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Quinoa is one of the crops well-adapted to high altitude regions that can grow relatively well under drought, humid, and high UV radiation conditions. This study was performed to investigate the effects of gamma-radiation on quinoa. Seeds were treated with various doses of 50 Gy, 100 Gy, 200 Gy, 300 Gy, 400 Gy, 600 Gy, 800 Gy, and 1000 Gy. We investigated germination, as well as plant height, chlorophyll content, and normalized difference vegetation index (NDVI) at 0, 30, 44, 58, and 88 days after transplanting (DAT) and panicle weight at 88 DAT. The plants grown from the seeds treated at radiation doses greater than 200 Gy showed reduced values in most growth and physiological characteristics. The germination rate and germination speed were higher in the 50 Gy-treated seeds than in 0 Gy-treated (control) seeds. Plant height and panicle weight were highest in the plants from 50 Gy-treated seeds. Chlorophyll content was higher in all treated samples than in the controls. NDVI value showed the highest value in 0 Gy controls and plants treated with 50 Gy. The antioxidant activity was also higher in the plants from the seeds treated with 50 Gy and 100 Gy, showing a steady increase as the radiation dose increased even at 200 Gy. The plants from seeds treated with 0 Gy showed higher expression of proteins related to photorespiration and tubulin chains. The plants from seeds treated with 50 Gy induced more stress-responsive proteins.

Soybean seed includes various bioactive substances. Also, they contain a variety of antinutritional factors including lipoxygenase, Kunitz trypsin inhibitor (KTI), lectin, and 7S α' subunit proteins. The genetic removal of these proteins will improve the nutritional value of soybean seed. The objective of this research was to breed new soybean with tetra recessive alleles (lox1lox2lox3/lox1lox2lox3‐ti/ti‐le/le‐cgy1/cgy1) for lipoxygenase, KTI, lectin, and 7S α' subunit proteins. Seven parents were used to breed tetra null strain. SDS‐PAGE and Western blot analysis were used to determine the presence or absence of lipoxygenase, 7S α' subunit, KTI, and lectin proteins in mature seed. Tetra null soybean line has a purple flower, determinate growth habit, tan pod, and yellow seed coat colour. Stem height of the breeding line was 62.3 cm. The 100‐seed weight of the breeding line was 27.1 g and yield (t/ha) was 2.84. This is the first soybean strain with lox1lox2lox3/lox1lox2lox3‐ti/ti‐le/le‐cgy1/cgy1 genotype (absence of lipoxygenase, KTI, lectin, and 7S α' subunit proteins).

The mutant allele of rice calmodulin-binding transcription activator OsCBT, oscbt-1, exhibits broad-spectrum resistance against rice pathogens. Previously, we reported that the strong resistance of the oscbt-1 mutant to pathogens was conferred by a constitutive upregulation of defense-related genes even under pathogen-free conditions. We also found strong induction of the hypersensitive response as a reaction to pathogen invasion. The results suggest that OsCBT acts as a negative regulator of basal resistance to pathogen attack. To identify the transcriptional network regulated by OsCBT, we compared global gene expression profiles between wild-type (WT) and oscbt-1 rice plants grown under pathogen-free conditions. The results of a 3′ tiling microarray revealed that in oscbt-1 plants, 81 genes are upregulated and 200 genes are downregulated when compared with the WT. A gene ontology analysis showed that differentially regulated genes in oscbt-1 were very closely associated with “death” GO term in a biological process category, and “catalytic activity” and “binding” GO terms in a molecular function category. A MapMan analysis indicated that the functions of these genes were associated with plant responses to biotic stress. Moreover, the results from quantitative real-time PCR in the oscbt-1 mutant showed a significant effect on the gene expression patterns of the fungal pathogen response. Our results suggested that the OsCBT regulates a rice defense response by modulating the expressions of various defense-related genes.

Soybean [Glycine max (L.) Merr.] seed is an important dietary source of protein, oil, carbohydrate, isoflavone and other various nutrients for humans and animals. However, there are anti-nutritional factors in the raw mature soybeans. Kunitz trypsin inhibitor (KTI) protein and stachyose are the main anti-nutritional factors in soybean seed. The α′-subunit of β-conglycinin protein exhibit poor nutritional and food processing properties. The genetic removal of the KTI and α′-subunit proteins will improve the nutritional value of the soybean seed. The objective of this research was to develop a new soybean strain with KTI and α′-subunit protein free (titicgy1cgy1 genotype) and proper agronomic traits. A breeding population was developed from the cross of the Bl-1 and 15G1 parents. A total of 168 F2 seeds from the cross of the BL-1 and 15G1 parents were obtained. The segregation ratios of 9: 3: 3: 1 (104 Ti_Cgy1_: 30 Ti_cgy1cgy1: 21 cgy1cgy1Ti_: 13 titicgy1cgy1) between the Ti and Cgy1 genes in the F2 seeds were observed (χ 2= 5.12, P=0.5-0.10). Two F4 plant strains with proper agronomical traits and titicgy1cgy1 genotype (free of both KTI and α′ -subunit protein) were selected and harvested. 2 strains (S1 and S2) had yellow seed coats and hilum. The plant height of the S1 strain was 65 centimeters. The 100-seed weight was 29.2 g. The plant height of the S2 strain was 66 centimeters and 100-seed weight was 26.2 g. The two strains selected in this research will be used to improve the new cultivar that will be free of the KTI and α′-subunit proteins.

Global warming has an impact on crop growth and development. Flowering time is particularly sensitive to environmental factors such as day length and temperature. In this study, we investigated the effects of global warming on flowering using an open-top Climatron chamber, which has a higher temperature and CO2 concentration than in the field. Two different soybean cultivars, Williams 82 and IT153414, which exhibited different flowering times, were promoted flowering in the open-top Climatron chamber than in the field. We more specifically examined the expression patterns of soybean flowering genes on the molecular level under high-temperature conditions. The elevated temperature induced the expression of soybean floral activators, GmFT2a and GmFT5a as well as a set of GmCOL genes. In contrast, it suppressed floral repressors, E1 and E2 homologs. Moreover, high-temperature conditions affected the expression of these flowering genes in a day length-independent manner. Taken together, our data suggest that soybean plants properly respond and adapt to changing environments by modulating the expression of a set of flowering genes in the photoperiod pathway for the successful production of seeds and offspring.

