Introduction
============

Cytochrome P450 monooxygenases (CYP450s) are a large and complex superfamily which can be found in almost all living organisms ([@ref-22]). Plant CYP450s are heme-containing enzymes that take part in a wide variety of reactions of both primary and secondary metabolism ([@ref-17]), including the production of fatty acids, sterols, plant hormones, flavonoids, terpenoids, lignin, signaling molecules, and other biological molecules ([@ref-32]).

*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 ([@ref-33]). There are more than 500 traditional Chinese medicine prescriptions containing *L. japonica* ([@ref-33]). 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 ([@ref-36]; [@ref-39]).

The active compounds of *L. japonica* have been extensively studied. Essential oils ([@ref-30]), phenolic acids ([@ref-19]), flavone ([@ref-5]), triterpenoid saponins ([@ref-4]), 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* ([@ref-7]).

As one of the most important secondary metabolites in plants, CGA is often used in medicines and foods for its high anti-oxidative activity ([@ref-43]). The biosynthetic pathway of CGA has been investigated in many plants and is catalyzed by a series of enzymes ([@ref-25]). Cinnamate 4-hydroxylase (C4H) and *p*-coumarate 3′-hydroxylase (C3H) are two CYP450s that participate in the two steps of hydroxylation in CGA biosynthetic pathway ([@ref-13]; [@ref-31]). In *L. japonica*, a CYP98A subfamily gene encoding LjC3H was isolated and characterized. By using heterologous expressed LjC3H *in vitro* assay, a recent study revealed that the recombinant protein was effective in converting *p*-coumaroylquinate to CGA ([@ref-28]). 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* ([@ref-41]).

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* ([@ref-40]; [@ref-15]). 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](https://www.ncbi.nlm.nih.gov/sra?term=SRR290309), [SRR342027](https://www.ncbi.nlm.nih.gov/sra?term=SRR342027), [SRR576924](https://www.ncbi.nlm.nih.gov/sra?term=SRR576924), [SRR576925](https://www.ncbi.nlm.nih.gov/sra?term=SRR576925) and [SRR766791](https://www.ncbi.nlm.nih.gov/sra?term=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 ([@ref-10]) 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 \>95% sequence similarity with reference sequences, they were classified into the same family, subfamily, or allelic variant, respectively ([@ref-23]).

The deduced amino acid sequences of *L. japonica* CYP450s were subjected to Multiple Expectation Maximization for Motif Elicitation (MEME, <http://meme-suite.org/>) ([@ref-1]) 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/>) ([@ref-9]) 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 ([@ref-11]). The result of alignment was imported to MEGA4 ([@ref-34]) 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/>) ([@ref-42]).

Gene ontology and KEGG pathway analysis
---------------------------------------

Blast2GO (<http://www.blast2go.com/>) ([@ref-8]) 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>) ([@ref-38]) 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 ([@ref-7]). 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 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% H~3~PO~4~ (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 manufacturer's instructions using SYBR® Premix Ex TaqTM II (Takara) and 2 µl of 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](#supp-1){ref-type="supplementary-material"}. 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](#table-1){ref-type="table"}). 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.

10.7717/peerj.3781/table-1

###### List of predicted CYP450s with complete cytochrome P450 domain from *L. japonica*.

![](peerj-05-3781-g008)

  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
  A       71     CYP81    CYP81B      m211982   non-A   85     CYP707   CYP707A     m35702
  A       71     CYP81    CYP81C      m12729    non-A   85     CYP707   CYP707A     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                                        

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 classified into two major branches, A-type (47%) and non-A type (53%) ([Fig. 1](#fig-1){ref-type="fig"}). 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 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.

![Phylogenetic analysis of predicted CYP450s in *L. japonica* and the representative members of CYP450 families.](peerj-05-3781-g001){#fig-1}

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 ([@ref-26]). 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](#fig-2){ref-type="fig"}). 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 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 ([@ref-6]).

![Weblogos of conserved motifs identified in A-type (A) and non-A type (B) CYP450s from *L. japonica*.](peerj-05-3781-g002){#fig-2}

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](#fig-3){ref-type="fig"}). 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 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*.

