Molecular Cloning and Characterization of an Anthocyanidin Synthase Gene in Prunus persica ( L . ) Batsch

To elucidate the effect of anthocyanidin synthase (ANS) gene on anthocyanin accumulation in fruit skin of Prunus persica (L.) Batsch cv. ‘Chunmei’, this study cloned and characterized an ANS gene (PpANS) from peach. PpANS (GenBank accession No. KX760117) was encoded by a 1074 bp-long open reading frame (ORF) corresponding to a polypeptide consisting of 358 amino acids with a molecular mass of 40.45 kD and an isoelectric point of 5.46. PpANS contains a conserved 2-oxoglutarateand iron-dependent dioxygenases and non-haem dioxygenase binding regions. PpANS shared high similarities to angiosperm ANS and displayed the closest genetic relationship to Prunus domestica. Real-time PCR analysis indicated that PpANS was highly expressed in fruit skin, flesh and flowers, and peach fruit skin showed the highest transcript level of PpANS. Anthocyanin accumulation analysis indicated that it was highly accumulated in fruit skin and flesh of peach. Changes in the transcript level were highly correlated with anthocyanin content in the different tissues of peach. Prokaryotic expression analysis showed PpANS that protein can be expressed correctly in E. coli, and the size of PpANS recombinant protein was consistent with its predicted size. In vitro enzyme activity assay revealed that recombinant PpANS protein could catalyze the formation the cyanidin from leucocyanidin.These results indicated that PpANS was responsible for anthocyanin accumulation


Introduction
Peach (Prunus persica (L.) Batsch) is a member of family Rosaceae.In terms of land area for production, peach is the third most cultivated fruit crop in temperate regions (Dirlewanger et al., 2002).Moreover, a peach tree offers high economic value.Candied fruit, dried fruit, and peach sauce are products that are very popular locally (Wisniewski et al., 2011).Peach fruit is a healthy food that contains nutrients, namely, amino acids, proteins, fats, sugars, essential oils, minerals, amygdalin, and vitamins, which are all essential to the body (Arndt et al., 2001;Jha et al., 2012).Improvement in fruit quality has recently become an important research field in pomology.The sugar-acid ratio is thus commonly used as a quality index, acid and sugar content and composition are major determinants of peach quality (Dirlewanger et al., 1999).External quality is mainly determined by fruit size and skin color.In fresh peach, the most important quality is skin color, which is easily perceived by consumers.Skin coloration is possibly the index that ultimately determines whether consumers will purchase a given batch of peaches.For this reason, skin color has been significantly improved in the past decades, and peach breeders have selected primarily for intense red color at early stage of maturity (Iglesias and Echeverría, 2009).
Anthocyanins, a class of flavonoids that play a major role in coloration of fruits and most flowers (Wang et al., 2013).Red coloration in plants is attributed to anthocyanin accumulation usually in skin and flesh of fruits.The degree of anthocyanin accumulation largely determines the quality of grape berries (Afifi et al., 2003;Yoshida et al., 2003).Fig. 1 shows the synthesis of anthocyanins in peach and of key precursors, including 4-coumaroyl-CoA and 3×malonyl-CoA, through the flavonoid biosynthetic pathway.Anthocyanidin synthase (ANS, EC. 1114111119) catalyzes the key synthesis reaction from leucoanthocyanidin to anthocyanidin.The following reaction will occur in the process described above, that is, oxidation, dehydration, and glycosylation of various leucoanthocyanidins produce corresponding anthocyanidins.Furthermore, glycosylation, methylation, and acylation are required to convert anthocyanin into cyanidin 3-glucosides (Holton and Cornish, 1995).

Cloning of full-length cDNA of PpANS
The specific primer pair ANS-FP (5′-ATGGTGAGCTCTGDTTCAGTGA-3′) and ANS-RP (5′-TCACTTGTTGAGCAAAGCTTCT-3′) were designed and synthesized (Shanghai Sangon, China) based on the transcriptome sequencing data for P. persica.The PCR reaction conditions were as follows: 95 °C for 3 min followed by 35 cycles at 95 °C for 30 s, 56 °C for 30 s, and 72 °C for 90 s with an extension at 72 °C for 10 min (Li et al., 2015).The amplified products were analyzed by 1% gel electrophoresis, purified using an AxyPrep DNA Extraction Kit, cloned into pMD19-T vector (Shanghai Sangon, China), and then sequenced (Flugge and Gao, 2005).

