Compensatory Modulation of Seed Storage Protein Synthesis and Alteration of Starch Accumulation by Selective Editing of 13 kDa Prolamin Genes by CRISPR-Cas9 in Rice.

Rice prolamins are categorized into three groups by molecular size (10, 13, or 16 kDa), while the 13 kDa prolamins are assigned to four subgroups (Pro13a-I, Pro13a-II, Pro13b-I, and Pro13b-II) based on cysteine residue content. Since lowering prolamin content in rice is essential to minimize indigestion and allergy risks, we generated four knockout lines using CRISPR-Cas9, which selectively reduced the expression of a specific subgroup of the 13 kDa prolamins. These four mutant rice lines also showed the compensatory expression of glutelins and non-targeted prolamins and were accompanied by low grain weight, altered starch content, and atypically-shaped starch granules and protein bodies. Transcriptome analysis identified 746 differentially expressed genes associated with 13 kDa prolamins during development. Correlation analysis revealed negative associations between genes in Pro13a-I and those in Pro13a-II and Pro13b-I/II subgroups. Furthermore, alterations in the transcription levels of 9 ER stress and 17 transcription factor genes were also observed in mutant rice lines with suppressed expression of 13 kDa prolamin. Our results provide profound insight into the functional role of 13 kDa rice prolamins in the regulatory mechanisms underlying rice seed development, suggesting their promising potential application to improve nutritional and immunological value.

Down-Regulation of Rice Glutelin by CRISPR-Cas9 Gene Editing Decreases Carbohydrate Content and Grain Weight and Modulates Synthesis of Seed Storage Proteins during Seed Maturation.

The glutelins are a family of abundant plant proteins comprised of four glutelin subfamilies (GluA, GluB, GluC, and GluD) encoded by 15 genes. In this study, expression of subsets of rice glutelins were suppressed using CRISPR-Cas9 gene-editing technology to generate three transgenic rice variant lines, GluA1, GluB2, and GluC1. Suppression of the targeted glutelin genes was confirmed by SDS-PAGE, Western blot, and q-RT-PCR. Transgenic rice variants GluA1, GluB2, and GluC1 showed reduced amylose and starch content, increased prolamine content, reduced grain weight, and irregularly shaped protein aggregates/protein bodies in mature seeds. Targeted transcriptional profiling of immature seeds was performed with a focus on genes associated with grain quality, starch content, and grain weight, and the results were analyzed using the Pearson correlation test (requiring correlation coefficient absolute value ≥ 0.7 for significance). Significantly up- or down-regulated genes were associated with gene ontology (GO) and KEGG pathway functional annotations related to RNA processing (spliceosomal RNAs, group II catalytic introns, small nucleolar RNAs, microRNAs), as well as protein translation (transfer RNA, ribosomal RNA and other ribosome and translation factors). These results suggest that rice glutelin genes may interact during seed development with genes that regulate synthesis of starch and seed storage proteins and modulate their expression via post-transcriptional and translational mechanisms.

Rice peroxygenase catalyzes lipoxygenase-dependent regiospecific epoxidation of lipid peroxides in the response to abiotic stressors.

The peroxygenase pathway plays pivotal roles in plant responses to oxidative stress and other environmental stressors. Analysis of a network of co-expressed stress-regulated rice genes demonstrated that expression of OsPXG9 is negatively correlated with expression of genes involved in jasmonic acid biosynthesis. DNA sequence analysis and structure/function studies reveal that OsPXG9 is a caleosin-like peroxygenase with amphipathic α-helices that localizes to lipid droplets in rice cells. Enzymatic studies demonstrate that 12-epoxidation is slightly more favorable with 9(S)-hydroperoxyoctadecatrienoic acid than with 9(S)-hydroperoxyoctadecadienoic acid as substrate. The products of 12-epoxidation are labile, and the epoxide ring is hydrolytically cleaved into corresponding trihydroxy compounds. On the other hand, OsPXG9 catalyzed 15-epoxidation of 13(S)-hydroperoxyoctadecatrienoic acid generates a relatively stable epoxide product. Therefore, the regiospecific 12- or 15-epoxidation catalyzed by OsPXG9 strongly depends on activation of the 9- or 13- peroxygenase reaction pathways, with their respective preferred substrates. The relative abundance of products in the 9-PXG and 13-PXG pathways suggest that the 12-epoxidation involves intramolecular oxygen transfer while the 15-epoxidation can proceed via intramolecular or intermolecular oxygen transfer. Expression of OsPXG9 is up-regulated by abiotic stimuli such as drought and salt stress, but it is down-regulated by biotic stimuli such as flagellin 22 and salicylic acid. The results suggest that the primary function of OsPXG9 is to modulate the level of lipid peroxides to facilitate effective defense responses to abiotic and biotic stressors.

