Soon-Kee Sung

Microarray analysis of apple gene expression engaged in early fruit development

To evaluate gene expressions mostly engaged in early development of apple fruit, we performed the identification of transcripts differentially expressed in young fruit by using microarrays spotted with 6,253 cDNAs collected from young and mature apple fruits of the cultivar Fuji (Malus domestica Borkh. cv. Fuji). A total of 3,484 cDNAs out of 6,253 were selected after quality control of microarray spots and analyzed for differential gene expression patterns between young fruit and other tissues (mature fruit, leaf and flower). Among them, 192 cDNAs displayed a signal value higher than twofold in young fruit compared with other tissues. Blast analysis of the 192 cDNA clones identified 88 non-redundant groups encoding proteins with known function and 50 non-redundant groups with unknown function. The putative protein products were classified into the following categories: photosynthesis (16.7%), protein synthesis (12.3%), cell proliferation and differentiation (10.9%), cell enlargement (5.8%), metabolism (8.0%), stress response (7.2%), others (2.9%), and unknown functions (32.2%). Furthermore, confirming the microarray data by reverse transcription-polymerase chain reaction revealed that the wide range of transcripts differentially expressed in young fruit was expressed in other organs but not in the mature fruit. The data presented suggested that apple fruit development depends on the tight regulation of the expression of a number of genes, which are also expressed in other organs.

DEVELOPMENTALLY REGULATED EXPRESSION OF TWO MADS-BOX GENES, MDMADS3 AND MDMADS4, IN THE MORPHOGENESIS OF FLOWER BUDS AND FRUITS IN APPLE

Abstract. Two MADS-box genes, MdMADS3 and MdMADS4, were isolated from the apple (Malus × 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.

Two polyphenol oxidases are differentially expressed during vegetative and reproductive development and in response to wounding in the Fuji apple

Abstract Polyphenol oxidase (PPO), a copper-containing metalloprotein, catalyzes the oxidation of phenolics to quinones which make brown pigments in wounded tissues. Because the phenomena decrease fruit quality, PPO has been regarded to be a critical enzyme in food technology. In the course of expressed sequence tags (ESTs) analysis of the Fuji apple ( Malus domesticus Borkh.), we identified two partial PPO cDNA clones; F114 corresponded to the previously isolated Granny Smith apple pAPO5 (Plant Mol. Biol. 27 (1995) 429), while F226 was a new clone. Using F226 as a probe, we isolated a full length PPO clone, pMD-PPO2, from a cDNA library prepared from young fruits of the Fuji apple. The deduced amino acid sequences of pMD-PPO2 and pAPO5 share 55% identity, and display a high degree of sequence identity (43–58%) with previously identified PPO from various species. RNA gel blot analysis using gene-specific probes showed that two apple PPO genes display unique patterns of expression in a tissue- and developmental-specific manner. In the process of flower development, the APO5 transcript was detectable only at the post-anthesis stage. In contrast, MD-PPO2 was expressed in all stages of flower development, with the abundance of mRNA being the highest at the pre-anthesis stage and then receding as the flower developed. Both genes are expressed in the early stages of fruit development. The expression was dramatically reduced as the fruit ripened. In leaf tissue, the APO5 gene was highly expressed in young and immature leaves, while MD-PPO2 was transcriptionally more active in both immature and mature leaves. Upon wounding, APO5 was significantly induced in leaves and fruits, whereas the level of MD-PPO2 mRNA was not affected by mechanical damage. Thus, it appears that two Fuji PPO genes are differentially expressed during vegetative and reproductive development and in response to wounding in the Fuji apple plants. The possible molecular mechanism of differential regulation of PPO gene expression in apple plant and its physiological significance are discussed.

Discovery of a novel cytoplasmic male-sterility and its restorer lines in radish (Raphanus sativus L.)

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.

Molecular cloning and characterization of constans-like cDNA clones of the fuji apple

Two cDNA clones,MdCOL1 andMdCOL2, encoding CONSTANS (CO)-like proteins were isolated from an apple (Malus domestica cv. Fuji) fruit cDNA library. Both proteins contain two zinc finger motifs at the amino terminal end and a putative nuclear localization domain at the carboxyl terminal end. Genomic DNA blot analysis suggests that theCO-like genes are members of a small multigene family. RNA blot and RT-PCR analyses revealed that these genes are expressed in every organ that was examined. However, the expression levels were higher in floral buds and fruits at their early developmental stages compared to late reproductive stages or vegetative organs. Such expression patterns are quite different from those of theCO-like genes fromArabidopsis, which show strong organ specificity in either roots, cauline leaves, or flowers. These results indicate that the appleCO-like genes are significantly different from theArabidopsis genes and that they appear to play important roles in reproductive organ development.

The salicylic acid-induced protection of non-climacteric unripe pepper fruit against Colletotrichum gloeosporioides is similar to the resistance of ripe fruit

The anthracnose fungus Colletotrichum gloeosporioides deleteriously affects unripe pepper fruit, but not ripe fruit. Here, we show that the induction of local acquired resistance (LAR) by salicylic acid (SA), 2,6-dichloroisonicotinic acid, or benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester pretreatment protects unripe pepper fruit against the fungus, while jasmonic acid (JA) does not. The SA-mediated LAR in the unripe fruit inhibited the fungal appressoria, resulting in protection against fungal infection. Microarray analysis revealed that 177 of 7,900 cDNA clones showed more than fourfold transcriptional accumulation in SA-treated unripe fruit. The reverse transcription-polymerase chain reaction showed that most of the SA-responsive genes (SRGs) were regulated by SA, but not by JA or ethylene-releasing ethephon. Furthermore, most of the SRGs were preferentially expressed in the ripe fruit. These results suggest that the SA-mediated transcriptional regulation of SRGs has a critical role in the resistance of ripe pepper fruit to fungal infection.