Microglia plays a critical role in the brain and protects neuronal cells from toxins. However, over-activation of microglia leads to deleterious effects. Lipopolysaccharide (LPS) has been reported to affect neuronal cells via activation of microglia as well as directly to initiate neuroinflammation. In the present study, we evaluated the anti-inflammatory and anti-oxidative effect of anthocyanins against LPS-induced neurotoxicity in an animal model and in cell cultures. Intraperitoneal injections of LPS (250 μg/kg/day for 1 week) induce ROS production and promote neuroinflammation and neurodegeneration which ultimately leads to memory impairment. However, anthocyanins treatment at a dose of 24 mg/kg/day for 2 weeks (1 week before and 1 week co-treated with LPS) prevented ROS production, inhibited neuroinflammation and neurodegeneration, and improved memory functions in LPS-treated mice. Both histological and immunoblot analysis indicated that anthocyanins reversed the activation of JNK, prevented neuroinflammation by lowering the levels of inflammatory markers (p-NF-kB, TNF-α, and IL-1β), and reduced neuronal apoptosis by reducing the expression of Bax, cytochrome c, cleaved caspase-3, and cleaved PARP-1, while increasing the level of survival proteins p-Akt, p-GSK3β, and anti-apoptotic Bcl-2 protein. Anthocyanins treatment increased the levels of memory-related pre- and post-synaptic proteins and improved the hippocampus-dependent memory in the LPS-treated mice. Overall, this data suggested that consumption of naturally derived anti-oxidant agent such as anthocyanins ameliorated several pathological events in the LPS-treated animal model and we believe that anthocyanins would be a safe therapeutic agent for slowing the inflammation-induced neurodegeneration in the brain against several diseases such as Alzheimer's disease and Parkinson's disease.

Soybean [Glycine max (L.) Merr.] is an important source for protein, oil, carbohydrates, isoflavones, and many other nutrients to humans and animals. Lipoxygenase protein is the main antinutritional factor in mature soybean seed. Soybean cultivar, "Choseon" with yellow medium seed size and free of lipoxygenase 2,3 protein was developed. "Choseon" was selected from the population derived from the cross between cultivar, "Jinpum 2" and germplasm, PI408155. "Choseon" has purple flowers, tawny pubescence, a determinate growth habit, and light yellow pods at maturity. The seed of "Choseon" has yellow hilum and yellow seed coat color. Seed protein and oil content on a dry weight basis were 34.2 and 16.9%, respectively. "Choseon" has shown a resistant reaction to soybean necrosis, soybean mosaic virus, Cercospora leaf spot and blight, black root rot, pod and stem blight, and bacterial pustule. "Choseon" matured in 5 October with plant height of 51 cm and a 100-seed weight of 15.1 g. Average yield of "Choseon" was 2.66 (Ton/ha) on the regional yield trials of 2013 at 4 locations. "Choseon" has been registered as a soybean cultivar (registration number: 5985, registration date: April 11, 2016) by Korea Seed & Variety Service, Republic of Korea.

Anthocyanins from the black soybean seed coat are known to have many pharmaceutical effects. However, black soybean seed contains antinutritional factors such as lipoxygenase, Kunitz trypsin inhibitor (KTI), lectin, and stachyose. The genetic removal of these components will improve the nutritional value of black soybean seed. The objective of this research was to breed a soybean strain with the black seed coat color, green cotyledon color, and tetra recessive allele (lox1lox2lox3/lox1lox2lox3-ti/ti-le/le-rs2/rs2) for lipoxygenase, KTI, lectin, and stachyose components. Eight parents were used to breed the tetra null strain. Analysis of lipoxygenase, KTI, lectin, and stachyose components in mature seeds was conducted by SDS-PAGE, Western blot, and HPLC. The soybean line with the black seed coat color, the green cotyledon color, a large seed size, and tetra recessive alleles has purple flowers, a determinate growth habit, and brown pods at maturity. The stem height of the breeding line was 52.3 cm. The 100-seed weight of the breeding line was 35.2 g and the yield (Ton/ha) was 2.50. The stachyose content of the breeding line was 3.30 g/kg. This is the first soybean strain with the black seed coat color, the green cotyledon color, a large seed size, and tetra null alleles (lox1lox2lox3/lox1lox2lox3-ti/ti-le/le-rs2/rs2, low content of stachyose, free of lipoxygenase, KTI, and lectin proteins).

Trampling stress in turfgrass fields caused by traffics often occurs in zoysiagrass (Zoysia japonica) fields including golf course. In order to know the influences of trampling stress on the growth and development of turfgrass, leaf and root growth, chlorophyll fluorescence, chlorophyll content and 2-DE protein analysis were conducted in the turfgrass plants subjected to various levels of trampling stress from 0 to 9,420 J day -1 day. Shoot growth was more highly inhibited by trampling stress than root growth. Although root growth was affected by trampling with weak intensity, the highest root length was observed in the turfgrass treated with weak trampling (1,570 J day -1 ). Chlorophyll fluorescence (Fv Fm -1 ) was drastically lowered by trampling stress with moderate intensity. Leaf number showed similar tendency with leaf greenness. The number was decreased as the trampling intensity was increased. Shoot dry weight was decreased showing a similar tendency with the result of shoot length. The specific protein expressions under weak trampling were related to the functions of stress amelioration. Heat shock 70 kDa protein 10 and oxygen-evolving enhancer protein 1 were the proteins increased positively under trampling stress.