Improvement of Bronchial Immune Hypersensitivity Reaction Using Extracts from Chrysanthemum morifolium Ramatuelle and Scutellaria baicalensis Georgi.

Chrysanthemum morifolium Ramatuelle and Scutellaria baicalensis Georgi (skullcap) have been used as safe raw materials for drinking or as traditional medicines in Korea. In this study, we investigated the potential therapeutic effects of ovalbumin-induced asthma in a mouse model. After establishing the model, mice were treated with a mixture of chrysanthemum and skullcap extracts at different mixing ratios (6 : 4, 7 : 3, and 8 : 2). Immune cell counts and the production of various inflammatory cytokines were measured using biochemical tests. Among the mixtures tested, the 7 : 3 ratio (CS73) showed the most pronounced effects. CS73 significantly reduced the levels of the inflammatory cytokines interleukin- (IL-) 1ß, IL-4, IL-5, IL-6, IL-10, IL-13, IL-17A, IL-17F, and IL-17E in the serum and bronchoalveolar lavage fluid of asthmatic mice. In addition, CS73 treatment significantly increased the production of IL-2 and interferon-γ and decreased the production of immunoglobulin E, histamine, and thymic stromal lymphopoietin in asthmatic mice compared to the control group. Our results suggest that the combination of chrysanthemum and skullcap extracts, especially at a 7 : 3 ratio, can be used to improve bronchial health and contribute to improved public health.

Proteomic Determination of Low-Molecular-Weight Glutenin Subunit Composition in Aroona Near-Isogenic Lines and Standard Wheat Cultivars.

The low-molecular weight glutenin subunit (LMW-GS) composition of wheat (Triticum aestivum) flour has important effects on end-use quality. However, assessing the contributions of each LMW-GS to flour quality remains challenging because of the complex LMW-GS composition and allelic variation among wheat cultivars. Therefore, accurate and reliable determination of LMW-GS alleles in germplasm remains an important challenge for wheat breeding. In this study, we used an optimized reversed-phase HPLC method and proteomics approach comprising 2-D gels coupled with liquid chromatography-tandem mass spectrometry (MS/MS) to discriminate individual LMW-GSs corresponding to alleles encoded by the Glu-A3, Glu-B3, and Glu-D3 loci in the 'Aroona' cultivar and 12 'Aroona' near-isogenic lines (ARILs), which contain unique LMW-GS alleles in the same genetic background. The LMW-GS separation patterns for 'Aroona' and ARILs on chromatograms and 2-D gels were consistent with those from a set of 10 standard wheat cultivars for Glu-3. Furthermore, 12 previously uncharacterized spots in 'Aroona' and ARILs were excised from 2-D gels, digested with chymotrypsin, and subjected to MS/MS. We identified their gene haplotypes and created a 2-D gel map of LMW-GS alleles in the germplasm for breeding and screening for desirable LMW-GS alleles for wheat quality improvement.

Prebiotics/Probiotics Mixture Induced Changes in Cecal Microbiome and Intestinal Morphology Alleviated the Loperamide-Induced Constipation in Rat.

