A MADS family gene, OsMADS6, was isolated from a rice (Oryza sativa L.) young flower cDNA library using OsAMDS1 as a probe. With this clone, various MADS box genes that encode for proteinto-protein interaction partners of the OsMADS6 protein were isolated by the yeast two-hybrid screening method. On the basis of sequence homology, OsMADS6 and the selected partners can be classified in the APETALA1/AGAMOUS-LIKE9 (AP1/AGL9) family. One of the interaction partners, OsMADS14, was selected for further study. Both genes began expression at early stages of flower development, and their expression was extended into the later stages. In mature flowers the OsMADS6 transcript was detectable in lodicules and also weakly in sterile lemmas and carpels, whereas the OsMADS14 transcript was detectable in sterile lemmas, paleas/ lemmas, stamens, and carpels. Using the yeast two-hybrid system, we demonstrated that the region containing of the 109th to 137th amino acid residues of OsMADS6 is indispensable in the interaction with OsMADS14. Site-directed mutation analysis revealed that the four periodical leucine residues within the region are essential for this interaction. Furthermore, it was shown that the 14 amino acid residues located immediately downstream of the K domain enhance the interaction, and that the two leucine residues within this region play an important role in that enhancement.

A MADS-box gene, MdMADS2, was isolated from the apple (Malus ؋ domestica Borkh.) var Fuji and its developmental expression pattern was studied during flower development. MdMADS2 shares a high degree of amino acid sequence identity with the SQUAMOSA subfamily of genes. RNA blot analysis showed that MdMADS2 is transcribed through all stages of flower development, and its transcription was seen in the four floral organs. RNA in situ hybridization revealed that the MdMADS2 mRNA is expressed both in the inflorescence meristem and in the floral meristem. The Md-MADS2 transcript was detected at all stages of flower development. Protein localization analysis showed that MdMADS2 protein was excluded from the stamen and carpel primordia, in which a considerable MdMADS2 mRNA signal was detected. This indicates that posttanscriptional regulation may be involved in the MdMADS2-mediated control of flower development. Transgenic tobacco expressing the MdMADS2 gene from the cauliflower mosaic virus 35S promoter showed early flowering and shorter bolts, but did not show any homeotic changes in the floral organs. These results suggest that MdMADS2 plays an important role during early stages of flower development.

A male-sterile (MS) radish (Raphanus sativus L.) was found in an accession collected from Uzbekistan. Unlike Ogura MS radishes in which no pollen grain is typically visible during anthesis, a small number of pollen grains stuck together in the dehiscing anthers was observed in the newly identified MS radish. Fluorescein diacetate tests and scanning electron micrographs showed that pollen grains in the new MS radish were severely deformed and non-viable. Cytological examination of pollen development stages showed a clear difference in the defective stage from that seen in Ogura male-sterility. Reciprocal cross-pollination with diverse male-fertile lines indicated that pollen grains of the new MS radish were completely sterile, and the female organs were fully fertile. When the new MS radish and Ogura MS lines were cross-pollinated with a set of eight breeding lines, all F1 progeny originating from crosses with the new MS radish were male-sterile. In contrast, most of the F1 progeny resulting from crosses with Ogura MS lines were male-fertile. These results demonstrated that factors associated with induction of the newly identified male-sterility are different from those of Ogura male-sterility. The lack of restorer lines for the newly identified male-sterility led us to predict that it might be a complete cytoplasmic male-sterility without restorer-of-fertility genes in nuclear genomes. However, cross-pollination with more diverse radish germplasm identified one accession introduced from Russia that could completely restore fertility, proving the existence of restorer-of-fertility gene(s) for the new male-sterility. Meanwhile, the PCR amplification profile of molecular markers for the classification of radish mitochondrial genome types revealed that the new MS radish contained a novel mitotype.

Two MADS-box genes, MdMADS3 and MdMADS4, were isolated from the apple (Malus x domestica Borkh.) cultivar Fuji, and their spatial and temporal expression patterns were studied during morphological differentiation of the flower buds and the fruits. Both MdMADS3 and MdMADS4 showed high sequence similarities to FBP2 from petunia, TM5 from tomato, and AGL2, AGL4 from Arabidopsis. Although MdMADS3 was expressed in the inner three whorls of the floral primordium, its expression was hardly detectable in developing fruit. The second gene, MdMADS4, was ubiquitously expressed in the inflorescence meristem, floral meristem, all four floral organs, and fruit. Moreover, MdMADS4 expression was high in the vascular bundles assigned to the floral tube and the carpellary vascular bundles in fruit at early developmental stages. The MdMADS4 transcript also accumulated in embryos of the developing seeds. These results suggest that MdMADS3 and MdMADS4 are involved in different functions, and that MdMADS4 may function in the important events controlling flower and fruit development.

A cDNA clone, MdMADS1, containing MADS domain was isolated from the Fuji apple. The gene was expressed in all floral organs and young fruits but not in leaves. The expression was higher at the early stages of flower and fruit developments, suggesting that MdMADS1 plays a major role in the initiation of reproductive organ developments.