Soybean is the main source of protein and oil for human and animal nutrition. However, antinutritional factors in the raw mature soybean are exist. Lipoxygenase protein is the main antinutritional factor in mature soybean seed. A new soybean cultivar, ‘Jinyang’ with yellow seed coat and free of lipoxygenase 1,2,3 protein was developed. It was selected from the population derived from the cross between ‘Jinpumkong2ho’ and GS301. ‘Jinyang’ has purple flowers, tawny pubescence, a determinate growth habit, oval leaflet shape, and light brown pods at maturity. The seed has yellow hilum and yellow seed coat color. Seed protein and oil content on a dry weight basis were 33.5% and 16.6%, respectively. It has shown a resistant reaction to soybean necrosis, soybean mosaic virus, cercospora leaf spot and blight, black root rot, pod and stem blight, and soybean pod borer. ‘Jinyang’ matured in 1 October with plant height of 65cm and a 100-seed weight of 21 g. Average yield of ‘Jinyang’ was 300 - 330 kg/10a in 2010 - 2012. ‘Jinyang’ has been registered as a new soybean cultivar (registration number: 4279, registration date: Dec. 28, 2012) by Korea Seed & Variety Service, Republic of Korea.

Polycyclic aromatic hydrocarbons (PAHs) are a group of ubiquitous hazardous pollutants derived from fossil fuel, various combustion sources and pyrolysis of a wide range of plastics. Because PAHs can be uptake into crop plants, the inhibitory effects on rice and soybean plants were examined in greenhouse and growth chamber experiment. Soil-applied PAHs (phenanthrene of 0, 10, 30, 100 ppm) slightly reduced the plant height and dry weight both in transplanted rice and soybean plant. The inhibitory effect on growth was greater in soybean than rice. Plant height of soybean plants treated by 100 ppm was 58.9 cm and this value was 87.2% of untreated plant. In rice plant, the plant height was less inhibited (96.0% of untreated plant) by 100 ppm at 80 days after treatment (DAT). However, leaf chlorophyll content and chlorophyll fluorescence were less inhibited by PAHs at late growth stage (after heading) although the photosynthesis-related parameters were slightly inhibited from 20 DAT to 70 DAT. In agar medium experiment with infant seedlings, inhibition of seedling length and fresh weight by phenanthrene at 100 ppm were greater as compared to the experiment with adult plant in pot. Seedling length and fresh weight were reduced by 54.2% and 33.3% for rice and 27.9% and 13.2% for soybean, respectively. The results reflected that PAHs were more inhibitory during juvenile stage than adult stage and more inhibitory to rice plant than soybean for juvenile stage.

농업생명과학연구원The quality characteristics and antioxidant activity of three kinds of tofu made from lipoxygenase (LOX)-free genotypes soybeans were compared to the Taekwang (LOX-present) tofu as the control. The mineral contents of Jinyang tofu were significantly higher than those of the control. The hardness and gumminess of tofu made from LOX-free genotypes were significantly higher than those of the control. Gaechuck#2 tofu showed higher sensory evaluation results than the other kinds of tofu in terms of taste, flavor and overall acceptability. The total isoflavone contents were higher in the LOX-free genotypes than in the control. The total phenol content was similar for the Gaechuck#1, #2 and Taekwang tofu. The flavonoid content was higher in Gaechuck#1 and #2 tofu than in the control. The antioxidant activities were the highest in Gaechuck#1 tofu, followed by the Gaechuck#2. During the 15-day storage at 4, the turbidity of the immersing water tended to increase, but Gaechuck#1 and #2 tofu were significantly lower than in the control after 15-day storage. Therefore it is suggested that Gaechuck#2 tofu could be the suitable genotype for tofu products because it is the most effective in terms of overall acceptability, antioxidant activity, and storage stability. Key words tofu, lipoxygenase-free genotypes, isoflavone, antioxidant activity

The properties of non-GM soybeans with lipoxygenase (LOX) free genotypes, such as Gaechuck#1 (black, LOX2,3-free), Gaechuck#2 (yellow, LOX2,3-free) and Jinyangkong (yellow, LOX1,2,3-free), were compared to Taekwangkong (yellow, LOX1,2,3-present) as control. Length and weight of soybeans were significantly longer in Gaechuck#1, but LOX free genotypes of yellow beans was smaller than Taekwangkong, in terms the size and weight. After soaking for 12 hr, increased ratios of soybean weight were 217.07～226.55%, and they were significantly higher in Gaechuck#2. The total color difference (ΔE) to Taekwangkong was the highest in Gaechuck#1, but another yellow soybean were similar. Crude lipids content was significantly higher in Taekwangkong (17.30%), and these LOX free genotypes (13.71～14.66%) did not show significant difference. Crude protein contents were significantly higher in Gaechuck#1 (34.79%) compared to the other soybeans (32.93～33.47%). Contents of glutamic acid among the detected composition amino acid were highest, and its total contents were highest in Gaechuck#1 (32.42 g/100 g), and the total content of Gaechuck#2 was similar to Taekwangkong. Contents of total isoflavone were higher in LOX free genotypes than Taekwangkong, especially significantly higher in Gaechuck#1 (1672.54 μg/g). Contents of total phenol were highest in Gaechuck#1 (276.91 mg/100 g), and flavonoids content of Gaechuck#1 (26.93 mg/100 g) was 2.6～2.8 times higher than the other samples. DPPH, ABTS radical scavenging activities and reducing power were significantly higher in Gaechuck#1. The yield of soybean curd from tested samples was the highest in Gaechuck#2. Gaechuck#2 as LOX free genotypes was similar to Taekwangkong with regard to physicochemical properties and antioxidant activities. Therefore, we propose that Gaechuck#2 is a more suitable genotype for soybean curd products.