Bioinformatics and molecular evolution analyses
BioEdit 7.0 and MEGA 6.0 were used to construct a phylogenetic tree through the neighbor-joining method and to perform multiple alignment analysis of the amino acid sequences of ANS, respectively (Kumar et al., 2004;Tamura et al., 2007).Sequence homology searches and analysis were implemented using online bioinformatics tools (http://www.ncbi.nlm.nih.gov).Oligo 6 software was utilized to design the primers used in the experiment.The online tool ExPASy-ProtParam (www.expasy. The main determinant of anthocyanin accumulation in fruit is a genetic factor.The reference for environmental factors is required.Environmental factors (light, temperature, environmental stress, and loss of nutrient elements) can upregulate or downregulate the expression levels of structural and regulatory genes and correspondingly promote or inhibit synthesis and accumulation of anthocyanin, resulting in different colorations in leaves or flowers (Koes et al., 2005;Lepiniec et al., 2006).ANS is one of the key enzymes in anthocyanin biosynthesis.ANS involved in later phase of anthocyanin biosynthetic and dependent on 2-ketoglutate acid and Fe 2+ in catalyzing conversion of colorless pigments into colored pigments in flowers (Heller et al., 1985;Koes et al., 2005).ANS is encoded by a mini-gene family in many plants.ANS gene have been cloned and functionally analyzed in Theobroma cacao (Liu et al., 2013), Brassica carinata (Yan et al., 2014), Brassica juncea (Yan et al., 2011), Fructus mori (Qi et al., 2014), Punica granatum L. (Ben-Simhon et al., 2015), Malus spectabilis (Zhang et al., 2015), and Pyrus communis L. (Yang et al., 2013); however, few reports on regulation of anthocyanin accumulation by ANS gene in peach fruit skin are available.In the present study, an ANS gene (PpANS) was isolated from peach fruit skin by using RT-PCR technique to determine the function of PpANS in peach fruit.We investigated the structure of the gene, as well as deduced the amino acid composition and the recombinant product of prokaryotic expression vector in E. coli.We identified the products of the incubation of recombinant PpANS protein with 3,4-cisleucocyanidin as substrate in vitro through high-performance liquid chromatography (HPLC).Moreover, we determined the transcript levels of PpANS and accumulation pattern of anthocyanins in roots, stems, leaves, flowers, and fruit skin and flesh of peach.

Plant material
This study used the six-year-old trees of peach cv.'Chunmei' (P.persica) and peach rootstock cultivars of 'Flordaguard' grown in deciduous fruit tree base in the College of Horticulture and Landscape Architecture, Yangtze University, Jingzhou, Hubei Province, China.Spatial expression profile of PpANS was measured from roots, stems, leaves, flowers, and fruit flesh and skin, which were immediately frozen in liquid nitrogen and stored at −80 °C until further analyses.

DNA and RNA extraction and cDNA synthesis
Different tissues of peach sample (1 g) were immediately pulverized in liquid nitrogen, filtered, and centrifuged at 12,000 × g for 10 min at 4 °C.Total RNA was extracted from the roots, stems, leaves, flowers, and fruit flesh and skin of peach by using the CTAB method as described by Xu et al. (2008).Genomic DNA of the peach fruit skin was extracted by using the CTAB method (Xu et al., 2014).Prior to the use of the total RNA and DNA, their purity, concentration, and quality were tested at OD260/280 absorbance ratio and by using spectrophotometer and 1% (w/v) agarose gel electrophoresis.First-strand cDNA was synthesized using PrimeScript TM II 1st Strand cDNA Synthesis Kit (TaKaRa, Dalian, China).The reaction mixture was 20 μl and consisted of Oligo dT Primer (1 μl), dNTP mixture (1 μl), total RNA (8 μl); 5 × Primescript buffer (4 μl), 29 Fig. 1.Anthocyanin biosynthesis pathway in peach.CHS chalcone synthase, CHI chalcone isomerase, F3H flavanone-3hydroxylase, DFR dihydroflavonol-4-reductase, ANS anthocyanidin synthase, UFGT glucoseflavonoid3-o-glucosyl transferase org/tools/protparam.html) was used to deduce the molecular weight and isoelectric point (pI) of proteins (Guex et al., 1997;Emanuelsson et al., 2000).