The aim of this study was to investigate the effect of a mixture of multi-strain probiotics and prebiotics on loperamide-induced constipation in Sprague-Dawley rats. A multi-strain probiotics alone (loperamide-induced group with multi-strain probiotics mixture group; Lop-Pro) and a mixture of multi-strain probiotics and prebiotics (loperamide-induced group with multi-strain probiotics and prebiotics mixture group; Lop-Pro/Pre) were administered orally after inducing constipation. The fecal water content was significantly higher (by 42%) in the Lop-Pro/Pre group (33.5%) than in the loperamide-induced group (Lop) (23.7%) (p<0.05). The intestinal mucosal thickness, crypt cell area, and interstitial cells of Cajal area were significantly higher in the Lop-Pro/Pre group compared to the Lop group by 16.4%, 20.6%, and 42.3%, respectively. Additionally, the total short-chain fatty acid content was significantly increased in the Lop-Pro and Lop-Pro/Pre groups by 56.4% and 54.2%, respectively, compared with the Lop group. The Lop-Pro and Lop-Pro/Pre groups recovered loperamide-induced alteration in Bacteroidetes and Verrucomicrobia abundance among intestinal microbiota, whereas the Lop-Pro/Pre group recovered Akkermansia, Lactobacillus, Clostridium, Bacteroides, and Oscillibacter abundance. Moreover, the relative abundance of Oscillibacter and Clostridium was significantly different in the Lop-Pro/Pre group compared to the Lop group. Collectively, administration of synbiotics rather than multi-strain probiotics alone is effective in alleviating constipation.

Immobilization of Soybean Lipoxygenase on Nanoporous Rice Husk Silica by Adsorption: Retention of Enzyme Function and Catalytic Potential.

Soybean lipoxygenase was immobilized on nanoporous rice husk silica particles by adsorption, and enzymatic parameters of the immobilized protein, including the efficiency of substrate binding and catalysis, kinetic and operational stability, and the kinetics of thermal inactivation, were investigated. The maximal adsorption efficiency of soybean lipoxygenase to the silica particles was 50%. The desorption kinetics of soybean lipoxygenase from the silica particles indicate that the silica-immobilized enzyme is more stable in an anionic buffer (sodium phosphate, pH 7.2) than in a cationic buffer (Tris-HCl, pH 7.2). The specific activity of immobilized lipoxygenase was 73% of the specific activity of soluble soybean lipoxygenase at a high concentration of substrate. The catalytic efficiency (kcat/Km) and the Michaelis-Menten constant (Km) of immobilized lipoxygenase were 21% and 49% of kcat/Km and Km of soluble soybean lipoxygenase, respectively, at a low concentration of substrate. The immobilized soybean lipoxygenase was relatively stable, as the enzyme specific activity was >90% of the initial activity after four assay cycles. The thermal stability of the immobilized lipoxygenase was higher than the thermal stability of soluble lipoxygenase, demonstrating 70% and 45% of its optimal specific activity, respectively, after incubation for 30 min at 45 °C. These results demonstrate that adsorption on nanoporous rice husk silica is a simple and rapid method for protein immobilization, and that adsorption may be a useful and facile method for the immobilization of many biologically important proteins of interest.

Comparison of MALDI-TOF-MS and RP-HPLC as Rapid Screening Methods for Wheat Lines With Altered Gliadin Compositions.

The wheat gliadins are a complex group of flour proteins that can trigger celiac disease and serious food allergies. As a result, mutation breeding and biotechnology approaches are being used to develop new wheat lines with reduced immunogenic potential. Key to these efforts is the development of rapid, high-throughput methods that can be used as a first step in selecting lines with altered gliadin contents. In this paper, we optimized matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and reversed-phase high-performance liquid chromatography (RP-HPLC) methods for the separation of gliadins from Triticum aestivum cv. Chinese Spring (CS). We evaluated the quality of the resulting profiles using the complete set of gliadin gene sequences recently obtained from this cultivar as well as a set of aneuploid lines in CS. The gliadins were resolved into 13 peaks by MALDI-TOF-MS. α- or γ-gliadins that contain abundant celiac disease epitopes and are likely targets for efforts to reduce the immunogenicity of flour were found in several peaks. However, other peaks contained multiple α- and γ-gliadins, including one peak with as many as 12 different gliadins. In comparison, separation of proteins by RP-HPLC yielded 28 gliadin peaks, including 13 peaks containing α-gliadins and eight peaks containing γ-gliadins. While the separation of α- and γ-gliadins gliadins achieved by RP-HPLC was better than that achieved by MALDI-TOF-MS, it was not possible to link peaks with individual protein sequences. Both MALDI-TOF-MS and RP-HPLC provided adequate separation of ω-gliadins. While MALDI-TOF-MS is faster and could prove useful in studies that target specific gliadins, RP-HPLC is an effective method that can be applied more broadly to detect changes in gliadin composition.

