Flower color is one of the most important characters of ornamental plants, and directly affects the ornamental value and economic value; creating with novelty colors has been the pursuit of plant breeders.
The flavonoids, a class of secondary plant metabolites, played the most important role in flower coloration of many flowering plants, increased and reduced anthocyanin content of petals and pericarp which are likely to make the color change   . In plants, flavonoids are synthesized via the phenylpropanoid and polyketide pathway, which starts with the condensation of one molecule of CoA-ester of cinnamic acid or derivatives such as coumaric or ferulic acid, and three molecules of malonyl-CoA, yielding a naringen in chalcone as major product. A number of enzymes are involved in the flavonoids biosynthesis; genes encoding these enzymes have been isolated from many plants  . Chalcone synthases (CHSs), the most well known representatives of this family, provide the starting materials for a diverse set of metabolites (flavonoids) which have different and important roles in providing floral pigments, auxin protection and transport and stress resistance     .
Sweet osmanthus (Osmanthus fragrans) is one of the important ornamental plants in the Oleaceae family. Owing to their pleasant scent, and potential medicinal value, Osmanthus fragrans is widely distributed in China, Japan, Thailand, and India, and was introduced in Europe late in the 18th century  . So far, there are a total of 166 cultivars that have been identified and divided into four groups based on traditional, morphological and agronomic traits (e.g., flower color): O. fragrans Asiaticus Group, O. fragrans Albus Group, O. fragrans Luteus Group, and O. fragrans Aurantiacus Group  .
Interestingly, flower color varies among the four groups and even at different stages of blossom development. The flower color in the Aurantiacus Group is mainly orange or orange-red, while in other three groups is ivory or yellow. Previous studies have shown that the major flower pigment compounds are flavonoids and carotenoids in sweet osmanthus  . However, there have been only few molecular studies of the anthocyanin biosynthesis pathway in sweet osmanthus. In this study, we cloned CHS cDNA from O. fragrans and analyzed CHS expression pattern of OfCHS in flowers and other tissues. We also detected the different expression of CHSin petals of Yingui and Dangui in different floral stages. These results provided basic information to study the function of OfCHS in O. fragrans flavonoids biosynthesis and metabolism.
2. Material and Methods
2.1. Plant Materials
Two varieties of O. fragrans, “Zi Yingui” and “Chenghong Dangui” used in this study were grew on the South campus of Shandong Agricultural University. Flowers from xianyan flowering stage (S1), initial flowering stage (S2) and full flowering stage (S3)  and mature leaves (L) were collected from sweet osmanthus trees, immediately frozen in liquid nitrogen and stored at −80˚C untilfurther used.
2.2. Isolation and Sequence Analysis of OfCHS
For cloning of CHS cDNA from O. fragrans, we aligned amino acid sequences of CHS from different plant species, and identified highly conserved regions of CHS from these species. Following the conserved sequence, two degenerate primers DCHSF1 and DCHSR were synthesized to amplify the partial sequence of the CHS gene (Table 1). RT-PCR was performed with the degenerate primer pair. After an initial 98˚C for 2 min denaturation step, 35 cycles were run each with 10 s of denaturation at 98˚C, followed by 15 s annealing at 55˚C, and 2 min extension at 68˚C using MightyAmp® DNA Polymerase Ver.2 (TAKARA). The PCR product of about 1000 bp was cloned into pMD18- T vector (TaKaRa) and confirmed by sequencing.
The rapid amplification of cDNA end (RACE) approach was used to isolate the 3’ and 5’ ends of OfCHS cDNA by use of 3’ SMARTer™ RACE cDNA Amplification Kit (Clontech) and 5’ RACE System for Rapid Amplification of cDNA Ends, Version 2.0 kits (Invitrogen). These primers for 5’ RACE (A001-1, A001-2 and A001-3) and 3’RACE (3’F001-1 and 3’F001-2) were listed in Table 1. All reactions were performed according to the manufacturer’s instructions. These amplified cDNA fragments were ligased into pMD18-T vector (TaKaRa) and sequenced. The CDS sequence of OfCHS and CHS sequences from other plant species was compared. These sequences were aligned by DNAMAN software and manually adjusted. Phylogenetic analysis was performed by use of DNAMAN version 6.0 (LynnonBio Soft company, USA).
