molecules Article Transcriptomic Analysis of Leaf in Tree Peony Reveals Differentially Expressed Pigments Genes Jianrang Luo, Qianqian Shi, Lixin Niu * and Yanlong Zhang * College of Landscape Architecture and Art, Northwest A & F University, Yangling 712100, Shaanxi, China; [email protected] (J.L.); [email protected] (Q.S.) * Correspondence: [email protected] (L.N.); [email protected] (Y.Z.); Tel.: +86-29-8708-2878 (Y.Z.), Fax: +86-29-8708-0269 (L.N. & Y.Z.) Academic Editor: Derek J. McPhee Received: 15 January 2017; Accepted: 13 February 2017; Published: 20 February 2017 Abstract: Tree peony (Paeonia suffruticosa Andrews) is an important traditional flower in China. Besides its beautiful flower, the leaf of tree peony has also good ornamental value owing to its leaf color change in spring. So far, the molecular mechanism of leaf color change in tree peony is unclear. In this study, the pigment level and transcriptome of three different color stages of tree peony leaf were analyzed. The purplish red leaf was rich in anthocyanin, while yellowish green leaf was rich in chlorophyll and carotenoid. Transcriptome analysis revealed that 4302 differentially expressed genes (DEGs) were upregulated, and 4225 were downregulated in the purplish red leaf vs. yellowish green leaf. Among these DEGs, eight genes were predicted to participate in anthocyanin biosynthesis, eight genes were predicted involved in porphyrin and chlorophyll metabolism, and 10 genes were predicted to participate in carotenoid metabolism. In addition, 27 MYBs, 20 bHLHs, 36 WD40 genes were also identified from DEGs. Anthocyanidin synthase (ANS) is the key gene that controls the anthocyanin level in tree peony leaf. Protochlorophyllide oxido-reductase (POR) is the key gene which regulated the chlorophyll content in tree peony leaf. Keywords: tree peony; transcriptome; flavonoids; chlorophyll; carotenoid; differentially expressed genes (DEGs) 1. Introduction Tree peony (Paeonia suffruticosa Andr.) is a woody shrub of the genus Paeonia and family Paeoniaceae, and is a traditional ornamental flower because of its large, showy, and colorful flowers [1]. Besides its beautiful flower, the leaf of tree peony has also good ornamental value owing to its leaf color change in spring. Coloration of plant organs such as flowers, fruit and leaves is governed by pigments in plants [2]. There are four main classes of pigments for coloration in plants: flavonoids/anthocyanins, chlorophylls, carotenoids, and betalains [3]. Flavonoids, particularly anthocyanins, acting as major pigments that responsible for the orange to blue colors found in many plant flowers, leaves, fruits, seeds, and other tissues [2–4]. Chlorophylls including chlorophyll a and chlorophyll b, the photosynthetic pigments that play essential roles in plant photosynthesis, are main pigments in plant green leaf [5]. Carotenoid belong to the isoprenoid pigments, which are usually responsible for yellow-orange colors [6,7]. Carotenoids are also photosynthetic pigments that usually exist in plant leaf with chlorophylls. Betalains are the yellow and violet pigments belong to the order Caryophyllales, and they only exist in very few plants such as Beta vulgaris and Suaeda salsa [8,9]. In plants, anthocyanins and betalains are usually mutual exclusive, which are never found together in the same plant [10]. Prior to advancements in this field, researchers mainly focused on floral flavonoid/anthocyanin pigment components detection [11–13], or flavonoid/anthocyanin genes of petal analysis in tree Molecules 2017, 22, 324; doi:10.3390/molecules22020324 www.mdpi.com/journal/molecules Molecules 2017, 22, 324 2 of 14 peonyMolecules [14–17 2017]. As, 22, far324 as we know, there has been no report on leaf pigments components detection2 of 14 and related gene analysis in tree peony. In the absence of a complete genome sequence in tree peony, peony [14–17]. As far as we know, there has been no report on leaf pigments components detection transcriptomic analysis is an effective method for gaining insights into differentially expressed genes and related gene analysis in tree peony. In the absence of a complete genome sequence in tree peony, (DEGs) between different color leaves. Transcriptome sequencing of the different leaf color stages transcriptomic analysis is an effective method for gaining insights into differentially expressed genes of tree(DEGs) peony between will provide different useful color leaves. insights Transcriptome into the molecular sequencing mechanisms of the differen oft leaf tree color peony stages leaf of color changestree inpeony spring. will Therefore, provide useful the leaf insights pigment into levelsthe molecular of different mechanisms stagesin of tree treepeony peony wasleaf analyzed.color The differentchanges in leaf spring. color Therefore, stage transcriptomes the leaf pigment were leve compared,ls of different and stages the DEGsin tree werepeony filtered. was analyzed. Our main objectiveThe different was to annotateleaf color stage and analyzetranscriptomes the DEGs were involvedcompared, in and flavonoid/anthocyanin the DEGs were filtered. Our biosynthesis, main chlorophyllobjective and was carotenoid to annotate biosynthesis and analyze tothe identifyDEGs involved the candidate in flavonoid/anthocyanin genes responsible biosynthesis, for tree peony purplishchlorophyll red leaf and in spring. carotenoid biosynthesis to identify the candidate genes responsible for tree peony purplish red leaf in spring. 