Quantitative real-time PCR (qRT-PCR) analysis
Roots, stems, leaves, flowers, and fruit flesh and skin were sampled from peach trees.Total RNA was isolated from each sample by using the CTAB method, first-strand cDNA was synthesized using a PrimeScriptTM RT Reagent Kit (Dalian TaKaRa, China).The resulting cDNA mixtures were used to prepare the templates for qRT-PCR.The qRT-PCR product was measured using a Bio-Rad iQ5 thermal cycler with SYBR Premix Ex Taq™ II Kit (Dalian TaKaRa, China) according to the manufacturer's protocol.Expression was normalized to a gene from peach (Pp18S), the reference genes and primer sequences for the qRT-PCR are Pp18S-F (5′-CGGCCCATTCGA GATAACAC -3′) and Pp18S-R (5′-AGACTTATGGGAG CCGATCC -3′); ANS-RTF (5′-AACGAAAATGTGAGA GAGAG -3′) and ANS-RTR (5′-GCATTGTTTGCAA GCTTGCT -3′) were designed and synthesized by Shanghai Sangon (China).Original data were analyzed by Light Cycler software and real-time PCR data on ANS gene were calculated using the 2 −ΔΔCT method as described by Livak and Schmittgen (2001).

Measurement of anthocyanin content
Anthocyanin content in the tissues of P. persica was assayed according to the method of Connor et al. (2002).One gram sample were frozen with liquid nitrogen, ground into a fine powder and extracted using 0.5 % HCl in methanol solution with shaking at 4 °C for 6 h.The extracts were centrifuged at 8,000 × g for 20 min; the supernatant was transferred into a sterile tube.Absorbance was measured at 530 nm and expressed as mg of cyanidin 3-O-glucoside equivalents per gram fresh weight (FW) of tissue.

PpANS expression in E. coli
The recombinant product of the prokaryotic expression vector pET32a-PpANS was constructed using the forward primer ANS-YF (5ʹ-GCCATGGCTGATATCGATCC ATGGTGAGCTCTGATTCAGTGA -3ʹ) and the reverse primer ANS-YR (5ʹ-CTCGAGTGCGGCCGCAAGCT TTCACTTGTTGAGCAAAGCTTCT -3ʹ); the underlined portion of the forward and reverse primers are the sites for enzymatic digestion, in which GGATCC was digested by BamH I and AAGCTT was digested by Hind III.The 1074 bplong ORF of the amplified cDNA fragments was doubly digested by BamH I and Hind III, and pET32a vector (Thermo Fisher, USA) was digested with the same restriction enzymes.The target gene of PpANS was subsequently inserted into the pET32a vector, generating the recombinant plasmid pET32a-PpANS.
The recombinant plasmid of pET32a-PpANS was transformed into E. coli BL21.The transformants were incubated overnight in 20 ml of LB liquid medium containing 50 μg/ml of ampicillin at 37 °C with shaking at 200 rpm.Overnight cultures were diluted by 100-fold in LB medium, placed into 50 mg/ml ampicillin, and cultivated at 37 °C for 4 h until an A600 of 0.5-0.6 was reached.The cultures were induced with 1.0 mM isopropyl-β-D-d-thiogalactoside (IPTG) and incubated at 30 °C with shaking at 200 rpm for 4 h.The suspension was incubated in 200 µl of buffer solution at 100 °C for 5 min and centrifuged at 12, 000 × g for 15 min (Zhu et al., 2014).Experimental result was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) by using 12% polyacrylamide separation gel and 5% stacking gel as described by Bradford (1976).The recombinant PpANS protein was purified using View-IP™ Calmodulin Red Separopore® (Agarose) 4B-CL (BioWORD Separopore) and then used for in vitro enzyme assay.

