Apple (Malus ©7 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 0́8dark treatment0́9 versus dark-grown fruit subjected to 0́814 h-light-exposure0́9 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 0́8Red Delicious0́9 than that in 0́8Golden Delicious0́9, 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.

This book covers information on the economics; botany, taxonomy, and origin; germplasm resources; cytogenetics and nuclear DNA; genetic improvement efforts of scion cultivars; genetic and genomic improvement efforts of rootstocks; genetic and physical mapping; genomic resources; genome and epigenome; regulatory sequences; utility of whole-genome sequencing and gene editing in trait dissection; flowering and juvenility; cold hardiness and dormancy; fruit color development; fruit acidity and sugar content; metabolomics; biology and genomics of the microbiome; apple domestication; as well as other ‘omics’ opportunities and challenges for genetic improvement of the apple. The cultivated apple (Malus x domestica Borkh.) is one of the most important tree fruit crops of temperate regions of the world. It is widely cultivated and grown in North America, Europe, and Asia. The apple fruit is a highly desirable fruit due to its flavor, sugar and acid content, metabolites, aroma, as well as its overall texture and palatability. Furthermore, it is a rich source of important nutrients, including antioxidants, vitamins, and dietary fiber.

Apple fruit acidity which considerably affects fruit taste and flavor is primarily determined by malate concentrations. Previous studies reported that apple fruit acidity was predominantly controlled by the major QTL Ma. To better understand apple fruit acidity, this study attempts to identify and characterize the gene underpinning Ma and its associated co-expression gene networks. To achieve the goal, three sets of experiments were conducted. The first set of experiments was designed to identify the candidate gene for Ma and was summarized in Chapter 1. This chapter presents that the Ma locus physically spans over 65kb and harbors two aluminum-activated malate transporter (ALMT) -like genes, designated Ma1 and Ma2. Other important findings include 1) only Ma1 was expressed in significant correlation with the variation of acid levels, and 2) there is a nucleotide mutation that leads to a pre-mature stop codon in Ma1. Overall, it concludes that Ma1 rather than Ma2 is the gene underlying Ma. The second set of experiments aims at identification of the Ma1 associated coexpression gene network regulating malate levels in developing fruit of Golden Delicious and was reported in Chapter 3. One finding of this chapter is that malatepyruvate interconversion, photosynthesis, mitochondrial electron transport, and amino acid degradation were important pathways for the change of malate levels in developing fruit. The other finding is that symplastic signal transduction, transcriptional regulation, post-translational modification and apoplastic roles were important in the regulation of fruit acidity. The third set of experiments was conducted to identify the Ma1 associated coexpression gene network controlling acidity in mature fruit of ten diverse apples and was described in Chapter 4. Data in this chapter suggested that calcium signaling was likely a crucial mechanism that regulates both the expression of Ma1 and the Ma1 associated co-expression gene network governing fruit acidity. Chapter 2 is about improving the current version of apple reference transcriptome due to its unaccep low coverage in mapping of RNA-seq reads. The improved apple reference transcriptome comprises 71,178 genes (17,524 are novel) and increases the coverage of RNA-seq reads from 37-46% to 62-82%. This chapter lays a foundation for data analysis in Chapters 3 and 4. In conclusion, this study takes the understanding of apple fruit acidity to a higher level and opens more grounds for further dedicated studies.

Plant polyphenols are secondary metabolites that constitute one of the most common and widespread groups of natural products. They express a large and diverse panel of biological activities including beneficial effects on both plants and humans. Many polyphenols, from their structurally simplest representatives to their oligo/polymeric versions (also referred to as vegetable tannins) are notably known as phytoestrogens, plant pigments, potent antioxidants, and protein interacting agents. Sponsored by Groupe Polyphénols, this publication, which is the third volume in this highly regarded Recent Advances in Polyphenol Research series, is edited by Véronique Cheynier, Pascale Sarni-Manchado, and Stéphane Quideau (the current President of Groupe Polyphénols). Like their predecessors, they have once again put together an impressive collection of cutting-edge chapters written by expert scientists internationally respected in their respective field of polyphenol sciences. This Volume 3 provides the latest information and opinion on the following major research topics about polyphenols: Organic chemistry and physical chemistry Biosynthesis, genetics and metabolic engineering The role of polyphenols in plants and ecosystems Health and nutrition Analysis and metabolomics Chemists, biochemists, plant scientists, pharmacognosists and pharmacologists, biologists, ecologists, food scientists and nutritionists will all find this book an invaluable resource. Libraries in all universities and research institutions where these disciplines are studied and taught should have copies on their bookshelves.

Fruits are an important part of the human diet as they provide the vitamins, minerals and phytochemicals that are essential for human health. Commercialization of the fruit has driven new frontiers for crop development. The study of model plant systems such as Arabidopsis and tomato has uncovered highly conserved complex regulatory networks involving MADS-box transcription factors for controlling critical aspects of flower and fruit development. In apple, the effects of suppressing the apple SEP1/2-like gene, MADS8, have shown that the function of MADS8 is likely involved in fruit development. Fruits of MADS8-suppressed apple lines had reduced fruit size due to a loss of flesh tissues and severely inhibited ripening. In this research, five independent MADS8-overexpressing (MADS8ox) apple lines were analyzed. It was found that changes in tree architecture when compared to wild-type were strongly correlated with MADS8-overexpression. Early in tree development, the overexpression of MADS8 was found to result in reprioritization of flowering over vegetative growth, thereby changing the overall architecture of the tree with detrimental effects observed in the following year. At the fruit level, MADS8 overexpression was found to increase fruit flesh development and accelerate ripening particularly as seen from an increased rate of degreening in MADS8ox fruits. The analysis of possible protein interactions with MADS8 showed the ability of MADS8 to homodimerize which suggests that this may be the stable or activating form of the MADS8 protein. Transformation of Arabidopsis thaliana for novel expression of MADS8 in floral tissues did not produce any significant change in floral phenotypes. Overall, the results from this research have provided clear support for MADS8 as a key genetic determinant in a both tree and fruit development in apple. Elucidation of the functions of MADS8 provides new opportunities for the development of apple lines with improved yield and fruit quality. The results from this research also adds to the growing body of work that shows the diversification of MADS-box protein functions in fruit development arising from a conserved genetic framework at floral development.