Expression and functional analysis of apple MdMADS13 on flower and fruit formation.

Apple MdMADS13 has a transcription factor with MADS domain. Moreover, it is expressed specifically at petals and carpels. The product forms a dimer with MdPISTILLATA (MdPI) protein as a class B gene for floral organ formation. Reportedly, in parthenocarpic cultivars of apple (Spencer Seedless, Wellington Bloomless, Wickson and Noblow) the MdPI function is lost by genome insertion of retrotransposon, which cultivars show a homeotic mutation of floral organs, petals to sepals and stamens to carpels. Apple fruit is pome from receptacle tissue, and MdSEPALLATA (MdMADS8/9) and AGAMOUS homologues MdMADS15/22 involved in the fruit development, the transgenic apple suppressed these gene showed poor fruit development and abnormal flower formation. This article describes that the MdMADS13 retained expression after blossom and small fruits of parthenocarpic cultivars. Yeast two-hybrid experiment showed specific binding between MdPI and MdMADS13 proteins. Furthermore, transgenic Arabidopsis with 35S::MdMADS13 have malformed stamens and carpels. These results suggest strongly that MdMADS13 is related to flower organ formation as a class B gene with MdPI.

The analysis of transgenic apples with down-regulated expression of MdPISTILLATA.

In parthenocarpic cultivars of apple (Malus×domestica Borkh.), MdPISTILLATA (MdPI) expression has been suppressed by retrotransposon insertion into the MdPI genome. In this study, transgenic apple lines were produced that exhibited the same level of MdPI depression. The 1P-2 promoter from the MdPI genome, which specifies its expression in the petals and stamens, was used for antisense-MdPI expression, and rolC:AtFT was included to accelerate flowering. The transgenic apple with rolC:AtFT/1P-2:antisense-MdPI showed homeotic changes in the floral organs, whereby petals and stamens were replaced with sepals and pistils, respectively. Line 9-2 of this transgenic apple also showed strong suppression of MdPI. Some individuals from this line had deformed floral organs, suggesting that the homeotic changes were incomplete. Other transformants of line 9-2 that had double sepals in the first and second whorls, and many pistils in the third and fourth whorls, as seen in apple cultivars with class B mutations, which demonstrated MdPI functioned for floral organs formation same as Arabidopsis PISTILLATA gene. The transgenic apples set parthenocarpic fruits (15.7%). However, precocious transgenic apples with rolC:AtFT exhibited more parthenocarpy (14-27%). This indicates that MdPI depression cannot explain fruit formation in parthenocarpic cultivars of apple, and so some other as yet unidentified genes must be responsible.

Homologous pairing activities of two rice RAD51 proteins, RAD51A1 and RAD51A2.

In higher eukaryotes, RAD51 functions as an essential protein in homologous recombination and recombinational repair of DNA double strand breaks. During these processes, RAD51 catalyzes homologous pairing between single-stranded DNA and double-stranded DNA. Japonica cultivars of rice (Oryza sativa) encode two RAD51 proteins, RAD51A1 and RAD51A2, whereas only one RAD51 exists in yeast and mammals. However, the functional differences between RAD51A1 and RAD51A2 have not been elucidated, because their biochemical properties have not been characterized. In the present study, we purified RAD51A1 and RAD51A2, and found that RAD51A2 robustly promotes homologous pairing in vitro. RAD51A1 also possesses homologous-pairing activity, but it is only about 10% of the RAD51A2 activity. Both RAD51A1 and RAD51A2 bind to ssDNA and dsDNA, and their DNA binding strictly requires ATP, which modulates the polymer formation activities of RAD51A1 and RAD51A2. These findings suggest that although both RAD51A1 and RAD51A2 have the potential to catalyze homologous pairing, RAD51A2 may be the major recombinase in rice.

The regulation of seasonal flowering in the Rosaceae.

Molecular mechanisms regulating the flowering process have been extensively studied in model annual plants; in perennials, however, understanding of the molecular mechanisms controlling flowering has just started to emerge. Here we review the current state of flowering research in perennial plants of the rose family (Rosaceae), which is one of the most economically important families of horticultural plants. Strawberry (Fragaria spp.), raspberry (Rubus spp.), rose (Rosa spp.), and apple (Malus spp.) are used to illustrate how photoperiod and temperature control seasonal flowering in rosaceous crops. We highlight recent molecular studies which have revealed homologues of terminal flower1 (TFL1) to be major regulators of both the juvenile to adult, and the vegetative to reproductive transitions in various rosaceous species. Additionally, recent advances in understanding of the regulation of TFL1 are discussed.

