Identification and analysis of CYP450 genes from transcriptome of Lonicera japonica and expression analysis of chlorogenic acid biosynthesis related CYP450s Xiwu Qi1 ,2 , Xu Yu1 ,2 , Daohua Xu1 , Hailing Fang1 ,2 , Ke Dong3 , Weilin Li1 ,2 and Chengyuan Liang1 ,2 1

Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing, China The Jiangsu Provincial Platform for Conservation and Utilization of Agricultural Germplasm, Nanjing, China 3 Department of Biological Sciences, College of Natural Sciences, Seoul National University, Seoul, South Korea 2

ABSTRACT

Submitted 21 April 2017 Accepted 17 August 2017 Published 12 September 2017 Corresponding authors Weilin Li, [email protected] Chengyuan Liang, [email protected] Academic editor David Hyten Additional Information and Declarations can be found on page 16 DOI 10.7717/peerj.3781 Copyright 2017 Qi et al. Distributed under Creative Commons CC-BY 4.0

Background. Lonicera japonica is an important medicinal plant that has been widely used in traditional Chinese medicine for thousands of years. The pharmacological activities of L. japonica are mainly due to its rich natural active ingredients, most of which are secondary metabolites. CYP450s are a large, complex, and widespread superfamily of proteins that participate in many endogenous and exogenous metabolic reactions, especially secondary metabolism. Here, we identified CYP450s in L. japonica transcriptome and analyzed CYP450s that may be involved in chlorogenic acid (CGA) biosynthesis. Methods. The recent availability of L. japonica transcriptome provided opportunity to identify CYP450s in this herb. BLAST based method and HMM based method were used to identify CYP450s in L. japonica transcriptome. Then, phylogenetic analysis, conserved motifs analysis, GO annotation, and KEGG annotation analyses were conducted to characterize the identified CYP450s. qRT-PCR was used to explore expression patterns of five CGA biosynthesis related CYP450s. Results. In this study, 151 putative CYP450s with complete cytochrome P450 domain, which belonged to 10 clans, 45 families and 76 subfamilies, were identified in L. japonica transcriptome. Phylogenetic analysis classified these CYP450s into two major branches, A-type (47%) and non-A type (53%). Both types of CYP450s had conserved motifs in L. japonica. The differences of typical motif sequences between A-type and non-A type CYP450s in L. japonica were similar with other plants. GO classification indicated that non-A type CYP450s participated in more molecular functions and biological processes than A-type. KEGG pathway annotation totally assigned 47 CYP450s to 25 KEGG pathways. From these data, we cloned two LjC3Hs (CYP98A subfamily) and three LjC4Hs (CYP73A subfamily) that may be involved in biosynthesis of CGA, the major ingredient for pharmacological activities of L. japonica. qRT-PCR results indicated that two LjC3Hs exhibited oppositing expression patterns during the flower development and LjC3H2 exhibited a similar expression pattern with CGA concentration measured by HPLC. The expression patterns of three LjC4Hs were quite different and the expression pattern of LjC4H3 was quite similar with that of LjC3H1.

OPEN ACCESS

How to cite this article Qi et al. (2017), Identification and analysis of CYP450 genes from transcriptome of Lonicera japonica and expression analysis of chlorogenic acid biosynthesis related CYP450s. PeerJ 5:e3781; DOI 10.7717/peerj.3781

Discussion. Our results provide a comprehensive identification and characterization of CYP450s in L. japonica. Five CGA biosynthesis related CYP450s were cloned and their expression patterns were explored. The different expression patterns of two LjC3Hs and three LjC4Hs may be due to functional divergence of both substrate and catalytic specificity during plant evolution. The co-expression pattern of LjC3H1 and LjC4H3 strongly suggested that they were under coordinated regulation by the same transcription factors due to same cis elements in their promoters. In conclusion, this study provides insight into CYP450s and will effectively facilitate the research of biosynthesis of CGA in L. japonica.

Subjects Molecular Biology, Plant Science Keywords Lonicera japonica, CYP450, CGA biosynthesis, C3H, C4H

INTRODUCTION Cytochrome P450 monooxygenases (CYP450s) are a large and complex superfamily which can be found in almost all living organisms (Nelson, 1999). Plant CYP450s are heme-containing enzymes that take part in a wide variety of reactions of both primary and secondary metabolism (Kumar et al., 2014), including the production of fatty acids, sterols, plant hormones, flavonoids, terpenoids, lignin, signaling molecules, and other biological molecules (Schuler & Werck-Reichhart, 2003). Lonicera japonica Thunb. is a perennial evergreen vine belonging to the family Caprifoliaceae. L. japonica is a medicinal plant of great importance in traditional Chinese medicine that has been used for thousands of years (Shang et al., 2011). There are more than 500 traditional Chinese medicine prescriptions containing L. japonica (Shang et al., 2011). Modern pharmacological studies have indicated that the extracts of L. japonica possess many biological and pharmacological activities, such as anti-inflammatory, antiviral, antibacterial, antioxidant, hepato-protective, anti-tumor, and other activities (Xiang et al., 2001; Yoo et al., 2008). The active compounds of L. japonica have been extensively studied. Essential oils (Schlotzhauer, Pair & Horvat, 1996), phenolic acids (Lu, Jiang & Chen, 2004), flavone (Chen et al., 2005), triterpenoid saponins (Chai, Li & Li, 2005), iridoilds and inorganic elements as the main compositions were isolated and identified in L. japonica. Among all these products, chlorogenic acid (CGA) is the major ingredient for pharmacological activities and its content is typically used as the main indicator of quality for evaluating L. japonica (Chinese Pharmacopoeia Commission, 2010). As one of the most important secondary metabolites in plants, CGA is often used in medicines and foods for its high anti-oxidative activity (Zucker & Levy, 1959). The biosynthetic pathway of CGA has been investigated in many plants and is catalyzed by a series of enzymes (Niggeweg, Michael & Martin, 2004). Cinnamate 4-hydroxylase (C4H) and p-coumarate 30 -hydroxylase (C3H) are two CYP450s that participate in the two steps of hydroxylation in CGA biosynthetic pathway (Gabriac et al., 1991; Schoch et al., 2001). In L. japonica, a CYP98A subfamily gene encoding LjC3H was isolated and characterized.

Qi et al. (2017), PeerJ, DOI 10.7717/peerj.3781

2/20

By using heterologous expressed LjC3H in vitro assay, a recent study revealed that the recombinant protein was effective in converting p-coumaroylquinate to CGA (Pu et al., 2013). Two C4Hs belonging to the CYP73A subfamily were also cloned in L. japonica. Expression and activity analysis suggested that LjC4H2 may be one of the critical genes that regulate CGA content in L. japonica (Yuan et al., 2014). The studies of L. japonica have been focused on the identification of active compounds and pharmacological activity assays. In recent years, with the technological advancement in molecular biology, especially the development of next-generation sequencing technology, great progress has been made in the identification of active compounds involved in the biosynthesis processes in L. japonica (Yuan et al., 2012; He et al., 2013). In this study, bioinformatics tools were used to identify and analyze the CYP450 genes based on transcriptome data of L. japonica. We identified two LjC3Hs and three LjC4Hs from the CYP450 candidate genes, which including one previously reported LjC3H and two LjC4Hs genes. We further cloned the five CYP450 genes and analyzed their transcriptional patterns in different developmental stages flowers. The results provided here will expand CYP450s information and could effectively facilitate CGA biosynthetic studies in L. japonica.

MATERIALS AND METHODS Identification of CYP450 genes in L. japonica The transcriptome data of L. japonica generated from different sequencing platforms including 454 GS-FLX, Illumina HiSeq2000, and Illumina GA II was downloaded from the NCBI SRA database with accession numbers SRR290309, SRR342027, SRR576924, SRR576925 and SRR766791. Four datasets were assembled and annotated. To identify putative CYP450 genes, both Hidden Markov Model (HMM) method and BLAST method were used. For HMM method, P450.hmm file which represents the Hidden Markov Model of the cytochrome P450 family was initially downloaded from Pfam (http://pfam.xfam.org/), and then, HMMER3 software (Eddy, 2011) was used to search P450.hmm against L. japonica deduced amino acid database. For BLAST method, 19,047 full length plant CYP450 sequences were retrieved from UniProt (http://www.uniprot.org/). These sequences were used as queries to tblastn against L. japonica transcriptome assembly with an E-value cutoff of 1e−5. After filtering out the repeated results, the coding sequences of the resultant subjects were retrieved. Finally, results from the two methods were integrated and corrected manually. The identification methods were conducted for the four datasets of L. japonica and the results were also integrated and corrected. The corrected L. japonica CYP450s were further submitted to NCBI Conserved Domain Search (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) to predict the conserved domain. Sequences with complete cytochrome P450 domain were selected for further analysis.

