Proanthocyanidin monomers and cyanidin 3o-glucoside accumulation in blood-flesh peach ( Prunus persica ( l . ) Batsch ) fruit

To better understand the characteristics and mechanisms of proanthocyanidin monomers and anthocyanin synthesis in blood-flesh peach (Prunus persica (L.) Batsch), the accumulation of catechin, epicatechin and cyanidin 3-O-glucoside was determined, and the expression patterns of structural genes associated with biosynthesis of those compounds were investigated in the blood-flesh peach fruit of cultivar “Dahongpao” during fruit development. Our results show that catechin concentration remained low and comparatively stable throughout fruit development. The concentration of epicatechin remained low at the early stages of fruit development and rapidly accumulated during ripening. Cyanidin 3-O-glucoside was not detected in the early stages. Epicatechin started to rapidly accumulate during the ripening period, reaching a maximum at the mature stage. The expressions of the early and common genes, phenylalanine ammonia-lyase and chalcone isomerase, were less associated with proanthocyanidin monomers and cyanidin 3-O-glucoside accumulation. The expression of other flavonoid ‘early’ biosynthetic genes, including chalcone synthase (CHS), flavanone 3-hydroxylase, dihydroflavonol 4-reductase (DFR) and leucoanthocyanidin dioxygenase (LDOX), were partly associated with proanthocyanidin monomers and cyanidin 3-O-glucoside levels, with expression quantities peaking synchronously at the mature stage. Leucoanthocyanidin reductase and anthocyanidin reductase, which were the key genes for proanthocyanidin monomer synthesis, correlated during fruit development with catechin and epicatechin accumulation respectively; UDP-glucose: flavonoid 3-O-glucosyltransferase (UGFT), the key gene for anthocyanin synthesis, was correlated with cyanidin 3-O-glucoside levels. The synchronous accumulation of epicatechin and cyanidin 3-O-glucoside in blood-flesh peach could not be explained by the current theory of competitive distribution mechanism of common substrate.


INTRODUCTION
Proanthocyanidin and anthocyanin in fruit are increasingly recognized as producing health beneficial effects in humans.At the same time, proanthocyanidin possesses astringency and flavor, and is a major quality factor for fruit, while anthocyanin improves visual appeal and enhances the commercial value of fruit [1,2].Therefore, data collection and theoretical research on the composition, accumulation and synthetic mechanism of proanthocyanidin and anthocyanin in fruit are of great significance for breeding new cultivars rich in beneficial ingredients.
In the current paper, the blood-flesh peach cultivar "Dahongpao" was researched to better understand the characteristics and mechanisms of proanthocyanidin monomers (catechin and epicatechin) and cyanidin 3-O-glucoside accumulation.We investigated in detail the accumulation of catechin, epicatechin and cyanidin 3-O-glucoside in blood-flesh peach during fruit development, and identified the expression of structural genes encoding the proanthocyanidin monomers and cyanidin 3-O-glucoside biosynthesis enzymes, including PAL, CHS, CHI, F3H, DFR, LDOX, UFGT, LAR and ANR.We discuss the relationship between the accumulation of these proanthocyanidin monomers and cyanidin 3-O-glucoside.The results of our research could be very useful for further study of the proanthocyanidin and anthocyanin synthesis mechanism in peach.

Plant materials and experimental treatments
Experiments were conducted using seven-year old trees of blood-flesh peach (cultivar "Dahongpao") maintained at the National Peach Germplasm Repository (Nanjing, China) in 2014.Fruit samples were collected from six trees every 7 days, beginning at 30 DAFB (days after full bloom).And each stage consisted 18 fruits with three replicates.Fruit was mechanically peeled and cored and the flesh cut into small sections.Fruit samples at each stage were mixed and immediately frozen in liquid nitrogen and stored at -75°C until use.According to the method of Lombardo et al [21], the sampling points were confirmed through the growth curve of a double-S form fitted to fruit weight.Specifically, the sampling points were 30 (S1), 58 (S2), 79 (S3), 93 (S4) and 100 (H) DAFB.

Extraction and measurement of catechin, epicatechin and cyanidin 3-O-glucoside
Catechin and epicatechin were extracted and measured according to Yan et al [11].Frozen fruit (1 g) was homogenized in 2 mL of methanol containing 0.1% H 3 PO 4 , and the extracts were centrifuged at 10000xg for 5 min at 4°C, and filtered through a 0.22µm filter for analysis in the Agilent 1100 series HPLC system (Agilent, USA).Samples (5 µL of extract) were analyzed using an Agilent ZORBAX SB-C18 column (4.6×250 mm, 5 μm) coupled with a UV detector at 280 nm, with ethanol (0.1% H 3 PO 4 ) and water (0.1% H 3 PO 4 ) as solvents: the flow rate was 1.0 mL•min -1 and temperature was 30°C.
Catechin, epicatechin and cyanidin 3-O-glucoside, and HPLC-grade methanol, acetic acid, phosphoric acid and trifluoroacetic acid were purchased from Sigma-Aldrich (Shanghai, China).Aqueous solutions were prepared using ultra-pure water purified by Milli-Q System (S.A.S. 67120, Millipore, Molsheim, France).Compounds were quantified by comparing the peak areas and presented as mg of catechin or epicatechin or cyanidin 3-O-glucoside per g of fresh tissue (mg•kg -1 FW).

