Nobuhiro Kotoda

Anthocyanin biosynthetic genes are coordinately expressed during red coloration in apple skin

Five genes of anthocyanin biosynthetic enzymes, chalcone synthase (CHS; EC 2.3.1.74), flavanone 3-hydroxylase (F3H; EC 1.14.11.9), dihydroflavonol 4-reductase (DFR; EC 1.1.1.219), anthocyanidin synthase (ANS; EC 1.14.11.X), and UDP glucose:flavonoid 3-O-glucosyltransferase (UFGluT; EC 2.4.1.X), were isolated, and their expression was investigated to elucidate the molecular mechanism for red coloration in apple 〚Malus sylvestris (L.) Mill. var. domestica (Borkh.) Mansf.〛 skin. In ‘Orin,’ a yellow apple cultivar, no significant levels of anthocyanin were detectable, whereas in ‘Jonathan’ and ‘Fuji,’ both red apple cultivars, anthocyanin concentrations increased during fruit development. At the ripe stage, the level of anthocyanin concentration was about three times higher in ‘Jonathan’ than in ‘Fuji.’ The accumulation of transcripts for the five genes was induced at the later developmental stages in all three cultivars. The levels for the expression of the five genes basically corresponded to the anthocyanin concentrations; that is, the induction of the genes in ‘Orin’ was less pronounced, and that in ‘Fuji’ and ‘Jonathan’ was notable, with much higher expression levels in ‘Jonathan’ than in ‘Fuji’. These results indicate that the five genes are coordinately expressed during fruit development and that their levels of expression are positively related to the degree of anthocyanin concentration. This is the first report that characterizes the relationship between the expression of anthocyanin biosynthetic genes and apple fruit coloration.

Molecular Characterization of FLOWERING LOCUS T-Like Genes of Apple (Malus × 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.

Apple has two orthologues of FLORICAULA/LEAFY involved in flowering

Two orthologues of FLORICAULA/LEAFY, AFL1 and AFL2 (apple FLO/LFY), were isolated from the floral buds of apple trees. Their expression was detected in various tissues and during differentiation of the floral buds. Furthermore, the flowering effectiveness of each gene was assessed with transgenic Arabidopsis. Both AFL1 and AFL2 showed high homology to each other (90%) and a high degree of similarity to PTLF and PEAFLO (70%), which are homologues of FLO/LFY from poplar and pea, respectively. RNA blot analysis showed that AFL1 was expressed only in the floral bud during the transition from vegetative to reproductive growth, whereas AFL2 was expressed in vegetative shoot apex, floral buds, floral organs and root. Genomic Southern analysis showed that apple had other homologues in addition to AFL1 and AFL2. The transgenic Arabidopsis with over-expressed AFL2 showed accelerated flowering and gave rise to several solitary flowers from rosette axils directly. AFL1 had similar effects, but the phenotypes of the transgenic Arabidopsis with AFL1 were weaker than those with AFL2. These results suggest that both genes are involved in flower differentiation in apple.

The FLOWERING LOCUS T/TERMINAL FLOWER 1 Family in Lombardy Poplar

Genes in the FLOWERING LOCUS T (FT) and TERMINAL FLOWER 1 (TFL1) family have been shown to be important in the control of the switch between vegetative and reproductive growth in several plant species. We isolated nine members of the FT/TFL1 family from Lombardy poplar (Populus nigra var. italica Koehne). Sequence analysis of the members of the FT/TFL1 family revealed considerable homology within their coding regions both among family members and to the members of the same family in Arabidopsis, tomato and grapevine. Moreover, members of this family in all four species examined display a common exon-intron organization. Phylogenetic analysis revealed that the genes fall into four different clades: two into the TFL1 clade; five into the FT clade; and one each into the MOTHER OF FT AND TFL1 and BROTHER OF FT AND TFL1 clades. One gene in the TFL1 clade, PnTFL1, is expressed in vegetative meristems, and transgenic Arabidopsis that ectopically expressed PnTFL1 had a late-flowering phenotype. The expression patterns of two genes in the FT clade, PnFT1 and PnFT2, suggested a role for them in the promotion of flowering, and transgenic Arabidopsis that ectopically expressed either PnFT1 or PnFT2 had an early-flowering phenotype.