![Gene ontology annotation of A-type and non-A type CYP450s in *L. japonica*.](peerj-05-3781-g003){#fig-3}

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](#fig-4){ref-type="fig"}). 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, CYP76A-m155830, CYP78A-m152788, CYP93B-m79556, CYP98A-m184946, CYP98A-m43608 and CYP736A-m18282) were found to be involved in the biosynthesis of phenolic compounds 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, CYP72A-m20456, 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.

![KEGG pathway analysis of predicted CYP450s in *L. japonica*.](peerj-05-3781-g004){#fig-4}

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](https://www.ncbi.nlm.nih.gov/nucleotide?term=KX845341)) and '*LjC3H2*' (GenBank accession number: [KX845342](https://www.ncbi.nlm.nih.gov/nucleotide?term=KX845342)), respectively. The *C4Hs* belonged to CYP73A subfamily and *C3Hs* belonged to CYP98A subfamily. Phylogenetic analysis indicated that two clades were clustered for C4Hs and C3Hs from *L. japonica* and other plants ([Fig. 5](#fig-5){ref-type="fig"}).

![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).](peerj-05-3781-g005){#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](#fig-6){ref-type="fig"}, 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](#fig-7){ref-type="fig"}). 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](#fig-7){ref-type="fig"}). Interestingly, the expression patterns of *LjPAL1*, *LjC4H3*, *LjC3H1* and *LjHQT* were quite similar, which 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.

![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.](peerj-05-3781-g006){#fig-6}

![Transcriptional analyses of CGA biosynthetic pathway genes in buds and flowers of *L. japonica* at different developmental stages.](peerj-05-3781-g007){#fig-7}

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 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 ([@ref-21]; [@ref-24]). In this study, ten CYP450s (CYP73A-m13469, CYP73A-m177245, 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 ([@ref-21]), 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 ([@ref-21]).

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 ([@ref-13]; [@ref-31]). In *L. japonica*, a gene encoding LjC3H has been isolated and characterized by [@ref-28], 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 [@ref-28], 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* by [@ref-41], 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 ([@ref-2]; [@ref-29]), which might be caused by functional divergence of both substrate and catalytic specificity during plant evolution ([@ref-16]; [@ref-37]). 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 ([@ref-18]). 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 ([@ref-35]). C3H was also a key enzyme in lignin biosynthesis ([@ref-3]). 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 ([@ref-2]). 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* ([@ref-12]). 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 ([@ref-20]). In pine and eucalyptus, xylem-associated MYB transcription factors could bind to the AC elements and activate the transcription of the lignin biosynthetic genes ([@ref-27]; [@ref-14]). 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 ([@ref-2]).

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*.

Supplemental Information
========================

10.7717/peerj.3781/supp-1

###### Supplemental Table 1

Primers used for qRT-PCR

###### 

Click here for additional data file.

10.7717/peerj.3781/supp-2

###### Sequences of LjC4H3 and LjC3H2

###### 

Click here for additional data file.

Additional Information and Declarations
=======================================

The authors declare there are no competing interests.

[Xiwu Qi](#author-1){ref-type="contrib"} performed the experiments, analyzed the data, wrote the paper, prepared figures and/or tables.

[Xu Yu](#author-2){ref-type="contrib"} and [Daohua Xu](#author-3){ref-type="contrib"} performed the experiments.

[Hailing Fang](#author-4){ref-type="contrib"} analyzed the data.

[Ke Dong](#author-5){ref-type="contrib"} contributed reagents/materials/analysis tools.

[Weilin Li](#author-6){ref-type="contrib"} and [Chengyuan Liang](#author-7){ref-type="contrib"} conceived and designed the experiments, reviewed drafts of the paper.

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](https://www.ncbi.nlm.nih.gov/nucleotide?term=KX845341) and [KX845342](https://www.ncbi.nlm.nih.gov/nucleotide?term=KX845342).

The following information was supplied regarding data availability:

The raw data used for transcriptomic assemblies was downloaded from NCBI SRA with accession numbers [SRR290309](https://www.ncbi.nlm.nih.gov/sra?term=SRR290309), [SRR342027](https://www.ncbi.nlm.nih.gov/sra?term=SRR342027), [SRR576924](https://www.ncbi.nlm.nih.gov/sra?term=SRR576924), [SRR576925](https://www.ncbi.nlm.nih.gov/sra?term=SRR576925) and [SRR766791](https://www.ncbi.nlm.nih.gov/sra?term=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>).