Classification and characterization of L. japonica CYP450 genes L. japonica CYP450s were classified into different families and subfamilies according to the sequence similarity using sequences from Cytochrome P450 Homepage as reference sequences. If the amino acid sequences of L. japonica CYP450s showed >40%, >55%, or

Qi et al. (2017), PeerJ, DOI 10.7717/peerj.3781

3/20

>95% sequence similarity with reference sequences, they were classified into the same family, subfamily, or allelic variant, respectively (Nelson, 2009). The deduced amino acid sequences of L. japonica CYP450s were subjected to Multiple Expectation Maximization for Motif Elicitation (MEME, http://meme-suite.org/) (Bailey et al., 2009) analysis for identification of conserved motifs. Sequences of the four conserved CYP450 motifs including heme-binding region, PERF motif, K-helix region and I-helix region were extracted and then subjected to WEBLOGO (http://weblogo.berkeley.edu/) (Crooks et al., 2004) to create the sequence logos.

Phylogenetic analysis of predicted CYP450 genes A total of 63 representative sequences from plant CYP450 families were selected for phylogenetic analysis with 151 L. japonica CYP450 sequences. Specifically, CYP450 sequences whose functions had already been identified were preferentially selected. Multiple sequence alignment was performed using MUSCLE 3.6 software (Edgar, 2004). The result of alignment was imported to MEGA4 (Tamura et al., 2007) and phylogenetic analysis was performed. The phylogenetic tree was constructed using the Neighbor-Joining algorithm with the Poisson model and pairwise deletion. Bootstrap testing with 1,000 replications was used to test the phylogenetic tree. The Newick format file of bootstrap consensus tree was exported and then modified using EvolView (http://www.evolgenius.info/evolview/) (Zhang et al., 2012).

Gene ontology and KEGG pathway analysis Blast2GO (http://www.blast2go.com/) (Conesa et al., 2005) was used to perform Gene ontology (GO) annotation of L. japonica CYP450s. These predicted genes were functionally categorized according to three different criterions including cellular component, molecular function and biological process. The GO terms of all L. japonica CYP450s were extracted and subjected to Web Gene Ontology Annotation Plot (WEGO, http: //wego.genomics.org.cn/cgi-bin/wego/index.pl) (Ye et al., 2006) to plot GO annotation results. KEGG annotation that maps the L. japonica CYP450s to possible KEGG pathway for biological interpretation of systemic functions was also conducted using Blast2GO.

Extraction and quantification of CGA The L. japonica used for this study was maintained at the Germplasm Nursery in Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing, Jiangsu Province. Flower buds and flowers samples for CGA and RNA extraction were collected at five stages: young alabastrum (YA, ≤1.5 cm), green alabastrum (GA, 2.0–3.0 cm), while alabastrum (WA, 3.2–4.4 cm), silvery flower (SF, about 5 cm), and golden flower (GF, about 5 cm). The extraction and quantification of CGA were conducted as described in Chinese Pharmacopoeia with minor modifications (Chinese Pharmacopoeia Commission, 2010). Briefly, dried buds or flowers were separately comminuted with a miler, and 0.2 g of each solid sample (40 mesh) was extracted with 25 mL of 50% aqueous methanol by ultrasonication (250 W, 35 kHz) for 30 min. After cooling to room temperature, the extracts were replenished to earlier weights with 50% aqueous methanol. Then, 5 mL of the extracts were diluted to 25 mL with 50% aqueous methanol and filtered with 0.45 µm

Qi et al. (2017), PeerJ, DOI 10.7717/peerj.3781

4/20

Millipore filter membranes. An Agilent 1200LC series HPLC system was used to analyze the CGA levels. Separations were performed on an Agilent TC-C18 reserved-phase column (5 µm, 250 mm × 4.6 mm) at 25 ◦ C. The mobile phase was composed of acetonitrile-0.4% H3 PO4 (13:87). The flow rate was 1 mL/min and fractions were monitored at 327 nm. Components were identified by comparison of the retention times of the eluting peaks to those of commercial standards under the same conditions.

RNA extraction and qRT-PCR Total RNA from five samples was extracted using RNAiso Plus (Takara, Tokyo, Otsu, Shiga, Japan) according to the manufacturer’s instructions. RNA quality and concentration were measured using a ND-1000 UV spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA). First-strand cDNA was synthesized using 3 µg of total RNA with M-MLV reverse transcriptase (Promega, USA) in a 25 µl reaction system. For quantitative real-time reverse transcriptional PCR (qRT-PCR), each reaction was prepared according to the R Premix Ex TaqTM II (Takara) and 2 µl of manufacturer’s instructions using SYBR diluted cDNA as a template. The qRT-PCR reactions were conducted on the qTOWER2.2 Real Time PCR Systems (Analytik, Jena, Germany). The L. japonica actin gene was used as a control to normalize the relative expression levels of target genes. Gene-specific primers used for qRT-PCR were listed on Table S1. All results were representative of three independent experiments.

RESULTS Identification and classification of CYP450 genes in L. japonica Cytochrome P450 is one of the most massive gene superfamilies that is comprised of a number of families and subfamilies. In the present study, by integrating the results from different datasets and manual correction, we in total identified 151 putative CYP450s with complete cytochrome P450 domain in L. japonica. Among them, nine CYP450s had been previously reported and the other 142 CYP450s were identified here for the first time in L. japonica. Based on sequence similarity, we classified the 151 CYP450 genes from L. japonica into 10 clans consisting of 45 families and 76 subfamilies (Table 1). Among them, the CYP71 clan, which represents the whole set of A-type CYP450 genes, contains 71 genes belonging to 19 families (CYP71, CYP73, CYP75–CYP84, CYP89, CYP92, CYP93, CYP98, CYP701, CYP706, and CYP736). The non-A type CYP450 genes of L. japonica contains the remaining 80 genes, which belongs to nine CYP clans (CYP51, 72, 74, 85, 86, 97, 710, 711, and 727) and 26 families (CYP51, CYP72, CYP714, CYP715, CYP721, CYP734, CYP749, CYP74, CYP85, CYP87, CYP88, CYP90, CYP707, CYP716, CYP722, CYP724, CYP728, CYP729, CYP86, CYP94, CYP96, CYP704, CYP97, CYP710, CYP711, and CYP727). The largest CYP family of L. japonica is CYP71 and CYP72, containing 17 and 18 members, respectively.

Phylogenetic analysis of predicted CYP450s in L. japonica Representative members of each plant CYP450 family were selected and used to conduct phylogenetic analysis with 151 CYP450s from L. japonica. The predicted CYP450s were

Qi et al. (2017), PeerJ, DOI 10.7717/peerj.3781

5/20

Table 1 List of predicted CYP450s with complete cytochrome P450 domain from L. japonica. Type

Clan

Family

Subfamily

Gene ID

Type

Clan

Family

Subfamily

Gene ID

non-A

51

CYP51

CYP51G

m183961

non-A

72

CYP72

CYP72A

m61801

non-A

51

CYP51

CYP51G

m52657

non-A

72

CYP72

CYP72A

m25640

A

71

CYP71

CYP71B

m62714

non-A

72

CYP72

CYP72A

m206268

A

71

CYP71

CYP71D

m153867

non-A

72

CYP72

CYP72A

m132911

A

71

CYP71

CYP71D

m20042

non-A

72

CYP72

CYP72A

m20456

A

71

CYP71

CYP71D

m123612

non-A

72

CYP72

CYP72A

m161676

A

71

CYP71

CYP71D

m203354

non-A

72

CYP72

CYP72A

m16935

A

71

CYP71

CYP71D

m30084

non-A

72

CYP72

CYP72A

m216352

A

71

CYP71

CYP71D

m75810

non-A

72

CYP72

CYP72A

m196797

A

71

CYP71

CYP71D

m124427

non-A

72

CYP72

CYP72A

m21341

A

71

CYP71

CYP71D

m94101

non-A

72

CYP72

CYP72A

m194714

A

71

CYP71

CYP71D

m117052

non-A

72

CYP72

CYP72A

m76011

A

71

CYP71

CYP71AP

m198376

non-A

72

CYP72

CYP72A

m178417

A

71

CYP71

CYP71AU

m112981

non-A

72

CYP72

CYP72D

m62754

A

71

CYP71

CYP71AU

m12680

non-A

72

CYP72

CYP72D

m75640

A

71

CYP71

CYP71AU

m33704

non-A

72

CYP714

CYP714A

m189781

A

71

CYP71

CYP71BC

m197010

non-A

72

CYP714

CYP714E

m200247

A

71

CYP71

CYP71BE

m79469

non-A

72

CYP714

CYP714E

m125702

A

71

CYP71

CYP71BG

m82900

non-A

72

CYP714

CYP714E

m205273

A

71

CYP73

CYP73A

m177245

non-A

72

CYP714

CYP714E

m19972

A

71

CYP73

CYP73A

m13469

non-A

72

CYP714

CYP714E

m17561

A

71

CYP73

CYP73A

m8810

non-A

72

CYP715

CYP715A

m34769

A

71

CYP75

CYP75B

m13120

non-A

72

CYP721

CYP721A

m85505

A

71

CYP76

CYP76A

m204536

non-A

72

CYP734

CYP734A

m842

A

71

CYP76

CYP76A

m184619

non-A

72

CYP749

CYP749A

m139970

A

71

CYP76

CYP76A

m18954

non-A

74

CYP74

CYP74A

m4277

A

71

CYP76

CYP76A

m155830

non-A

74

CYP74

CYP74A

m50137

A

71

CYP76

CYP76A

m197465

non-A

74

CYP74

CYP74A

m67219

A

71

CYP76

CYP76B

m31162

non-A

74

CYP74

CYP74B

m52145

A

71

CYP76

CYP76B

m1881

non-A

74

CYP74

CYP74B

m19408

A

71

CYP76

CYP76B

m156655

non-A

85

CYP85

CYP85A

m206529

A

71

CYP76

CYP76B

m122126

non-A

85

CYP85

CYP85A

m42514

A

71

CYP76

CYP76Y

m139737

non-A

85

CYP87

CYP87D

m191193

A

71

CYP76

CYP76Y

m19896

non-A

85

CYP88

CYP88A

m84626

A

71

CYP77

CYP77A

m194721

non-A

85

CYP90

CYP90A

m21741

A

71

CYP77

CYP77B

m148606

non-A

85

CYP90

CYP90B

m8267

A

71

CYP78

CYP78A

m187124

non-A

85

CYP90

CYP90C

m119587

A

71

CYP78

CYP78A

m152788

non-A

85

CYP90

CYP90D

m88205

A

71

CYP79

CYP79D

m32635

non-A

85

CYP707

CYP707A

m212742

A

71

CYP79

CYP79D

m230122

non-A

85

CYP707

CYP707A

m213600

A

71

CYP80

CYP80C

m37356

non-A

85

CYP707

CYP707A m47109 (continued on next page)