An Anthocyanin-Enriched Extract from Vaccinium uliginosum Improves Signs of Skin Aging in UVB-Induced Photodamage.

Accumulating evidence indicates that botanical extracts affect skin biophysical parameters, such as hydration, transepidermal water loss (TEWL), melanin index, erythema index, and wrinkle development. Vaccinium uliginosum extract contains a high level of anthocyanins as antioxidant and is ideal for use in dietary skin care products. Here, we assessed the photoprotective effects of dietary V. uliginosum extract in ultraviolet B (UVB)-irradiated hairless mice. Quantitative analysis of anthocyanin composition in the ethanol-extracted V. uliginosum sample was performed using high-performance liquid chromatography (HPLC). Skin parameter analysis and hematoxylin and eosin (H&E) staining were conducted on skin samples from UVB-irradiated hairless mice to evaluate the effects of V. uliginosum extract on skin conditions. In addition, skin mRNA and protein expression were assessed to characterize the molecular mechanisms underlying the effects of the anthocyanin-enriched extract on skin appearance and condition. Administration of the ethanol-extracted V. uliginosum sample caused significant changes in skin water-holding capacity, TEWL, wrinkle-related parameters, and epidermal thickness in UVB-irradiated hairless mice. In addition, oral administration of V. uliginosum attenuated the gene expression of matrix metalloproteinase (MMP) and increased levels of tissue inhibitor of metalloproteinase (TIMP) and antioxidant-related genes. Further, V. uliginosum administration downregulated inflammatory cytokine levels and UVB-induced phosphorylation of extracellular signaling regulated kinase (ERK), as well as Jun N-terminal kinase (JNK) and p38 protein levels. Oral administration of anthocyanin-enriched V. uliginosum extract can improve the appearance and condition of the skin following UV irradiation.

Development of an Optimized MALDI-TOF-MS Method for High-Throughput Identification of High-Molecular-Weight Glutenin Subunits in Wheat.

Because high-molecular-weight glutenin subunits (HMW-GS) are important contributors to wheat end-use quality, there is a need for high-throughput identification of HMW-GS in wheat genetic resources and breeding lines. We developed an optimized method using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to distinguish individual HMW-GS by considering the effects of the alkylating reagent in protein extraction, solvent components, dissolving volume, and matrix II components. Using the optimized method, 18 of 22 HMW-GS were successfully identified in standard wheat cultivars by differences in molecular weights or by their associations with other tightly linked subunits. Interestingly, 1Bx7 subunits were divided into 1Bx7 group 1 and 1Bx7 group 2 proteins with molecular weights of about 82,400 and 83,000 Da, respectively. Cultivars containing the 1Bx7 group 2 proteins were distinguished from those containing 1Bx7OE using well-known DNA markers. HMW-GS 1Ax2* and 1Bx6 and 1By8 and 1By8*, which are difficult to distinguish due to very similar molecular weights, were easily identified using RP-HPLC. To validate the method, HMW-GS from 38 Korean wheat varieties previously evaluated by SDS-PAGE combined with RP-HPLC were analyzed by MALDI-TOF-MS. The optimized MALDI-TOF-MS method will be a rapid, high-throughput tool for selecting lines containing desirable HMW-GS for breeding efforts.

