CLEMENT: genomic decomposition and reconstruction of non-tumor subclones.

Genome-level clonal decomposition of a single specimen has been widely studied; however, it is mostly limited to cancer research. In this study, we developed a new algorithm CLEMENT, which conducts accurate decomposition and reconstruction of multiple subclones in genome sequencing of non-tumor (normal) samples. CLEMENT employs the Expectation-Maximization (EM) algorithm with optimization strategies specific to non-tumor subclones, including false variant call identification, non-disparate clone fuzzy clustering, and clonal allele fraction confinement. In the simulation and in vitro cell line mixture data, CLEMENT outperformed current cancer decomposition algorithms in estimating the number of clones (root-mean-square-error = 0.58-0.78 versus 1.43-3.34) and in the variant-clone membership agreement (∼85.5% versus 70.1-76.7%). Additional testing on human multi-clonal normal tissue sequencing confirmed the accurate identification of subclones that originated from different cell types. Clone-level analysis, including mutational burden and signatures, provided a new understanding of normal-tissue composition. We expect that CLEMENT will serve as a crucial tool in the currently emerging field of non-tumor genome analysis.

Overexpression of R2R3-MYB IbMYB1a induces anthocyanin pigmentation in soybean cotyledon.

KEY MESSAGE: This is the first report showing anthocyanin accumulation in the soybean cotyledon via genetic transformation of a single gene. Soybean [Glycine max (L.) Merrill] contains valuable components, including anthocyanins. To enhance anthocyanin production in Korean soybean Kwangankong, we utilized the R2R3-type MYB gene (IbMYB1a), known for inducing anthocyanin pigmentation in Arabidopsis. This gene was incorporated into constructs using two promoters: the CaMV 35S promoter (P35S) and the ß-conglycinin promoter (Pß-con). Kwangankong was transformed using Agrobacterium, and the presence of IbMYB1a and Bar transgenes in T0 plants was confirmed through polymerase chain reaction (PCR), followed by gene expression validation. Visual inspection revealed that one P35S:IbMYB1a and three Pß-con:IbMYB1a lines displayed seed color change. Pß-con:IbMYB1a T1 seeds accumulated anthocyanins in cotyledon outer layers, whereas P35S:IbMYB1a and non-transgenic black soybean (Cheongja 5 and Seum) accumulated anthocyanins in the seed coat. During the germination and growth phase, T1 seedlings from Pß-con:IbMYB1a lines exhibited anthocyanin pigmentation in cotyledons for up to 1 month without growth aberrations. High-performance liquid chromatography confirmed cyanidin-3-O-glucoside as the major anthocyanin in the Pß-con:IbMYB1a line (#3). We analyzed the expression patterns of anthocyanin biosynthesis genes, chalcone synthase 7,8, chalcone isomerase 1A, flavanone 3-hydroxylase, flavanone 3'-hydroxylase, dihydroflavanol reductase 1, dihydroflavanol reductase 2, anthocyanidin synthase 2, anthocyanidin synthase 3, and UDP glucose flavonoid 3-O-glucosyltransferase in transgenic and control Kwangankong and black soybean (Cheongja 5 and Seum) seeds using quantitative real-time PCR. We conclude that the induction of gene expression in transgenic plants in comparison with Kwangankong was attributable to IbMYB1a transformation. Notably, flavanone 3-hydroxylase, flavanone 3'-hydroxylase, and dihydroflavanol reductase 1 were abundantly expressed in black soybean seed coat, distinguishing them from transgenic cotyledons.

Mutation of GmIPK1 Gene Using CRISPR/Cas9 Reduced Phytic Acid Content in Soybean Seeds.

