Min Jeong Hong

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.

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.

The effects of chronic gamma irradiation on oxidative stress response and the expression of anthocyanin biosynthesis-related genes in wheat (Triticum aestivum)

Abstract Purpose: To investigate the mechanisms of adaptation and tolerance to ionizing radiation using chronic radiation in wheat. Materials and methods: We exposed wheat plants to chronic gamma irradiation (50 Gy) for 2, 4, and 6 weeks and measured various biological parameters. Results: Plant height was reduced by exposure to gamma irradiation; this effect increased with increasing exposure time. Photosynthetic pigment levels decreased with increasing exposure time, while anthocyanin levels significantly increased after exposure to gamma rays. The activities of antioxidant enzymes (superoxide dismutase [SOD], ascorbate peroxidase [APX], catalase [CAT], and peroxidase [POD]) and malondialdehyde (MDA) levels increased with increasing duration of exposure to gamma irradiation. Electron spin resonance (ESR) signals were strongly detected in wheat that was gamma-irradiated for two weeks and then gradually decreased with increasing exposure time. The expression of anthocyanin biosynthesis genes (flavanone 3-hydroxylase [F3H], dihydroflavonol reductase [DFR], anthocyanin reductase [ANS], and UDPG-flavonoid glucosyl transferase [UFGT]) and sugar contents increased after exposure to gamma rays. Conclusions: This suggests that exposure to ionizing radiation according to increase of exposure time has led to efficient induction of anthocyanin and antioxidant enzyme activities. This study indicates that reactive oxygen species (ROS) is eliminated by biosynthesis of anthocyanin and antioxidant enzymes. This study helps elucidate the biological effects of various durations of low-dose exposure to chronic gamma radiation in wheat plants.

The effects of chronic radiation of gamma ray on protein expression and oxidative stress in Brachypodium distachyon.

Abstract Purpose: To compare the effects of gamma-irradiation on biochemical responses and growth, six-week-old Brachypodium plants were chronically exposed to gamma-irradiation for 30 days at various dosages. Materials and methods: Growth surveys of Brachypodium plants in response to different dosages of gamma-irradiation were conducted to compare physiological changes between irradiated and non-irradiated plants. Photosynthetic pigments, soluble sugar content, activities of antioxidant enzymes, and malonaldehyde (MDA) induced by reactive oxygen species (ROS) production were also measured. Results: Gamma-irradiation had a negative influence on the average plant height, leaf length, leaf width, and fresh weight. Photosynthetic pigment levels decreased with increasing dosages of gamma-irradiation, while soluble sugar content slightly increased. Gamma-irradiation responsive proteins were detected and identified by two-dimensional gel electrophoresis (2D-PAGE) and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF). The proteins had a role in photosynthetic carbon fixation, anabolic pathway glycolysis, mitochondrial ATP production, and oxidative stress response regulation. MDA levels and activities of antioxidant enzymes such as superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), and peroxidase (POD) increased with the increase in gamma-irradiation dosage level. Conclusions: This study provides some basic information regarding responses to gamma-irradiation, and provides valuable physiological and biological data on the effects of different gamma-irradiation dosages on Triticeae species.

Wheat F-box protein recruits proteins and regulates their abundance during wheat spike development

F-box proteins, components of the Skp1–Cullin1–F-box (SCF) protein E3 ubiquitin ligase complex, serve as the variable component responsible for substrate recognition and recruitment in SCF-mediated proteolysis. F-box proteins interact with Skp1 through the F-box motif and with ubiquitination substrates through C-terminal protein interaction domains. F-box proteins regulate plant development, various hormonal signal transduction processes, circadian rhythm, and cell cycle control. We isolated an F-box protein gene from wheat spikes at the onset of flowering. The Triticum aestivum cyclin F-box domain (TaCFBD) gene showed elevated expression levels during early inflorescence development and under cold stress treatment. TaCFBD green fluorescent protein signals were localized in the cytoplasm and plasma membrane. We used yeast two-hybrid screening to identify proteins that potentially interact with TaCFBD. Fructose bisphosphate aldolase, aspartic protease, VHS, glycine-rich RNA-binding protein, and the 26S proteasome non-ATPase regulatory subunit were positive candidate proteins. The bimolecular fluorescence complementation assay revealed the interaction of TaCFBD with partner proteins in the plasma membranes of tobacco cells. Our results suggest that the TaCFBD protein acts as an adaptor between target substrates and the SCF complex and provides substrate specificity to the SCF of ubiquitin ligase complexes.

Effect of gamma radiation on growth and lignin content in Brachypodium distachyon

Brachypodium distachyon has been highlighted as a model monocot plant with small genome and short life cycle. Biofuels are being developed as renewable energy sources to replace fossil fuels. Bioethanol production is negatively correlated with lignin content. Here, Brachypodium was acutely or chronically irradiated at doses of 50, 100, 150, 200, and 250 Gy. The effect of radiation on plant growth and generation of mutant populations was explored. The lethal effect of radiation was higher in acutely irradiated M0 populations. A dose-dependent negative effect in plant height, tiller number, floral spikelet, and total seed number was observed, with a positive effect in days to heading. The phenotype of 1,773 M1 plants was evaluated, with 417 plants being selected to construct the M2 population. The 31 M2 plants that showed the least staining with phloroglucinol were selected. These mutants could be useful materials for studies such as identification of nucleotide substitutions in genes involved in lignin biosynthesis pathway, monitoring of mutant physiological traits, and evaluation of fitness for bioethanol production. As biological resources, the M2 populations generated in this work will contribute to studies of functional genomics of Brachypodium and to the breeding of grass crops.

Employment of hordein subunit polymorphisms in establishing selection criteria for high quality malting barley (Hordeum vulgare L.)