Protective effects of combination of Stauntonia hexaphylla and Cornus officinalis on testosterone-induced benign prostatic hyperplasia through inhibition of 5α- reductase type 2 and induced cell apoptosis.

Benign prostatic hyperplasia (BPH) is a progressive pathological condition associated with proliferation of prostatic tissues, prostate enlargement, and lower-urinary tract symptoms. However, the mechanism underlying the pathogenesis of BPH is unclear. The aim of this study was to investigate the protective effects of a combination of Stauntonia hexaphylla and Cornus officinalis (SC extract) on a testosterone propionate (TP)-induced BPH model. The effect of SC extract was examined in a TP-induced human prostate adenocarcinoma cell line. Male Sprague-Dawley rats were randomly divided into 5 groups (n = 6) for in vivo experiments. To induce BPH, all rats, except those in the control group, were administered daily with subcutaneous injections of TP (5 mg/kg) and orally treated with appropriate phosphate buffered saline/drugs (finasteride/saw palmetto/SC extract) for 4 consecutive weeks. SC extract significantly downregulated the androgen receptor (AR), prostate specific antigen (PSA), and 5α-reductase type 2 in TP-induced BPH in vitro. In in vivo experiments, SC extract significantly reduced prostate weight, size, serum testosterone, and dihydrotestosterone (DHT) levels. Histologically, SC extract markedly recovered TP-induced abnormalities and reduced prostatic hyperplasia, thereby improving the histo-architecture of TP-induced BPH rats. SC extract also significantly downregulated AR and PSA expression, as assayed using immunoblotting. Immunostaining revealed that SC extract markedly reduced the 5α-reductase type 2 and significantly downregulated the expression of proliferating cell nuclear antigen. In addition, immunoblotting of B-cell lymphoma 2 (Bcl-2) family proteins indicated that SC extract significantly downregulated anti-apoptotic Bcl-2 and markedly upregulated pro-apoptotic B cell lymphoma-associated X (Bax) expression. Furthermore, SC treatment significantly decreased the Bcl-2/Bax ratio, indicating induced prostate cell apoptosis in TP-induced BPH rats. Thus, our findings demonstrated that SC extract protects against BPH by inhibiting 5α-reductase type 2 and inducing prostate cell apoptosis. Therefore, SC extract might be useful in the clinical treatment of BPH.

Anti-inflammatory action of herbal medicine comprised of Scutellaria baicalensis and Chrysanthemum morifolium.

Various mixtures were prepared depending on the mixing ratio of Scutellaria baicalensis hot water extract (SB-HW), and Chrysanthemum morifolium ethanol extract (CM-E) and their anti-inflammatory activity were compared. Among them, SB-HW (80 µg/mL)/CM-E (120 µg/mL) or SB-HW (40 µg/mL)/CM-E (160 µg/mL) significantly inhibited LPS-stimulated NO and IL-6 levels in RAW 264.7 cells. The SB-HW (80 µg/mL)/CM-E (120 µg/mL) mixture, which was determined as active mixture, significantly reduced MUC5AC secretion in PMA and LPS-induced NCI-H292 cells. The active mixture also reduced the production of PGE2 and IL-8 in PMA-induced A549 cells. LC-MS/MS analysis showed that the active mixture was composed of high contents of flavone glycosides, such as baicalin and cynaroside. Western blot analysis indicated that the active mixture suppressed phosphorylation of ERK, JNK, and p38, associating with the inhibition of MAPK signaling. Taken together, our results suggest that the active mixture could be applied as a new anti-inflammatory herbal medicine. ABBREVIATIONS: JNK: c-Jun N-terminal kinases; COPD: chronic obstructive pulmonary disease; CM: Chrysanthemum morifolium; COX-2: cyclooxygenase-2; ERK: extracellular-signal-regulated kinase; IL-6: interleukin-6; IL-8: interleukin-8; IL-12: interleukin-12; LPS: lipopolysaccharide; MAPK: mitogen-activated protein kinase; NO: nitric oxide; NK- κB: nuclear factor kappa B; p38: p38 mitogen-activated protein kinases; PBS: phosphate buffered saline; PMA: phorbol-12-myristate-13-acetate; SB: Scutellaria baicalensis; PGE2: prostaglandin E2; TBST: Tris-buffered saline containing 0.1% Tween 20; TIC: total ion chromatogram; TNF-α: tumor necrosis factor-alpha.