Phytic acid (PA) acts as an antinutrient substance in cereal grains, disturbing the bioavailability of micronutrients, such as iron and zinc, in humans, causing malnutrition. GmIPK1 encodes the inositol 1,3,4,5,6-pentakisphosphate 2-kinase enzyme, which converts myo-inopsitol-1,3,4,5,6-pentakisphosphate (IP5) to myo-inositol-1,2,3,4,5,6-hexakisphosphate (IP6) in soybean (Glycine max L.). In this study, for developing soybean with low PA levels, we attempted to edit the GmIPK1 gene using the CRISPR/Cas9 system to introduce mutations into the GmIPK1 gene with guide RNAs in soybean (cv. Kwangankong). The GmIPK1 gene was disrupted using the CRISPR/Cas9 system, with sgRNA-1 and sgRNA-4 targeting the second and third exon, respectively. Several soybean Gmipk1 gene-edited lines were obtained in the T0 generation at editing frequencies of 0.1-84.3%. Sequencing analysis revealed various indel patterns with the deletion of 1-9 nucleotides and insertions of 1 nucleotide in several soybean lines (T0). Finally, we confirmed two sgRNA-4 Gmipk1 gene-edited homozygote soybean T1 plants (line #21-2: 5 bp deletion; line #21-3: 1 bp insertion) by PPT leaf coating assay and PCR analysis. Analysis of soybean Gmipk1 gene-edited lines indicated a reduction in PA content in soybean T2 seeds but did not show any defects in plant growth and seed development.

Antimicrobial Activity of Smilax china L. Root Extracts against the Acne-Causing Bacterium, Cutibacterium acnes, and Its Active Compounds.

The root of Smilax china L. is used in traditional Korean medicine. We found that the Smilax china L. root extract has strong antimicrobial activity against two Cutibacterium acnes strains (KCTC 3314 and KCTC 3320). The aim of this study was to identify the beneficial properties of Smilax china L. extracts for their potential use as active ingredients in cosmetics for the treatment of human skin acne. The high-performance liquid chromatography (HPLC) and liquid chromatography-hybrid quadrupole time-of-flight mass spectrometry (LC/QTOF/MS) methods were used to obtain the profile of secondary metabolites from the ethyl acetate-soluble fraction of the crude extract. Agar diffusion and resazurin-based broth microdilution assays were used to evaluate antimicrobial activity and minimum inhibitory concentrations (MIC), respectively. Among the 24 metabolites, quercetin, resveratrol, and oxyresveratrol were the most potent compounds against Cutibacterium acnes. Minimum inhibitory concentrations of quercetin, resveratrol, and oxyresveratrol were 31.25, 125, and 250 µg/mL, respectively.

Elucidation of genomic organizations of transgenic soybean plants through de novo genome assembly with short paired-end reads.

Elucidation of the genomic organizations of transgene insertion sites is essential for the genetic studies of transgenic plants. Herein, we establish an analysis pipeline that identifies the transgene insertion sites as well as the presence of vector backbones, through de novo genome assembly with high-throughput sequencing data in two transgenic soybean lines, AtYUCCA6-#5 and 35S-UGT72E3/2-#7. Sequencing data of approximately 28× and 29× genome coverages for each line generated by high-throughput sequencing were de novo assembled. The databases generated from the de novo assembled sequences were used to search contigs that contained putative insertion sites and their flanking sequences (integration sites) of transgene fragments using transgenic vector sequences as queries. The predicted integration site sequences, which are located at three annotated genes that might regulate plant development or confer disease resistance, were then confirmed by local alignment against the soybean reference genome and PCR amplification. As results, we revealed the precise transgene-flanking sequences and sequence rearrangements at insertion sites in both the transgenic lines, as well as the aberrant insertion of a transgene fragment. Consequently, relative to experimental or enrichment technologies, our approach is straightforward and time-effective, providing an alternative method for the identification of insertion sites in transgenic plants.

Comparison of the seed nutritional composition between conventional varieties and transgenic soybean overexpressing Physaria FAD3-1.

