Biotechnology and apple breeding in Japan.

Apple is a fruit crop of significant economic importance, and breeders world wide continue to develop novel cultivars with improved characteristics. The lengthy juvenile period and the large field space required to grow apple populations have imposed major limitations on breeding. Various molecular biological techniques have been employed to make apple breeding easier. Transgenic technology has facilitated the development of apples with resistance to fungal or bacterial diseases, improved fruit quality, or root stocks with better rooting or dwarfing ability. DNA markers for disease resistance (scab, powdery mildew, fire-blight, Alternaria blotch) and fruit skin color have also been developed, and marker-assisted selection (MAS) has been employed in breeding programs. In the last decade, genomic sequences and chromosome maps of various cultivars have become available, allowing the development of large SNP arrays, enabling efficient QTL mapping and genomic selection (GS). In recent years, new technologies for genetic improvement, such as trans-grafting, virus vectors, and genome-editing, have emerged. Using these techniques, no foreign genes are present in the final product, and some of them show considerable promise for application to apple breeding.

C-type lectin receptor SIGNR1 expressed on peritoneal phagocytic cells with an immature dendritic cell-like phenotype is involved in uptake of oligomannose-coated liposomes and subsequent cell maturation.

The mannose-binding C-type lectin receptor SIGNR1 appears to be a structural and functional murine homologue of human DC-SIGN, but expression of SIGNR1 and its function in induction of immune responses in dendritic cell (DC) lineages remains unclear. In this study, we demonstrated expression and function of SIGNR1 on mouse peritoneal phagocytic cells with an immature DC-like phenotype. Analysis of these cells with a series of cell lineage markers indicated that CD11b(+)F4/80(-) phagocytic cells expressed costimulatory molecules, the DC marker CD83, and MHC class II, suggesting an immature DC-like phenotype. These immature peritoneal DC-like cells expressed low levels of SIGNR1, in addition to another mannose-binding C-type lectin, CD206. The immature peritoneal DC-like cells ingested oligomannose- or Lewis antigen-coated liposomes in vitro through SIGNR1. Following in vitro uptake of oligomannose-coated liposomes, SIGNR1, but not CD206, disappeared rapidly from the surface of the cells. In response to in vitro uptake of OMLs, the peritoneal DC-like cells matured with increasing expression of CD11c, CD86, and MHC class II. Thus, low levels of SIGNR1 expressed on mouse peritoneal phagocytic cells with an immature DC-like phenotype are primarily involved in uptake of mannose- or fucose-decorated particles, and this uptake leads to cell maturation.

Distribution of MdACS3 null alleles in apple (Malus × domestica Borkh.) and its relevance to the fruit ripening characters.

Expression of MdACS3a, one of the ripening-related ACC synthase genes, plays a pivotal role in initiating the burst of ethylene production by MdACS1 in apple fruit. Although previous studies have demonstrated the presence of MdACS3a-null alleles through deficiency of transcription activity or loss of enzyme activity due to amino acid substitution, which may affect the storage properties of certain fruit cultivars, an overall picture of these null alleles in cultivars is still lacking. The present study investigated the distribution of null allelic genes in 103 cultivars and 172 breeding selections by using a simple sequence repeat (SSR) marker linked to them. The results indicated that both allelic genes were widely distributed throughout the examined cultivars and selections, some occurring as the null genotype, either homozygously or heterozygously, with each null allele. The implications of MdACS3a distribution results and the influence of its null allelotypes in fruit characters are discussed.

Development of peritoneal macrophage along a dendritic cell lineage in response to uptake of oligomannose-coated liposomes.

In this study, we investigate the potential of peritoneal macrophages to differentiate into dendritic cell (DCs) in response to preferential uptake of oligomannose-coated liposomes (OMLs). About 30% of peritoneal cells (PECs) preferentially took up OMLs that were administered into the peritoneal cavity. The OML-ingesting cells expressed CD11b and F4/80, but lacked CD11c expression, indicating that the OML-ingesting PECs with a CD11b(high)CD11c(-) phenotype are resident peritoneal macrophages. During in vitro cultivation, CD11c(+) cells arose among the PECs with ingested OMLs. CD11c(+) cells also developed among enriched peritoneal CD11b(high)CD11(-) cells from OML-treated mice, and the resulting CD11c(+) cells expressed co-stimulatory molecules and MHC class II. In addition, OML-ingesting CD11b(high)CD11c(+) cells were found in spleen after the enriched peritoneal macrophages with ingested OMLs were transplanted in the peritoneal cavity of mice. These results show that a fraction of peritoneal macrophages can differentiate into mature DCs following uptake of OMLs.

Molecular characterization of FLOWERING LOCUS T-like genes of apple (Malus x domestica Borkh.).