Protective effect of Prunella vulgaris var. L extract against blue light induced damages in ARPE-19 cells and mouse retina.

Age-related macular degeneration (AMD) is one of leading causes that induce severe visual impairment and loss in the elderly. Previous studies have suggested that blue light (BL) could induce retinal degeneration, which is a major cause of the onset and development of severe AMD. In the retinal pigment epithelium (RPE) cells, A2E, a lipofuscin fluorophore, is accumulated with aging. When A2E is exposed to BL, it is easily oxidized to A2E-epoxides, leading to oxidative stress and inflammatory response in retina. The aim of this study was to investigate protective effect of Prunella vulagris (P.V) extract against oxidative stress and inflammation caused by BL, and to elucidate the underlying mechanisms in the cultured RPE cells and balb-c mice. In both model studies, P.V extract activated NF-E2 related factor 2 (Nrf-2)/hemeoxygenase-1 (HO-1) signaling pathway, followed by inhibition of ROS/MDA production, GSH depletion and reduction in SOD activity. Furthermore, P.V extract inhibited upregulation of inflammatory related genes (interlukin (IL)-1beta, IL-6, monocyte chemoattractant protein-1 (MCP-1), vascular endothelial growth factor A (VEGF A)) and BL induced RPE cell death, determined by cell viability and histological analyses. The mechanism of protection against inflammation by P.V extract involves inhibition of nuclear translocation of nuclear factor kappa beta (NF-kB) along with degradation of NF-kB inhibitor alpha (IkB alpha). The results suggest that P.V extract could be a potential intervention to prevent the onset and development of severe AMD.

The therapeutic effects of Stauntonia hexaphylla in benign prostate hyperplasia are mediated by the regulation of androgen receptors and 5α-reductase type 2.

ETHNOPHARMACOLOGICAL RELEVANCE: Stauntonia hexaphylla (Lardizabalaceae, S. hexaphylla) is traditionally used as a folk remedy for alleviating fever and for its anti- inflammatory and analgesic properties. In Korea and China, S. hexaphylla has been used as a traditional medicine that acts as diuretic and analgesic. S. hexaphylla has also been reported to inhibit osteoporosis and aldose reductase activity. AIM OF THE STUDY: The study aimed to evaluate the therapeutic effects of an extract of S. hexaphylla on testosterone induced benign prostate hyperplasia (BPH) models and to observe its mechanism of action. MATERIALS AND METHODS: To induce a BPH model in vitro and in vivo, a testosterone-treated LNCaP cell line and Sprague Dawley (SD) rats were used, respectively. Androgen receptors (ARs) and prostate-specific antigens (PSA), which are typical BPH-related proteins, were evaluated using western blotting. Prostate weights and dihydrotestosterone (DHT) levels were measured in vivo, and histopathology of the prostate examined using hematoxylin and eosin staining. Proliferating cell nuclear antigen (PCNA) and 5α-reductase type 2 were also evaluated via immunohistochemistry (IHC). In addition, TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) staining and LC3 staining of IHC were performed to evaluate apoptosis and autophagy. RESULTS: S. hexaphylla reduced prostates weights and the thickness of prostate epithelial cells. In vivo and in vitro, PSA and ARs were downregulated following S. hexaphylla treatment. The S. hexaphylla extracts also reduced DHT and 5α-reductase type 2 expression. In addition, the expression of PCNA was reduced, and in the TUNEL staining and IHC of LC3, the number of positive cells was increased in the groups treated with S. hexaphylla. CONCLUSIONS: It was observed that extracts of S. hexaphylla inhibited both 5α -reductase type 2 and ARs. The results indicate that the use of S. hexaphylla extract in BPH is probably beneficial through 5α-reductase inhibition and α-adrenergic receptor blockade. In addition, apoptosis and autophagy were induced, and PCNA was downregulated after S. hexaphylla treatment. Therefore, it can be concluded that S. hexaphylla has a therapeutic effect on BPH.

