Chang Jae Oh

Organization of nif gene cluster in Frankia sp. EuIK1 strain, a symbiont of Elaeagnus umbellata

The nucleotide sequence of a 20.5-kb genomic region harboring nif genes was determined and analyzed. The fragment was obtained from Frankia sp. EuIK1 strain, an indigenous symbiont of Elaeagnus umbellata. A total of 20 ORFs including 12 nif genes were identified and subjected to comparative analysis with the genome sequences of 3 Frankia strains representing diverse host plant specificities. The nucleotide and deduced amino acid sequences showed highest levels of identity with orthologous genes from an Elaeagnus-infecting strain. The gene organization patterns around the nif gene clusters were well conserved among all 4 Frankia strains. However, characteristic features appeared in the location of the nifV gene for each Frankia strain, depending on the type of host plant. Sequence analysis was performed to determine the transcription units and suggested that there could be an independent operon starting from the nifW gene in the EuIK strain. Considering the organization patterns and their total extensions on the genome, we propose that the nif gene clusters remained stable despite genetic variations occurring in the Frankia genomes.

Postembryonic Seedling Lethality in the Sterol-Deficient Arabidopsis cyp51A2 Mutant Is Partially Mediated by the Composite Action of Ethylene and Reactive Oxygen Species

Seedling-lethal phenotypes of Arabidopsis (Arabidopsis thaliana) mutants that are defective in early steps in the sterol biosynthetic pathway are not rescued by the exogenous application of brassinosteroids. The detailed molecular and physiological mechanisms of seedling lethality have yet to be understood. Thus, to elucidate the underlying mechanism of lethality, we analyzed transcriptome and proteome profiles of the cyp51A2 mutant that is defective in sterol 14α-demethylation. Results revealed that the expression levels of genes involved in ethylene biosynthesis/signaling and detoxification of reactive oxygen species (ROS) increased in the mutant compared with the wild type and, thereby, that the endogenous ethylene level also increased in the mutant. Consistently, the seedling-lethal phenotype of the cyp51A2 mutant was partly attenuated by the inhibition of ethylene biosynthesis or signaling. However, photosynthesis-related genes including Rubisco large subunit, chlorophyll a/b-binding protein, and components of photosystems were transcriptionally and/or translationally down-regulated in the mutant, accompanied by the transformation of chloroplasts into gerontoplasts and a reduction in both chlorophyll contents and photosynthetic activity. These characteristics observed in the cyp51A2 mutant resemble those of leaf senescence. Nitroblue tetrazolium staining data revealed that the mutant was under oxidative stress due to the accumulation of ROS, a key factor controlling both programmed cell death and ethylene production. Our results suggest that changes in membrane sterol contents and composition in the cyp51A2 mutant trigger the generation of ROS and ethylene and eventually induce premature seedling senescence.

Isolation and Characterization of a Root Nodule-Specific Cysteine Proteinase cDNA from Soybean

We have determined that a nodule-specific cDNA clone (GmCysP1), obtained from a soybean root nodule-specific EST pool, encodes cysteine proteinase. Its amino acid sequence homology, as well as the conservation of typical motifs and amino acid residues involved in active site formation, shows that GmCysP1 can be classified as a legumain (C13) family cysteine proteinase, belonging to clan CD. Moreover, based on its expression patterns,GmCysP1 is a nodule-specific cysteine proteinase gene that is possibly associated with nodule development or senescence. Our genomic Southern analysis also suggests thatGmCysP1 is a member of a multigene family. Therefore, we propose that GmCysP1 is the first to be identified as a nodule-specific and senescence-related cysteine proteinase that belongs to the legumain family from soybean.

A Type-A chalcone isomerase mRNA is highly expressed in the root nodules ofElaeagnus umbellate

We have used the hybridization-competition method to isolateEuNOD-CHI from a root nodule cDNA library ofElaeagnus umbellate. This cDNA clone encodes chalcone isomerase (CHI) for a protein of 256 amino-acid residues and a mature molecular mass of 28 kDa. Multiple sequence alignment and phylogenetic analysis have demonstrated that EuNOD-CHI can be classified as Type I. Moreover, northern hybridization shows that theEuNOD-CHI gene is highly expressed in root nodules, with levels increasing during nodule development The highest level of expression is at 6 to 8 weeks after inoculation, decreasing thereafter. Genomic Southern hybridization also demonstrates thatEuNOD-CHI has as many as two copies in theE umbellate genome. Taken together with the previous results, we propose that the higher expression level of theEuNOD-CHI gene in root nodules is likely associated with this species’ defense mechanism against infection byFrankia.