RNA extraction and quantitative real-time PCR (qRT-PCR) analysis
RNA was extracted from frozen flesh obtained from "Dahongpao" using a modified CTAB method [14].After the removal of DNA by DNase I, the concentration of total RNA was measured and the first cDNA strand was synthesized from 1 µg of total RNA using Supersmo III M-MuLV Reverse Transcriptase (Bioteke Corporation, Beijing, China) primed with oligo (dT) 18.The cDNA was diluted 50× and 2 µL of the diluted cDNA was used as the template for qRT-PCR analysis.We designed qRT-PCR primers with Oligo 7.37 and Primer-Blast (http://www.ncbi.nlm.nih.gov/tools/primer-blast/) using the obtained sequences of cDNA fragments.RT-PCR analysis was performed using the primers presented in Table 1 [14,18,23].qRT-PCR was performed on an ABI 7500 System (Applied Biosystems, Foster, CA, USA) using the SYBR Premix Ex Taq TM (Takara, Japan).The PCR reaction consisted of 10 μL of SYBR Green PCR Master Mix, 0.8 μL of forward and reverse primer (10 μM), 0.4 μL of ROX Reference dye (50x; all from Takara), 6.0 μL of dH 2 O and 2.0 μL of 1:50-diluted template cDNA in a total volume of 20 μL.The two-step RT-PCR program was initiated with a preliminary step at 95°C for 30 s, followed by 40 cycles at 95°C for 5 s and 60°C for 34 s.The assay included a no-template control for each primer pair.All qRT-PCR reactions were normalized using the Ct value corresponding to actin.Three measurements for each biological replicate sample were performed.

Statistical analysis
Figures were drawn with Microsoft Excel 2010 (Microsoft corp., Northampton, MA, USA), and least significant differences were calculated for mean separations using a t-test of the Data Processing System (DPS, version 14.10, Zhejiang University, Hangzhou, China).

Catechin, epicatechin and cyanidin 3-O-glucoside accumulation during fruit development
In the blood-flesh peach "Dahongpao", the concentration ranges of catechin, epicatechin and cyanidin 3-O-glucoside were 28.00-44.42,13.45-268.20 and 0-254.42mg•kg -1 FW, respectively (Fig. 2).This result pointed to significant differences in proanthocyanidin and anthocyanin concentrations in blood-flesh peach.Moreover, the dynamic change trends of catechin and epicatechin concentrations significantly differed.At the early and middle stages of fruit development, the concentrations of catechin were slightly higher than those of epicatechin, while in the fruit maturation period the concentration of epicatechin was significantly higher than that of catechin (p<0.01).The catechin concentration remained low throughout fruit development, and even declined at stage S2 (p<0.01); it increased during the middle stages (p<0.01)and remained stable in the ripening period, with a slight but non-significant increase.The concentration of epi-catechin remained low at the early stages of fruit development, exhibiting rapid accumulation from stage S3 to a peak at stage H, with a concentration of 268.20 mg•kg -1 FW (p<0.01).The dynamic change trend of cyanidin 3-O-glucoside was only slightly different from that of epicatechin.Cyanidin 3-O-glucoside was not detected at the early stages of fruit development, however, it started to accumulate at stage S3 and rapidly accumulated from stage S3 to a peak at H, with a concentration of 254.42 mg•kg -1 FW (p<0.01).

Expression of structural genes during fruit development
There were significant differences in the levels of expression of structural genes during fruit development in "Dahongpao" (Fig. 3).The upstream region of genes PAL, CHS, CHI, F3H, DFR and LDOX will be discussed first.The expression of PAL and CHI was low and at about equivalent levels at different stages.
The level of expression of CHS at the transcriptional level decreased gradually from stage S1 to a minimum that was observed at S3 (p<0.01); it then increased sharply at the ripening stages and attained a maximum at stage H (p<0.01).The level of F3H expression was similar to that of CHS, with a slight decrease at the middle stage and a significant rise at stage H (p<0.01).
The level of DFR expression increased slightly and remained stable at the middle stage.As for CHS and F3H, there was a significant rise at stage H (p<0.01).The levels of both LDOX and UFGT expression increased gradually from stage S1 and attained a maximum at stage H (p<0.01).The level of LAR expres- sion did not significantly differ from stage S1 to S3; it slightly decreased at stages S2 and S3 and markedly increased from stage S4 to H (p<0.01).The expression of ANR significantly increased with the stages of fruit development, and reached a stable maximum in the ripening period (p<0.01).