BACKGROUND: PfFAD3 transgenic soybean expressing omega-3 fatty acid desaturase 3 of Physaria produces increased level of α-linolenic acid in seed. Composition data of non-transgenic conventional varieties is important in the safety assessment of the genetically-modified (GM) crops in the context of the natural variation. RESULTS: The natural variation was characterized in seed composition of 13 Korean soybean varieties grown in three locations in South Korea for 2 years. Univariate analysis of combined data showed significant differences by variety and cultivation environment for proximates, minerals, anti-nutrients, and fatty acids. Percent variability analysis demonstrated that genotype, environment and the interaction of environment with genotype contributed to soybean seed compositions. Principal component analysis and orthogonal projections to latent structure discriminant analysis indicated that significant variance in compositions was attributable to location and cultivation year. The composition of three PfFAD3 soybean lines for proximates, minerals, anti-nutrients, and fatty acids was compared to a non-transgenic commercial comparator (Kwangankong, KA), and three non-transgenic commercial varieties grown at two sites in South Korea. Only linoleic and linolenic acids significantly differed in PfFAD3-1 lines compared to KA, which were expected changes by the introduction of the PfFAD3-1 trait in KA. CONCLUSION: Genotype, environment, and the interaction of environment with genotype contributed to compositional variability in soybean. PfFAD3-1 soybean is equivalent to the conventional varieties with respect to these components. © 2020 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

OsNF-YC2 and OsNF-YC4 proteins inhibit flowering under long-day conditions in rice.

MAIN CONCLUSION: OsNF-YC2 and OsNF-YC4 proteins regulate the photoperiodic flowering response through the modulation of three flowering-time genes ( Ehd1, Hd3a , and RFT1 ) in rice. Plant NUCLEAR FACTOR Y (NF-Y) transcription factors control numerous developmental processes by forming heterotrimeric complexes, but little is known about their roles in flowering in rice. In this study, it is shown that some subunits of OsNF-YB and OsNF-YC interact with each other, and among them, OsNF-YC2 and OsNF-YC4 proteins regulate the photoperiodic flowering response of rice. Protein interaction studies showed that the physical interactions occurred between the three OsNF-YC proteins (OsNF-YC2, OsNF-YC4 and OsNF-YC6) and three OsNF-YB proteins (OsNF-YB8, OsNF-YB10 and OsNF-YB11). Repression and overexpression of the OsNF-YC2 and OsNF-YC4 genes revealed that they act as inhibitors of flowering only under long-day (LD) conditions. Overexpression of OsNF-YC6, however, promoted flowering only under LD conditions, suggesting it could function as a flowering promoter. These phenotypes correlated with the changes in the expression of three rice flowering-time genes [Early heading date 1 (Ehd1), Heading date 3a (Hd3a) and RICE FLOWERING LOCUS T1 (RFT1)]. The diurnal and tissue-specific expression patterns of the subsets of OsNF-YB and OsNF-YC genes were similar to those of CCT domain encoding genes such as OsCO3, Heading date 1 (Hd1) and Ghd7. We propose that OsNF-YC2 and OsNF-YC4 proteins regulate the photoperiodic flowering response by interacting directly with OsNF-YB8, OsNF-YB10 or OsNF-YB11 proteins in rice.

The effect of ionized collagen for preventing postoperative adhesion.

BACKGROUND: Collagen exhibits ideal multifactorial action for preventing tissue adhesions. This study examined the efficacy of ionized collagen in preventing tissue adhesion after surgical procedures. MATERIALS AND METHOD: Ionized collagen was prepared using the esterification technique of natural collagen. Three forms of collagen materials (membrane, film, and gel) were compared with three commercialized materials (oxidized regenerated cellulose membrane [OC membrane], hyaluronic acid and carboxymethylcellulose film, and gel [HC film and HC gel]) in a rat cecum abrasion model. Antiadhesive activity and histologic findings were assessed. RESULT: The incidence of adhesion was reduced significantly in all test groups compared to the sham-operated control group (100% in control, 14.3% in collagen membrane, 63.6% in collagen film, 25.0% in collagen gel, 55.6% in OC membrane, 75% in HC film, and 83.3% in HC gel). All collagen materials of the three forms exhibited a significant reduction in adhesion grade compared with the sham operation, whereas no significant difference was found among these three different forms. The collagen membrane showed significantly less adhesion grade, less inflammation and more regenerative features compared to widely used conventional materials. CONCLUSIONS: This preclinical investigation indicated that ionized collagen materials readily formed clinically suitable shapes for easy handling without the need for any complex processing and effectively reduced postoperative tissue adhesion profiles compared to conventional antiadhesive agents.