Isolation, structural elucidation, and insights into the anti-inflammatory effects of triterpene saponins from the leaves of Stauntonia hexaphylla.

Using various chromatographic techniques, 23 triterpene saponins (1-23) were isolated from an ethanol extract of Stauntonia hexaphylla, including two new compounds (12 and 15). Their chemical structures were established by comprehensive spectroscopic methods such as 1D- and 2D-NMR, and HR-ESI-MS, and chemical reactions. The anti-inflammatory activities of the isolated saponins were determined using the nitric oxide (NO) assay. Compound 13 exhibited the greatest inhibitory effect (IC50 = 0.59 µM). In addition to NO, compound 13 suppressed the secretion of PGE2, IL-1ß, and IL-6, but not TNF-α, and inhibited the protein expression of iNOS and COX-2 in LPS-activated RAW264.7 cells. The chemical derivatives of the isolated compounds were studied using structure-activity relationships. The results suggested that compound 13 isolated from S. hexaphylla might be useful for treating inflammation. This is the first comprehensive study of saponins from the leaves of S. hexaphylla based on anti-inflammatory extract screening guidelines.

Overexpressing wheat low-molecular-weight glutenin subunits in rice (Oryza sativa L. japonica cv. Koami) seeds.

Genes encoding wheat low-molecular-weight glutenin subunits (LMW-GSs) that confer dough strength and extensibility were previously identified from Korean wheat cultivars. To improve low viscoelasticity of rice (Oryza sativa L.) dough caused by the lack of seed storage proteins comparable to wheat gluten, two genes, LMW03 and LMW28, encoding LMW-GSs are cloned from Korean wheat cultivar Jokyoung. The LMW genes are inserted into binary vectors under the control of the rice endosperm-specific Glu-B1 promoter. Transgenic rice plants expressing LMW03 or LMW28 in their seeds are generated using Agrobacterium-mediated transformation. The expression of recombinant wheat LMW-GS in the transgenic rice seeds was confirmed by SDS-PAGE and immunoblot analysis. Their accumulation in the endosperm and aleurone layers of rice seeds was observed through in situ immuno-hybridization.

Structural Evidence for the Substrate Channeling of Rice Allene Oxide Cyclase in Biologically Analogous Nazarov Reaction.

Allene oxide cyclase (AOC) is a key enzyme in the jasmonic acid (JA) biosynthetic pathway in plants, during which it catalyzes stereospecific conversion of 12,13(S)-epoxy-9(Z),11,15(Z)-octadecatrienoic acid (12,13-EOT) to cis(+)-12-oxophytodienoic acid. Here, rice allene oxide cyclase (OsAOC) was localized to the chloroplast and its native oligomeric structure was analyzed by gel electrophoresis in the absence and presence of a protein-crosslinking reagent. The results suggest that OsAOC exists in solution as a mixture of monomers, dimers, and higher order multimers. OsAOC preferentially exists as dimer at room temperature, but it undergoes temperature-dependent partial denaturation in the presence of SDS. A heteromeric 2:1 complex of OsAOC and rice allene oxide synthase-1 (OsAOS1) was detected after cross-linking. The yield of cis(+)-12-oxophytodienoic acid reached maximal saturation at a 5:1 molar ratio of OsAOC to OsAOS1, when OsAOC and OsAOS1 reactions were coupled. These results suggest that the OsAOC dimer may facilitate its interaction with OsAOS1, and that the heteromeric 2:1 complex may promote efficient channeling of the unstable allene oxide intermediate during catalysis. In addition, conceptual similarities between the reaction catalyzed by AOC and Nazarov cyclization are discussed.

Proteomic Profiling and Epitope Analysis of the Complex α-, γ-, and ω-Gliadin Families in a Commercial Bread Wheat.