Assay of PpANS activity
The enzymatic activity of recombinant PpANS was assayed using the method described by Pang et al. (2007) with slight modification.ANS activity was measured in a final volume of 500 μl containing 8 mmol/l sodium ascorbate, 400 mmol/l sodium chloride (NaCl), 2 mmol/l 2-oxoglutaric acid, 20 mmol/l maltose, 10 mmol/l dithiothreitol (DTT), 1 mmol/L FeSO4, 1 μmol/L 3,4-cis-leucocyanidin, 40 mmol/L KPi buffer (pH 7.5), and 100 μg of purified PpANS protein.After incubation at 30 °C for 1 h, the reaction was terminated by addition of 8 μl of 18% hydrochloric acid (HCl) and 40 μl of methyl alcohol (Tanner and Kristiansen, 1993).The sample aqua was centrifuged at 12,000 × g for 5 min, and then the supernatant was passed through a 0.22 μm syringe filter according to the method described by Lockhart and Winzeler (2000).The enzymatic products were analyzed using an Agilent 1100 HPLC (Agilent Technology Co., Ltd.USA) with XDB-C18 reverse phase column (25 cm × 4.6 mm × 25 μm) (Agilent ZORBAX Eclipse, USA).The binary mobile phase used in the experiment consisted of 1% phosphoric acid and 50% methyl cyanide.Flow rate was maintained at 1 ml/min, and experimental data were measured at 530 nm.Cyanidin concentration was calculated based on a 3,4-cis-leucocyanidin standard (Sigma-Aldrich, USA).Empty pET32a vector from induced BL21 was used as control.

Cloning of cDNA of PpANS
Based on the sequences of the 5ʹ-and 3ʹ-RACE products, full-length cDNA was deduced and amplified through PCR by using the primer pair ANS-FP and ANS-RP.The cloned fulllength ORF of the PpANS was 1074 bp long.The G/C content in PpANS nucleotide sequence was 49.16% and encodes 358 amino acid with a molecular mass of 40.45 kD and a pI of 5.46.The nucleotide sequence of PpANS was highly similar to that of the ANS of other plants (Table 1).The nucleotide sequence of PpANS was 98%, 98%, 97%, 97%, 97%, 97%, 97%, 89%, 91%, and 91% identical to that of the ANS genes of P. cerasifera, P. salicina var.cordata, P. avium, P. domestica, P. cerasus, Pyrus communis, Malus domestica, Rosa rugosa, Pyrus x bretschneideri, and Pyrus pyrifolia, respectively, implying that PpANS is a member of the ANS gene family.Furthermore, the homology of ANS gene sequence among different species suggested that ANS was strongly conserved during molecular evolution.

Analysis of the molecular evolution of PpANS
A phylogenetic tree was constructed using BioEdit 7.0 and MEGA 6.0 software to further explore the evolutionary relationships among PpANS and other proteins involved in anthocyanidin biosynthesis in plants.Phylogenetic analysis (Fig. 3) indicated that ANS genes are clearly divided into five distinct families, namely, Rosaceae, Vitaceae, Paeoniexpertae, Convolvulaceae, and Solanaceae; in addition, all of these families descended from a common progenitor.PpANS is closely related to PcLDOX (AKV89246) and clustered into a subgroup that includes PaANS (AEO79983), PcANS (ABB70119), PbANS (AJD00703), and PsANS (AMH93692), which are found in plants that also belong to Rosaceae.PpANS is less closely related to IpANS (ABW69684), IbANS (BAA75305), and IhANS (ACS71531), which are found in members of Convolvulaceae, and to StANS (AEJ90548), SmANS (ACJ02088), IcANS (AIY22762), LrANS (AHH55331), ScANS (AEJ90547), and ScANS (AEJ90547), which are found in members of Solanaceae.