Functional conservation of rice OsNF-YB/YC and Arabidopsis AtNF-YB/YC proteins in the regulation of flowering time.

KEY MESSAGE: Rice Os NF - YB and Os NF - YC complement the late flowering phenotype of Arabidopsis nf - yb double and nf - yc triple mutants, respectively. In addition, OsNF-YB and OsNF-YC interact with AtNF-YC and AtNF-YB, respectively. Plant NUCLEAR FACTOR Y (NF-Y) transcription factors play important roles in plant development and abiotic stress. In Arabidopsis thaliana, two NF-YB (AtNF-YB2 and AtNF-YB3) and five NF-YC (AtNF-YC1, AtNF-YC2, AtNF-YC3, AtNF-YC4, and AtNF-YC9) genes regulate photoperiodic flowering by interacting with other AtNF-Y subunit proteins. Three rice NF-YB (OsNF-YB8, OsNF-YB10, and OsNF-YB11) and five rice OsNF-YC (OsNF-YC1, OsNF-YC2, OsNF-YC4, OsNF-YC6, and OsNF-YC7) genes are clustered with two AtNF-YB and five AtNF-YC genes, respectively. To investigate the functional conservation of these NF-YB and NF-YC genes in rice and Arabidopsis, we analyzed the flowering phenotypes of transgenic plants overexpressing the respective OsNF-YB and OsNF-YC genes in Arabidopsis mutants. Overexpression of OsNF-YB8/10/11 and OsNF-YC2 complemented the late flowering phenotype of Arabidopsis nf-yb2 nf-yb3 and nf-yc3 nf-yc4 nf-yc9 mutants, respectively. The rescued phenotype of 35S::OsNF-YC2 nf-yc3 nf-yc4 nf-yc9 plants was attributed to the upregulation of FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1). In vitro and in planta protein-protein analyses revealed that OsNF-YB8/10/11 and OsNF-YC1/2/4/6/7 interact with AtNF-YC3/4/9 and AtNF-YB2/3, respectively. Our data indicate that some OsNF-YB and OsNF-YC genes are functional equivalents of AtNF-YB2/3 and AtNF-YC3/4/9 genes, respectively, and suggest functional conservation of Arabidopsis and rice NF-Y genes in the control of flowering time.

Management of late-onset portal vein complications in pediatric living-donor liver transplantation.

The purpose of this study was to evaluate retrospectively the results of PTA for late-onset PV complications after pediatric LDLT and to assess whether a meso-Rex shunt is a viable option for treating restenosis of the PV after PTA in selected cases. Seventy-five children who underwent adult-to-child LDLT were included in this study, and there were six late-onset PV complications (8.0%). The initial therapeutic approach was PTA, with or without stent: PTA with balloon dilation for three children, PTA with stent placement for one child, and failure to cannulate the occluded PV for two children. A meso-Rex shunt was performed in the two children after failed PTA: One suffered complete obstruction of the main PV, and the other, restenosis with total thrombosis after PTA with stent. The PTA was a technical and clinical success in four with PV stenosis of the six patients (66.7%), and successful application of a meso-Rex shunt in the other two children resulted in restoration of PV flow. In conclusion, PTA is a safe and effective procedure for treating late-onset PV stenosis after pediatric LDLT. However, in growing pediatric recipients with restenosis of the PV after PTA or chronic PV thrombosis, a meso-Rex shunt may be a better choice for late-onset PV complications.