Anthocyanins are known to have antioxidant and antiinflammatory effects. We hypothesized that anthocyanins would enhance wound healing in Sprague-Dawley rats. The purpose of this study was to evaluate our hypothesis and investigate the mechanism of wound healing enhancement. The cytoprotective effect of an immortalized epidermal keratinocyte cell line (HaCaT) and human neonatal dermal fibroblasts in response to various concentrations of anthocyanins was determined. Vascular endothelial growth factor (VEGF) and thrombospondin 1 (TSP1) of HaCaT were measured by Western blot analysis. Anthocyanins were applied to the wounds in rats, and the healing ratio was calculated. Tissue VEGF, TSP1, CD31, nuclear factor-κB, and phosphorylation of IκBα were measured. The viability of the HaCaT cell line and human neonatal dermal fibroblasts increased under cytotoxicity by H2O2 in the anthocyanin-treated groups. The VEGF in the anthocyanin-treated groups increased, whereas TSP1 decreased. Wounds in the experimental groups healed faster, and VEGF and CD31 increased in the experimental groups, whereas TSP1 decreased. Anthocyanins inhibited the translocation of nuclear factor-κB (p65) from cytosol to nucleus and also prevented the phosphorylation of IκBα. Anthocyanins enhance wound healing through a cytoprotective effect, enhancement of angiogenesis, and an antiinflammatory effect.

Soybean cyst nematode (Heterodera glycines Ichinohe) is one of the serious soybean [Glycine max (L.) Merr.] pests in major soybean producing countries. The objective of this study was to investigate of Heterodera glycines type using the five SCN infested soybean field soils and was to evaluate resistance to the soybean cyst nematode HG 2.5.7 type on soybean varieties and germplasms. The five SCN contaminated soil samples were collected from the three provinces on November 2011 in Korea, and eggs were cultured on early spring season in 2012. For the second study, a total fifty nine soybean varieties and germplasms were tested by infestation of HG type 2.5.7 in the greenhouse. Soybean cyst nematode HG types were investigated from five locations, HG 2 (race 1) type at Donghae, HG 2.5 (race 1) type at Jeongseon and Hapcheon, HG type 2.5.7 (race 1 or 5) at Yeongwol, and HG 1.2.7 (race 5) type at Haenam locations in present study. No Korean soybean varieties and germplasms were observed with SCN resistant trait to the HG type 2.5.7. Average SCN female index were calculated with 82.7% in 59 plant materials. Our results could be provided useful information to develop a SCN resistant cultivar in Korea.

BACKGROUND Soybean sprouts (Kongnamool) are one of the most popular and nutritive traditional vegetables in East Asia. Anthracnose caused by Colletotrichum gloeosporioides is one of the most serious diseases of soybean sprouts. In order to obtain basic information for breeding and/or selecting soybean genotypes with increased natural defense against anthracnose, phenolic compounds were profiled for healthy and infected soybean (Glycine max Merr.) sprouts by using high-performance liquid chromatography coupled with tandem mass spectrometry. RESULTS Tryptophan and eight phenolic compounds (daidzin, genistin, malonyldaidzin, malonylgenistin, daidzein, glycitein, genistein and coumestrol) were determined from healthy and inoculated sprouts. Total identified phenolic content was 40.02 ± 0.03 mg kg−1, 99.4% of which was isoflavones. CONCLUSION The monitoring suggested that de novo induced glycitein appeared to act as a phytoalexin in the defence mechanism of the soybean sprouts against C. gloeosporioides, and constitutively formed seven phenolic components that functioned as phytoanticipins in the diseased soybean sprouts. © 2013 Society of Chemical Industry

dlm mutant newly identified displays necrotic spots with chlorotic halo despite the absence of any detectable pathogens in soybean. Kunitz trypsin inhibitor (KTI) protein of mature soybean seed is a main anti-nutritional factor and is controlled by Ti allele in soybean seed. The objective of this research was to determine the linkage or independent assortment between dlm allele and ti allele. F2 population was made by crossing between "Gaechuck2ho" (DlmDlmtiti) and T363 (dlmdlmTiTi) parents. The normal leaf or disease lesion mimic leaf trait of parents and F2 individual plants was recorded at R2 and R3 growth stage from field. The random seeds from individual F2 plants harvested were analysed electrophoretically to determine the presence of the KTI protein. The disease lesion mimic trait was matched to 3 : 1 ratio at the 0.01 levels of probability. The absence of the KTI protein was controlled by a single recessive gene. In F2 generation, the ratios of 102 Ti_Dlm_: 21 Ti_dlmdlm : 24 titiDlm_ : 4 titidlmdlm were observed. Segregation ratio gave a good fit to the 9 : 3 : 3 : 1 ratio based on F2 generation at the 0.01 levels of probability. The data confirmed that dlm allele controlling disease lesion mimic trait was inherited independently with the ti allele controlling absence of KTI protein in soybean.

Native of soybean aphid (Aphis glycines Matsumura) is an Asia and aphid is one of the dangerous pests in soybean [Glycine max (L.) Merr.]. High density aphid populations can reduce crop production by causing severe damage. The objective of this study was evaluation of resistance to the soybean aphid in soybean cultivars and germplasms. A total of fifty five soybean cultivars or germplasms, including two susceptible and two resistant check varieties, were infested to evaluate their resistance in the field cage and greenhouse test by aphid colonies which derived from wild collected one soybean aphid biotype in Korea. The average number of reproduced soybean aphid was evaluated with 62.7 aphids in the resistant check variety PI 567598B and also estimated with 1,807 aphids for susceptible check variety Williams 82. In soybean varieties and germplasms, the average reproduced soybean aphid populations ranged from the lowest 497 aphids for Junjeori to the highest 3,862 aphids for Mansu. About seventy six percent of soybean cultivars and germplasms were shown high density soybean aphid populations when compared with another susceptible check variety PI 567543C in the field cage test. From the greenhouse test to evaluate aphid index, 87.3% of soybean cultivars or germplasms presented aphid index with 9.0. No soybean cultivars and germplasms were observed with soybean resistant phenotype when regarded a aphid resistant level as less than 10% aphid reproductions compared with susceptible check Williams 82. Although no Korean soybean cultivars were identified with resistant trait to the soybean aphid, we found one great resistant genetic resource PI 567598B in this study. This result will be helpful to further study for providing useful genetic information for soybean researchers.

