Elsevier

Plant Science

Volume 170, Issue 3, March 2006, Pages 571-578
Plant Science

Expression analysis of anthocyanin biosynthetic genes in apple skin: Effect of UV-B and temperature

https://doi.org/10.1016/j.plantsci.2005.10.009Get rights and content

Abstract

We investigated the expression of the five anthocyanin biosynthetic genes, chalcone synthase (CHS), flavanone 3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), and UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGluT) during ripening in five early ripening apple cultivars (‘Iwai’, ‘Sansa’, ‘Tsugaru’, ‘Homei-Tsugaru’, and ‘Akane’), as well as under UV-B and temperature treatments. Anthocyanin biosynthesis increased progressively during ripening, with the onset of rapid accumulation occurring at 107 days after full bloom (DAFB) in the four apple cultivars except ‘Iwai’. Generally, increase in the expression levels of all five biosynthetic genes in fruit skin coincided with increase of anthocyanin concentration. When the fruit was bagged about 1 month prior to commercial harvest to prevent anthocyanin biosynthesis, the expression of CHS, ANS and UFGluT was substantially depressed in fruit skin, whereas that of all five genes including CHS, ANS and UFGluT was enhanced by UV-B and low temperature treatments with the accumulation of anthocyanins. The accumulation level of the main anthocyanin pigment, cyanidin 3-galactoside, in the fruits under UV-B and low temperature treatment, was less than that in the field-ripened fruits, while cyanidin 3-arabinoside was relatively the same level. Our results showed that UV-B and low temperature were important factors for anthocyanin accumulation in apple fruit skin by inducing the expression of the anthocyanin biosynthetic genes, especially CHS, ANS and UFGluT genes.

Introduction

Among apple fruit quality characters, the red coloration of the fruit skin is of major importance and significantly determines the market value of the produce. Although the pigments that compose fruit color vary with fruits, the amount and composition of anthocyanins that belong to a class of flavonoids, are the major determinants of apple fruit skin reddening [1], [2]. Anthocyanin biosynthesis in flowers and fruits including apple fruit skin has been well characterized genetically and biochemically [2], [3], [4]. A generalized scheme of the pathway in apple fruit skin is shown in Fig. 1. The anthocyanins in apples are predominantly glycosylated cyanidins [2]. The main cyanidin pigment which accounts for the red color in apple fruit skin is cyanidin (cy) 3-galactoside [idaein (∼80%)], while cy 3-arabinoside, cy 3-rutinoside, cy 3-xyloside and cy 3-glucoside exist only in minor amounts in some apple cultivars [2], [5].

Anthocyanin biosynthesis in apple fruit is developmentally regulated and occurs at two peaks. The first peak occurs at the fruitlet stage in both red and non-red cultivars, and is not economically important [2]. The second peak takes place later, at the ripening fruit stage only in red cultivars. The intensity and extent of the reddening at the mature fruit stage are most important, as they impact greatly on the marketable value of the produce and have direct bearing in the economics of apple production. The accumulation at the second peak is markedly affected by the environmental factors, including temperature and light. Temperature is one of the major factors affecting anthocyanin synthesis in apple skin. It has been known from experience that cool temperature conditions at the ripening stage lead to redder fruits than warm conditions. In early ripening cultivars, poor pigmentation in fruit skin often becomes a serious problem since the fruits of these cultivars are harvested at a season of relatively high temperature (usually in September, late-summer season, in Japan). In addition to a cool temperature requirement, light intensity and quality are also important factors affecting anthocyanin biosynthesis in apples [2]. In red-fruited cultivars, anthocyanin accumulation is induced by light in the UV-B region (wavelengths from 280 to 320 nm) when applied in combination with infrared (IR) [6]. Ju et al. [7] have shown in ‘Fuji’ apples that light-reflecting mulches increase anthocyanin concentration and the enzyme activity of UDP-galactose:flavonoid-3-O-glucosyl transferase (UFGalT) in anthocyanin biosynthetic pathway, and that the effect is greater for those mulches with a higher reflectivity in UV and IR.

Previous reports on anthocyanin accumulation during ripening of apples were based mainly on enzyme assays [7], [8], [9], except for a few reports on apple flowers [10] and fruit skin [1], [11], [12], [13] using the anthocyanin biosynthetic genes for the expression analysis. These recent studies at the molecular level have revealed that the regulation of the expression of the biosynthetic genes is one of the causes that determine the anthocyanin accumulation in apple skin during fruit ripening. However, there is still a dearth of knowledge on the expression of the biosynthetic genes under varying temperature and UV-B conditions. To further investigate the regulation of the pathway in apple fruit skin, we investigated the effect of UV-B and temperature on the expression of the anthocyanin biosynthetic genes in apple skin of early ripening cultivars utilizing apple cDNAs for chalcone synthase (CHS), flavanone 3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), and UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGluT).

Section snippets

Plant materials and fruit development stages

All apple (Malus × domestica) samples were obtained from the orchard of the National Institute of Fruit Tree Science at Morioka, Japan. Five early ripening apple cultivars: ‘Iwai’ (English name; ‘American Summer Pearmain’), ‘Sansa’, ‘Tsugaru’, ‘Homei-Tsugaru’, and ‘Akane’ were used for this study. ‘Iwai’ is actually a pale red cultivar, but regarded as a green cultivar commercially because the fruit is harvested at the immature stage. ‘Tsugaru’ is a pale red cultivar, while ‘Sansa’,

Anthocyanin accumulation during apple fruit ripening

Anthocyanin concentrations were measured from the first to the last harvesting date in the five cultivars used in this study: ‘Iwai’, ‘Sansa’, ‘Tsugaru’, ‘Homei-Tsugaru’ and ‘Akane’ (Fig. 2). Two ripening stages were used for ‘Iwai’, and the pattern of anthocyanin accumulation in four ripening stages was evaluated for the other four cultivars. In ‘Iwai’, there was no detectable accumulation of anthocyanin at 83 and 96 DAFB. In ‘Sansa’ and ‘Tsugaru’, no noticeable increase in anthocyanin

Discussion

In apple, changes in the levels of anthocyanin accumulation have been well known to occur during fruit development [1], [2], [18]. The ripening phase is an important aspect of fruit development that is initiated after the completion of maturation, and during which tissue softening and accumulation of pigments occur [19]. We report here a study on the expression of the anthocyanin biosynthetic genes at the ripening stage of maximal anthocyanin accumulation in different early ripening apple

Acknowledgements

The Inoue Research Foundation for Science of Japan is gratefully acknowledged for the award of a visiting research fellowship to Dr. B.E. Ubi. This work was partially supported by a grant from the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN) to Dr. C. Honda. We thank Dr. Ji-Hong Liu for his critical reading of this manuscript.

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