Improving the Traits of Perilla frutescens (L.) Britt Using Gene Editing Technology.

Plant breeding has evolved significantly over time with the development of transformation and genome editing techniques. These new strategies help to improve desirable traits in plants. Perilla is a native oil crop grown in Korea. The leaves contain many secondary metabolites related to whitening, aging, antioxidants, and immunity, including rosmarinic acid, vitamin E, luteolin, anthocyanins, and beta-carotene. They are used as healthy and functional food ingredients. It is an industrially valuable cosmetics crop. In addition, perilla seeds are rich in polyunsaturated fatty acids, such as α-linolenic acid and linoleic acid. They are known to be effective in improving neutral lipids in the blood, improving blood circulation, and preventing dementia and cardiovascular diseases, making them excellent crops whose value can be increased through improved traits. This research will also benefit perilla seeds, which can increase their stock through various methods, such as the increased production of functional substances and improved productivity. Recently, significant attention has been paid to trait improvement research involving gene-editing technology. Among these strategies, CRISPR/Cas9 is highly adaptable, enabling accurate and efficient genome editing, targeted mutagenesis, gene knockouts, and the regulation of gene transcription. CRISPR/Cas9-based genome editing has enormous potential for improving perilla; however, the regulation of genome editing is still at an early stage. Therefore, this review summarizes the enhancement of perilla traits using genome editing technology and outlines future directions.

Risk factors for acute kidney injury after radical nephrectomy and inferior vena cava thrombectomy for renal cell carcinoma.

OBJECTIVE: The objectives of the present study are to estimate the incidence of postoperative acute kidney injury (AKI) after radical nephrectomy with inferior vena cava (IVC) thrombectomy for renal cell carcinoma (RCC) based on the Acute Kidney Injury Network (AKIN) criteria, to investigate the risk factors for postoperative AKI, and to define the association between postoperative AKI and clinical outcome in patients undergoing such a surgery. METHODS: We retrospectively analyzed 76 patients (22 women; mean age, 56.9 years; range, 29-83 years) with RCC and IVC thrombus who underwent radical nephrectomy and IVC thrombectomy at our institute between January 2003 and December 2011. Postoperative AKI was diagnosed after surgery based on the AKIN criteria. Logistic regression was used to model the association between preoperative factors and the risk of AKI after surgery. The relationship between postoperative AKI and clinical outcomes, including chronic kidney disease (CKD), mortality, and days in hospital, was investigated. RESULTS: Postoperative AKI was diagnosed in 41 patients (53.9%) based on the AKIN criteria (stage 1, n = 34; stage 2, n = 2; and stage 3, n = 5). Multivariate analysis demonstrated an independent association between postoperative AKI and male gender (odds ratio 4.79, 95% confidence interval: 1.13-20.39; P = .034), and IVC clamping time lasting more than 20 minutes (odds ratio 6.60, 95% confidence interval: 1.48-29.42; P = .013). Development of AKI was associated with an increased rate of postoperative CKD (43.9% vs 20.0%; P = .031) and prolonged hospitalization (17.7 vs 12.2 days; P = .047). Only one patient who had postoperative AKI required renal replacement therapy. There was no 30-day mortality during the study period and no difference in mortality between AKI and non-AKI patients (4.9% vs 5.7%; P = .859). CONCLUSIONS: The incidence of postoperative AKI in patients with RCC and IVC thrombus was considerable. Intraoperative management seems to influence the risk of AKI after surgery; particularly, the longer the IVC clamping time, the higher the risk of postoperative AKI. Postoperative AKI was associated with postoperative CKD (P = .031), prolonged hospitalization (P = .047), and increased long-term mortality (1 year after surgery).

Genome-edited HEADING DATE 3a knockout enhances leaf production in Perilla frutescens.

