Yong Weon Seo

Selection of 5-methyltryptophan resistant rice mutants from irradiated calli derived from embryos

For increasing the contents of specificfree amino acids in rice (Oryzasativa L.) cultivar, Donganbyeo, mutantcell lines resistant to growth inhibitionby 5-methyltryptophan (5MT) were selectedfrom embryo cultured callus irradiated with50 Gy of gamma rays. Two lines, M2-1and M2-2, were obtained from theregenerated plants by selfing. Thesegregation ratios of resistance andsensitivity in these lines fitted 9:7 and3:1 ratios, respectively. Considering theagronomic traits, M2-1 was about thesame as the original variety in culm lengthand a little longer or higher in paniclelength and number of tillers. However, thefertility of M2-1 significantlyincreased compared to the originalvariety. M2-2 had an extremely highculm length and tillering capacity. Four5MT-resistant homozygous M3lines (MRclasses), M3-1-40 and M3-1-116from M2-1 (MR I),and M3-2-8 and M3-2-12from M2-2 (MR II), were obtained inthe same manner as with the M2generation. Protein contents of brown ricewere increased about 19% and 32% and the total contents of 9 free essentialamino acids were 71% and 34% greater thanthe original variety in the MR I andMR II groups, respectively. However, forthe free essential amino acids of polishedrice, there was no difference from theoriginal variety.

Development of AFLP and STS markers for identifying wheat-rye translocations possessing 2RL

Wheat-rye translocation line (2BS/2RL) has been developed for resistance to biotype L of Hessian fly and agronomically useful traits. AFLP analysis using 64 primer pairs was conducted in order to identify 2RL-specific polymorphisms between “Coker 797” (non-2RL), near-isogenic line (NIL) carrying 2RL, and “Hamlet”. Nine primer combinations identified twelve reproducible polymorphic fragments in the NIL carrying 2RL. These twelve fragments were cloned and sequenced with an aim towards converting AFLP markers into sequence tagged sites (STS). A comparison of the 12 sequences with non-redundant accessions in the NCBI database using the BLAST search option indicated that one fragment of approximately 200 bp in length (amplified using primer combination E+AAC / M+CTA) was highly homologous with the rye-specific repetitive sequence R173-1 and Wis-2-lA, a retrotransposon-like element in wheat. Two STS primers (SJ07 and SJ09) out of twelve STS primer sets enabled the detection of polymorphisms between Coker 797 and NIL carrying 2RL. In order to verify whether the polymorphism detected by primers SJ07 and SJ09 was in fact the result of the presence of 2RL, additional plant material was examined. Amplified products of about 260 bp fragment with the SJ07 primer set were generated in rye cvs.“Chilbohomil” and “Jochunhomil”, triticale experimental line Suwon 15, and wheat experimental line K-14 (1AL/1RS & 2BS/2RL), as well as NIL carrying 2RL and Hamlet, but not in Coker 797 (non-2RL), “Keumgangmil” (non-translocation wheat), KS92WGRC17 (PI592729 /;/ 6BS/6BL-6RL), KS92WGRC19 (P1592731 /;/ 4BS/4BL-6RL), “TAM200” (1AL/1RS), and “Siouxland” (1BL/1RS). Our data suggest that primer set SJ07 amplifies a “2RL-specific” fragment of diagnostic value.

Identification of Kunitz trypsin inhibitor mutations using SNAP markers in soybean mutant lines

The Kunitz trypsin inhibitor (KTi) in soybean has several polymorphic types that are controlled by multiple alleles, which behave in a co-dominant fashion. Of these, Tia and Tib, which differ by nine amino acids, are the predominant types. In order to develop a single nucleotide amplified polymorphism (SNAP) marker for the classification of the predominant KTi types, Tia and Tib, and evaluate KTi activities by differing KTi type total 451 soybean mutant lines (M12–M16 generation) were incorporated in this study. Among 451 soybean mutants, 144 and 13 mutant lines showed decreased and increased trypsin inhibitor activity when compared with the original cultivars, respectively. To identify the KTi type, we designed a SNAP marker. Among 451 mutant lines from 12 soybean cultivars and landraces, 8 mutant lines derived from cvs. Baekwoon, Paldal and Suwon115 showed a change in KTi type when compared with the original cultivars using the SNAP marker. Five mutant lines in Suwon115 changed from Tib to Tia, while two mutant lines derived from cv. Baekwoon and one mutant line derived from cv. Paldal were changed from Tia to Tib. These changes of KTi types were confirmed by sequencing of the KTi genes and non-denaturing polyacrylamide gel electrophoresis of the KTi proteins. To identify the effect of KTi activity based on the change in KTi type, we measured the KTi activity using the three cultivars and eight mutant lines that showed changes in KTi type. Two mutant lines (BW-1 and 7-2) derived from cv. Baekwoon and one mutant line (PD-5-10) from cv. Paldal that changed from Tia to Tib showed lower activity than the original cultivar. In cv. Suwon115, five mutant lines that changed from Tib to Tia showed higher activity than the original cultivar. These results indicate that the designed SNAP marker was capable of identifying the KTi type and that Tia activity was higher than Tib activity in soybean.