Wheat gliadins are a complex group of proteins that contribute to the functional properties of wheat flour doughs and contain epitopes that are relevant for celiac disease (CD) and wheat-dependent exercise-induced anaphylaxis (WDEIA). In this study, we extracted ethanol-soluble gliadin fractions from flour of the Korean bread wheat cultivar Keumkang. Proteins were separated by 2-dimensional gel electrophoresis (2-DE) using a pI range of 6-11 in the first dimension and subjected to tandem mass spectrometry. α-, γ-, and ω-gliadins were identified as the predominant proteins in 31, 28, and one 2-DE spot, respectively. An additional six ω-gliadins were identified in a separate experiment in which a pI range of 3-11 was used for protein separation. We analyzed the composition of CD- and WDEIA-relevant epitopes in the gliadin sequences from Keumkang flour, demonstrating the immunogenic potential of this cultivar. Detailed knowledge about the complement of gliadins accumulated in Keumkang flour provides the background necessary to devise either breeding or biotechnology strategies to improve the functional properties and reduce the adverse health effects of the flour.

Covalent immobilization of oxylipin biosynthetic enzymes on nanoporous rice husk silica for production of cis(+)-12-oxophytodienoic acid.

Soybean lipoxygenase, recombinant rice allene oxide synthase-1 and rice allene oxide cyclase were covalently immobilized on nanoporous rice husk silica using two types of linkers: glutardialdehyde and polyethylene glycol. The immobilization efficiency achieved using glutardialdehyde-linked rice husk silica was higher than that achieved using polyethylene glycol-linked rice husk silica (50-92% and 25-50%, respectively). Immobilization on both types of matrices significantly decreased the specific activities of the immobilized enzymes. Solid-phase reaction yields of the enzymes were determined relative to the yields observed for the solution-phase reactions. Yields of the solid-phase reactions catalyzed by immobilized soybean lipoxygenase, rice allene oxide synthase-1, and rice allene oxide cyclase ranged from 50% to 230% and were dependent on both the enzymes and linkers used. Production of cis(+)-12-oxophytodienoic acid from α-linolenic acid by consecutive reactions using all three enzymes in a co-immobilization system resulted in 83.6% and 65.1% yields on glutardialdehyde-linked and epichlorohydrin-polyethylene glycol-linked rice husk silica, respectively. Our results suggest that immobilization of biosynthetic enzymes of the octadecanoid pathway on rice husk silica may be an efficient method for the in vitro production of oxylipins. Additionally, enzyme immobilizations on rice husk silica matrices may be more broadly applicable for producing physiologically important compounds in other biosynthetic pathways.

Cellular Localization of Wheat High Molecular Weight Glutenin Subunits in Transgenic Rice Grain.

Rice (Oryza sativa L.) is a primary global food cereal. However, when compared to wheat, rice has poor food processing qualities. Dough that is made from rice flour has low viscoelasticity because rice seed lacks storage proteins that are comparable to gluten protein from wheat. Thus, current research efforts aim to improve rice flour processing qualities through the transgenic expression of viscoelastic proteins in rice seeds. In this study, we characterized the transgenic expression of wheat glutenin subunits in rice seeds. The two genes 1Dx5_KK and 1Dy10_JK, which both encode wheat high-molecular-weight glutenin subunits that confer high dough elasticity, were cloned from Korean wheat cultivars KeumKang and JoKyung, respectively. These genes were inserted into binary vectors under the control of the rice endosperm-specific Glu-B1 promoter and were expressed in the high-amylose Korean rice cultivar Koami (Oryza sativa L.). Individual expression of both glutenin subunits was confirmed by SDS-PAGE and immunoblot analyses performed using T3 generation of transgenic rice seeds. The subcellular localization of 1Dx5_KK and 1Dy10_JK in the rice seed endosperm was confirmed by immunofluorescence analysis, indicating that the wheat glutenin subunits accumulate in protein body-II and novel protein body types in the rice seed. These results contribute to our understanding of engineered seed storage proteins in rice.