Environmental cues regulate the transition of many plants from vegetative to flowering development. Day length, or photoperiod, is one cue that synchronizes flowering by changing seasons. Consequently, the molecular mechanism of flowering control is prominent in Arabidopsis and rice, where essential genes like FLOWERING LOCUS T (FT) homolog, HEADING DATE 3a (Hd3a), have been connected to flowering regulation. Perilla is a nutrient-rich leaf vegetable, and the flowering mechanism remains largely elusive. We identified flowering-related genes under short-day conditions using RNA sequencing to develop an enhanced leaf production trait using the flowering mechanism in the perilla. Initially, an Hd3a-like gene was cloned from the perilla and defined as PfHd3a. Furthermore, PfHd3a is highly rhythmically expressed in mature leaves under short-day and long-day conditions. Ectopic expression of PfHd3a in Atft-1 mutant plants has been shown to complement Arabidopsis FT function, resulting in early flowering. In addition, our genetic approaches revealed that overexpression of PfHd3a in perilla caused early flowering. In contrast, the CRISPR/Cas9 generated PfHd3a-mutant perilla showed significantly late flowering, resulting in approximately 50% leaf production enhancement compared to the control. Our results suggest that PfHd3a plays a vital role in regulating flowering in the perilla and is a potential target for molecular breeding in the perilla.

Ectopic expression of ubiquitin-conjugating enzyme gene from wild rice, OgUBC1, confers resistance against UV-B radiation and Botrytis infection in Arabidopsis thaliana.

A previously unidentified gene encoding ubiquitin-conjugating enzyme was isolated from leaves of wild rice plant treated with wounding and microbe-associated molecular patterns. The OgUBC1 gene was composed of 148 amino acids and contained a typical active site and 21 ubiquitin thioester intermediate interaction residues and 4 E3 interaction residues. Both exogenous application of salicylic acid and UV-B irradiation triggered expression of OgUBC1 in leaves of wild rice. Recombinant OgUBC1 proteins bound to ubiquitins in vitro, proposing that the protein might act as E2 enzyme in planta. Heterologous expression of the OgUBC1 in Arabidopsis thaliana protected plants from cellular damage caused by an excess of UV-B radiation. A stable expression of chalcone synthase gene was detected in leaves of OgUBC1-expressing Arabidopsis, resulting in producing higher amounts of anthocyanin than those in wild-type Col-0 plants. Additionally, both pathogenesis-related gene1 and 5 were transcribed in the transgenic Arabidopsis in the absence of pathogen infection. The OgUBC1-expressing plants were resistant to the infection of Botrytis cinerea. Taken together, we suggested that the OgUBC1 is involved in ubiquitination process important for cellular response against biotic and abiotic stresses in plants.

Molecular cloning and characterization of OsUPS, a U-box containing E3 ligase gene that respond to phosphate starvation in rice (Oryza sativa).

The ubiquitin-26S proteasome system is important in the quality control of intracellular proteins. The ubiquitin-26S proteasome system includes the E1 (ubiquitin activating), E2 (ubiquitin conjugating), and E3 (ubiquitin ligase) enzymes. U-box proteins are a derived version of RING-finger domains, which have E3 enzyme activity. Here, we present the isolation of a novel U-box protein, U-box containing E3 ligase induced by phosphate starvation (OsUPS), from rice (Oryza sativa). The cDNA encoding the O. sativa U-box protein (OsUPS) comprises 1338 bp, with an open reading frame of 445 amino acids. The amino acid sequence of OsUPS cDNA shows 41-79% identity with other plant U-box homologous genes. The open reading frame of the OsUPS protein is comprised of notable domains: a single ~70-amino acid domain and a GKL domain that contains conserved glycine, lysine/arginine residues and leucine-rich feature. We found that full-length expression of OsUPS was up-regulated in both rice plants and cell culture in the absence of inorganic phosphate (P(i)). A self-ubiquitination assay indicated that the bacterially expressed OsUPS protein had E3 ligase activity, and subcellular localization results showed that OsUPS was located in the chloroplast. These results support the notion that OsUPS plays an important role in the P(i) signaling pathway through the ubiquitin-26S proteasome system.

