Characterization of MADS box genes from hot pepper.

The cDNA clone, CanMADS1, was isolated from young flower buds of the hot pepper (Capsicum annuum L.) by screening a cDNA library using the OsMADS1 rice MADS-box gene as a probe. We used a yeast two-hybrid screening method to investigate interaction partners of the protein product of CanMADS1. A MADS-box gene, CanMADS6, was isolated from young flower buds using the region containing the K domain and 15 amino acid residues of the C-terminal region of CanMADS1 as a bait. CanMADS1 and CanMADS6 showed high amino acid sequence similarities to members of the AGL2 subfamily and the SQUA subfamily, respectively. CanMADS1 transcript was expressed in flower buds and fruits, and the transcription signal was the strongest in the stage of the fruit set (2 d after anthesis). CanMADS6 showed the same expression pattern as CanMADS1. CanMADS1 and CanMADS6 were not expressed in leaves. These results suggest that a regulatory role for flower and fruit development of the hot pepper may be accomplished through an interaction of the protein products of the two MADS-box genes, CanMADS1 and CanMADS6.

Analysis of expressed sequence tags of Porphyra yezoensis.

Single direct partial sequencing of anonymous cDNA clones was performed to obtain genetic information on red algae Porphyra yezoensis of which genetic information is not available. This expressed sequence tags (EST) analysis revealed 81 clones (42%) had significant homologies to known genes in GenBank. Of these clones, eight are related to known algal genes, whereas above 90% of the EST clones were newly identified in algae. Putative functional categories of these clones showed that the most abundant genes were involved in stress and defense mechanisms and that the next abundant genes were associated with protein synthetic pathways.

Developmentally regulated expression of two MADS-box genes, MdMADS3 and MdMADS4, in the morphogenesis of flower buds and fruits in apple.

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.

Characterization of MdMADS2, a member of the SQUAMOSA subfamily of genes, in apple.

A MADS-box gene, MdMADS2, was isolated from the apple (Malus x 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 MdMADS2 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.

Determination of the motif responsible for interaction between the rice APETALA1/AGAMOUS-LIKE9 family proteins using a yeast two-hybrid system.

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 protein-to-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.

Expressed sequence tags of fruits, peels, and carpels and analysis of mRNA expression levels of the tagged cDNAs of fruits from the Fuji apple.

In order to understand molecular events during fruit development and provide genetic resources for molecular breeding, 430 expressed sequence tags (ESTs) were generated from randomly selected clones of cDNA libraries prepared from young fruits, peels of mature fruits, and carpels of the Fuji apple (Malus domestica Borkh.). Database comparisons of the ESTs revealed that 180 non-redundant clones showed a high similarity with previously identified genes. Among these, 138 clones exhibited a homology with previously identified plant genes and 12 were identical to genes that were previously identified from apples. The deduced amino acid sequences of 42 clones had a homology to proteins that have not been reported from plants. Eighteen cDNA clones from the young fruit library were selected for studying expression levels and patterns in reproductive organs and leaves. This study revealed that the clones can be classified into 3 different groups based on their expression levels. The first 9 clones were expressed strongly in at least one reproductive organ. Eight of these clones (vacuolar processing protease, sucrose phosphate synthase, arabinogalactan protein, UDP-glucose glucosyl transferase, major allergen D1, cystein proteinase inhibitor, lipoxygenase, and protease subunit SUG2) were highly expressed in mature flowers and young fruits, whereas one clone (z-carotene desaturase protein precursor) was preferentially expressed in mature flowers but weakly in young fruits. The second group includes 6 cDNA clones (glucose transport protein, aminomethyl transferase precursor protein, dTDP-D-glucose-4,6-dehydrogenase, 2 types of protein kinase, and selenium binding protein) that were weakly expressed. These clones were characterized by their preferential expression patterns in mature flowers and young fruits. The transcripts of 3 cDNA clones in the third group (vacuolar aminopetidase, beta-galactosidase, and EREBP-4) were detectable only by RT-PCR and they were preferentially expressed in young fruits. These results indicate that most ESTs that were isolated from young fruits are preferentially expressed in reproductive organs and thereby play important roles during reproductive organ development.

Molecular cloning and characterization of a MADS-box cDNA clone of the Fuji apple.

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.