Qi et al. (2017), PeerJ, DOI 10.7717/peerj.3781

6/20

Table 1 (continued) Type

Clan

Family

Subfamily

Gene ID

Type

Clan

Family

Subfamily

Gene ID

A A

71 71

CYP81 CYP81

CYP81B CYP81C

m211982 m12729

non-A non-A

85 85

CYP707 CYP707

CYP707A CYP707A

m35702 m17557

A

71

CYP81

CYP81E

m131282

non-A

85

CYP716

CYP716A

m191349

A

71

CYP81

CYP81E

m61839

non-A

85

CYP716

CYP716A

m57776

A

71

CYP81

CYP81E

m61297

non-A

85

CYP716

CYP716A

m12551

A

71

CYP82

CYP82C

m99205

non-A

85

CYP716

CYP716C

m77065

A

71

CYP82

CYP82C

m76311

non-A

85

CYP716

CYP716C

m23342

A

71

CYP82

CYP82D

m169021

non-A

85

CYP716

CYP716D

m141170

A

71

CYP82

CYP82D

m39884

non-A

85

CYP716

CYP716E

m153199

A

71

CYP82

CYP82D

m211151

non-A

85

CYP716

CYP716E

m200248

A

71

CYP82

CYP82U

m215270

non-A

85

CYP722

CYP722A

m120593

A

71

CYP82

CYP82U

m56636

non-A

85

CYP722

CYP722C

m202676

A

71

CYP82

CYP82U

m82069

non-A

85

CYP724

CYP724A

m206239

A

71

CYP82

CYP82U

m61602

non-A

85

CYP728

CYP728B

m166264

A

71

CYP83

CYP83F

m86843

non-A

85

CYP729

CYP729A

m77833

A

71

CYP83

CYP83F

m64245

non-A

86

CYP86

CYP86A

m71018

A

71

CYP83

CYP83F

m109275

non-A

86

CYP86

CYP86A

m6298

A

71

CYP83

CYP83F

m91426

non-A

86

CYP86

CYP86C

m120347

A

71

CYP84

CYP84A

m176881

non-A

86

CYP94

CYP94A

m145786

A

71

CYP84

CYP84A

m124488

non-A

86

CYP94

CYP94B

m38093

A

71

CYP89

CYP89A

m131845

non-A

86

CYP94

CYP94C

m59371

A

71

CYP92

CYP92A

m14848

non-A

86

CYP94

CYP94C

m6650

A

71

CYP92

CYP92B

m61326

non-A

86

CYP94

CYP94D

m102827

A

71

CYP93

CYP93B

m79556

non-A

86

CYP94

CYP94D

m100765

A

71

CYP98

CYP98A

m184946

non-A

86

CYP94

CYP94F

m175121

A

71

CYP98

CYP98A

m43608

non-A

86

CYP96

CYP96A

m794

A

71

CYP701

CYP701A

m27329

non-A

86

CYP96

CYP96A

m21366

A

71

CYP701

CYP701A

m150262

non-A

86

CYP704

CYP704A

m94230

A

71

CYP706

CYP706C

m115920

non-A

97

CYP97

CYP97A

m56546

A

71

CYP736

CYP736A

m18282

non-A

97

CYP97

CYP97B

m17072

A

71

CYP736

CYP736A

m135731

non-A

97

CYP97

CYP97C

m3461

A

71

CYP736

CYP736A

m182725

non-A

710

CYP710

CYP710A

m92981

non-A

72

CYP72

CYP72A

m51504

non-A

711

CYP711

CYP711A

m201472

non-A

72

CYP72

CYP72A

m55535

non-A

727

CYP727

CYP727B

m144680

non-A

72

CYP72

CYP72A

m11850

classified into two major branches, A-type (47%) and non-A type (53%) (Fig. 1). There were 10 clans in plants CYP450s. Four clans contained multiple families and were designated by their lowest-numbered family members, CYP71, CYP72, CYP85 and CYP86. The other six clans were designated by their only family, CYP51, CYP74, CYP97, CYP710, CYP711 and CYP727. In L. japonica, all 10 clans were identified. Genes belonging to same clan clustered as one clade. For example, the 72 clan, which comprised 28 CYP450s belonged to six families, were clustered as one clade with the eight representative CYP450s. The CYP71

Qi et al. (2017), PeerJ, DOI 10.7717/peerj.3781

7/20

CYP72A-m 216352|Lj CYP72 A-m 16935 |Lj 676 |Lj CYP72A-m 161 178 417 |Lj CYP72A-m 10 |At CYP7 2A 6| Lj -m 20 45 j CYP7 2A 29 11 |L -m 13 |Lj CYP7 2A 62 68 A-m 20 5 0 4 |Lj CYP72 51 j 2 A-m 5 6 4 0 |L 2 CYP7 j |L -m 1 72A 180 CYP -m 6 8 5 0 |Lj 72A 1 j CYP -m 1 5 3 5 |L j 5 72A CYP 2 A-m 5 7 5 4 |L j |L 7 62 CYP 2 D -m 5 6 4 0 7 P7 |At | Lj -m CY A1 2D 21 5505 P7 P7 CY CY -m 8 1 | At | Lj A 4A 42 21 P7 YP7 3 -m 8 | At CY C 3 4 A B1 g j P7 7 0 9 2 2 | P 0 | L 7 P CY A CY 4 9 3 9 9 | At 1 t 1 j P7 CY A-m 3 5 A 1 | A 9 | L j 4 9 P7 5 A 7 6 t P7 CY P7 1 3 4 1 | A 1 | L m CY 8 CY A- 1 4 A 9 7 15 7 18 P7 CYP -m CY A 14 P7 CY

cla 72

n

n

CY CY P7 P 1 CY 7 1 4 E4 CY P7 E- m 1 CY P7 1 4 m 2 2 5 P7 1 4 E-m 0 0 7 0 CY 1 4 E E-m 1 9 2 4 2 | L P9 -m 1 9 7 7 | j CY 7 B 2 0 7 5 2 | Lj P9 -m 5 2 6 1 Lj 7 1 7 7 3 | Lj C CCY 0 P9 YP9 m 3 4 7 2 | Lj |L 7 7 CY A-m A3 | 6 1 | L j A P 5 j CY 7 3 2 6 5 4 t P A 6 CY 7 3 1 1 | O | Lj A1 s P7 | 3 O CY CYP7 0 A1 |O s P7 0 04 A1 s 4A |At -m 9 CYP CYP8 4 2 3 0 6 86C |Lj -m 1 B1 |At CYP 86A 20347 -m 6 CYP |Lj 86A -m 7 2 9 8 |Lj CYP9 6 A-m 1 0 1 8 |L j CYP96 2 1 3 6 6 |L j A18 |V CYP9 v 6A-m 79 CYP9 4F -m 17 51 4| Lj 21 |Lj CYP9 4C -m 59 37 1|Lj CYP94 C1 |At CYP94C-m 665 0|Lj CYP9 4B-m 3809 3|Lj CYP94A-m 145786|Lj CYP94D-m 100765|Lj CYP94D-m 102827|Lj CYP77 B1|At 606 |Lj CYP77B-m 148 194 721 |Lj CYP77A-m A3 |Os CYP7 23 2| At 9A CYP8 j 18 45 |L -m 13 CYP8 9A 71 9A1| Cj CYP |At j 0 1 A3 CYP7 5 0 2 6 2 |L j 1 2 9 |L j A-m 701 273 CYP A-m 8 8 1 0 |L j 701 -m 9 |L A CYP 6 3 4 7 CYP A-m 1 3 |At A5 |Lj 73 CYP CYP7 3 7 2 4 5 t 17 3 |A Lj -m 3A 8A 946| j 7 9 P L P CY CY 1 8 4 6 0 8 | 3 -m t 8 A -m 4 5 | A | Lj 9 P 4 A A CY P9 8 P7 8 7 1 2 8 | Lj j CY CY 1 8 2 7 8 2 | L j -m 1 5 1 2 | L 5 8A 0 P7 A-m 2 3 6 3 CY 7 8 -m 3 2 | At t j P 2 D m CY 7 9 D - 9 A 2 | A 6 | L Lj P 7 A 9 2 | CY YP7 CYP 0 3 2 1 5 5 C P7 1 2 6 6 CY B-m 1 5 6 -m P7 6 B CY P7 CY

