Identifying and characterizing genes and transcription factors involved in coloration of apple fruit

Apple (Malus × domestica Borkh.) is one of the most highly cultivated fruit crops grown around the world and apple consumption has been increasing over the years. One of the most important determinants of fruit quality is skin color. Red coloration in apple fruit is attributed to anthocyanin accumulation. Anthocyanins are encoded by structural genes, in the anthocyanin biosynthetic pathway, which are highly regulated by transcription factors. In this thesis, studies were conducted to identify and characterize structural genes and associated transcription factors involved in the anthocyanin biosynthetic pathway. Three genes encoding apple anthocyanin reductase gene (MdANR) were isolated, designated as MdANR1, MdANR2a, and MdANR2b. It is found that MdANR2a, and MdANR2b are in fact allelic. MdANR1 and MdANR2 were mapped to the apple linkage map on linkage groups (LG) 10 and 5, respectively. The functionality of MdANR gene was investigated following its overexpression in tobacco and found to influence flower color pigmentation and pattern. Overexpression of MdANR influenced other genes in the flavonoid biosynthesis pathway by down-regulating chalcone isomerase (CHI), dihydroflavonol reductase (DFR), and leucoanthocyanidin reductase (LAR) genes. Moreover, the observed loss of flower color in transgenic tobacco lines was attributed to reduction of anthocyanin pigments. This was likely due to down-regulation of tobacco CHI and DFR genes that are important in anthocyanin production. In addition, a new floral pigmented pattern was generated by incomplete inhibition of anthocyanin production. As expected, the epicatechin accumulated at higher levels in transgenic tobacco than in wild-type tobacco. However, higher amounts of catechin but lower levels of LAR, responsible for synthesis of catechin, were found in transgenic lines when compared to wild-type tobacco. Thus, it has been proposed that ANR plays a redundant role to that of LAR. A novel MYB transcription factor (TF) gene, designated as MdMYB11, was isolated and genetically mapped onto LG15 of the apple genetic map. Alignment of deduced amino acid sequences of MdMYB11 to those of other R2R3 MYB TFs revealed that this new apple transcription factor contains the R2R3 conserved domain. Moreover, this TF is highly similar to Arabidopsis MYB subgroup 4, such as AtMYB3, 4, and 6, by which they negatively regulate genes involved in monolignol biosynthesis. Functional analysis of MdMYB11 was conducted via ectopic expression in tobacco. Expression of MdMYB11 increased anthocyanin production in tobacco flowers by inducing several anthocyanin biosynthesis pathway genes, particularly those of CHI, chalcone synthase (CHS), and UDP-glucose: flavonoid 3-o-glucosyltransferase (UFGT). In addition, this TF functioned as a repressor of both cinnamate-4-hydroxylase (C4H) and 4-coumaroyl:CoA-ligase (4CL) genes, both important in lignin biosynthesis, and possibly contributing to modulation of floral morphogenesis. Moreover, transgenic flowers had longer styles than those of wild-type flowers, suggesting that the MdMYB11 gene might be involved in pistil development. New candidate TF genes regulating apple fruit coloration were identified following global gene expression analysis of the apple transcriptome using an apple microarray. Comparison of gene expression in fruit peel of apple cv. Red Delicious subjected to continuous ‘dark treatment’ versus dark-grown fruit subjected to ‘14 h-light-exposure’ identified 815 genes that were modulated. Following annotation (to the Arabidopsis Gene Ontology), these genes were classified into 19 categories, and were mostly involved in primary metabolism (17%) and transcription (12%). Of these, 18 genes encoded for putative TFs. Further identification of color-related TFs was conducted by comparison of expression profiles of fruit of red skinned apple cv. Red Delicious and non-red skinned apple cv. Golden Delicious, and using quantitative real-time (RT)-PCR (qRT-PCR). Two putative TF genes were found to be expressed at higher levels in fruit of ‘Red Delicious’ than that in ‘Golden Delicious’, thus suggesting that these TFs might be involved in fruit coloration. Altogether, these findings have provided novel information and knowledge of the role(s) of genes and transcription factors involved in the anthocyanin biosynthesis pathway. Moreover, the regulator mechanism of fruit coloration has been further elucidated following transcriptome analysis of the apple genome and functional analysis of selected genes and transcription factors