P34 has long been known as one of major allergenic proteins in soybean. Recently, two low-P34 soybean accessions, PI603570A and PI567476, were identified. In order to understand the molecular mechanism of a significant reduction of P34 protein level in PI567476, we performed comparative analysis of the P34 cDNAs and genomic sequences from low-P34 and normal soybean accessions. While no sequence variation was detected in P34 coding regions of the soybean accessions tested, the P34 gene from PI567476 contains ‘ATGT’ 4-bp insertion in front of the start codon. We also found the length polymorphism of ‘TA’ repeats in the P34 promoter regions among accessions. Northern and western blot analysis revealed that, even though mRNA levels of P34 are similar in low-P34 and normal soybean accessions, lower accumulation of P34 protein is only detected in the low-P34 accession, suggesting that the genetic polymorphisms in P34 promoters have a more significant effect on translation efficiency than transcription of the P34 gene. Transient expression analysis showed that the P34 promoter of PI567476 has significantly reduced activity compared to that of normal accession, cv. Clark. In addition, the chimeric promoter generated by introducing the 4-bp insertion in front of the start codon of the P34 gene from normal Clark accession showed markedly reduced activity. The results suggest that lower accumulation of P34 protein in low-P34 accession is largely due to the reduced translation efficiency caused by the 4-bp insertion in the P34 promoter, thus providing direct evidence for the molecular mechanism to explain the regulation of P34 gene expression in various soybean accessions.

Ischemia-reperfusion injury is a phenomenon that occurs when tissues are subjected to ischemia for a variable period of time, and then reperfused. Inflammatory reaction has been implicated as one of the most important mechanism of ischemia-reperfusion injury. The purpose of this study was to evaluate the anti-inflammatory effects of anthocyanins from black soybean seed coat on keratinocytes in vitro and ischemia-reperfusion injury in vivo. We investigated the inhibition, by anthocyanins, of the expression of various inflammatory genes associated with ischemia-reperfusion injury in the tumor necrosis factor-alpha-treated (TNF-α) immortalized epidermal keratinocyte cell line (HaCaT). We also investigated the effects of anthocyanins on the survival of skin flaps after ischemia-reperfusion injury in the rats. According to Western blot analysis and a luciferase activity assay, anthocyanins inhibited TNF-α-induced intercellular adhesion molecule-1 and cyclooxygenase-2 (COX-2) levels through the NF-κB-dependent pathway. Administration of anthocyanins (50 and 100 mg/kg) significantly improved the flap area survival in the 10-hour ischemic model from 62% to 74.5% and 83%, respectively (P = 0.001). The related cytokines in skin flap also changed as the same pattern as in vitro. Our results indicate that anthocyanins from black soybean seed coat had anti-inflammatory effects on the HaCaT cell line and increase the survival of skin flaps through anti-inflammatory properties against ischemia-reperfusion injury.

Phospholipids (PLs) in 57 varieties of soybeans were profiled by high-performance liquid chromatography–electrospray ionization-tandem mass spectrometry and principal component analysis (PCA) to discriminate PL-rich soybeans. The PL calibration curves showed linearity with correlation coefficients >0.9964. The recoveries at 5 mg/L spiked level ranged from 72.8 to 86.7% and those at 12.5 mg/L from 78.2 to 85.1%. The repeatability at a 5 mg/L spiked level ranged from 2.5 to 7.0% and those at 12.5 mg/L from 1.2 to 3.9%. The average total PL content in the 57 soybean varieties was about 35.3 mg/kg. The total PL content was the highest in Aodaiz (35, 48.7 ± 1.4 mg/kg) and the lowest in Poongsannamul (56, 16.0 ± 0.7 mg/kg). The PCA showed that RS-78sun (42), Gyeongsang #1 (3) and Aodaiz (35) are the most improved varieties of the investigated 57 varieties from the viewpoint of PL content. Copyright © 2012 John Wiley & Sons, Ltd.

Soybean (Glycine max (L.) Merr.) protein is a high quality source for food and feed. But, antinutritional factors in the raw mature soybean are exist. Kunitz trypsin inhibitor (KTI) protein is a main antinutritional factor in soybean seed. Also, P34 protein, referred as Gly m Bd 30K, has been identified as a predominant immunodominant allergen. Genetic relationship between KTI protein and P34 protein could be useful in soybean breeding program for the genetic elimination or reduction of these factors. The objective of this study was to determine the independent inheritance or linkage between KTI protein and P34 protein in soybean seed. A total of 479 F2 seeds were obtained from the cross of 07B1 and PI567476 parents. KTI protein and relative amount of P34 protein were analysed from F2 seeds harvested from the F1 plants by using SDS-PAGE and Western blot analysis. The segregation ratios of 3 : 1 for KTI protein (353 KTI protein present : 126 KTI protein absent) and relative amount of P34 protein (363 normal amount of P34 protein : 116 low amount of P34 protein). The segregation ratio of 3 : 1 suggested that KTI protein and relative amount of P34 protein in mature soybean seed were controlled by a single major gene. The segregation ratios of 9 : 3 : 3 : 1 (266 KTI protein present, normal amount of P34 protein: 88 KTI protein present, low amount of P34 protein: 102 KTI protein absent, normal amount of P34 protein: 23 KTI protein absent, low amount of P34 protein) and Chi-square value (χ 2 =3.31, P=0.346) were observed in F2 seeds. This data showed that KTI protein was inherited independently with relative amount of P34 protein in soybean. These results will be helpful in breeding program for selecting the line with lacking KTI protein and reduced amount of P34 protein

Soybean (Glycine max L. Merr.) is a major source of protein for human and animal nutrients. However, soybean seed also contains a number of allergenic proteins, which limit its extensive usage in the food industry. P34 (Gly m Bd 30K), a low-leveled but highly conserved seed storage protein, has been described as a major allergen in soybean seed. Recently, two low P34 soybean accessions, PI603570A and PI567476, were identified by extensive screening of soybean germplasm. We have surveyed spatiotemporal expression pattern of P34 protein in low P34 accession, PI567476, and cultivated soybean accession, Clark, using antibody against P34. Immunoblot analysis showed not only seed specific expression pattern of P34, but also significantly reduced expression of P34 in PI567476 compared to Clark. Additionally, we performed proteomic analysis to compare protein compositions between PI567476 and Clark accessions. Although the overall distribution pattern of the protein spots was similar in both soybean accessions, 19 protein spots showing different intensity between both accessions were detected and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). PI567476 accession exhibited altered expression of several seed storage proteins including allergens and seed maturation-related proteins. The detailed analysis for composition of seed storage proteins of the PI567476 accession provides valuable information in use of this soybean germplasm for breeding of low allergenic elite soybean lines, which is expected to enhance food safety and make soy products available to allergen-sensitive individuals.