Four TFL1/CEN-Like Genes on Distinct Linkage Groups Show Different Expression Patterns to Regulate Vegetative and Reproductive Development in Apple (Malus×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.

Apple FLOWERING LOCUS T proteins interact with transcription factors implicated in cell growth and organ development

Understanding the flowering process in apple (Malus × domestica Borkh.) is essential for developing methods to shorten the breeding period and regulate fruit yield. It is known that FLOWERING LOCUS T (FT) acts as a transmissible floral inducer in the Arabidopsis flowering network system. To clarify the molecular network of two apple FT orthologues, MdFT1 and MdFT2, we performed a yeast two-hybrid screen to identify proteins that interact with MdFT1. We identified several transcription factors, including two members of the TCP (TEOSINTE BRANCHED1, CYCLOIDEA and PROLIFERATING CELL FACTORs) family, designated MdTCP2 and MdTCP4, and an Arabidopsis thaliana VOZ1 (Vascular plant One Zinc finger protein1)-like protein, designated MdVOZ1. MdTCP2 and MdVOZ1 also interacted with MdFT2 in yeast. The expression domain of MdTCP2 and MdVOZ1 partially overlapped with that of MdFT1 and MdFT2, most strikingly in apple fruit tissue, further suggesting a potential interaction in vivo. Constitutive expression of MdTCP2, MdTCP4 and MdVOZ1 in Arabidopsis affected plant size, leaf morphology and the formation of leaf primordia on the adaxial side of cotyledons. On the other hand, chimeric MdTCP2, MdTCP4 and MdVOZ1 repressors that included the ethylene-responsive transcription factors (ERF)-associated amphiphilic repression (EAR) domain motif influenced reproduction and inflorescence architecture in transgenic Arabidopsis. These results suggest that MdFT1 and/or MdFT2 might be involved in the regulation of cellular proliferation and the formation of new tissues and that they might affect leaf and fruit development by interacting with TCP- and VOZ-family proteins. DDBJ accession nos. AB531019 (MdTCP2a mRNA), AB531020 (MdTCP2b mRNA), AB531021 (MdTCP4a mRNA), AB531022 (MdTCP4b mRNA) and AB531023 (MdVOZ1a mRNA).

Genetic studies on resistance to Valsa canker in apple: genetic variance and breeding values estimated from intra- and inter-specific hybrid progeny populations

Malus sieboldii Rehd. exhibits high levels of resistance to Valsa canker caused by Valsa ceratosperma (Tode ex Fr.) Maire while cultivated apples (Malus domestica Borkh.) are susceptible to the disease. In this study, progenies from 23 full-sib families derived from both inter- and intra-specific hybridization among 16 Malus genotypes as parents were assessed for resistance to V. ceratosperma (Vc) for two seasons using an excised shoot assay to determine the pattern of inheritance of the resistance and to also estimate the variance components, narrow-sense heritability, and breeding values of parental genotypes. Generally, M. sieboldii × M. domestica and its reciprocal crosses had more resistant progenies to Vc than intra-specific crosses of M. domestica. Resistance to Vc expressed as the relative lesion length among progenies showed continuous variation irrespective of cross, suggesting the quantitative nature of the resistance to the three virulent isolates of Vc that were tested. Resistance to Vc using the progeny population was analyzed using a mixed linear model based on restricted maximum likelihood. The parental effect (general combining ability (GCA)) was significant while the interaction effect between parents (specific combining ability (SCA)) was relatively small and non-significant. The ratio of SCA/GCA variance was about 32%, suggesting that additive genetic variance had a major contribution to the total genetic variance for resistance to Vc. There was a positive correlation (r = 0.49, p < 0.01) between mid-parental GCA and SCA predictions among 23 full-sib families for the resistance. Narrow-sense heritability estimated by sib analysis was moderate

RAD-Seq analysis of typical and minor Citrus accessions, including Bhutanese varieties

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