Expression profile of PpANS and Anthocyanin content in different tissues
The expression pattern of PpANS in different tissues of P. persica was examined by real-time PCR.Total RNA was extracted from roots, stems, leaves, flowers, and fruit flesh and skin.As shown in Fig. 4A, PpANS was expressed constitutively in most plant organs but most highly expressed in fruit skin and moderately expressed in fruit flesh.By contrast, PpANS was weakly expressed in flowers, and the lowest expression level was observed in leaves.The stems and roots of P. persica did not show detectable PpANS transcripts.
Fig. 4B shows the anthocyanin concentration of roots, stems, leaves, flowers, and fruit skin and flesh.The anthocyanins were accumulated abundantly in fruit skin, followed in the fruit flesh and flowers, and were not detected in roots.The anthocyanin content showed a good correlation with transcript level of PpANS in different tissues of P. persica.These data suggests that the expression of PpANS gene is one of the critical factors which determine the anthocyaninpigmentation pattern in P. persica.protein band of 39.2-66.2kDa (Lane 3, Fig. 5), and this band was not observed in other control, consistent with the predicted molecular mass of the translated sequences.These results suggested that recombinant PpANS proteins were expressed as inclusion bodies in E. coli cells.The activity of the purified recombinant PpANS protein was assayed by using 3,4-cis-leucocyanidin as substrate in the presence of ferrous iron and 2-oxoglutaric acid.As shown in Figs.6A and 6C, recombinant PpANS was detected by HPLC at 530 nm, and its identity confirmed as cyanidin by comparison of retention time and UV/Vis spectrum with those of authentic standards.By contrast, the empty vector control for the incubations with protein extract showed that no 3,4-cis-leucocyanidin was converted into cyanidin (Fig. 6B).

Discussion
In plants, ANS gene plays an important role in regular developmental processes, as well as involved in adaptation to all environmental conditions.Many ANS genes participating in anthocyanin biosynthesis have been cloned from plants (Reddy et al., 2007); however, few reports on ANS proteins in P. persica are available.The present study reports on one ANS gene (PpANS) isolated from the fruit skin of P. persica.Amino acid sequence analysis showed that PpANS protein contains a conserved structural domain consisting of 2-oxoglutarate and combination sites for iron ions and belonging to the 2OG-Fe 2+ -OXY superfamily.This structure is composed of a conserved 2-oxoglutarate-and Fe 2+ -dependent oxidation region, containing conserved arginine related to the combination of 2-oxoglutarate and the Fe 2+ combinationrelated amino acid residues as both conserved histidine and aspartate.The structural characteristic of ANS gene is

