Phenylpropanoid metabolites and expression of key genes involved in anthocyanin biosynthesis in the shaded peel of apple fruit in response to sun exposure

doi:10.1016/j.plaphy.2013.04.020

Plant Physiology and Biochemistry

Volume 69, August 2013, Pages 54-61

https://doi.org/10.1016/j.plaphy.2013.04.020 Get rights and content

Highlights

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Sun exposure of shaded peel up-regulated the expression of MdMYB10 & structural genes.
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This up-regulation led to higher anthocyanins & flavonols in the original shaded peel.
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Fruit turning also elevated the levels of flavonols in the original sun-exposed peel.
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Turned fruit had better color and higher total phenolics in the peel than the control.

Abstract

The shaded peel of ‘Fortune’ (a red cultivar) and ‘Mutsu’ (a yellow/green cultivar) apple (Malus domestica Borkh.) was exposed to full sun by turning fruit 180° at about one week before harvest to determine the expression of key genes involved in anthocyanin synthesis in response to sunlight exposure and their relationships with the levels of anthocyanins and other phenolics. For the unturned (control) fruit, the shaded peel had lower expression levels of MdMYB10 (a transcriptional factor in the regulation of anthocyanin biosynthesis) and seven structural genes in anthocyanin synthesis (MdPAL, MdCHS, MdCHI, MdF3H, MdDFR1, MdLDOX, and MdUFGT), and lower levels of anthocyanins and flavonols than the sun-exposed peel in both cultivars. Exposure of the shaded peel to full sun caused marked up-regulation of the expression of MdMYB10 and all seven structural genes, which peaked between 6 h and 30 h after fruit turning, consequently leading to higher levels of anthocyanins, flavonols, and total phenolics than in the shaded peel and even in the sun-exposed peel of control fruit. Interestingly, the levels of flavonols were higher in the shaded peel of turned fruit (the original sun-exposed peel) than in the sun-exposed peel of both control and turned fruit in both cultivars, suggesting that competition for substrates exists in different branches of the phenylpropanoid pathway. These results indicate that sunlight exposure stimulates the expression of MdMYB10 and structural genes in anthocyanin synthesis, thereby elevating the levels of anthocyanins and other phenolic compounds in both red and yellow/green cultivars.

Introduction

Anthocyanins are a group of phenolic compounds derived from the phenylpropanoid pathway. Other phenolic compounds derived from other branches of the same pathway include phenolic acids, flavonols, dihydrochalcones, and flavanols. All these phenolic compounds are considered as main factors contributing to the internal and external qualities of apple fruit [1]. Generally, apple peel has much higher concentrations of polyphenols than the flesh [2], [3], [4]. Cyanidin 3-galactoside is the main anthocyanin in red apple peel [5]. In addition to aiding seed dispersal [6], anthocyanins play a role in protecting fruit from photooxidative damage [7], [8], and other environmental stress such as high temperature under high light [9]. From a human nutrition perspective, polyphenolics in apple have been demonstrated to have the potential to improve human health and reduce risks of many diseases, including cancer, cardiovascular disease, diabetes, pulmonary disorders, Alzheimer's disease, and other degenerative diseases [10], [11].

The biosynthetic pathway for anthocyanins has been studied extensively. Phenylalanine is the precursor for the synthesis of many polyphenols, and is enzymatically converted to anthocyanins via many steps. The enzymes involved in this pathway include phenylalanine ammonialyase (PAL, EC: 4.3.1.24), chalcone synthase (CHS, EC: 2.3.1.74), chalcone isomerase (CHI, EC: 5.5.1.6), flavanone 3-hydroxylase (F3H, EC: 1.14.11.19), dihydroflavonol 4-reductase (DFR, EC: 1.1.1.219), leucoanthocyanidin dioxygenase (LDOX, EC: 1.14.11.19) and UDP-glucose: flavonoid 3-O-glucosyltransferase (UFGT, EC: 2.4.1.91) [12]. As a transcriptional factor involved in the regulation of anthocyanin biosynthesis, MdMYB10, and its allelic genes, MdMYB1 and MdMYBA controls apple red color development via regulating the expression of structural genes in anthocyanin biosynthesis [13], [14], [15], [16]. The expression levels of these biosynthetic genes correspond to anthocyanin concentrations and fruit coloration in apple fruit, and the transcript levels of these genes in ‘Orin’, a yellow cultivar, were less than those in ‘Fuji’ and ‘Jonathan’, two red cultivars [17]. Apple skin patterning is also associated with differential expression of MYB10. Both ‘Honeycrisp’ and ‘Royal Gala’ had higher mRNA levels of MYB10 and biosynthetic genes CHS, CHI, F3H, DFR1, LDOX, and UFGT in red stripes compared to green stripes [18].