Impact of Exercise Intensity on Systemic Oxidative Stress, Inflammatory Responses, and Sirtuin Levels in Healthy Male Volunteers.

Exercise can induce anti-inflammatory and antioxidant effects, for which regulation of sirtuins (SIRTs) may be a major consideration for exercise prescription. The purpose of this study was to investigate the effects of acute aerobic exercise, in particular its intensity, on systemic oxidative stress, inflammatory responses, and SIRT levels. Twenty healthy, untrained males were recruited and randomly assigned to moderate-intensity (MI, 65% VO2max, n = 10) and high-intensity (HI, 85% VO2max, n = 10) exercise. Blood samples were obtained pre-, immediately post-, and 1 h post-exercise for measurements of malonaldehyde (MDA), superoxide dis-mutase (SOD), interleukin (IL)-1ß, IL-6, tumor necrosis factor (TNF)-α, SIRT-1, SIRT-2, and SIRT-3. Overall, MDA, SOD, IL-6, SIRT-1, and SIRT-3 levels were significantly increased at post-exercise compared with pre-exercise regardless of exercise intensity (p < 0.05). The HI group had significantly higher MDA, SOD, and IL-6 levels than the MI group at post-exercise (p < 0.05), whereas no significant differences were observed in the IL-1ß, TNF-α, and SIRT-2 levels (p > 0.05). Altogether, these findings suggest that exercise-induced oxidative stress and inflammatory responses may be dependent on exercise intensity. Moreover, activation of inflammatory cytokines and SIRT family members may be dependent on the intensity of the exercise.

RNA-seq Gene Profiling Reveals Transcriptional Changes in the Late Phase during Compatible Interaction between a Korean Soybean Cultivar (Glycine max cv. Kwangan) and Pseudomonas syringae pv. syringae B728a.

Soybean (Glycine max (L) Merr.) provides plant-derived proteins, soy vegetable oils, and various beneficial metabolites to humans and livestock. The importance of soybean is highly underlined, especially when carbon-negative sustainable agriculture is noticeable. However, many diseases by pests and pathogens threaten sustainable soybean production. Therefore, understanding molecular interaction between diverse cultivated varieties and pathogens is essential to developing disease-resistant soybean plants. Here, we established a pathosystem of the Korean domestic cultivar Kwangan against Pseudomonas syringae pv. syringae B728a. This bacterial strain caused apparent disease symptoms and grew well in trifoliate leaves of soybean plants. To examine the disease susceptibility of the cultivar, we analyzed transcriptional changes in soybean leaves on day 5 after P. syringae pv. syringae B728a infection. About 8,900 and 7,780 differentially expressed genes (DEGs) were identified in this study, and significant proportions of DEGs were engaged in various primary and secondary metabolisms. On the other hand, soybean orthologs to well-known plant immune-related genes, especially in plant hormone signal transduction, mitogen-activated protein kinase signaling, and plant-pathogen interaction, were mainly reduced in transcript levels at 5 days post inoculation. These findings present the feature of the compatible interaction between cultivar Kwangan and P. syringae pv. syringae B728a, as a hemibiotroph, at the late infection phase. Collectively, we propose that P. syringae pv. syringae B728a successfully inhibits plant immune response in susceptible plants and deregulates host metabolic processes for their colonization and proliferation, whereas host plants employ diverse metabolites to protect themselves against infection with the hemibiotrophic pathogen at the late infection phase.

Gibberellic acid sensitive dwarf encodes an ARPC2 subunit that mediates gibberellic acid biosynthesis, effects to grain yield in rice.