Expression and assay of recombinant PpANS
To induce PpANS expression in E. coli BL21 cells, we cloned the coding region of PpANS into pET32a, yielding pET32a-PpANS.The constructed expression vector was checked for in-frame fusion through restriction enzyme digestion (HindIII and BamHI) and DNA sequencing.Expression of the recombinant protein containing the 1074 bp-long ORF of PpANS was induced for 3 h with 0.5 mmol/l IPTG under stirring in the early log-phase cultures of positive clones; E. coli cells were subsequently recovered through centrifugation and lysed via a traditional physical-chemical process.The expected molecular mass of pET32a-PpANS protein was 40.45 kDa.SDS-PAGE profile revealed a clear obviously consistent with those of the dioxygenase enzyme family, which was speculated to catalyze the formation of anthocyanidins and flavonoids in plants (Rosati et al., 1999).These typical features are consistent with those of ANS gene of other plants.The results of the sequence analysis suggest that PpANS facilitates the catalytic synthesis of anthocyanins.Multiple alignment of the amino acid sequence of PpANS with that of P. cerasifera, P. avium, and P. salicina showed that PpANS shared high identity with the homologous member of Rosaceae (Fig. 3).The evolution of PpANS resulted in distinct characteristics at the genus and species levels.Purified recombinant PpANS protein showed that PpANS can catalyze conversion of cyanidin into leucocyanidin (Fig. 6), confirming its participation in anthocyanidin biosynthetic pathway.Based on the results of the structural, functional, and sequence analyses of PpANS, PpANS is speculated to play the role of multifunctional dioxygenases and mediates different dioxygenase activities in anthocyanidin biosynthetic pathway in P. persica, consistent with previous studies in ginkgo (Xu et al., 2008).
The expression levels of PpANS in different tissues of peach trees varied (Fig. 4A).PpANS expression was higher in fruit skin and flesh, and flowers, and this phenomenon is possibly related to anthocyanin synthesis.Scientific research shows that these changes in transcript levels and anthocyanin accumulation at different developmental stages in the skins of peach were positively related to each other (Tsuda et al., 2004).PpANS is possibly involved in molecular regulations of coloration in peach flowers and fruits, in which large amounts of PpANS expression are required.This finding is consistent with that in grapes as reported by Wang et al. (2010).However, no PpANS expression was detected in stem.This result may be attributed to the use of young parts as experimental materials; these parts were still in the phase of leucoanthocyanidin synthesis, and PpANS remains unexpressed.These data showed that the expression levels of PpANS, the contents of anthocyanin were significantly consistency (Fig. 4).Our recently work showed that the dynamic changes of anthocyanin content in peach peel were similar to the changes of ANS gene expression levels (Ye et al., 2017).The changes in abundance of PpANS expression in different tissues of peach were synergistic with the changes in anthocyanin content, suggesting that PpANS is a key factor in anthocyanin synthesis in peach.Anthocyanin content changed with enzyme activity, which was controlled by abundance of ANS gene expression.Aharoni et al. (2001) inhibited ANS gene expression in strawberries; consequently, anthocyanin accumulation significantly decreased and then the color of corolla changed from pink to white.This study showed that the catalytic process induced by ANS in the transition from colorless anthocyanin into colored anthocyanin was one of the dramatically decreased in peach leaves, resulting in a significant decrease of anthocyanin accumulation (Zhou et al., 2013).Nakamura et al. (2006) inhibited ANS gene expression in Torenia fournieri by using RNAi and found that anthocyanin content in T. fournieri strains was greatly reduced.ANS gene was mainly regulated by effect of various signals on transcription, thereby influencing the anthocyanin synthesis in plants; this finding is consistent with the results for Litchi chinensis as reported by Wei et al. (2011).To sum up, ANS gene exerts a regulatory function in anthocyanin synthesis.
In anthocyanin biosynthetic pathway of plant, enzymes involved in anthocyanin synthesis can catalyze conversion of colorless anthocyanin into colored anthocyanin, and the byproducts further demonstrate coupled reaction with 3-Oglucose transferase, and the coupling product is subsequently sent to the vacuole where colored 3-O-glycosylated anthocyanins form; hence, anthocyanin plays a very important role in color formation in plants (Nakajima et al., 2001).All the biosynthetic pathway genes, including ANS, showed significantly higher level of expression in red leaves than in green leaves (Zhou et al., 2014).ANS act as a key enzyme necessary in anthocyanin synthesis and in the metabolic pathways of anthocyanin in peach.However, little is known about the links between phenotype and genotype in coloration of flowers or fruits.In this study, the PpANS gene that expresses the enzyme for anthocyanin synthesis was obtained from peach fruit skin, and prokaryotic expression of its protein was performed in vitro to obtain purified recombinant protein showing clear target bands.This study found that in vitro expression of PpANS is possible.In further study, gene expression carriers for ANS will be established for genetic transformation and function validation to further investigate the mechanism of the role of PpANS in anthocyanin metabolism in peach skin and ultimately lay the foundation for molecular modification of peach fruit colors.

Conclusions
This study isolated and characterized PpANS from P. persica, and the results suggested that post-transcriptional RNA processing controls color changes in fruit skin.Although we have not yet sufficiently demonstrated the function of PpANS, the higher transcript levels of PpANS in fruit skin and flesh than in other plant tissues indicate that PpANS protein plays an active role in fruit coloration.Further characterization of the specific gene necessary to establish a color development model for PpANS in P. persica, as well as identification of specific means to improve fruit coloring by utilizing molecular breeding in P. persica, is a significant endeavor in future research.

Fig. 5 .Fig. 6 .
Fig. 5. SDS-PAGE electrophoresis of PpANS expressed in E. coli BL21.After IPTG induction, E. coli BL21cells containing pET32a-PpANS were grown at 30 °C for 6 h.Supernatants and pellets were examined by SDS-PAGE analysis followed by Coomassie Brilliant Blue R250 (CBB) staining.Lane Mprotein molecular markers; Lane 1 -Proteins of total cells containing pET32a after 4 h induction by IPTG; Lane 2 -Proteins of total cells containing pET32a without IPTG induction; Lane 3 -Proteins of total cells containing pET32a-PpANS after 4h induction by IPTG; Lane 4 -Proteins of total cells containing pET32a-PpANS without IPTG induction.Lane 5 -Purified recombinant PpANS protein used for enzyme activity assay

Table 1 .
Nucleotide sequence of PpANS similarity to the ANS genes of other plant species