74

cla

n

71

0

Lj 4| Tc 6 1| 62 5 A 1 6 Pt j 2 m | |L P7 B- D 2 93 CY 7 2 8 2 8 | At 0 5 j P P7 1 8 12 t |L CY CY P7 A-m 1 | A 6 7 6 CY 2 2 2 A 2 0 2 P7 P7 2 -m | Pt | Lj Y 7 C CY 2 C A1 5 5 2 3 17 P7 7 3 t j CY CYP 7 A-m 1 | A 2|L 7 0 7 0 7 A 3 5 7 0 2 |Lj P CY CYP A-m 1 2 7 4 |Lj 2 00 07 P7 7 A-m 2 1 3 6 |Lj Y 0 C -m P7 109 CY 7 0 7 A -m 4 7 j P 6 |L CY 7 0 7 A 8 4 6 2 P CY 8 8 A-m |At j 3 3 3 |L CYP YP8 8 A 778 C -m A |Os 729 CYP P7 2 9 A1 t CY A1 |A 2 0 At CYP7 8A2| CYP70 2| At CYP8 7A 93 |Lj -m 19 11 CYP8 7D 20 65 29 |Lj -m CYP8 5A 425 14| Lj CYP85A-m CYP85A1|At CYP72 0A1|At CYP90D-m 88205|Lj CYP90C-m 119587|Lj CYP90A-m 21741|Lj CYP9 0B-m 8267 |Lj CYP90B1|At CYP72 4A -m 206 239 |Lj CYP7 24 A1 |At

97

cla

51

CYP72A-m 196797|Lj CYP72A-m 21341|Lj CYP7 2A-m 1947 14|Lj CYP72A-m 760 11|Lj CYP72 7B -m 144 680 |Lj CYP7 27 B2 |Pt CYP7 11 A-m 20 14 72 |L CYP7 j 11 CYP51 A1 |At G2| CYP5 1 G-m At CYP 1839 5 6 1 |L CYP 1 G-m 5 j 265 74A 7 |Lj CYP -m 5 CYP 7 4 A-m 0 1 3 7 |L j 672 74A 1 9 |L -m C j CY YP7 4 4 2 7 7 P7 |L A |A j 4 CY t B-m CY P7 4 B 52 1 P7 1 0 m 1 9 4 5 |L j CY CYP A-m 4 0 8 7 CY P7 1 1 0 A 9 2 9 |Lj CY P7 1 6 A-m 1 | A 8 1 | L j t P7 6 A 57 1 CY CY 6 A m 1 7 7 6 C P7 P7 1 -m 1 2 5 5 | Lj C YP7 1 6 6 A 9 1 1 | L C YP7 1 6 D -m 1 | A 3 4 9 j |L CY YP7 1 6 C-m 1 4 t j 11 P7 1 6 C7 1 6 E- m 2 7 0 6 7 0 E- m 1 3 3 5 | | Lj m 5 3 4 2 Lj 20 |L 1 02 99 j 4 8 | Lj |L j

727 71 1

non-A type

85

cla

8 6 cl an

n

j |L 2 7 Lj | 44 0 j 12 81 4|L j -m 7 5 0 8 2 | L j 1 D -m 3 0 6 1 4 | L P7 1 D -m 1 2 3 3 5 j CY YP7 1 D -m 0 3 2 | L Lj C P7 1 D m 2 0 0 4 7 | CY P7 1 D - m 2 3 8 6 15 CY 7 l D P E 1 CY P7 D -m A1 | j CY P7 1 7 2 6 2 | Os 8 2 | L CY CYP 9 9 A 1 8 2 j P m Pg CY 3 6 A- A1 2 | 7 3 1 |L P7 7 3 6 1 3 5 2 5 |Lj P CY 7 Y 2 C A- m 3 6 -m 1 8 P7 j CY 7 3 6 A 1 |At A P 8 1 |L CY YP8 4 1 7 6 8 j C 8 8 |L -m 244 84A 1 8 |Lj CYP 4 A-m 8 484 CYP 2 A-m 1 t 9 CYP P9 2 A2 |N 6 |Lj CY 6132 2 B-m 0| Lj CYP9 B-m 13 12 CYP75 5B1| At CYP7 A5 |At CYP7 05 A1 |At CYP7 12 795 56| Lj CYP93B-m CYP93D 1|At CYP81 E-m 61839 |Lj CYP81E-m 61297|Lj

C CY YP C P7 7 6 CY YP 6 B B-m P7 7 6 -m 1 Y 6 Y -m 3 1 8 8 -m 1 1 1 | CY 6 P8 CYP 1 3 9 8 9 2 | Lj 0 C 8 0 9 7 6 Lj CY CY -m C1 3 7 | Lj P CY 7 6 A P7 3 7 3 | Pt | Lj 6 P CY 7 6 A -m 1 G1 | 5 6 | L A 9 P j CY 7 6 A m 2 0 7 4 6 t -m P7 45 5| CY 6 A-m 1 8 4 3 6 | Lj 6 P7 L 6A 155 19| j Lj 8 m 3 C CY P7 0 YP7 0 1 8 9 5 0 | Lj 6 6 4 CYP C-m 1 A1 |A |Lj t 1 8 CYP 2 U-m 5 9 2 0 82 |L 82U -m 5 0 6 9 |L j CYP 6 82U j -m 6 6 3 6 |L CYP j 82U 160 -m 2 2 |Lj CYP8 1 2 C-m 5 2 7 0 |L j 7 CYP82 6 3 1 1 |Lj C4| A CYP8 t 2C-m 99 20 CYP8 2D -m 16 90 5| Lj 21 |Lj CYP8 2D -m 21 11 51 |Lj CYP82 D-m 39 88 4|Lj CYP81B-m 211 982 |Lj CYP8 1C-m 1272 9|Lj CYP81D1 1|At CYP81E-m 131282|Lj

C CYP YP8 3 B 1 CY 8 3 F-m |At P CY 8 3 F-m 1 0 9 2 P8 7 5 |L 9 3F CY j -m 1 4 2 6 P 8 64 |Lj CY 3F 2 CY P7 1 B -m 8 6 4 5 |L j CY P7 1 C-m 8 4 3 |Lj CY P7 1 B-m 1 9 7 P7 BG 6 2 7 0 1 0 1 | 1 CY CY AP- m 8 2 4 | L Lj 90 j CY P7 P7 1 m 1 C P7 1 AU A1 2 9 8 3 0 | Lj CY YP7 1 A -m | At 7 6 | Lj C P7 1 A U-m 1 2 CY YP7 1 B U-m 1 1 6 8 0 P7 1 D E3 29 |L 1 D -m m 7 3 7 8 1 j - m 1 1 9 4 0 4 | | Lj L 6 j 94 70 9| 1 0 5 2 Lj 1 | | Lj Lj

7 1 clan

A type

Figure 1 Phylogenetic analysis of predicted CYP450s in L. japonica and the representative members of CYP450 families.

clan that comprised 71 members belonging to 19 families was the largest clan. Three clans, CYP710, CYP711 and CYP727, had only one member identified for each clan.

Conserved motifs analysis of L. japonica CYP450s Plant CYP450s shared some typical conserved motifs including heme-binding region, PERF motif, K-helix region and I-helix region, which were important for catalytic activities (Paquette, Jensen & Bak, 2009). The L. japonica CYP450s were divided into A-type and non-A type according to phylogenetic analysis. The deduced amino acid sequences were subjected to MEME to analyze the conserved motifs. The consensus sequences of the heme-binding region, also known as ‘‘P450 signature’’, were ‘‘PFGXGRRXCPG’’ and ‘‘XFXXGXRXCXG’’ for A-type and non-A type CYP450s, respectively (Fig. 2). The cysteine residues in this motif of two types of CYP450s were universally conserved, which links the heme iron to the apoprotein. The consensus sequences of the PERF motif were

