; bars with the same uppercase letters within a genotype indicate non-significant differences by Fisher’s least significant difference test at P＜0.01.

5. Conclusion

Cytochrome P450s play important roles in herbicide metabolism in maize. Malathion increased nicosulfuron injury and hampered maize growth. There are 314 full length CYP genes in maize, which belong to 2 types and 10 clans consisting of 44 families. The 168 A-type CYP genes belonging to the single clan CYP71 are represented by 17 families, and 146 non-A-type genes in nine clans are distributed into 27 families. ‘PFGXGRRXCPG’ and‘FXXGPRXCXG’ are the consensus sequences of the hemebinding regions for A-type and non-A-type P450s. There are 53 differentially expressed CYP genes in nicosulfurontreated and untreated HBR and HBS samples. This study will further our understanding of nicosulfuron metabolism in maize.

Acknowledgements

This research was funded by the National Natural Science Foundation of China (31501660), the Technology Research and Development Program of Hebei, China (17226507D)and the Foundation of Institute of Cereal and Oil Crops of Hebei Academy of Agriculture and Forestry, China(LYS2015001).