Comprehensive analysis of AHL homologous genes encoding AT-hook motif nuclear Localized protein in rice

The AT-hook motif is a small DNA-binding protein motif that has been found in the high mobility group of non-histone chromosomal proteins. The Arabidopsis genome contains 29 genes encoding the AT-hook motif DNA-binding protein (AHL). Recent studies of Arabidopsis genes (AtAHLs) have revealed that they might play diverse functional roles during plant growth and development. In this report, we mined 20 AHL genes (OsAHLs) from the rice genome database using AtAHL genes as queries and characterized their molecular features. A phylogenetic tree revealed that OsAHL proteins can be classified into 2 evolutionary clades. Tissue expression pattern analysis revealed that all of the OsAHL genes might be functionally expressed genes with 3 distinct expression patterns. Nuclear localization analysis using transgenic Arabidopsis showed that several OsAHL proteins are exclusively localized in the nucleus, indicating that they may act as architectural transcription factors to regulate expression of their target genes during plant growth and development.

KiwiPME1 encoding pectin methylesterase is specifically expressed in the pollen of a dioecious plant species, kiwifruit (Actinidia chinensis)

Pectin methylesterases (PMEs) mediate demethylesterification of pectic polysaccharides such as homogalacturonan, a major component of the primary plant cell wall, in the apoplasm. The PMEs are implicated in a number of developmental processes, including pollen development and pollen tube growth, through the fine tuning of the methylesterification status of pectin. In this study, we isolated a full-length cDNA (KiwiPME1) encoding PME from kiwifruit and characterized its molecular features. Analyses of the primary protein structure and gene structure revealed that KiwiPME1 encodes a pre-pro-PME protein that is predicted to localize to the outside of the cell and belongs to group 2 (formerly type 1). The KiwiPME1 expression was highly detected in pollen grains of kiwifruit but not in vegetative tissues investigated. Expression pattern analysis of KiwiPME1 among different floral tissues of male and female plants revealed that KiwiPME1 was expressed specifically in the stamens of flower buds in male and female plants, whereas its expression was detected only in the stamens of male plants when the flowers opened. Expression analysis of KiwiPME1 promoter fused to the GUS reporter gene in Arabidopsis displayed a very similar pattern to that in kiwifruit. Our study suggests that the cell wall-localized KiwiPME1 is likely implicated in the pollen development and pollen tube growth of a dioecious species kiwifruit.

Molecular cloning of a cDNA encoding glutamine synthetase from root nodules ofElaeagnus umbellate

We analyzed a cDNA clone encoding cytosolic glutamine synthetase,EuNOD-GS1, isolated from a root nodule cDNA library ofElaeagnus umbellata. This clone has an insert size of 1359 bp and encodes a protein for 355 amino-acid residues, with a molecular weight of 39.2 kDa. Its expression is slightly higher in the root nodules than in the leaves or uninfected roots. Analysis of the deduced amino acid sequences and phytogeny revealed thatEuNOD-GS1 is clustered with cytosolic GS-α isoenzymes. Therefore, based on this and previous results, we propose that the main physiological role ofEuNOD-GS1 is the assimilation of ammonia from secondary and, in part, primary sources.

Molecular cloning and characterization of adr and ivd genes from Frankia EuIK1 strain

The nucleotide sequences of adrenodoxin reductase (adr) gene and isovaleryl-CoA dehydrogenase (ivd) gene and the expression pattern of adr from Frankia EuIK1 strain, symbiont of Elaeagnus umbellata, were determined. 5.5-kb NotI, 5.5-kb SacI, 1.3-kb SacI restriction fragments of pEuAR1, a cosmid clone hybridized with a squalene-hopene cyclase (shc) DNA probe, were subcloned and partially sequenced. Sequence analysis showed three fragments to overlap and harbor adr and ivd genes but not the targeted shc gene. The deduced amino acid sequence of AdR, consisting of 487 amino acids, showed sequence similarity of about 55% with other AdRs, and that of ivd, consisting of 384 amino acids, showed about 60% similarity with others. RT-PCR experiments revealed that the expression of adr was in low level at 6 weeks after inoculation (WAI), reached peak at 8 WAI, and decreased to some extent at 10 WAI. AdR is a probable redox partner of [2Fe–2S] ferredoxin involved in the biosynthesis of Fe/S cluster for cellular Fe/S proteins and also could be involved in nitrogen-fixation. It is the first report about adr and ivd in Frankia.