Qi et al. (2017), PeerJ, DOI 10.7717/peerj.3781

8/20

18

17

16

15

14

13

12

11

9

10

8

7

6

5

4

3

2

13

12

11

9

8

7

6

5

3

2

10

C

MAS V

N

E F G I H V

I

F

T

16

15

14

S

13

12

11

10

S

9

8

7

6

5

4

3

Y

T

A

D

A

C

LTW

SL VA

S

TT

I

T

F S FA

A I S M LC A AVS I SVG M T

Y G S

V

V

S

M

N

L

C

S

L

16

I

I

A

15

A

H

14

A K

N

S

13

I

Y

V

S A

E

Q

H

R

V

M

F A

12

I

M M

11

I

0 N

10

bits

T

Y

V

AG TT

YF FL L V

C

S

I-helix region

1L

I

Q

SM

Q

9

C

A

M

E LRL P

Y

S

C

K

A F

8

Q

C

W

D

L

N

AV

K

7

W

D

T

18

I

I V I L

G

K

A

6

L

W

T

V

Y

S

W S

R

M

T

3 2

R

M

Q

TQ

4

I G T T V FV

17

A

E

V

Y N

T

H

R

C

F

Q

R

S D

T

P

4

I M

H

P

12

9

R T

A

S

16

S

N

G

A

S

7

4

3

2

1

V

10

I N N E

F

8

I

V

S

6

I

5

F

0 N

I

N

Q

M

A H N F S E L R V S C

F

EA

T

V

LS V VT

15

A F I T V M TS M

14

GAS

V

I L NV

W

S E

V TS TL SA 13

F DL 1L M I L ME

1

bits bits

16

11

T

D AGTE

2

11

3

N

L Y V I

LK

0 N

V

9

6

5

4

1

R R

I-helix region

4

M

2

I

M

M

P

R

K-helix region

3

L S V F T T

R

L

Y

D

E

K N E

L K

R

3

L

Q

3

2

1

V

M

L M

15

N

Y

14

C

N

F

I

K M

F

Y D SK I G M

S H

L K

A

A

W N

2

A R F S

L

M

F M

A S

H

L N

M

G

H

T

L

KN

N

V

S

Q

C K V S

E K P

4

A V AG S

13

R

E

10

0F N

KC S

I

12

Q

1

KETLRLHPP

AVI VI

8

2

R

L

1

12

11

10

0 N

C

H

H

QE

I P

V

L

T

Q

D

1

T

V

Y

Y

9

4

3

2

1

YL

3

2

2

F

G

V

Y

K T

R

H

L

T

T

E

G

D

15

W

R

S

D

I

E N K

Q S

N

14

D

F

A

D E M G N

D

Q

S

C

13

S

C

N

I

I

A

G

L

S

T

M

V

S

N

Q

PERF motif

3

L

K

R

K

A

SA

L NW

S

5

bits 21

20

19

18

17

C

4

F PERF

EE

LT

N

E

D A M Q S S

P

V

0 N

A

L

G

F

LA

V

T

S

Q

16

15

14

G

F GRCG A WG D EF P RF

FY P G P

I M P L

H

1

A

G

L

13

7

A

R

2

I

M

M

Q

L

T

1

L

AT

K-helix region

4 bits

6

5

3

2

1

W

P

D

K

D S

bits

K

I

MS

F

I V

3

PERF motif

E

0 N

M

T

A L F

L P

A

A

8

1

V

7

2

M S

V

7

bits

3

G

G

6

4

ST

4

0 N

A S A

V M

V

12

H Q

A

S

11

G

9

K

RYM

Y H L

E

L

I

10

D N

S A S

Heme-binding region

4

PFG GRR CPG

I EL FL

8

F

2

5

bits

3

non-A type

4

Heme-binding region

4

1

B

A-type

1

A

C

Figure 2 Weblogos of conserved motifs identified in A-type (A) and non-A type (B) CYP450s from L. japonica.

also different for two types of CYP450s in L. japonica, which are ‘‘PERF’’ for A-type and ‘‘PXRX’’ for non-A type. The R residues in the PERF motif and E and R residues in the K-helix region were universally conserved, which form a salt bridge that has been proposed to be involved in locking the Cys-pocket in position and assuring the stable association of heme with the protein. The threonine residues in the I-helix region which is involved in oxygen activation was highly conserved in both A-type and non-A type CYP450s. In general, sequences of the typical motifs were conserved in L. japonica CYP450s, and the differences between A-type and non-A type CYP450s in L. japonica were similar with other plants (Chen et al., 2014).

Gene ontology classification of L. japonica CYP450s Gene ontology (GO) is a classification system for standardized gene functions which classifies genes into three main independent GO categories: cellular component, molecular function and biological process. In this study, GO assignments were conducted to classify the functions of CYP450s from L. japonica using Blast2GO. Results indicated that all 151 CYP450s were mapped to one or more GO terms, of which 145 were assigned to the ‘‘cellular component’’, 151 to the ‘‘molecular function’’, and 151 to the ‘‘biological process’’ (Fig. 3). Of these categories, cell, binding, catalytic, and metabolic process were the largest subcategories. Comparison of the GO classification between the A-type and non-A type CYP450s, we found that non-A type CYP450s participated in more molecular

Qi et al. (2017), PeerJ, DOI 10.7717/peerj.3781

9/20

75

71 80 53 60

50

35 40

25

17 20

0

0 0

macr o

mole

Number of genes

A type non-A type

c cell ell enve part cular lo comp pe le orga nelle organe x lle mem br orga nelle ane pa bindi rt ng c dem atalytic ethy hydr lase olase oxido l redu yase struc c t u ral m tase anat olecu omic al str t l uctur ransfera e se cellu e b f i o o r l cellu lar comp ogical re mation lar co g o mpo nent bio ulation nent g orga enesis deve cellula nization r estab lo lishm pmental process ent o p f loca rocess lizati o gro n multi meta localiza wth cellu lar o rgan bolic proction isma l p ess pigm rocess en repro reprod tation ducti uctio resp onse ve proce n ss to sti mulu s

Percent of genes

100

Cellular Component Molecular Function

Biological Process

Figure 3 Gene ontology annotation of A-type and non-A type CYP450s in L. japonica.

functions and biological processes than A-type. For example, GO terms of non-A type CYP450s in molecular function category included demethylase, hydrolase, lyase, and transferase; however, no A-type CYP450s was assigned to these subcategories. In biological process category, non-A type CYP450s participated in more biological processes than A-type, including anatomical structure formation, cellular component organization, developmental process, establishment of localization, growth, localization, multicellular organismal process, and reproduction. The GO annotation provided a valuable clue to investigate the functions of CYP450s in L. japonica.

KEGG pathway analysis of L. japonica CYP450s In order to further understand the biological functions of CYP450s in L. japonica, pathway-based analysis was performed. Given that a CYP450 could be assigned to one or more KEGG pathways as well as GO terms, 47 (31.1%) CYP450s were totally assigned to 25 KEGG pathways (Fig. 4). The 25 pathways could be mainly grouped into six classes, including lipid metabolism, amino acid metabolism, metabolism of cofactors and vitamins, metabolism of terpenoids and polyketides, biosynthesis of other secondary metabolites, and xenobiotics biodegradation and metabolism. In the class of ‘biosynthesis of other secondary metabolites’, after removing duplicate hits, ten CYP450s (CYP73A-m13469, CYP73A-m177245, CYP73A-m8810, CYP75B-m13120, CYP76Am155830, CYP78A-m152788, CYP93B-m79556, CYP98A-m184946, CYP98A-m43608 and CYP736A-m18282) were found to be involved in the biosynthesis of phenolic compounds

Qi et al. (2017), PeerJ, DOI 10.7717/peerj.3781

10/20

0

Lipid metabolism

Amino acid metabolism Metabolism of cofactors and vitamins Metabolism of terpenoids and polyketides

Biosynthesis of other secondary metabolites

Xenobiotics biodegradation and metabolism

2

4

6

8

10

12

Fatty acid degradation Steroid biosynthesis Steroid hormone biosynthesis Arachidonic acid metabolism Linoleic acid metabolism alpha-Linolenic acid metabolism Phenylalanine metabolism Tryptophan metabolism Cyanoamino acid metabolism Retinol metabolism Ubiquinone and other terpenoid-quinone biosynthesis Monoterpenoid biosynthesis Diterpenoid biosynthesis Carotenoid biosynthesis Phenylpropanoid biosynthesis Stilbenoid, diarylheptanoid and gingerol biosynthesis Flavonoid biosynthesis Flavone and flavonol biosynthesis Isoflavonoid biosynthesis Indole alkaloid biosynthesis Caffeine metabolism Aminobenzoate degradation Metabolism of xenobiotics by cytochrome P450 Drug metabolism - cytochrome P450 Biosynthesis of antibiotics

KEGG pathway

Number of sequence

Figure 4 KEGG pathway analysis of predicted CYP450s in L. japonica.

including phenylpropanoid, stilbenoid, diarylheptanoid and gingerol, flavonoid, flavone and flavonol, and isoflavonoid. All ten CYP450s belonged to CYP71 clan. In the class of ‘metabolism of terpenoids and polyketides’, nine CYP450s (CYP72A-m132911, CYP72Am20456, CYP72A-m206268, CYP72D-m62754, CYP72D-m75640, CYP714A-m189781, CYP714E-m17561, CYP714E-m205273 and CYP734A-m842) were found to be involved in ‘monoterpenoid biosynthesis’, all of which belonged to CYP72 clan. Three CYP450s (CYP701A-m150262, CYP701A-m27329 and CYP728B-m166264) were found to be involved in ‘diterpenoid biosynthesis’, among them, two belonged to CYP71 clan and one belonged to CYP85 clan. Five CYP450s (CYP707A-m213600, CYP707A-m35702, CYP707A-m47109, CYP707A-m212742 and CYP728B-m166264) were found to be involved in ‘carotenoid biosynthesis’, all of which belonged to CYP85 clan.