2. Results 2. Results 2.1. Pigments Level in the Leaf of Three Developmental Stages in Tree Peony 2.1. Pigments Level in the Leaf of Three Developmental Stages in Tree Peony In spring, as the tree peony leaf developed two major physiological changes, color fading and In spring, as the tree peony leaf developed two major physiological changes, color fading and yellowish greening were visually apparent (Figure1a). The tree peony leaf color was purplish red yellowish greening were visually apparent (Figure 1a). The tree peony leaf color was purplish red at S1,at then S1, then changed changed to to half half purplish purplish redred and half half yellowish yellowish green green at S2, at fina S2,lly finally yellowish yellowish green at green S3. at S3. AsAs shown shown inin FigureFigure1 1b,b, thethe anthocyaninanthocyanin contents contents were were decreased decreased from from S1 S1to S3. to S3.While While the thetotal total flavonoid,flavonoid, carotenoid, carotenoid, and and chlorophyll chlorophyll contents contents werewere increased from from S1 S1 to toS3. S3. These These indicated indicated that that the changethe change in leaf in colorleaf color phenotypes phenotypes of treeof tree peony peon ‘Many ‘Man Yuan Yuan Chun Chun Guang’ Guang’ waswas mainlymainly caused by by the decreasethe decrease of anthocyanin of anthocyanin content content and increase and increase of chlorophyll of chlorophyll and and carotenoid. carotenoid. FigureFigure 1. Images 1. Images of tree of tree peony peony leaf leaf and and pigments pigments contentscontents at at three three stages. stages. (a) ( aImages) Images of tree of treepeony peony leaf leaf at threeat three different different stages; stages; (b) contents(b) contents of anthocyanin,of anthocyanin, chlorophyll, chlorophyll, total total flavonoid, flavonoid, andand carotenoid in in the the leaf of three different stages. leaf of three different stages. Molecules 2017, 22, 324 3 of 14 2.2. Library Construction and Transcriptome Sequencing To understand the molecular basis of leaf color change in tree peony, the leaves of S1, S2, and S3Molecules were used 2017, 22 to, 324 build three libraries for high-throughput sequencing. 64,530,684;3 of 67,008,409; 14 and 69,283,538 raw reads were obtained from S1, S2, and S3 sequencing libraries, respectively. After 2.2. Library Construction and Transcriptome Sequencing removal of adaptor sequences, ambiguous reads, and low-quality reads, 62,835,627; 65,363,790; and 67,681,919 highTo understand quality cleanthe molecular reads comprisingbasis of leaf color 9.43, ch 9.80,10.15ange in tree Gb peony, nucleotides the leaves withof S1, Q20S2, and > 98%S3 were were used to build three libraries for high-throughput sequencing. 64,530,684; 67,008,409; and 69,283,538 generatedraw from reads S1, were S2, obtained and S3 transcriptomefrom S1, S2, and sequencing. S3 sequencing In libraries, total, 53,323 respectively. unigenes After with removal an N50 of of 1215 were generatedadaptor sequences, byTrinity ambiguous 2.1.1 (Broad reads, Institute, and low-qua Cambridge,lity reads, 62,835,627; MA, USA). 65,363,790; The min and length, 67,681,919 max length and averagehigh quality length clean of these reads unigenescomprising were 9.43, 201,9.80,10. 13121,15 Gb andnucleotides 801 nt, with respectively. Q20 > 98% Thewere size generated distributions of thesefrom unigenes S1, S2, areandshown S3 transcriptome in Supplementary sequencing. Materials In total, 53,323 Figure unigenes S1. with an N50 of 1215 were generated byTrinity 2.1.1 (Broad Institute, Cambridge, MA, USA). The min length, max length and 2.3. De Novoaverage Assembly length of andthese Gene unigenes Annotation were 201, 13121, and 801 nt, respectively. The size distributions of these unigenes are shown in Supplementary Materials Figure S1. A total of 28,439 unigenes (53.33% of all 53,323 cleaned unigenes) were annotated based on BLAST+2.3. 2.4.0 De Novo (http://www.ncbi.nlm.nih.gov/BLAST/ Assembly and Gene Annotation ) (E-value < 1 × 10−5) searches of four public databases. AmongA total of them,28,439 unigenes 28,296 unigenes(53.33% of all (53.06%); 53,323 cleaned 20,710 unigenes) (38.84%); were 17,537 annotated (32.89%); based on 11,204 BLAST+ (21.01%) could be2.4.0 annotated (http://www.ncbi.nlm.nih. to the NCBIgov/BLAST/) nr database (E-value released < 1 31× 10 August−5) searches 2016 of (http://www.ncbi.nlm.nih.four public databases. gov), theAmong Swiss-Prot them, 28,296 protein unigenes database (53.06%); (http://www.expasy.ch/sprot 20,710 (38.84%); 17,537 (32.89%); ),11,204 the (21.01%) COG/KOG could databasesbe (http://www.ncbi.nlm.nih.gov/COGannotated to the NCBI nr database), released and the 31 KEGGAugust database2016 (http://www.