Expression of Structural Genes Related to Anthocyanin Biosynthesis of Vitis amurensis  

Quan Zhao1,2 , Fei He 1 , Reeves J. Malcolm 3 , Pan Qiuhong1 , Changqing Duan1 , Jun Wang 1
1 Center for Viticulture and Enology, College of Food Science & Nutritional Engineering, China Agricultural University, Beijing, 100083, P.R.China
2 Traditional Chinese Medicine Department, Jilin Agricultural Science and Technology College, Jilin, 132101 , P.R.China
3 Faculty of Applied Science, Business and Computing, Eastern Institute of Technology, Napier 4142, New Zealand
Author    Correspondence author
Molecular Plant Breeding, 2013, Vol. 4, No. 31   doi: 10.5376/mpb.2013.04.0031
Received: 08 Jul., 2013    Accepted: 27 Sep., 2013    Published: 30 Sep., 2013
© 2013 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:

Quan et al., Expression of Structural Genes Related to Anthocyanin Biosynthesis of Vitis amurensis, Molecular Plant Breeding, Vol.4, No.31 254-264 (doi: 10.5376/mpb.2013.04.0031)

Abstract

The accumulation of anthocyanins in skins is one of the main features of red grape berry ripening. This research was designed to assess the changes in anthocyanin content in grape skins of V. amurensis and to explore mRNA transcriptions of 11 structural genes (PAL, CHS3, CHI1, F3H2, F3'H, F3'5'H, DFR, LDOX, UFGT, OMT and GST) related to anthocyanin biosynthesis during grape berry development, by the use of HPLC-MS/MS and real-time Q-PCR analysis. Accumulation of anthocyanins began at veraison, continued throughout the later berry development and reached a peak at maturity. Veraison is the time when the berries turn from green to purple. Expression of PAL, CHI1, and LDOX were up-regulated from 2 to 4 weeks after flowering (WAF), down-regulated from 6 WAF to veraison, whereas DFR was up-regulated at 8 WAF, and then up-regulated from veraison to maturity. The expression of these genes at the transcriptional level had a positive correlation with the content of anthocyanins from veraison to maturity. The CHS3, F3'5'H, UFGT, GST, and OMT were down-regulated from 2 WAF to veraison, and then up-regulated from veraison to maturity. The transcriptional expressions of the 11 structural genes also showed positive correlations with the anthocyanin content from veraison to maturity. Positive correlations were also observed between OMT transcriptional level and the content of methoxyl-anthocyanins and between F3'5'H transcriptional level and the content of delphinidin anthocyanins. F3H2 and F3'H expression was up-regulated at 2 WAF. F3H2 expression was down-regulated from 4 WAF to veraison and then up-regulated again from veraison to maturity. F3'H expression was down-regulated at 4 WAF and then up-regulated again from 6 WAF to maturity. F3'H transcriptional level was correlated positively with the cyanidin anthocyanin concentration from veraison to maturity. These results indicate that the onset of anthocyanin synthesis during berry development coincides with a coordinated increase in the expression of a number of genes in the anthocyanin biosynthetic pathway.

Keywords
Vitis amurensis; Anthocyanins; HPLC-MS/MS; Real-time Q-PCR; Gene expression

Anthocyanins are group of important phenolics compounds that play a crucial role in the color of red grapes and wines (Garcia-Alonso et al., 2009; Gómez- Plaza et al., 2006). Anthocyanins and their related genes (PAL, CHS, CHI, F3H, DFR, LDOX and UFGT) are all biosynthesized through the flavonoid pathways(Figure 1). (Boss et al.,1996a; 1996b; Koes et al., 2005; Matus et al., 2009; He et al., 2010; Sparvoli et al., 1994). Generally, in red V. vinifera grapes or red young wines, the five monoglucosidic anthocyanidins (cyanidin, peonidin, delphinidin, petunidin and malvidin) and their acylated derivatives are the main color components (Mattivi et al., 2006; Pomar et al., 2005). Their accumulation in the skins starts at veraison and reaches at a maximum around harvest (Downey et al., 2004). In non-V. vinifera grapes or wines, 3-O-monoglucoside (Wang et al., 2003; Castillo-Muñoz et al., 2009; He et al., 2010) or 3,5-O-diglucoside (Zhao et al., 2010) of pelargonidin have also been detected. In the present study, the accumulation of anthocyanins and the expression of the related 11 biosynthetic genes (PAL, CHS3, CHI1, F3H2, F3'H, F3'5'H, DFR, LDOX, UFGT, OMT and GST) at different development stages of V. amurensis grapes were analyzed by HPLC-MS/MS and real-time Q-PCR, to provide a general overview of the anthocyanin biosynthesis in such unique Chinese grape species.


Figure 1 Simplified schematic of the anthocyanin biosynthetic pathway


1.
Results and Discussion
1.1 Anthocyanin accumulation during berry development of Shuang Hong
In the skins of V. amurensis cv. Shuang Hong, 11 anthocyanins were identified by using HPLC-ESI-MS/MS. The principal individual anthocyanins were 3-O-monoglucosides and 3,5-O-diglucosides of delphinidin, cyanidin, petunidin, peonidin and malvidin. Additionally pelargonidin-3,5-O-diglucoside was detected at a trace level (Zhao et al., 2010). Soluble solids, measured as °Brix, increased after veraison reaching a value of 13 °Brix at maturity (Figure 2 A). No anthocyanins were detected before veraison increased in the skin after veraison. The anthocyanin content initially increased slowly until the berries were 10% colored and much faster once 50% colored was reached. The total anthocyanin content at 100% colored was 8.3981 mg/g and this increased by only 0.5494 mg/g at maturity (Figure 2 B). The content of cyanidins anthocyanins varied differently to the total anthocyanin content. They increased quickly from veraison until 50% colored, and then decreased to maturity (Figure 2 C). The content of delphinidins anthocyanins, the methoxylated anthocyanins (peonidins, petunidins and malvidins) and anthocyanin monoglucosides all varied similarly to the total anthocyanins (Figure 2 D; Figure 2 E; Figure 2 F). The concentration of delphinidin anthocyanins, cyanidin anthocyanins, methoxyl anthocyanins and total anthocyanins at maturity were 1.2795, 0.3294, 7.3386, 8.9475 mg/g, respectively.The fact that the methoxyl anthocyanins make up the vast majorityof the total anthocyanins means that of course the pattern of accumulation of such anthocyanins will correlate highly with the total. The fact that the monoglucosides anthocyanins reached on 2.3653 mg/g compared with the total at maturity of about 8.9475 mg/g implies that the diglucosides reached about 6.5822 mg/g, that is significantly more than the monoglucosides.


Figure 2 Changes of color and soluble solids during berry development of Shuang Hong

1.2 Structural gene expression related to biosynthesis of anthocyanins during berry development of Shuang Hong
The expression of 11 anthocyanins biosynthetic genes (PAL, CHS3, CHI1, F3H2, F3'H, F3'5'H, DFR, LDOX, UFGT, OMT and GST) during the development of the grape berries is presented in Figures 3~Figures 14, and Table 1.


Table 1 Regression equations and correlation coefficients relating the cumulative expression of the anthocyanin biosynthetic genes during berry development, from 2 WAF to maturity, with the corresponding anthocyanin concentration (mg/g) in skins of Shuang Hon
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