CYP450s involved in CGA biosynthesis CGA is the most major active ingredient in L. japonica and the biosynthetic pathway of CGA has been investigated in many plants. In CGA biosynthetic pathway, C4H and C3H are the two CYP450-encoded enzymes that participate in the two steps of hydroxylation. In the present study, three C4H and two C3H genes were identified and cloned from L. japonica. Among them, two LjC4Hs and one LjC3H have been previously reported. The newly identified C4H and C3H were designated as ‘LjC4H3’ (GenBank accession number: KX845341) and ‘LjC3H2’ (GenBank accession number: KX845342), respectively. The C4Hs belonged to CYP73A subfamily and C3Hs belonged to CYP98A subfamily. Phylogenetic

Qi et al. (2017), PeerJ, DOI 10.7717/peerj.3781

11/20

47 100 53 55 100 80 66

81 100 91 92 98 57

C4H|Ms C4H|Ca C4H|Ps C4H|Gm C4H|Cr C4H3|Lj C4H1|Lj C4H|At C4H2|Lj C3dH1-2|Sm C3H1|Cc C3H1|Lj CYP98A3|At CYP98A44|Tp C3H2|Lj C3dHi2|Ob 100 C3dH|Ot

C4H

C3H

0.05

Figure 5 Phylogenetic analysis of C3Hs and C4Hs from L. japonica and other plants. LjC3Hs were labeled by black dots and LjC4Hs were labeled by black triangles. Protein sequences were downloaded from UniProt with accession numbers as follows: C3H1|Cc (A4ZKM5), CYP98A3|At (O22203), C3dH1-2|Sm (D8SCG3), C3dHi2|Ob (Q8L5H7), CYP98A44|Tp (C9EGT6), C3dH|Ot (T1NXG3), C4H|At (P92994), C4H|Cr (P48522), C4H|Ps (Q43067), C4H|Ms (P37114), C4H|Ca (O81928), C4H|Gm (Q42797).

analysis indicated that two clades were clustered for C4Hs and C3Hs from L. japonica and other plants (Fig. 5). Because CGA was mainly accumulated in flower bud of L. japonica, buds and flowers in different developmental stages were selected to explore the relationship of C4H and C3H expressions and CGA contents. HPLC analysis was used to measure CGA concentrations in different developmental stages of buds and flowers. As shown in Fig. 6, the percentage of CGA contents decreased during the flower development. Nevertheless, with the increase of bud or flower weights, the total CGA contents increased from young alabastrum (YA) to while alabastrum (WA) stage and reached peak at the WA stage. After flowering, the total CGA contents decreased quickly during flower development. Furthermore, qRT-PCR was conducted to analyze the transcriptional levels of CGA biosynthetic genes in the different developmental stages of buds and flowers, including the five CYP450s identified in this study. The two LjC3Hs exhibited oppositing expression patterns, the transcriptional levels of LjC3H1 increased but that of LjC3H2 decreased during the flower development (Fig. 7). The expression patterns of three LjC4Hs were quite different and the relative expression levels of LjC4H3 was obviously higher than those of the other two (Fig. 7). Interestingly, the expression patterns of LjPAL1, LjC4H3, LjC3H1 and LjHQT were quite similar, which

Qi et al. (2017), PeerJ, DOI 10.7717/peerj.3781

12/20

8

CGA contents (%)

7 6 5 4 3 2 1 0 YA

GA

WA

SF

GF

Figure 6 CGA contents of buds and flowers in different developmental stages L. japonica. YA-young alabastrum, GA-green alabastrum, WA-white alabastrum, SF-silvery flower, and GF-golden flower.

0.02

SF

F

0.01 0.005

GA

WA

SF

WA

SF

0.2 0.15 0.1 0.05

Relative expression

0.02 0.01

GA

WA

SF

GF

SF

GF

LjC4H1

0.06 0.05 0.04 0.03 0.02 0.01 0

GA

WA

SF

GF

YA

H

Lj4CL2

0.05 0.04 0.03 0.02 0.01 0

YA

J

LjC3H2

0.035 0.03 0.025 0.02 0.015 0.01 0.005 0

0.03

YA

G

LjC4H3

0.25

GF

0.04

GF

0 GA

0.05

0 YA

GF

Relative expression

0.04

D

LjPAL3

0.06

Relative expression

WA

LjC4H2

0.015

YA

Relative expression

Relative expression

0.06

Relative expression

GA

Rellative expression

Relative expression

0.1 0.08

0

I

C

LjPAL2

0.12

0 YA

E

B

LjPAL1

0.35 0.3 0.25 0.2 0.15 0.1 0.05 0

Relative expression

Relative expression

A

YA

GA

WA

SF

GF

GA

WA

SF

GF

SF

GF

LjC3H1

0.2 0.15 0.1 0.05 0 YA

GA

WA

LjHQT

0.8 0.6 0.4 0.2 0

YA

GA

WA

SF

GF

YA

GA

WA

Figure 7 Transcriptional analyses of CGA biosynthetic pathway genes in buds and flowers of L. japonica at different developmental stages.

exhibited a trend of decreasing first and then increasing. Considering the gene expressions with CGA contents, only LjC3H2 exhibited a similar pattern with CGA concentrations.

DISCUSSION L. japonica is an important medicinal plant that has been widely used in traditional Chinese medicine for thousands of years. The pharmacological activities of this medicinal plant are mainly due to its rich natural active ingredients, most of which are secondary

Qi et al. (2017), PeerJ, DOI 10.7717/peerj.3781

13/20

metabolites. CYP450s are a large, complex, and widespread superfamily that participate in many metabolic reactions, especially secondary metabolism. The identification and characterization of CYP450s in L. japonica will effectively facilitate the study of natural active compounds biosynthesis. In this study, we identified 151 putative CYP450s with complete cytochrome P450 domain from transcriptome data of L. japonica. According to the classification criteria, the 151 CYP450s were classified into 10 clans consisting of 45 families and 76 subfamilies. Next, we conducted phylogenetic analysis, conserved motifs analysis, GO annotation, and KEGG annotation to characterize the identified CYP450s. As mentioned above, nine CYP450s have been previously reported in L. japonica, which were also identified among the 151 CYP450s of this study. These results indicated that the identified CYP450s from the L. japonica transcriptome data in this study were quite comprehensive. The evolution of plant CYP450s can be divided into three major groups: CYP450s involved in sterol and carotenoid biosynthesis were the most ancient, CYP450s involved in adaptation to land environment were the next oldest, and CYP450s involved in biosynthesis of plant secondary metabolites were the most recent to evolve (Morant et al., 2007; Nelson et al., 2008). In this study, ten CYP450s (CYP73A-m13469, CYP73Am177245, CYP73A-m8810, CYP75B-m13120, CYP76A-m155830, CYP78A-m152788, CYP93B-m79556, CYP98A-m184946, CYP98A-m43608, and CYP736A-m18282) were annotated to participate in the biosynthesis of phenolic compounds, a most common type of secondary metabolite in plants, including phenylpropanoid, stilbenoid, flavonoid, and isoflavonoid. All ten CYP450s belonged to CYP71 clan. As earlier reported, the most recently evolved CYP450 group comprises the highly proliferated clan 71. This clan includes CYP450s involved in the biosynthesis of the majority of plant secondary metabolites involved in adaptation to abiotic and biotic stress (Morant et al., 2007), with which our present findings are in agreement. Five CYP450s (CYP707A-m213600, CYP707A-m35702, CYP707A-m47109, CYP707A-m212742, and CYP728B-m166264) were found to be involved in carotenoid biosynthesis, all of which belonged to the CYP85 clan. These CYP450s belonged to the oldest group with a function that preceded the colonization of land by plants (Morant et al., 2007). CGA is the major active ingredient in L. japonica, and the biosynthetic pathway of CGA has been investigated in many plants. In CGA biosynthetic pathway, C4H and C3H are two CYP450 encoded enzymes that participate in the two steps of hydroxylation (Gabriac et al., 1991; Schoch et al., 2001). In L. japonica, a gene encoding LjC3H has been isolated and characterized by Pu et al. (2013), and was identified as CYP98A subfamily member. In vitro assay using heterologous expressed LjC3H revealed that the recombinant protein was effective in converting p-coumaroylquinate to CGA. Southern blotting suggested that the gene was present in the genome in two copies, but unfortunately, only one copy of LjC3H was obtained. In this study, two LjC3Hs were identified and cloned from L. japonica, both of which belonged to the CYP98A subfamily. Among the two LjC3Hs, one was same as the LjC3H reported by Pu et al. (2013), the other is a newly identified gene and is hereby designated LjC3H2. These results suggested that the newly identified LjC3H2 was the other copy of LjC3H in the genome of L. japonica. Two C4Hs were also cloned in L. japonica