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).

Expression patterns of several floral genes during flower initiation in the apical buds of apple (Malus × domestica Borkh.) revealed by in situ hybridization.

The apple (Malus × domestica Borkh.) is one of the commercially important fruit crops in the worldwide. The apple has a relatively long juvenile period (up to 4 years) and a long reproductive period between the flower initiation and the mature fruit (14-16 months), which prevent the fruit breeding. Therefore, the understanding of the flowering system is important to improve breeding efficiency in the apple. In this study, to examine the temporal and spatial expression patterns of the floral genes, MdTFL1, MdAP1 (MdMASD5), AFL2, and MdFT, we conducted in situ hybridization analysis in the apple shoot apex. In vegetative phase, MdTFL1 was expressed on the rib meristem zone. When vegetative meristem began converting into inflorescence meristem, the expression level of MdTFL1 was drastically decreased. At the early stage of inflorescence meristem, the expression levels of AFL2, MdFT, and MdAP1 were up-regulated in the leaf primordia and the upper region of cell layers on the shoot apex. In late stage, the expression levels of AFL2 and MdAP1 were up-regulated in the young floral primordia. At a more advanced stage, high expression of MdAP1 was observed in the inflorescence primordium through the inner layer of sepal primordia and the outer layer of receptacle primordia and floral axis. Our results suggest that AFL2, MdFT, and MdAP1 affect to convert from the vegetative meristem into the inflorescence meristem after the decline of MdTFL1 expression. After that, AFL2 and MdAP1 promote the formation of the floral primordia and floral organs.

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.

Constitutive expression of two apple (Malus x domestica Borkh.) homolog genes of LIKE HETEROCHROMATIN PROTEIN1 affects flowering time and whole-plant growth in transgenic Arabidopsis.

Fruit trees, such as apple (Malus x domestica Borkh.), are woody perennial plants with a long juvenile phase. The biological analysis for the regulation of flowering time provides insights into the reduction of juvenile phase and the acceleration of breeding in fruit trees. In Arabidopsis, LIKE HETEROCHROMATIN PROTEIN1 (LHP1) is involved in epigenetic silencing of the target genes such as flowering genes. We isolated and characterized twin apple LHP1 homolog genes, MdLHP1a and MdLHP1b. These genes may have been generated as a result of ancient genome duplication. Although the putative MdLHP1 proteins showed lower similarity to any other known plant LHP1 homologs, a chromo domain, a chromo shadow domain, and the nuclear localization signal motifs were highly conserved among them. RT-PCR analysis showed that MdLHP1a and MdLHP1b were expressed constantly in developing shoot apices of apple trees throughout the growing season. Constitutive expression of MdLHP1a or MdLHP1b could compensate for the pleiotropic phenotype of lhp1/tfl2 mutant, suggesting that apple LHP1 homolog genes are involved in the regulation of flowering time and whole-plant growth. Based on these results, LHP1 homolog genes might have rapidly evolved among plant species, but the protein functions were conserved, at least between Arabidopsis and apple.

Two alternatively spliced transcripts generated from OsMUS81, a rice homolog of yeast MUS81, are up-regulated by DNA-damaging treatments.

OsMUS81, a rice homolog of the yeast MUS81 endonuclease gene, produced two alternative transcripts, OsMUS81alpha and OsMUS81beta. OsMus81alpha contained a Helix-hairpin-Helix (HhH) motif at the N- and C-termini, and a conserved XPF-like motif in the center, while the OsMus81beta isoform lacked the second HhH motif by alternative splicing of a cryptic intron generating a truncated protein. The two transcripts were induced after DNA-damaging treatments such as high intensity light, UV-C and gamma-radiation. The yeast two-hybrid assay detected a strong interaction between OsMus81 and OsRad54 recombinational repair proteins. These findings suggest that OsMus81 functions in maintaining genome integrity through homologous recombination.