DISCUSSION
The structure, function and synthetic mechanism of plant secondary metabolites have attracted attention in recent years.Anthocyanin function and biosynthesis and gene organization, expression and regulation have been investigated, however, there has been little research into proanthocyanidin [6,24].We determined the accumulation of proanthocyanidin monomers (catechin and epicatechin) and cyanidin 3-O-glucoside in blood-flesh peach "Dahongpao", and found that the concentration of catechin remained low and almost stable throughout fruit development.The concentration of epicatechin remained low at the early stages of fruit development and it rapidly accumulated to a maximum at the matured stage, while cyanidin 3-O-glucoside was not detected in the early stages of fruit development.As for epicatechin, it accumulated rapidly during the ripening period to a maximum at the mature stage.The developmental profiles of catechin and epicatechin accumulation were different from those reported in previous research, in which their accumulation decreased with fruit maturation [18,19].Cyanidin 3-O-glucoside accumulation increased with fruit maturation, and the result was consistent with previous research [14].
Until now, there has been no clear definition of the major genes regulating the synthesis of proanthocyanidins and anthocyanins in peach.Tsuda et al [17] found that CFS and DFR are the key genes regulating the synthesis of anthocyanins in blood-flesh peach.Zhao et al [13] suggested that PAL was the key enzyme for anthocyanin synthesis.Daniela et al [18] established that only UFGT was weakly correlated with anthocyanin level, while the expression of structural genes CHS, CHI, FSH, DFR, LDOX, UFGT, ANR and LAR correlated with proanthocyanidin accumulation.Jiao et al [14] found that cyanidin 3-O-glucoside accumulation in the fruit of blood-flesh peach "Banjintao" was closely related to the coordinated expression of UFGT and ANS.In white-flesh peach, Daniela et al [18] proposed that the expression of the genes encoding enzymes of the flavonoid pathway, especially LAR and ANR, correlated with proanthocyanidin concentration.Zhou et al [19] suggested that MYB7 activated transcription of LAR, but not ANR; however, in the same research, the expression of LAR and ANR could not adequately explain the dynamic changes in catechin and epicatechin concentrations during fruit development.In our research on blood-flesh peach "Dahongpao", the expression of the upstream region of PAL and CHI were less associated with proanthocyanidin monomers and cyanidin 3-O-glucoside accumulations, while the expression of other genes, including CHS, F3H, DFR and LDOX, were partly associated with proanthocyanidin monomers.Cyanidin 3-O-glucoside levels and the expression levels peaked synchronously in ripe fruit.Moreover, the expression of UFGT, the key gene for anthocyanin synthesis, was highly correlated with cyanidin 3-O-glucoside levels, while the expression of LAR and ANR, the key genes for proanthocyanidin synthesis, correlated with catechin and epicatechin accumulation during fruit development, respectively.We conclude that LAR, ANR and UFGT are key genes regulating the synthesis of proanthocyanidin monomers and cyanidin 3-O-glucoside because the levels of their expression correlated with the levels of these compounds.However, the expression of the upstream region of common genes such as CHS, DFR and LDOX, significantly increased with fruit development and maturation.This could give rise to the accumulation of a common substrate used in proanthocyanidin monomer and cyanidin 3-O-glucoside synthesis.Thus, CHS, DFR and LDOX might be major genes regulating the synthesis of epicatechin and cyanidin 3-O-glucoside in blood-flesh peach.Interestingly, we found synchronous accumulation of epicatechin and cyanidin 3-O-glucoside during the fruit-ripening period, which was very different from other fruits and could not be explained by the current mechanism of competitive distribution of a common substrate [25].There were obviously negative correlations between the accumulation of epicatechin and cyanidin 3-O-glucoside during fruit development in previous research conducted on colored fruits: grape [26], blueberry [27], bilberry [28], strawberry [29] and blackberry [30].Cyanidin 3-O-glucoside, which remained at a low concentration during the early developing stage, increased dramatically as the fruit matured.In contrast, epicatechin exhibited a continuously decreasing pattern.Accordingly, the transcript levels of genes specifically controlling either of these two compounds, UFGT anthocyanin and LAR and ANR proanthocyanidin, generally coordinated with the changing patterns of products.These findings imply that the mechanism of synchronous accumulation of epicatechin and cyanidin 3-O-glucoside in bloodflesh peach should be researched further.

Table 1 .
Primers for quantitative real-time PCR