Qi et al. (2017), PeerJ, DOI 10.7717/peerj.3781

14/20

by Yuan et al. (2014), which belonged to the CYP73A subfamily. Expression and activity analysis suggested that LjC4H2 may be one of the critical genes that regulate CGA content in L. japonica. In our study, three C4Hs were identified and cloned from L. japonica, including the previously reported two genes. The newly identified LjC4H was designated as LjC4H3, which showed high degree of sequence homology with LjC4H1. Phylogenetic analysis showed that LjC4H1 and LjC4H3 clustered to one clade. This result suggested that these two genes may be generated by recent gene duplication. In the present study, the expression patterns of two LjC3Hs and three LjC4Hs were quite different during the flower development. This phenomenon that different members of the same family exhibit different expression patterns during development was also observed in other plants (Bi et al., 2011; Qi et al., 2014), which might be caused by functional divergence of both substrate and catalytic specificity during plant evolution (Helariutta et al., 1996; Xu et al., 2009). Considering the gene expressions with CGA contents, only LjC3H2 exhibited a similar pattern with CGA concentrations in our study. This result was similar with that of coffee (Lepelley et al., 2007). In coffee, transcriptional levels of CGA biosynthetic genes and CGA contents were measured during grain development and C3H1 showed a similar expression pattern with CGA concentrations. Both the CGA concentrations and C3H expression pattern were similar with those of L. japonica, respectively. However, in this study, the expression patterns of LjC3H1 and three LjC4Hs were inconsistent with CGA contents during flower development. The reason for this phenomenon could be that C3H and C4H not only participated in CGA biosynthesis, but were also involved in other metabolites. The product catalyzed by C4H was a common precursor in phenylpropanoid metabolism, including flavonoids, anthocyanins, condensed tannins, and isoflavonoids (Winkel-Shirley, 2001). C3H was also a key enzyme in lignin biosynthesis (Boerjan, Ralph & Baucher, 2003). It is likely that the complexity of the metabolic pathways led to the inconsistency between gene expressions and product contents. In this study, the expression patterns of LjPAL1, LjC4H3, LjC3H1 and LjHQT were quite similar during flower development. The co-expression patterns of these four genes strongly suggested that they were under coordinated regulation by the same transcription factors due to similar cis elements in their promoters (Bi et al., 2011). In apple, anthocyanin biosynthetic genes including CHS, CHI, F3H, DFR, LDOX and UFGT showed similar expression patterns during fruit development, which were coordinately regulated by a MYB transcription factor, MdMYB10 (Espley et al., 2007). Fruit-specific ectopic expression of AtMYB12 in tomato led to upregulation of all biosynthetic genes required for the production of flavonols and their derivatives, including PAL, C4H, 4CL, CHS, CHI, F3H, F3’H, FLS, ANS, C3H, HCT, HQT, GT, and RT ; and, in addition, led to the increase of flavonols and their derivatives (Luo et al., 2008). In pine and eucalyptus, xylem-associated MYB transcription factors could bind to the AC elements and activate the transcription of the lignin biosynthetic genes (Patzlaff et al., 2003; Goicoechea et al., 2005). Moreover, the rice genome sequence analysis revealed that ACII motif existed in the promoters of many lignin biosynthetic genes, including PAL, 4CL, C4H, C3H, CCoAOMT, CCR, and CAD, suggesting that they were under coordinated regulation by the same transcription factors (Bi et al., 2011).

Qi et al. (2017), PeerJ, DOI 10.7717/peerj.3781

15/20

CONCLUSIONS In this study, we identified 151 putative CYP450s with complete cytochrome P450 domain in L. japonica transcriptome, 142 of which were identified here for the first time. According to the classification criteria, the 151 CYP450s were classified into 10 clans consisting of 45 families and 76 subfamilies. Next, we conducted phylogenetic analysis, conserved motifs analysis, GO annotation, and KEGG annotation to characterize the identified CYP450s. From these data, we cloned two LjC3Hs (CYP98A subfamily) and three LjC4Hs (CYP73A subfamily) genes that may be involved in biosynthesis of CGA, including the newly identified LjC3H2 and LjC4H3. Furthermore, qRT-PCR and HPLC results indicated that only LjC3H2 exhibited a similar expression pattern with CGA concentration. Different members of the same family exhibited different expression patterns during development that may be due to functional divergence of both substrate and catalytic specificity during plant evolution. The co-expression pattern of LjPAL1, LjC4H3, LjC3H1 and LjHQT strongly suggested that they were under coordinated regulation by the same transcription factors due to same cis elements in their promoters. In conclusion, this study provides insight into CYP450s and will effectively facilitate the research of biosynthesis of CGA in L. japonica.

ADDITIONAL INFORMATION AND DECLARATIONS Funding The work was supported by the National Natural Science Foundation of China (31500249), Six Talent Peaks Project in Jiangsu Province (2015-NY-032), the Natural Science Foundation of Jiangsu Province (BK20161381), and the Foundation of Jiangsu Key Laboratory for the Research and Uti1ization of Plant Resources (SQ201401). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Grant Disclosures The following grant information was disclosed by the authors: National Natural Science Foundation of China: 31500249. Six Talent Peaks Project in Jiangsu Province: 2015-NY-032. Natural Science Foundation of Jiangsu Province: BK20161381. Foundation of Jiangsu Key Laboratory for the Research and Uti1ization of Plant Resources: SQ201401.

Competing Interests The authors declare there are no competing interests.

Author Contributions • Xiwu Qi performed the experiments, analyzed the data, wrote the paper, prepared figures and/or tables. • Xu Yu and Daohua Xu performed the experiments. • Hailing Fang analyzed the data.

Qi et al. (2017), PeerJ, DOI 10.7717/peerj.3781

16/20

• Ke Dong contributed reagents/materials/analysis tools. • Weilin Li and Chengyuan Liang conceived and designed the experiments, reviewed drafts of the paper.

DNA Deposition The following information was supplied regarding the deposition of DNA sequences: The LjC4H3 and LjC3H2 sequences described here are accessible via GenBank accession numbers KX845341 and KX845342.

Data Availability The following information was supplied regarding data availability: The raw data used for transcriptomic assemblies was downloaded from NCBI SRA with accession numbers SRR290309, SRR342027, SRR576924, SRR576925 and SRR766791 (https://ftp.ncbi.nih.gov/sra/sra-instant/reads/ByRun/sra/SRR/SRR290/SRR290309/ SRR290309.sra https://ftp.ncbi.nih.gov/sra/sra-instant/reads/ByRun/sra/SRR/SRR342/SRR342027/ SRR342027.sra https://ftp.ncbi.nih.gov/sra/sra-instant/reads/ByRun/sra/SRR/SRR576/SRR576924/ SRR576924.sra https://ftp.ncbi.nih.gov/sra/sra-instant/reads/ByRun/sra/SRR/SRR576/SRR576925/ SRR576925.sra https://ftp.ncbi.nih.gov/sra/sra-instant/reads/ByRun/sra/SRR/SRR766/SRR766791/ SRR766791.sra).

Supplemental Information Supplemental information for this article can be found online at http://dx.doi.org/10.7717/ peerj.3781#supplemental-information.

REFERENCES Bailey TL, Boden M, Buske FA, Frith M, Grant CE, Clementi L, Ren J, Li WW, Noble WS. 2009. MEME SUITE: tools for motif discovery and searching. Nucleic Acids Research 37:W202–W208 DOI 10.1093/nar/gkp335. Bi C, Chen F, Jackson L, Gill BS, Li W. 2011. Expression of lignin biosynthetic genes in wheat during development and upon infection by fungal pathogens. Plant Molecular Biology Reporter 29:149–161 DOI 10.1007/s11105-010-0219-8. Boerjan W, Ralph J, Baucher M. 2003. Lignin biosynthesis. Annual Review of Plant Biology 54:519–546 DOI 10.1146/annurev.arplant.54.031902.134938. Chai XY, Li SL, Li P. 2005. Quality evaluation of Flos Lonicerae through a simultaneous determination of seven saponins by HPLC with ELSD. Journal of Chromatography A 1070:43–48 DOI 10.1016/j.chroma.2005.02.031. Chen J, Li SL, Li P, Song Y, Chai XY, Ma DY. 2005. Qualitative and quantitative analysis of active flavonoids in Flos Lonicerae by capillary zone electrophoresis coupled with solid–phase extraction. Jorunal of Separation Science 28:365–372 DOI 10.1002/jssc.200400024.