Dwarfness and Kunitz trypsin inhibitor (KTI) protein in soybean is useful traits for basic studies. df2 and ti gene control dwarfness and the expression of Kunitz trypsin inhibitor (KTI) protein in soybean, respectively. The objective of this research was to verify genetic linkage or independent inheritance of dj2 and ti loci in soybean. The F₂ population was made by cross combination between "Gaechuck#2" (Df2Df2titi genotype, KTI protein absence and a normal growth type) and T210 (df2df2TiTi genotype, a dwarf growth type and KTI protein present). A total of 258 F₂ seeds were analyzed for the segregation of KTI protein using SDS-PAGE. And so, 198 F₂ plants were recorded for the segregation of dwarfness. The segregation ratio of 3 : 1 for Ti locus (20t Ti_ : 57 titi) and Df2 locus (143 Df2_ : 55 df2df2) was observed. Segregation ratio of 9 : 3 : 3 : 1 (116 Ti_Df2_: 44 Ti_df2df2: 27 titiDf2_: 11 titidf2df2) between df2 gene and ti gene was observed (x²= 3.53, P = 0.223). These results showed that df2 gene was inherited independently with the ti gene in soybean.

Crimson clover (Trifolium incarnatum), hairy vetch (Vicia villosa) and rye (Secale cereale) are common leguminous cover crops. Because they contain water-soluble allelopathic substances that show a variable level in tissue depending on growth stage of the plants, the weed inhibition effects are dependent on the growth stage of cover crops. This study investigated the allelopathic effects of crimson clover, hairy vetch and rye on soybean, radish, oilseed rape and lettuce seeds germination and seedling growth. We used extract that were prepared from the shoots of crimson clover, hairy vetch and rye at different growth stages, vegetative growth stage, flowering stage, and fruiting stage. Applications of aqueous extracts from three growth stage of cover crops strongly affected to oilseed rape and lettuce seeds germination but the treatment resulted in a slight inhibition of the germination in soybean and radish. Radical length was more sensitive to aqueous extracts than seed germination rate. Especially, three cover crops extracts at vegetative growth stage highly inhibited seedling root growth of oilseed rape and lettuce by over 80% and 90% respectively. Furthermore, the GR50 values were lowest in the treatment of extracts from vegetative growth stage and the level of phenolics was decreased by the order of vegetative growth stage, flowering stage and fruiting stage.

Special interest has been focused on the variation in the composition and content of phospholipids (PLs) in soybeans following genetic modification, due to their beneficial effects on human health. As part of the attempt to discover PL-rich soybeans, a total of 12 cultivars were bred and their PL contents were determined by LC–MS/MS. In the LC, 0.1 M ammonium formate in methanol was essential to obtain good peak separation with minimum tailing. The LC–MS/MS method operating in the multiple reaction monitoring (MRM) mode, with two transitions monitored for each PL, provided good selectivity and sensitivity for the determination of PLs. All the calibration curves were linear, with correlation coefficients higher than 0.990. The extraction recoveries ranged from 75.6% to 89.9%. The limit of quantitations (LOQs) were within the range of 0.16–16.7 μg/kg. The average content of total PLs in the 12 soybean cultivars was about 75.7 mg/kg. The total PL and PC (phosphatidylcholine) contents were highest in the Gaechuck#2 cultivar. From the viewpoint of the PL content, Gaechuck#2 was demonstrated to be the most improved variety in the 12 cultivars investigated.

Aging is a major factor involved in neurological impairments, decreased anti-oxidant activities, and enhanced neuroinflammation. D-galactose (D-gal) has been considered an artificial aging model which induces oxidative stress and inflammatory response resulting in memory and synaptic dysfunction. Dietary supplementation exerts valuable effects against oxidative stress and neuroinflammation. Polyphenolic flavonoids, such as anthocyanins, have been reported as an anti-inflammatory and anti-oxidant agents against various neurodegenerative diseases. Recently, our group reported anthocyanin neuroprotection of the developing rat brain against ethanol-induced oxidative stress and neurodegenaration and ethanol-induced neuronal apoptosis via GABAB1 receptor intracellular signaling in prenatal rat hippocampus. Here, we examined the protective effect of anthocyanin neuroprotection against D-gal-induced oxidative and inflammatory response in the hippocampus and cortex regions and explore the potential mechanism of its action. Our results indicated that anthocyanins treatment significantly improved behavioral performance of D-gal-treated rats in Morris water maze and Y-maze tests. One of the potential mechanisms of this action was decreased expression of the receptor for advance glycation end product, reduced level of reactive oxygen species (ROS) and lipid peroxidation as well as markers of the Alzheimer's disease. Furthermore, the results also indicated that anthocyanins inhibited activated astrocytes and neuroinflammation via suppression of various inflammatory markers including p-NF- K B, inducible nitric oxide synthase (iNOS), and tumor necrosis factor-alpha (TNF-α) in the hippocampus and cortex regions of D-gal-treated rats brain. Moreover, anthocyanins abrogated neuroapoptosis via C-jun N-terminal kinase (p-JNK) suppression and improved deregulated synaptic proteins including synaptophysin, synaptosomal-associated protein (SNAP)-23, SNAP-25, and phosphorylated CREB. This data suggests that anthocyanins could be a safe and promising anti-oxidant and anti-neuroinflammatory agent for age-related neurodegenerative diseases such as Alzheimer's disease.