The two FLOWERING LOCUS T (FT)-like genes of apple (Malus x domestica Borkh.), MdFT1 and MdFT2, have been isolated and characterized. MdFT1 and MdFT2 were mapped, respectively, on distinct linkage groups (LGs) with partial homoeology, LG 12 and LG 4. The expression pattern of MdFT1 and MdFT2 differed in that MdFT1 was expressed mainly in apical buds of fruit-bearing shoots in the adult phase, with little expression in the juvenile tissues, whereas MdFT2 was expressed mainly in reproductive organs, including flower buds and young fruit. On the other hand, both genes had the potential to induce early flowering since transgenic Arabidopsis, which ectopically expressed MdFT1 or MdFT2, flowered earlier than wild-type plants. Furthermore, overexpression of MdFT1 conferred precocious flowering in apple, with altered expression of other endogenous genes, such as MdMADS12. These results suggest that MdFT1 could function to promote flowering by altering the expression of those genes and that, at least, other genes may play an important role as well in the regulation of flowering in apple. The long juvenile period of fruit trees prevents early cropping and efficient breeding. Our findings will be useful information to unveil the molecular mechanism of flowering and to develop methods to shorten the juvenile period in various fruit trees, including apple.

Null mutation of the MdACS3 gene, coding for a ripening-specific 1-aminocyclopropane-1-carboxylate synthase, leads to long shelf life in apple fruit.

Expression of MdACS1, coding for 1-aminocyclopropane-1-carboxylate synthase (ACS), parallels the level of ethylene production in ripening apple (Malus domestica) fruit. Here we show that expression of another ripening-specific ACS gene (MdACS3) precedes the initiation of MdACS1 expression by approximately 3 weeks; MdACS3 expression then gradually decreases as MdACS1 expression increases. Because MdACS3 expression continues in ripening fruit treated with 1-methylcyclopropene, its transcription appears to be regulated by a negative feedback mechanism. Three genes in the MdACS3 family (a, b, and c) were isolated from a genomic library, but two of them (MdACS3b and MdACS3c) possess a 333-bp transposon-like insertion in their 5' flanking region that may prevent transcription of these genes during ripening. A single nucleotide polymorphism in the coding region of MdACS3a results in an amino acid substitution (glycine-289 --> valine) in the active site that inactivates the enzyme. Furthermore, another null allele of MdACS3a, Mdacs3a, showing no ability to be transcribed, was found by DNA sequencing. Apple cultivars homozygous or heterozygous for both null allelotypes showed no or very low expression of ripening-related genes and maintained fruit firmness. These results suggest that MdACS3a plays a crucial role in regulation of fruit ripening in apple, and is a possible determinant of ethylene production and shelf life in apple fruit.

Four TFL1/CEN-like genes on distinct linkage groups show different expression patterns to regulate vegetative and reproductive development in apple (Malus x domestica Borkh.).

Recent molecular analyses in several plant species revealed that TERMINAL FLOWER1 (TFL1) and CENTRORADIALIS (CEN) homologs are involved in regulating the flowering time and/or maintaining the inflorescence meristem. In apple (Malusxdomestica Borkh.), four TFL1/CEN-like genes, MdTFL1, MdTFL1a, MdCENa and MdCENb, were found and mapped by a similar position on putatively homoeologous linkage groups. Apple TFL1/CEN-like genes functioned equivalently to TFL1 when expressed constitutively in transgenic Arabidopsis plants, suggesting that they have a potential to complement the TFL1 function. Because MdTFL1 and MdTFL1a were expressed in the vegetative tissues in both the adult and juvenile phases, they could function redundantly as a flowering repressor and a regulator of vegetative meristem identity. On the other hand, MdCENa was mainly expressed in fruit receptacles, cultured tissues and roots, suggesting that it is involved in the development of proliferating tissues but not in the control of the transition from the juvenile to the adult phase. In contrast, MdCENb was silenced in most organs probably due to gene duplication by the polyploid origin of apple. The expression patterns of MdTFL1 and MdCENa in apple were also supported by the heterologous expression of beta-glucuronidase fused with their promoter regions in transgenic Arabidopsis. Our results suggest that functional divergence of the roles in the regulation of vegetative meristem identity may have occurred among four TFL1/CEN-like genes during evolution in apple.

Isolation and functional analysis of a MYB transcription factor gene that is a key regulator for the development of red coloration in apple skin.

Red coloration of apple (Malus x domestica) skin is an important determinant of consumer preference and marketability. Anthocyanins are responsible for this coloration, and their accumulation is positively correlated with the expression level of anthocyanin biosynthetic genes. Regulation of expression of these genes is believed to be controlled by MYB transcription factors, and the MYB transcription factors involved in the activation of anthocyanin biosynthetic genes have been isolated in various plants. In the present study, we isolated and characterized a MYB transcription factor gene (MdMYBA) from apple skin. Characterization of MdMYBA demonstrated that (i) MdMYBA expression was specifically regulated depending on the tissue and cultivar/species; (ii) its expression level was much higher in a deep-red cultivar ('Jonathan') than in a pale-red cultivar ('Tsugaru'); (iii) when cauliflower mosaic virus 35S::MdMYBA was introduced into the cotyledons of apple seedlings by means of a transient assay, reddish-purple spots were induced, and MdMYBA also induced anthocyanin accumulation in reproductive tissues of transgenic tobacco; (iv) the expression of MdMYBA was induced by UV-B irradiation and low-temperature treatment, both of which are known to be important in the promotion of anthocyanin accumulation in apple skin; (v) MdMYBA bound specifically to an anthocyanidin synthase (MdANS) promoter region in a gel-shift assay; and (vi) MdMYBA was mapped to the near region of the BC226-STS (a1) marker for the red skin color locus (R(f)). These results suggest that MdMYBA is a key regulatory gene in anthocyanin biosynthesis in apple skin.