The dependence of apple coloration on light was recognized a long time ago [19], [20]. It is known that fruit from the top of the tree canopy have higher levels of cyanidin 3-galactoside and quercetin 3-glycosides than those from the canopy interior [21]. For varieties that do not typically develop red color (yellow/green varieties), bagging fruit first for a period of time, and then removing the bag can induce red color development [22], [23]. Only until recently, however, did the underlying molecular mechanism on light regulation of anthocyanin synthesis begin to be understood. MYB transcriptional factors (such as MdMYB1 and MdMYBA) are induced by light [16], [24], [25], and the stability of MdMYB1 is regulated by MdCOPs in response to light [26]. The expression of the down-stream anthocyanin biosynthetic genes is responsive to light [24], [27]. Fruit bagging and debagging has been used to study the responses of key genes involved in anthocyanin synthesis to light, but bagging fruit represents more of an extreme situation where fruit receives very little light. It remains unclear how the expression of the key genes in anthocyanin synthesis in the peel of apple fruit that have been adapted to a low level of light in the canopy respond to sudden exposure to full sun. This sudden change of light exposure happens when the gain of fruit weight on a branch alters the orientation of the branch or summer pruning opens up the tree canopy. In addition, very few studies have looked at how other classes of polyphenolics, in addition to anthocyanins, in apple peel respond to changes in light exposure.

Apple fruit has a sun-exposed side and a shaded side due to self-shading during fruit development. Compared with the sun-exposed side, the shaded side of apple fruit has lower xanthophyll cycle-dependent thermal dissipation and antioxidants of the ascorbate–glutathione pathway [28]. Exposure of the shaded side to full sun leads to up-regulation of both the xanthophyll cycle and the ascorbate–glutathione cycle [29]. This was accompanied by higher anthocyanin levels in the peel, suggesting anthocyanin synthesis is up-regulated. It appears that exposing the shaded peel to full sun by fruit turning provides a highly relevant experimental system for understanding the effects of light exposure on the synthesis of anthocyanins and other phenolic compounds. In addition, quantifying the effects of fruit turning on the levels of individual phenolics and total phenolics in apple peel may allow us to develop a new way of improving apple fruit quality in terms of total polyphenolic content and coloration for maximizing health benefits and consumer appeal of apple. In this study, we used two apple cultivars, red ‘Fortune’ and yellow/green ‘Mutsu’ to determine the expression levels of key genes involved in anthocyanin biosynthesis in the shaded peel of apple fruit in response to full sun exposure, and their relationships with the levels of anthocyanins and other phenolic compounds.

Section snippets

Expression of key genes in anthocyanin biosynthesis

For the control fruit at 0 h, the transcript levels of MdMYB10, MdPAL, MdCHS, MdCHI, MdF3H, MdDFR1, MdLDOX and MdUFGT were much lower in the shaded peel than those in the sun-exposed peel in both ‘Fortune’ and ‘Mutsu’ (Table 1). The transcript levels of MdMYB10 in the shaded peel were about 1/11 and 1/17 of those in the sun-exposed peel in ‘Fortune’ and ‘Mutsu’, respectively (Table 1). Comparing the two cultivars at 0 h, the transcript levels of MdMYB10 in the sun-exposed peel and the shaded

Discussion

Our results show that, in response to sunlight exposure, the expression levels of MdMYB10 and seven structural genes (MdPAL, MdCHS, MdCHI, MdF3H, MdDFR1, MdLDOX and MdUFGT) involved in anthocyanin synthesis were markedly up-regulated in the original shaded peel of both ‘Fortune’ and ‘Mutsu’ apple fruit, followed by red color development and corresponding increases in the level of cyanidin-3-galactoside (Figs. 1 and 2). These findings are consistent with the results obtained on other apple

Plant material and sampling

Two apple (Malus domestica Borkh.) cultivars, ‘Fortune’, a red apple, and ‘Mutsu’, a yellow/green apple were used in this study. They were on M.9 rootstocks and planted at a spacing of 1.8 × 4.2 m, and 2.1 × 4.2 m, respectively, at Cornell Orchards in Ithaca (42°26′N, 76°29′W; elevation 500 m), New York, USA. The trees were 15- and 14-year old, respectively, and trained as a central leader system. They received standard horticultural practices, and pest and disease control. These two cultivars

Acknowledgments

The authors would like to thank Mr. Richard Raba and Dr. Zhenchang Liang for technical assistance with HPLC.

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¹: These authors contributed equally to this work.

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Research articlePhenylpropanoid metabolites and expression of key genes involved in anthocyanin biosynthesis in the shaded peel of apple fruit in response to sun exposure

Highlights

Abstract

Introduction

Section snippets

Expression of key genes in anthocyanin biosynthesis

Discussion

Plant material and sampling

Acknowledgments

Sci. Hort.

Phytochemistry

Plant Sci.

Adv. Nutr.

Plant Physiol. Biochem.

Sci. Hort.

Trends Plant Sci.

Plant Sci.

Plant Sci.

Plant Sci.

Sci. Hort.

Postharvest Biol. Technol.

Food Chem.

Biosynthesis of phenolic compounds and its regulation in apple

Plant Growth Regul.

Activity and contents of polyphenolic antioxidants in the whole fruit, flesh and peel of three apple cultivars

Arch. Latinoam. Nutr.

Colour accumulation patterns and the anthocyanin biosynthetic pathway in ‘Red Delicious’ apple variants

J. Hort. Sci. Biotech.

The shaded side of apple fruit becomes more sensitive to photoinhibition with fruit development

Physiol. Plant

Red ‘Anjou’ pear has a higher photoprotective capacity than green ‘Anjou’

Physiol. Plant

Research article
Phenylpropanoid metabolites and expression of key genes involved in anthocyanin biosynthesis in the shaded peel of apple fruit in response to sun exposure