Soybean is a major source of protein meal in the world. Soybean kunitz trypsin inhibitor (SKTI) protein is a responsible for the inferior nutritional quality of unheated or incompletely heated soybean meal. The primary objective of this research was to identify DNA markers linked to the Ti locus controlling presence and absence of kunitz trypsin inhibitor protein. Two mapping populations were developed. Population 1 was derived from a cross between cultivar Jinpumkong2 (TiTi) and C242 (titi). Population 2 was made from a mating between cultivar Clark (TiTi) and C242. The F1 plants were grown in the greenhouse to produce F2 seeds. Each F2 seed from F1 plants was analyzed electrophoretically to determine the presence of the SKTI protein band. One-thousand RAPD primers, 342 AFLP primer sets, and 35 SSR primers were used to map Ti locus in population 1 and 2. The presence of SKTI protein was dominant to the lack of a SKTI protein and kunitz trypsin inhibit protein band was controlled by a single locus. Twelve DNA markers (4 RAPD, 4 AFLP, and 3 SSR) and Ti locus were found to be genetically linked in population 1 consisted with 94 F2 individual plants. Three SSR markers (Satt409, Satt228, and Satt429) were linked with Ti locus within 10 cM. Satt228 marker was tightly linked with Ti locus. Satt228 marker was tightly linked within 0–3.7 cM of the Ti locus and may be useful in a marker assisted selection program.

The dlm (disease lesion mimic) mutant of soybean (Glycine max L. Merr) shows the similar lesion of a soybean disease caused by a fungus, Corynespora cassilcola. The lesion was examined at cellular and molecular level. Trypan blue staining result indicated that cell death was detectable in the entire region of leaves excluding veins when the lesions had already been developed. We found that the mesophyll cells of palisade layer in the dim mutant appeared to be wider apart from each other. The chloroplasts of the dim mutant cells contained bigger starch granules than those in normal plants. We also found that the lesion development of dlm plant was light-dependent and the starch degradation during the dark period of diurnal cycle was impaired in the mutant. Three soybean pathogenesis-related genes, PR-1a, PR-4, and PR-10, were examined for their expression patterns during the development of disease lesion mimic. The expression of all three genes was up-regulated to some extent upon the appearance of the disease lesion mimic. Although the exact function of DLM protein remains elusive, our data would provide some insight into mechanism underling the cell death associated with the dim mutation.

Soybeans (Glycine max (L.) Merill) are popularly known as a healthy food in many Asian countries and are mostly consumed as soymilk, tofu, and fermented products such as miso, temph, and sufu. The objective of this study was to determine the variation and composition of phenolic compounds and isoflavone contents in soybean seeds [Glycine max (L.) Merill] and sprouts [Kongnamul] grown under dark conditions (producing yellow soybean sprouts) and in green and yellow boxes (producing green soybean sprouts). In seven soybean cultivars, the total phenolic content ranged from 6.67 μg−1 in Pureunkong to 72.33 μg−1 in Poongsannamulkong. The average total phenolic content in the green soybean sprouts (48.33 μg−1) was higher than in the yellow soybean sprouts (29.75 μg−1). The total phenolic content in the yellow soybean sprouts varied from 9.88 μg−1 to 47.71 μg−1, and the total phenolic content in the green soybean sprouts varied from 29.21 μg−1 to 79.70 μg−1. Only four phenolic compounds, p-hydroxybenzoic acid, salicylic acid, p-coumaric acid, and ferulic acid, were detected in all soybean cultivars. Syringic acid was not detected in yellow soybean sprouts, and myricetin was only detected in yellow soybean sprouts (4.65 μg−1) from the Pureunkong cultivar grown under dark conditions. The total isoflavone content in soybean seeds ranged from 2.1 μg−1 in Sowonkong to 33.0 μg−1 in Pureunkong, and the mean total isoflavones was 10.61 μg−1. Green soybean sprouts had higher average total isoflavones (1389.4 μg−1) than yellow soybean sprouts (559.2 μg−1), and the total isoflavone content was highest in the Pureunkong yellow soybean sprouts (756.3 μg−1) and the Sowonkong green soybean sprouts (2791.6 μg−1). In soybean sprouts, the higher the (malonyl)-daidzin or (malonyl)-genistein content, the higher the total isoflavone level. Our study suggests that producing soybean sprouts enriched in isoflavones under coloured-light sources is feasible.

Male germ cell apoptosis has been extensively explored in rodents. In contrast, very little is known about the susceptibility of developing germ cells to apoptosis in response to busulfan treatment. Spontaneous apoptosis of germ cells is rarely observed in the adult mouse testis, but under the experimental conditions described here, busulfan-treated mice exhibited a marked increase in apoptosis and a decrease in testis weight. TdT-mediated dUTP-X nicked end labeling analysis indicates that at one week following busulfan treatment, apoptosis was confined mainly to spermatogonia, with lesser effects on spermatocytes. The percentage of apoptosis-positive tubules and the apoptotic cell index increased in a time-dependent manner. An immediate effect was observed in spermatogonia within one week of treatment, and in the following week, secondary effects were observed in spermatocytes. RT-PCR analysis showed that expression of the spermatogonia-specific markers c-kit and Stra 8 was reduced but that Gli I gene expression remained constant, which is indicative of primary apoptosis of differentiating type A spermatogonia. Three and four weeks after busulfan treatment, RAD51 and FasL expression decreased to nearly undetectable levels, indicating that meiotic spematocytes and post-meiotic cells, respectively, were lost. The period of germ cell depletion did not coincide with increased p53 or Fas/FasL expression in the busulfan-treated testis, although p110Rb phosphorylation and PCNA expression were inhibited. These data suggest that increased depletion of male germ cells in the busulfan-treated mouse is mediated by loss of c-kit/SCF signaling but not by p53- or Fas/FasL-dependent mechanisms. Spermatogonial stem cells may be protected from cell death by modulating cell cycle signaling such that E2F-dependent protein expression, which is critical for G1 phase progression, is inhibited.