Expression of MdCAS1 and MdCAS2, encoding apple β-cyanoalanine synthase homologs, is concomitantly induced during ripening and implicates MdCASs in the possible role of the cyanide detoxification in Fuji apple (Malus domestica Borkh.) fruits

Fruit ripening involves complex biochemical and physiological changes. Ethylene is an essential hormone for the ripening of climacteric fruits. In the process of ethylene biosynthesis, cyanide (HCN), an extremely toxic compound, is produced as a co-product. Thus, most cyanide produced during fruit ripening should be detoxified rapidly by fruit cells. In higher plants, the key enzyme involved in the detoxification of HCN is β-cyanoalanine synthase (β-CAS). As little is known about the molecular function of β-CAS genes in climacteric fruits, we identified two homologous genes, MdCAS1 and MdCAS2, encoding Fuji apple β-CAS homologs. The structural features of the predicted polypeptides as well as an in vitro enzyme activity assay with bacterially expressed recombinant proteins indicated that MdCAS1 and MdCAS2 may indeed function as β-CAS isozymes in apple fruits. RNA gel-blot studies revealed that both MdCAS1 and MdCAS2 mRNAs were coordinately induced during the ripening process of apple fruits in an expression pattern comparable with that of ACC oxidase and ethylene production. The MdCAS genes were also activated effectively by exogenous ethylene treatment and mechanical wounding. Thus, it seems like that, in ripening apple fruits, expression of MdCAS1 and MdCAS2 genes is intimately correlated with a climacteric ethylene production and ACC oxidase activity. In addition, β-CAS enzyme activity was also enhanced as the fruit ripened, although this increase was not as dramatic as the mRNA induction pattern. Overall, these results suggest that MdCAS may play a role in cyanide detoxification in ripening apple fruits.

Allelic discrimination of the Restorer-of-fertility gene and its inheritance in peppers (Capsicum annuum L.).

Cytoplasmic male sterility (CMS), one of the most important traits in crop breeding, is used for commercial F1-hybrid seed production in peppers (Capsicum annuum L.). A nuclear gene, Restorer-of-fertility (Rf), can induce normal pollen production in CMS plants resulting in fertility. Since the first report of fertility restoration in peppers, various inheritance modes have been suggested, including the presence of a third haplotype of the locus. The pepper Rf gene has not been cloned, and calculated genetic distances of linked markers have varied between research groups. A more precise allelic test and additional genetic mapping are needed to accurately select recombinants for use in marker-assisted backcrossing (MAB). Therefore, the reliability and application of these markers for allelic selection of the Rf gene was tested. Two different F2 populations, Buja and Tamna, were used for the construction of a linkage map. From these linkage groups, a new closely linked flanking marker of the Rf gene were identified. Previous allelic testing revealed the existence of a third haplotype, Rfls7701, which can function as dominant (Rf) or recessive (rf). In a previous report, Rfls7701 was considered to be linked to unstable male sterility (MS). However, our results suggest that unstable MS was induced by a gene residing at another locus rather than by Rfls7701 haplotype-linked allele.

Identification of mitochondrial genome rearrangements unique to novel cytoplasmic male sterility in radish (Raphanus sativus L.)

A novel cytoplasmic male-sterility (CMS) radish (Raphanus sativus L.) and its associated mitotype (DCGMS) were previously identified; however, no mtDNA fragments flanking the atp6 gene were found in the DCGMS mitotype. Unlike three other mitotypes in this study, a unique mtDNA organization, atp6–nad3–rps12, was found to be the major mtDNA structure associated with this mitotype. This organization may have arisen from short repeat sequence-mediated recombination events. The short repeat clusters involved in the mtDNA rearrangement around the atp6 gene also exist as repetitive sequences in the complete mitochondrial genomes of other members of the Brassicaceae family, including rapeseed and Arabidopsis. These sequences do not exist as repetitive elements in the mtDNA of tobacco, sugar beet, or rice. While studying the regions flanking atp6, we identified a truncated atp6 mtDNA fragment which consists of the 5′ part of the atp6 gene linked to an unidentified sequence. This mtDNA structure was present in all mitotypes; however, a single nucleotide insertion mutation leading to a frame-shift was identified only in the DCGMS mitotype. Although this truncated atp6 organization was transcribed, there was no significantly different expression between male-sterile and fertile segregating individuals from the BC1F1 population originating from a cross between male-sterile and restorer parents. Comprehensive survey of the single base-pair insertion showed that it was maternally inherited and unique to the DCGMS mitotype. Therefore, this single nucleotide polymorphism (SNP) in the coding sequence of the mtDNA will be a useful molecular marker for the detection of the DCGMS mitotype.

Isolation and promoter analysis of anther-specific genes encoding putative arabinogalactan proteins in Malus × domestica

In this study, we searched for anther-specific genes involved in male gametophyte development in apple (Malus × domestica Borkh. cv. Fuji) by differential display-PCR. Three full-length cDNAs were isolated, and the corresponding genomic sequences were determined by genome walking. The identified genes showed intronless 228- to 264-bp open reading frames and shared 82–90% nucleotide sequence. Sequence analysis identified that they encoded a putative arabinogalactan protein (AGP) and were designated MdAGP1, MdAGP2, and MdAGP3, respectively. RT (reverse transcriptase)-PCR revealed that the MdAGP genes were selectively expressed in the stamen. Promoter analysis confirmed that the MdAGP3 promoter was capable of directing anther- or pollen-specific expression of the GUS reporter in tobacco and apple. Furthermore, expression of ribosome-inactivating protein under the control of the MdAGP3 promoter induced complete sporophytic male sterility as we had expected.