Evolution of non-specific lipid transfer protein (nsLTP) genes in the Poaceae family: their duplication and diversity.

Previously, the genes encoding non-specific lipid transfer proteins (nsLTPs) of the Poaceae family appear to evidence different genomic distribution and somewhat different shares of EST clones, which is suggestive of independent duplication(s) followed by functional diversity. To further evaluate the evolutionary fate of the Poaceae nsLTP genes, we have identified Ka/Ks values, conserved, mutated or lost cis-regulatory elements, responses to several elicitors, genome-wide expression profiles, and nsLTP gene-coexpression networks of both (or either) wheat and rice. The Ka/Ks values within each group and between groups appeared to be similar, but not identical, in both species. The conserved cis-regulatory elements, e.g. the RY repeat (CATGCA) element related to ABA regulation in group A, might be reflected in some degree of long-term conservation in transcriptional regulation postdating speciation. In group A, wheat nsLTP genes, with the exception of TaLTP4, evidenced responses similar to those of plant elicitors; however, the rice nsLTP genes evidenced differences in expression profiles, even though the genes of both species have undergone purifying selection, thereby suggesting their independent functional diversity. The expression profiles of rice nsLTP genes with a microarray dataset of 155 gene expression omnibus sample (GSM) plates suggest that subfunctionalization is not the sole mechanism inherent to the evolutionary history of nsLTP genes but may, rather, function in concert with other mechanism(s). As inferred by the nsLTP gene-coexpression networks, the functional diversity of nsLTP genes appears not to be randomized, but rather to be specialized in the direction of specific biological processes over evolutionary time.

Inverse relationship between boron toxicity tolerance and boron contents of barley seed and root

ABSTRACT A filter-paper bioassay method was used to investigate the differential response of 23 barley (Hordeum vulgare L.) genotypes to boron (B) toxicity. Two-day-old seedlings with equal radicles were treated with 10 (B10) or 100 (B100) ppm B for 10 d. Root and shoot growth was not affected by B10, but root growth was significantly reduced by B100. The shoot growth at B100 was either unaffected or affected to a smaller extent than the root. A significant inverse correlation was found between B content of seed and seed germination, and between root growth and B contents of root and shoot under B100. The barley genotypes with lower B contents in seeds had a higher germination, longer root length, and accumulated less B in roots and shoots when treated with B100. The B-tolerant genotypes with longer roots had lower B contents in their seed, root, and shoot and vice versa. These results suggest that a great variation exists among barley genotypes in response to high B application. There was no difference between naked (uncovered) and covered varieties in response to B100. The B tolerance could be attributed to the lower B content of seed and lower uptake or accumulation of B in the root and shoot.

SKP1-like-related genes interact with various F-box proteins and may form SCF complexes with Cullin-F-box proteins in wheat

S-phase kinase-associated protein 1 (SKP1), a core component of the SKP1–Cullin–F-box (SCF) E3 ubiquitin ligase complex, functions as an adaptor protein, connecting cullin and F-box proteins. SKP1 plays crucial roles in cell-cycle progression, transcriptional regulation, flower formation, signal transduction, and many other cellular processes. SKP1-like genes have been largely unstudied in wheat. Here, we isolated six wheat SKP1-like (TaSKP) genes from common wheat (Triticum aestivum) and analyzed the expression patterns of these six genes using reverse transcription-polymerase chain reaction (RT-PCR). Based on gene expression patterns, we divided the genes into two groups. Our data demonstrated that green fluorescent protein-tagged TaSKP proteins were targeted to the plasma membrane or cytoplasm in plant cells. In a yeast two-hybrid system, all TaSKP proteins interacted with TaCFBD, TaSKP1, and TaSKP5, while TaSKP6 interacted with RA and RLK. A BiFC assay suggested that specific combinations of TaSKP and F-box proteins may influence localization patterns in plant cells. TaSKP1, TaSKP5, and TaSKP6 interacted with TaCullin, while TaSKP2, TaSKP3, and TaSKP4 were not found to interact with TaCullin in the yeast two-hybrid system. This evidence indicated that some TaSKP proteins may have the ability to form SCF complexes. Taken together, these data suggested that TaSKP1, TaSKP5, and TaSKP6 proteins may act as a bridge between various F-box proteins and cullin proteins and that TaSKP genes may be involved in various growth and flower development processes.