Soybean sprouts (Kongnamool) are one of the most popular and nutritive traditional vegetables in East Asia. Anthracnose caused by Colletotrichum gloeosporioides is one of the most serious diseases of soybean sprouts. In order to obtain basic information for breeding and/or selecting soybean genotypes with increased natural defense against anthracnose, phenolic compounds were profiled for healthy and infected soybean (Glycine max Merr.) sprouts by using high-performance liquid chromatography coupled with tandem mass spectrometry.Tryptophan and eight phenolic compounds (daidzin, genistin, malonyldaidzin, malonylgenistin, daidzein, glycitein, genistein and coumestrol) were determined from healthy and inoculated sprouts. Total identified phenolic content was 40.02 ± 0.03 mg kg⁻¹, 99.4% of which was isoflavones.The monitoring suggested that de novo induced glycitein appeared to act as a phytoalexin in the defence mechanism of the soybean sprouts against C. gloeosporioides, and constitutively formed seven phenolic components that functioned as phytoanticipins in the diseased soybean sprouts.

Phospholipids (PLs) in 57 varieties of soybeans were profiled by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry and principal component analysis (PCA) to discriminate PL-rich soybeans. The PL calibration curves showed linearity with correlation coefficients0.9964. The recoveries at 5 mg/L spiked level ranged from 72.8 to 86.7% and those at 12.5 mg/L from 78.2 to 85.1%. The repeatability at a 5 mg/L spiked level ranged from 2.5 to 7.0% and those at 12.5 mg/L from 1.2 to 3.9%. The average total PL content in the 57 soybean varieties was about 35.3 mg/kg. The total PL content was the highest in Aodaiz (35, 48.7 ± 1.4 mg/kg) and the lowest in Poongsannamul (56, 16.0 ± 0.7 mg/kg). The PCA showed that RS-78sun (42), Gyeongsang #1 (3) and Aodaiz (35) are the most improved varieties of the investigated 57 varieties from the viewpoint of PL content.

Hyperglycemia, abnormal lipid and antioxidant profiles are the most usual complications in diabetes mellitus. Thus, in this study, we investigated the anti-diabetic and anti-oxidative effects of anthocyanins (ANT) from black soybean seed coats in streptozotocin (STZ)-induced diabetic rats. The administration of ANT markedly decreased glucose levels and improved heart hemodynamic function (left ventricular end diastolic pressure, +/-dp/dt parameters). ANT not only enhanced STZ-mediated insulin level decreases, but also decreased the triglyceride levels induced by STZ injection in serum. Diabetic rats exhibited a lower expression of glucose transporter 4 proteins in the membrane fractions of heart and skeletal muscle tissues, which was enhanced by ANT. In addition, ANT activated insulin receptor phosphorylation, suggesting an increased utilization of glucose by tissues. Moreover, ANT protected pancreatic tissue from STZ-induced apoptosis through regulation of caspase-3, Bax, and Bcl-2 proteins. Furthermore, ANT significantly suppressed malondialdehyde levels and restored superoxide dismutase and catalase activities in diabetic rats. Interestingly, the observed effects of ANT were superior to those of glibenclamide. Taken together, ANT from black soybean seed coat have anti-diabetic effects that are due, in part, to the regulation of glucose transporter 4 and prevention of insulin resistance and pancreatic apoptosis, suggesting a possible use as a drug to regulate diabetes.

Adhesion molecules have a key role in pathological inflammation. Thus, we investigated the effect of anthocyanins on the induction of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) by TNF-alpha and the possible molecular mechanisms by which anthocyanins differentially regulate ICAM-1 and VCAM-1 expression. Stimulation of cells with TNF-alpha increased ICAM-1 and VCAM-1 expression, and pretreatment with anthocyanins inhibited VCAM-1 expression, but not ICAM-1 expression. We found that IRF-1 and GATAs, especially GATA-4 and -6, were involved in the TNF-alpha-mediated expression of VCAM-1 but not ICAM-1, and anthocyanins decreased nuclear levels of GATA-4 and GATA-6 as well as IRF-1. Moreover, pretreatment with a Jak/STAT inhibitor decreased TNF-alpha-induced VCAM-1 expression and nuclear GATA-4, GATA-6, and IRF-1 levels. Furthermore, anthocyanins efficiently inhibited the phosphorylation of STAT-3. This suggests that anthocyanins differentially regulate TNF-alpha-mediated expression of VCAM-1 and ICAM-1 through modulation of the GATA and IRF-1 binding activity via Jak/STAT-3 activation.

Wound healing is a complex process that includes inflammation, tissue formation, and remodeling. While wound healing is accompanied by inflammatory reactions, chronic inflammation impairs acute wound healing. In this study, we investigated whether anthocyanins from black soybean seed coats could stimulate wound healing while preventing excessive inflammation. At 24h of treatment with anthocyanins, fibroblasts showed a significant increase in migration at 100 microg/mL whereas the migration of keratinocytes increased significantly at 50 and 100 microg/mL compared to control. Treatment of anthocyanins for 48 h significantly stimulated the migration of both human dermal fibroblasts and keratinocytes at 50 and 100 microg/mL concentrations. Treatment of cells with anthocyanins stimulated wound-induced VEGF production in fibroblasts and keratinocytes. However, anthocyanins inhibited ROS accumulation and VEGF production in TNF-alpha-stimulated endothelial cells. Furthermore, treatment of anthocyanins reduced, in a dose-dependent manner, the adhesion of inflammatory monocytes to endothelial cells. Anthocyanins also blocked both the translocation of nuclear factor-kappa B (NF-kappaB) p65 into the nucleus and the phosphorylation of the inhibitory factor kappaBalpha (IkappaBalpha). Thus, treatment with anthocyanins from black soybean seed coats may be a potential therapeutic strategy to promote wound healing and to prevent inflammation in a persistent inflammatory condition.