2.3. Quantitative Real-Time PCR Analysis
The first strand cDNA was synthesized from 200 ng purified RNA using random hexamers at 37˚C for 60 min and RevertAid First Strand cDNA Synthesis Kit (THERMO SCIENTIFIC). For endogenous control, the 18sRNA probe was used. The real-time PCR was carried out with the Power SYBR® Green PCR Master Mix (Applied Biosystems) using theBIO-RAD CFX Connect™ instrument (Applied Biosystems). The PCR program included a 5 min denaturation step at 95˚C and then 45 cycles of 10 s of denaturation at 95˚C and 20 s of hybridization at 55˚C and 20 s of polymerization at
Table 1. Primers used to isolate and analyze the expression of OfCHS gene.
72˚C. The relative expression ratios were calculated using the BIO-RAD CFX manage software vertion 3.1 sequence detection software (Applied Biosystems) and normalized using the 18S ribosomal RNA results. Real-time quantitative PCR was performed in three replicates for each sample. The primers used for OfCHS, and 18srRNA genes were listed in Table 1.
2.4. Analysis of Anthocyanin Relative Content
The ralative content and analysis of anthocyanin were carried out following the method described by Rabino and Mancinelli, 1986. Flower petals were obtained from different flowering stage of each cultivar. Three repetitions were performed. In brief, 0.3 g flower petals were ground in liquid nitrogen, and then extracted with appropriate solvent of 1% (hydrochloricacid: methanol) to 50 mL, immediately in 530, and 657 nm range scanning, and the total amount of anthocyanins ((A530-0.25A657)/g) was determined  .
3.1. Molecular Cloning and Sequence Analysis of OfCHS
Using RT-PCR and RACE approaches, we isolated OfCHS cDNA (GenBank accession number KR604813). The cDNA was 1383 bp long and a coding sequence (CDS) of 1173 bp encoding a polypeptide of 391 amino acids with an estimated molecular mass of 39.9 kDa. The theoreticalisoelectric point was 6.23. The protein sequences had three catalytic residues: Cysl64-His303-Asn336, and two Phe activeresidue (Phe215 and Phe265) and the characteristics peptide sequence of chalcone synthasefamily (RFMMYQQGCFAGG- TVLR and GVLFGFGPGL) were included  (Figure 1(a)). Amino acid sequence alignment of CHS from different plant species showed OfCHS shared 95% identity with the protein from Oleaeuropaea, and high similarity with those from other plants, such as Rhododendron simsii (89%), Gossypium hirsutum (79%), Hibiscus cannabinus (89%), Malus domestica (87%), Sorbus aucuparia (88%), Camellia sinensis (90%) (Figure 1(b)). The phylogenetic analysis demonstrated similar patterns, which indicated that CHSs from Solenostemon scutellarioides, Perillaf rutescens, Antirrhinum majusand Digitalis lanatacan be categorized into one group (Figure 1(b)). These results suggested that an evolutionary link did exist among these plants.
3.2. Expression of OfCHS
We analysed the expression pattern of OfCHS by Real-time quantification RT-PCR (qRT-PCR) approach. The expression pattern indicated that OfCHSs showed the highest transcript abundance in matureleaves, the lowest levels in flowers, which was very different toward several ornamental trees such as Tree Peony  (Figure 2). The expression of the initial flower stage (S2) was a little higher than that in full flowering stage (S3) (Figure 2). We also investigated the expression levels of OfCHS in different floral stage in two cultivars and showed that the transcripts in “Zi Yingui” petals were clearly higher than that in “Chenghong Dangui” at three stages especially at xiaoyan stage (S1) and there were no significant difference between the two cultivars in the full flowering stage (S3) (Figure 3).
