Mapping of an anthocyanin-regulating MYB transcription factor and its expression in red and green pear, Pyrus communis

Abstract

‘Max Red Bartlett’ is a red bud mutation of the yellow pear (Pyrus communis L.) cultivar ‘Williams’ (known as ‘Bartlett’ in North America). Anthocyanins are the most important pigments for red colour in fruits. Synthesis of anthocyanins is mediated by a number of well-characterized enzymes that include chalcone synthase (CHS), flavanone-3-hydroxylase (F3H), dihydroflavonol-4-reductase (DFR), anthocyanidin synthase (ANS), and UDP-glucose:flavonoid-3-O-glucosyltransferase (UFGT). Expression of the genes encoding these five enzymes was examined in pear fruit skin in order to elucidate the molecular mechanism for red coloration. In addition, the gene PcMYB10, encoding an R2R3 MYB transcription factor involved in anthocyanin biosynthetic pathway regulation, was isolated from both ‘Williams’ and ‘Max Red Bartlett’. Analysis of the deduced amino acid sequence suggests that this gene is an ortholog of anthocyanin regulators known in other plant species. Its expression level was significantly higher in ‘Max Red Bartlett’ (red pear) compared with the original yellow variety ‘Williams’. Although the map position of PcMYB10 corresponds to that of MdMYBa and MdMYB10, which control pigmentation of apple fruit skin, PcMYB10 is not directly responsible for red versus yellow colour in the two pear varieties, as the mutation underlying this difference maps to a different region of the pear genome.

Introduction

The European pear (Pyrus communis L.) cultivar ‘Williams’ was selected and commercialised in Europe in the eighteenth century; it is also known as ‘Bartlett’ or ‘Yellow Bartlett’ in North America. ‘Max Red Bartlett’ was discovered in the past century from a bud mutation of ‘Williams’ which caused a red skin pigmentation. The ‘Williams’ fruit is green at maturity, with a little blush on the sun-facing side, and then turns yellow when fully ripe. In contrast, ‘Max Red Bartlett’ fruit is dark red, almost purple, throughout maturation, but then the intensity of this coloration decreases and the fruit turns bright scarlet when ripe. This mutation is unstable and is known to revert from the ‘Max Red Bartlett’ to the ‘Williams’ phenotype in whole branches, or individual fruits, where it may involve sectors of fruit skin or its whole surface [1].

Anthocyanins, in combination with carotenoids and chlorophylls, are responsible for almost all fruit coloration. Anthocyanins belong to the diverse group of ubiquitous secondary metabolites known as flavonoids. These compounds, and polyphenols, in general, are of great importance in plants. Apart from their many biological functions, for example in pollinator attraction, male fertility, UV protection, regulation of polar auxin transport, establishment of microbial symbioses, and pathogen defence, polyphenols contribute to or even determine additional features that are of special relevance in fruit crops [2].

Anthocyanin biosynthetic genes as well as the regulatory proteins involved in activation (or repression) of pigmentation are highly conserved in higher plants [3], [4]. Recent studies, particularly in Petunia hybrida, Zea mays, and Arabidopsis thaliana, have revealed the complexity of the regulatory networks. In addition to anthocyanin and proanthocyanidin production, these networks control the development of trichomes, root hairs, and seed coat mucilage in Arabidopsis [5], [6], as well as the morphology of seed coat epidermal cells and the vacuolar pH of petals in Petunia [7], [8].

The structural genes encoding enzymes in the anthocyanin biosynthetic pathway have been cloned from a wide variety of plant species. In apple and pear, genes have been isolated for all of the key pathway enzymes, chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonol synthase (FLS), dihydroflavonol-4-reductase (DFR), anthocyanidin synthase/leucoanthocyanidin dioxygenase (ANS/LDOX), and UDP-glucose:flavonoid-3-O-glucosyltransferase (UFGT) [9], [10]. With the exception of ANS and UFGT, the enzymes are encoded by small gene families ranging in size from two to nine or more members. The sequences appear to be highly conserved among apple cultivars, as efforts to develop SNP markers for several of these genes in a ‘M.9’ × ‘Robusta 5’ cross were unsuccessful [11]. Moreover, the pear genes were isolated based on homology with apple sequences, underscoring the high sequence similarity of the genes in these two species [10].

Expression of flavonoid genes has been shown to be correlated with anthocyanin accumulation in the skin during fruit development in apple, grape, peach, and more recently in Mangosteen (Garcinia mangostana L.) [9], [12], [13], [14], [15]. Several recent studies have focused on the underlying mechanisms regulating the expression of these genes in fruit skin. Studies on diverse plant species revealed that anthocyanin expression is controlled, at least in part, at the transcriptional level, usually by an R2R3 MYB and/or a basic helix-loop-helix (bHLH) transcription factor [16]. In grape the red coloration of the berries depends on a coordinated increase in expression of a number of genes in the anthocyanin biosynthetic pathway during ripening [12], that is controlled by a single genetic locus [17], [18] containing four MYB genes; at least two of them are mutated in white grapes. In pepper fruit, anthocyanin accumulation appears to involve both MYBA and a myc (bHLH) gene [19]. In apple (Malus × domestica) three different groups have identified the R2R3 MYB transcription factors responsible for anthocyanin accumulation [20], [21], [22]. Very recently a Myb10 gene has been characterized in several species belonging to Rosaceae family. Among them a pear Myb10 (PcMYB10) has been sequenced and its functionality in activating an Arabidopsis DFR promoter was verified by transient expression assay [23]. This gene is thus likely to be responsible for the regulation of anthocyanin biosynthetic pathway also in European pear. MdMyb10 is known to be located in apple linkage group 9 (LG9) [11] but, in the mutated sport ‘Max Red Bartlett’ the red colour behaves as a dominant character that mapped to LG4 [24].

Here we report for the first time the expression levels of PcMYB10 in ‘Max Red Bartlett’ and the yellow variety ‘Williams’ along with that of the structural genes encoding CHS, CHI, F3H, DFR, ANS and UFGT, and, finally its position on the ‘Abbé Fétel’ × ‘Max Red Bartlett’ linkage map. The results confirm that although PcMYB10 is homologous to one of the flavonoid regulatory genes previously described in other rosaceous species [23], it does not appear to be directly responsible for the pigmentation differences between ‘Max Red Bartlett’ and ‘Williams’.