Cereal seed storage proteins are encoded by complex multigene families and their subunit profiles are highly related to end-use qualities. Each fraction of albumin and hordein was extracted and its subunit profile was evaluated in related to malt and grain quality parameters. The purpose of this study was to provide selection criteria for high quality malting barley using grain and malt quality parameters and biochemical-genetic information. Grain and malt quality of 13 local adaptability test (LAT) lines were evaluated for malting process. A total of 16 germplasm accessions of high or low seed storage protein content were also evaluated for biochemicalgenetic analysis. The correlation coefficients among quality parameters were analyzed. Several important quality parameters in brewing process showed significant positive or negative correlations. Seed storage protein subunits of albumin and hordein of all tested lines and accessions were evaluated using 12% 1D SDS-PAGE. Scored data of protein subunit’s presence or absence was applied to Agglomerative Hierarchical Clustering (AHC) for statistical analysis and showed specific grouping patterns among tested lines. Clustered lines with subunit information were highly related with agricultural performance and grain and malt qualities. Based on the profiles of seed storage protein subunits, association of hordein subunit of 38, 43, and 65 kDa with high malt scored lines was found. The obtained results would provide improved selection criteria for high quality malting barley in the malting barley breeding program.

Interactions between wheat Tubby-like and SKP1-like proteins.

Tubby proteins are highly conserved in a wide range of multicellular organisms. The Tubby gene family was first verified in obese mice. In plants, 11 Tubby genes have been identified in Arabidopsis, 14 in rice, and 11 in poplar. However, there is very little information about Tubby-like proteins in wheat. In this study, we identified four Tubby-like protein genes (TaTULP1-TaTULP4) in wheat. A comparison of the gene structure showed a conserved exon number pattern in TaTULPs, although the length of the introns differed. With the exception of TaTULP2, TaTULPs had four exons. To identify the chromosome localization of TaTULPs, BLASTn analyses were performed using the URGI database to predict the chromosomal location of TaTULP genes. TaTULP1, 2, 3 and 4 genes were localized on chromosomes 4, 5, 7 and 2. All TaTULPs harbor a Tubby domain in their C-terminal region and an F-box domain in the N terminus. We investigated protein-protein interactions between the F-box domain of TaTULPs and various wheat SKP1-like proteins (TaSKPs) using the yeast two-hybrid system. TaTULP1, TaTULP3 and TaTULP4 were found to interact with TaSKP1, TaSKP3 and TaSKP6, whereas TaTULP2 showed no interaction with TaSKP proteins. TaTULP proteins tagged with green fluorescent protein were targeted to the Golgi apparatus in plant cells. Our analysis of TaTULPs will aid in understanding the functions of TaTULPs in plants.

Agrobacterium-mediated transformation of Brachypodium distachyon inbred line Bd21 with two binary vectors containing hygromycin resistance and GUS reporter genes

Brachypodium distachyon (Brachypodium) is a novel model plant for structural and functional genomic studies of temperate grasses. Brachypodium as a model plant has many favorable features, such as small size, small genome, short life cycle, self-fertility, and simple growth requirements. The genome sequence of the standard line Bd21 has been released and genomic resources have been developed.It is imperative to develop a method for efficient Agrobacterium-mediated Brachypodium transformation. Yellowish and compact embryogenic calli derived from immature embryos of the Bd21 were transformed with the Agrobacterium strain AGL1. Seven- and nine-week-old calli were used for transformation with Agrobacterium carrying either pCAMBIA 1301 and pCAUGH. Transformation efficiencies were assessed through histochemical GUS assay. The efficiency of transformation with pCAMBIA 1301 (based on the number of callus lines producing GUS-detected plantlets and the number of calli used for transformation) reached 20.1% (7-week-old calli) and 1.7% (9-week-old calli), and with pCAUGH (based on the number of GUS-detected plantlets and the number of regenerants) 90 and 87% for 7- and 9-week-old calli, respectively. High selection pressure was obtained by using pCAUGH, which is preferred for saving labor and time consumption during the callus selection.

Characterization of 4 TaGAST genes during spike development and seed germination and their response to exogenous phytohormones in common wheat

Gibberellic acid (GA) is involved in the regulation of plant growth and development. We defined GA-stimulated transcript (GAST) gene family and characterized its four members (TaGAST1, 2, 3, and 4) in wheat spikes. Triticum aestivum whole spikes were collected at ten developmental stages and dehulled spikelets were obtained at various days after flowering. Expression of TaGAST1, 2, 3, and 4 was analyzed using RT-PCR at inflorescence development stages, in different tissues, and after phytohormones application. To identify proteins interacting with TaGAST1, yeast two-hybridization was performed and BiFC analysis was used for verification. TaGAST1 was expressed at the inflorescence stage and only expressed in seedlings under abscisic acid (ABA) treatment after phytohormone treatment. TaGAST2 and TaGAST3 showed moderate expression in the spike, vigorous transcript accumulation in the seedling, and up-regulation by exogenous GA in early germination stages. TaGAST4 was predominantly expressed in the seedling. Wheat cyclophilin A-1 (TaCypA1), identified as a TaGAST1-interacting protein, showed opposite expression pattern in the developing spike to TaGAST1. TaCypA1 transcript was slightly up-regulated by GA, slightly down-regulated by paclobutrazol, and was maintained after ABA treatment. The interaction of TaGAST1 with TaCypA1 is targeted to the plasma membrane. TaGAST1 was specifically expressed in the wheat spike and was stimulated by exogenous GA treatment. TaGAST2 and TaGAST3 expression in germinating seeds and seedlings was higher than that in the spike stage. TaGAST4 was not expressed in all developmental stages. TaGAST1 and TaCypA1 might be expressed antagonistically during wheat spike development.