Figure 1. Sequence analysis of OfCHS. (a) Deduced amino acid sequences of OfCHS. The characteristics peptide sequence of chalcone synthasefamily (RLMMYQQGCFAGGTVLR and GVLFGFGPGL) are double-underlined. Two Phe active residue were marked by Asterisk Start. The catalytic triad sites C164, H303 and N336 are boxed; (b) Phylogenetic analysis of OfCHS and other plant species. Accession number: Olea europaea, AHK07000.1; Osmanthus fragrans, KR604813; Rhododendron simsii, CAC88858.1; Gossypium hirsutum, ABS52573.1; Hibiscus cannabinus, AIC75908.1; Sorbus aucuparia, ABB89213.1; Prunus persica, XP_007223025.1; Malus domestica, XP_008380609.1; Camellia sinensis, P48387.1; Pyrus pyrifolia, AFQ92052.1; Solenostemon scutellarioides, ABP57071.1; Perilla frutescens, AA19548.1; Antirrhinum majus, P06515.1; Digitalis lanata, CAA05512.1; The tree is determined using the DNAMAN version 6.0.
Figure 2. The relative expression of OfCHS gene in different flowering stages and leaves of Osmanthus fragrans. S1: xiangyan stage; S2: initial flowering stage; S3: full flowering stage; L: leaves.
Figure 3. Relative quantity of OfDFR expression in different floral stages in “Chenghong Dangui” and “Zi Yingui”. S1: xiangyan stage; S2: initial flowering stage; S3: full flowering stage.
3.3. Anthocyanin Content of Flower Petals
The results showed that total relative anthocyanin content was 0.74 U・g−1, 1.60 U・g−1 and 0.41 U・g−1, each at xiangyan stage, initial flowering stage and full flowering stage in “Dangui” flower petals and 0.34 U・g−1, 0.51 U・g−1, and 0.28 U・g−1 in “Yingui” flower petals (Figure 4). Statistical analysis showed that the anthocyanin content of “Dangui” was clearly higher than that in “Yingui” petals of three flowering stage (correlation is significant at the 0.05 level. P < 0.05).
Figure 4. Relative contents of anthocyanin in the two cultivar petals. S1: xiangyan stage; S2: initial flowering stage; S3: full flowering stage.
Chalcone synthases (CHSs) catalyze the condensation of p-coumaroyl-CoA and three malonyl-CoA molecules to form the naringenin chalcone, which is the first committed step in the flavonoids pathway of plants, leading to the biosynthesis of flavonoids, isoflavonoids, and anthocyanins  . Chalcone synthases have been extensively studied in higher plants especially in herbaceous plants.
In this study, a full length of cDNA encoding CHS protein of O. frangrans (OfCHS) was cloned by using RT-PCR and RACE PCR. This is the first time the gene and the expression of CHS in sweet osmanthus was analysed. The sequence analysis showed that the CHS has similar structure with that of higher plants.
The expression pattern indicated that OfCHS showed the highest transcript abundance in mature leaves, the lowest levels in S3 (Figure 2). The result was different from other ornamental plants which CHS expression was highest in petals. Moreover the transcripts in “Zi Yingui” petals were clearly higher than those in “Chenghong Dangui” at three stages especially at S1 and there was no significant difference between the two cultivars in S3, but the anthocyanin content of “Dangui” was clearly higher than that in “Yingui” petals of three flowering stage and this showed that the OfCHS did not result in the accumulation of total anthocyanin in “Dangui” (Figure 3 & Figure 4).
Additional studies are needed to clarify the contribution of other enzymes or regulatory factors to the color formation in O. frangrans.
This research was supported by the National Natural Science Foundation of Shandong Province (Grant No. ZR2013CQ008) and Shandong agricultural seeds engineering major issue (LuNongNo.6).
*These authors contributed equally to this work.
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