Qi et al. (2017), PeerJ, DOI 10.7717/peerj.3781

17/20

Chen H, Wu B, Nelson DR, Wu K, Liu C. 2014. Computational identification and systematic classification of novel cytochrome P450 genes in Salvia miltiorrhiza. PLOS ONE 9:e115149 DOI 10.1371/journal.pone.0115149. Chinese Pharmacopoeia Commission. 2010. The pharmacopoeia of the People’s Republic of China, 2010 ed. Beijing: China Medical Science Press, 205–206. Conesa A, Götz S, García-Gómez JM, Terol J, Talón M, Robles M. 2005. Blast2go: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 21:3674–3676 DOI 10.1093/bioinformatics/bti610. Crooks GE, Hon G, Chandonia JM, Brenner SE. 2004. WebLogo: a sequence logo generator. Genome Research 14:1188–1190 DOI 10.1101/gr.849004. Eddy SR. 2011. Accelerated profile HMM searches. PLOS Computational Biology 7:e1002195 DOI 10.1371/journal.pcbi.1002195. Edgar RC. 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Research 32:1792–1797 DOI 10.1093/nar/gkh340. Espley RV, Hellens RP, Putterill J, Stevenson DE, Kutty-Amma S, Allan AC. 2007. Red colouration in apple fruit is due to the activity of the MYB transcription factor, MdMYB10. Plant Journal 49:414–427 DOI 10.1111/j.1365-313X.2006.02964.x. Gabriac B, Werck-Reichhart D, Teutsch H, Durst F. 1991. Purification and immunocharacterization of a plant cytochrome p450: the cinnamic acid 4-hydroxylase. Archives of Biochemistry and Biophysics 288:302–309 DOI 10.1016/0003-9861(91)90199-S. Goicoechea M, Lacombe E, Legay S, Mihaljevic S, Rech P, Jauneau A, Lapierre C, Pollet B, Verhaegen D, Chaubet-Gigot N, Grima-Pettenati J. 2005. EgMYB2, a new transcriptional activator from Eucalyptus xylem, regulates secondary cell wall formation and lignin biosynthesis. Plant Journal 43:553–567 DOI 10.1111/j.1365-313X.2005.02480.x. He L, Xu X, Li Y, Li C, Zhu Y, Yan H, Sun Z, Sun C, Song J, Bi Y, Shen J, Cheng R, Wang Z, Xiao W, Chen S. 2013. Transcriptome analysis of buds and leaves using 454 pyrosequencing to discover genes associated with the biosynthesis of active ingredients in Lonicera japonica Thunb. PLOS ONE 8:e62922 DOI 10.1371/journal.pone.0062922. Helariutta Y, Kotilainen M, Elomaa P, Kalkkinen N, Bremer K, Teeri TH, Albert VA. 1996. Duplication and functional divergence in the chalcone synthase gene family of Asteraceae: evolution with substrate change and catalytic simplification. Proceedings of the National Academy of Sciences of the United States of America 93:9033–9038 DOI 10.1073/pnas.93.17.9033. Kumar MS, Babu PR, Rao KV, Reddy VD. 2014. Organization and classification of cytochrome P450 genes in castor (Ricinus communis L.). Proceedings of the National Academy of Sciences India Section B 84:131–143 DOI 10.1007/s40011-013-0192-8. Lepelley M, Cheminade G, Tremillon N, Simkin A, Ciaillet V, McCarthy J. 2007. Chlorogenic acid synthesis in coffee: an analysis of CGA content and real-time RTPCR expression of HCT, HQT, C3H1, and CCoAOMT1 genes during grain development in C. canephora. Plant Science 172:978–996 DOI 10.1016/j.plantsci.2007.02.004.

Qi et al. (2017), PeerJ, DOI 10.7717/peerj.3781

18/20

Lu HT, Jiang Y, Chen F. 2004. Application of preparative high-speed counter-current chromatography for separation of chlorogenic acid from Flos Lonicerae. Journal of Chromatography A 1026:185–190 DOI 10.1016/j.chroma.2003.11.004. Luo J, Butelli E, Hill L, Parr A, Niggeweg R, Bailey P, Weisshaar B, Martin C. 2008. AtMYB12 regulates caffeoyl quinic acid and flavonol synthesis in tomato: expression in fruit results in very high levels of both types of polyphenol. Plant Journal 56:316–326 DOI 10.1111/j.1365-313X.2008.03597.x. Morant M, Jørgensen K, Schaller H, Pinot F, Møller BL, Werck-Reichhart D, Bak S. 2007. CYP703 is an ancient cytochrome P450 in land plants catalyzing in-chain hydroxylation of lauric acid to provide building blocks for sporopollenin synthesis in pollen. Plant Cell 19:1473–1487 DOI 10.1105/tpc.106.045948. Nelson DR. 1999. Cytochrome P450 and the individuality of species. Archives of Biochemistry and Biophysics 369:1–10 DOI 10.1006/abbi.1999.1352. Nelson DR. 2009. The cytochrome P450 homepage. Human Genomics 4:59–65 DOI 10.1186/1479-7364-4-1-59. Nelson DR, Ming R, Alam M, Schuler MA. 2008. Comparison of cytochrome P450 genes from six plant genomes. Tropical Plant Biology 1:216–235 DOI 10.1007/s12042-008-9022-1. Niggeweg R, Michael AJ, Martin C. 2004. Engineering plants with increased levels of the antioxidant chlorogenic acid. Nature Biotechnology 22:746–754 DOI 10.1038/nbt966. Paquette SM, Jensen K, Bak S. 2009. A web-based resource for the Arabidopsis P450, cytochromes b5, NADPH-cytochrome P450 reductases, and family 1 glycosyltransferases (http://www.P450.kvl.dk). Phytochemistry 70:1940–1947 DOI 10.1016/j.phytochem.2009.08.024. Patzlaff A, McInnis S, Courtenay A, Surman C, Newman LJ, Smith C, Bevan MW, Mansfield S, Whetten RW, Sederoff RR, Campbell MM. 2003. Characterisation of a pine MYB that regulates lignification. Plant Journal 36:743–754 DOI 10.1046/j.1365-313X.2003.01916.x. Pu G, Wang P, Zhou B, Liu Z, Xiang F. 2013. Cloning and characterization of Lonicera japonica p-coumaroyl ester 3-hydroxylase which is involved in the biosynthesis of chlorogenic acid. Bioscience Biotechnology and Biochemistry 77:1403–1409 DOI 10.1271/bbb.130011. Qi X, Shuai Q, Chen H, Fan L, Zeng Q, He N. 2014. Cloning and expression analyses of the anthocyanin biosynthetic genes in mulberry plants. Molecular Genetics and Genomics 289:783–793 DOI 10.1007/s00438-014-0851-3. Schlotzhauer WS, Pair SD, Horvat RJ. 1996. Volatile constituents from the flowers of Japanese honeysuckle (Lonicera japonica). Journal of Agricultural and Food Chemistry 44:206–209 DOI 10.1021/jf950275b. Schoch G, Goepfert S, Morant M, Hehn A, Meyer D, Ullmann P, Werck-Reichhart D. 2001. CYP98A3 from Arabidopsis thaliana is a 30 -hydroxylase of phenolic esters, a missing link in the phenylpropanoid pathway. Journal of Biological Chemistry 276:36566–36574 DOI 10.1074/jbc.M104047200.

Qi et al. (2017), PeerJ, DOI 10.7717/peerj.3781

19/20

Schuler MA, Werck-Reichhart D. 2003. Functional genomic of P450s. Annual Review of Plant Biology 54:629–667 DOI 10.1146/annurev.arplant.54.031902.134840. Shang X, Pan H, Li M, Miao X, Ding H. 2011. Lonicera japonica Thunb.: ethnopharmacology, phytochemistry and pharmacology of an important traditional Chinese medicine. Journal of Ethnopharmacology 138:1–21 DOI 10.1016/j.jep.2011.08.016. Tamura K, Dudley J, Nei M, Kumar S. 2007. MEGA4: molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Molecular Biology and Evolution 24:1596–1599 DOI 10.1093/molbev/msm092. Winkel-Shirley B. 2001. Flavonoid biosynthesis. a colorful model for genetics, biochemistry, cell biology, and biotechnology. Plant Physiology 126:485–493 DOI 10.1104/pp.126.2.485. Xiang T, Xiong QB, Ketut AI, Tezuka Y, Nagaoka T, Wu LJ, Kadota S. 2001. Studies on the hepatocyte protective activity and the structure-activity relationships of quinic acid and caffeic acid derivatives from the flower buds of Lonicera bournei. Planta Medica 67:322–325 DOI 10.1055/s-2001-14337. Xu Z, Zhang D, Hu J, Zhou X, Ye X, Reichel KL, Stewart NR, Syrenne RD, Yang X, Gao P, Shi W, Doeppke C, Sykes RW, Burris JN, Bozell JJ, Cheng MZ, Hayes DG, Labbe N, Davis M, Stewart Jr CN, Yuan JS. 2009. Comparative genome analysis of lignin biosynthesis gene families across the plant kingdom. BMC Bioinformatics 10(Suppl 11):S3 DOI 10.1186/1471-2105-10-S11-S3. Ye J, Fang L, Zheng H, Zhang Y, Chen J, Zhang Z, Wang J, Li S, Li R, Bolund L, Wang J. 2006. WEGO: a web tool for plotting GO annotations. Nucleic Acids Research 34:W293–W297 DOI 10.1093/nar/gkl031. Yoo HJ, Kang HJ, Song YS, Park EH, Lim CJ. 2008. Anti-angiogenic, antinociceptive and anti-inflammatory activities of Lonicera japonica extract. Journal of Pharmacy & Pharmacology 60:779–786 DOI 10.1211/jpp.60.6.0014. Yuan Y, Song L, Li M, Liu G, Chu Y, Ma L, Zhou Y, Wang X, Gao W, Qin S, Yu J, Wang X, Huang L. 2012. Genetic variation and metabolic pathway intricacy govern the active compound content and quality of the Chinese medicinal plant Lonicera japonica thumb. BMC Genomics 13:195 DOI 10.1186/1471-2164-13-195. Yuan Y, Wang Z, Jiang C, Wang X, Huang L. 2014. Exploiting genes and functional diversity of chlorogenic acid and luteolin biosyntheses in Lonicera japonica and their substitutes. Gene 534:408–416 DOI 10.1016/j.gene.2012.09.051. Zhang H, Gao S, Lercher MJ, Hu S, Chen WH. 2012. EvolView, an online tool for visualizing, annotating and managing phylogenetic trees. Nucleic Acids Research 40:W569–W572 DOI 10.1093/nar/gks576. Zucker M, Levy CC. 1959. Some factors which affect the synthesis of chlorogenic acid in disks of potato tuber. Plant Physiology 34:108–112 DOI 10.1104/pp.34.2.108.

Qi et al. (2017), PeerJ, DOI 10.7717/peerj.3781

20/20