Expressional diversity of wheat nsLTP genes: evidence of subfunctionalization via cis -regulatory divergence

Previously, the wheat non-specific lipid transfer proteins (TaLTP), members of a small multigene family, were reported to evidence a complex pattern of expression regulation. In order to assess further the expression diversity of the TaLTP genes, we have attempted to evaluate their expression profiles in responses to abiotic stresses, using semi-quantitative RT-PCR. The expression profiles generated herein revealed that the TaLTP genes in group A evidenced highly similar responses against abiotic stresses, whereas differential expression patterns among genes in each group were also observed. A total of seven promoters were fused to a GUS reporter gene and the recombinants were introduced into Arabidopsis, while three promoters evidenced non-detectible GUS activity. The promoters of TaLTP1, TaLTP7, and TaLTP10 included in group A drove strong expressions during plant development with overlapping patterns, in large part, but also exhibited distinct expression pattern, thereby suggesting subfunctionalization processing over evolutionary time. However, only trace expression in cotyledons, young emerged leaves, and epidermal cell layers of flower ovaries was driven by the promoter of TaLTP3 of group B. These results indicate that their distinct physiological functions appear to be accomplished by a subfunctionalization process involving degenerative mutations in regulatory regions.

Wheat truncated hemoglobin interacts with photosystem I PSK-I subunit and photosystem II subunit PsbS1

Recently, the truncated hemoglobin gene (trHb) was discovered in plant species, however, its role has not yet been determined. In this study, the gene expression of wheat trHb (TatrHb) was analyzed under various biotic and abiotic stresses. TatrHb transcript levels increased in NaCl-treated leaves and gibberellic acid (GA3)-treated roots. In addition, sodium nitroprusside (SNP), a nitric oxide donor, induced an increase in TatrHb transcript levels in roots and leaves. A yeast two-hybrid assay (YIIH) was used to screen a hypoxia-treated wheat seedling library with the goal of determining the putative function of TatrHb. In this YIIH assay, photosynthesis-related genes that showed high homology to the Hordeum vulgare chloroplast photosystem I PSK-I subunit and Zea mays photosystem II subunit PsbS1 were detected and their interactions with TatrHb were confirmed. Subcellular localization of a TatrHb-green fluorescent protein (GFP) fusion protein and bimolecular fluorescence complementation (BiFC) assay suggested that TatrHb is involved in photosynthesis. The TatrHb-GFP fusion protein was localized in the plastids and the yellow fluorescent protein signal indicated that the TatrHb protein interacted with PSK-I and PsbS1 in the chloroplast.

Biochemical characterization of a putative wheat caffeic acid O-methyltransferase

A wheat (Triticum aestivum L., near isogenic line of Hamlet) O-methyltransferase (OMT) was previously reported as a putative caffeic acid OMT (TaCOMT1), involved in lignin biosynthesis, based on its high sequence similarity with a number of graminaceous COMTs. The fact that the putative TaCOMT1 exhibits a significantly high sequence homology to another recently characterized wheat flavone-specific OMT (TaOMT2), and that molecular modeling studies indicated several conserved amino acid residues involved in substrate binding and catalysis of both proteins, prompted an investigation of its appropriate substrate specificity. We report here that TaCOMT1 exhibits highest preference for the flavone tricetin, and lowest activity with the lignin precursors, caffeic acid/5-hydroxyferulic acid as the methyl acceptor molecules, indicating that it is not involved in lignin biosynthesis. We recommend its reannotation to a flavone-specific TaOMT1 that is distinct from TaOMT2.

cDNA-AFLP analysis reveals differential gene expression in response to salt stress in Brachypodium distachyon

Environmental stresses such as drought, salinity, cold, and heat negatively affect the growth of plants and productivity of crops. The mechanism of salt tolerance is one of the most important fields in plant science, and our understanding of this process must be improved in order to increase agricultural crop production. In our study, we identified salt stress-responsive transcripts using the cDNA-AFLP technique. The obtained transcripts were further analyzed by semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) under various abiotic stresses and hormone treatments. Among 87 transcript-derived fragments (TDFs) that were classified based on their presence or absence (qualitative variants) or differential expression (quantitative variants), we identified 32 TDFs that corresponded to Brachypodium genes with locus name. These clones are involved in various molecular functions and have transferase, protein binding, nucleotide binding, transporter, protein kinase, catalytic, hydrolase, RNA binding, and enzyme regulation activities. Further, the expression patterns of up-regulated 9 salt stress-response genes in cDNA-AFLP experiments were evaluated through semi-quantitative RT-PCR. Those genes were involved in signaling cascades [the casein kinase I (CKI)-like protein], regulation of enzyme activity [protein phosphatase 2C (PP2C) gene], phospholipid asymmetry [aminophospholipid ATPase (ALA)], cellular ion homeostasis [calcium-transporting ATPase, potassium transporter, calcium-binding protein (CBP)], and plant growth and development [pentatricopeptide repeat-containing protein (PPRP), 2-oxoglutarate (2OG) and Fe(II)-dependent oxygenases] and the putative roles of the identified TDFs involved in salt stress mechanisms are discussed. A better understanding of the mechanisms of salt stress tolerance and salt stress response genes in Brachypodium would be very useful for the breeding and genetic engineering of salt tolerance varieties in other Poaceae families, including wheat, barley, and rice.