The plant hormone gibberellic acid (GA) is important for plant growth and productivity. Actin-related proteins (ARPs) also play central roles in plant growth, including cell elongation and development. However, the relationships between ARPs and GA signaling and biosynthesis are not fully understood. Here, we isolated OsGASD, encoding an ARP subunit from rice (Oryza sativa), using the Ac/Ds knockout system. The osgasd knockout (Ko) mutation reduced GA3 content in shoots as well as plant growth and height. However, GA application restored the plant height of the osgasd Ko mutant to a height similar to that of the wild type (WT). Rice plants overexpressing OsGASD (Ox) showed increased plant height and grain yield compared to the WT. Transcriptome analysis of flag leaves of OsGASD Ox and osgasd Ko plants revealed that OsGASD regulates cell development and the expression of elongation-related genes. These observations suggest that OsGASD is involved in maintaining GA homeostasis to regulate plant development, thereby affecting rice growth and productivity.

Molecular characterization of phenylalanine ammonia lyase gene from Cistanche deserticola.

We cloned the gene, CdPAL1, from Cistanche deserticola callus using RACE PCR with degenerate primers that were designed based on a multiple sequence alignment of known PAL genes from other plant species. The gene shows high homology to other known PAL genes registered in GenBank. The recombinant protein exhibited Michaelis-Menten kinetics with a Km of 0.1013 mM, Vmax of 4.858 µmol min(-1), Kcat of 3.36 S(-1), and Kcat/Km is 33,168 M(-1) S(-1). The enzyme had an optimal pH of 8.5 and an activation energy of 38.92 kJ mol(-1) when L-Phenylalanine was used as a substrate; L-tyrosine cannot be used as substrate for this protein. The optimal temperature was 55°C, and the thermal stability results showed that, after a treatment at 70°C for 20 min, the protein retained 87% activity, while a treatment at 75°C for 20 min resulted in a loss of over 85% of the enzyme activity. Treatment with heavy metal ions (Hg2+, Pb2+, and Zn2+) showed remarkable inhibitory effects. Among the intermediates from the lignin (cinnamyl alcohol, cinnamyl aldehyde, coniferyl aldehyde, coniferyl alcohol), phenylpropanoid (cinnamic acid, coumaric acid, caffeic acid, and chlorogenic acid) and phenylethanoid (tyrosol and salidroside) biosynthetic pathways, only cinnamic acid showed strong inhibitory effects against CdPAL1 activity with a Ki of 8 µM. Competitive inhibitor AIP exhibited potent inhibition with Ki=0.056 µM.

Effects of 2-aminoindan-2-phosphonic acid treatment on the accumulation of salidroside and four phenylethanoid glycosides in suspension cell culture of Cistanche deserticola.

2-Aminoindan-2-phosphonic acid (AIP), a specific competitive phenylalanine ammonia lyase (PAL) inhibitor was applied to a suspension cell culture of Cistanche deserticola. The effects of AIP treatment on cell growth, PAL activity, contents and yields of total phenolic compound, salidroside and four phenylethanoid glycosides (PheGs) are investigated. The results demonstrated that, 0.5 and 2.0 µM AIP treatments had similar effects on the measurements investigated in this study. AIP treatment resulted in significant decreases in PAL activity, total phenolic compounds content, and PheGs content. Linear regression analysis showed that PAL activity had a high correlation coefficient with the total phenolic compound content and the four PheGs contents. Total PAL activity-time area under curve (AUC) had a high correlation coefficient with the total phenolic compound yield and the yields of five tested compounds in untreated cell samples. In AIP-treated cells, total PAL activity-time AUC retained a high correlation with the total phenolic compound yield and the yields of three tested compounds, echinacoside, acteoside, and tubuloside A, but not salidroside and cistanoside A. The difference could be caused by the different biosynthetic origins of each of the tested compounds. These results demonstrate the important role of PAL in the biosynthesis of PheGs in the suspension cell culture of C. deserticola.