Genome-wide analysis of KIX gene family for organ size regulation in soybean (Glycine max L.).

The KIX domain, conserved among various nuclear and co-activator factors, acts as a binding site that interacts with other transcriptional activators and co-activators, playing a crucial role in gene expression regulation. In plants, the KIX domain is involved in plant hormone signaling, stress response regulation, cell cycle control, and differentiation, indicating its potential relevance to crop productivity. This study aims to identify and characterize KIX domains within the soybean (Glycine max L.) genome to predict their potential role in improving crop productivity. The conservation and evolutionary history of the KIX domains were explored in 59 plant species, confirming the presence of the KIX domains in diverse plants. Specifically, 13 KIX domains were identified within the soybean genome and classified into four main groups, namely GmKIX8/9, GmMED15, GmHAC, and GmRECQL, through sequence alignment, structural analysis, and phylogenetic tree construction. Association analysis was performed between KIX domain haplotypes and soybean seed-related agronomic traits using re-sequencing data from a core collection of 422 accessions. The results revealed correlations between SNP variations observed in GmKIX8-3 and GmMED15-4 and soybean seed phenotypic traits. Additionally, transcriptome analysis confirmed significant expression of the KIX domains during the early stages of soybean seed development. This study provides the first characterization of the structural, expression, genomic haplotype, and molecular features of the KIX domain in soybean, offering a foundation for functional analysis of the KIX domain in soybean and other plants.

Sargassum horneri (Turner) C. Agardh Extract Regulates Neuroinflammation In Vitro and In Vivo.

Previously, we reported that Sargassum horneri (Turner) C. Agardh (S. horneri) is a brown algae species that exerts anti-inflammatory activity toward murine macrophages. However, the anti-neuroinflammatory effects and the mechanism of S. horneri on microglia cells are still unknown. We investigated the anti-neuroinflammatory effects of S. horneri extract on microglia in vitro and in vivo. In the present study, we found that S. horneri was not cytotoxic to BV-2 microglia cells and it significantly decreased lipopolysaccharide (LPS)-induced NO production. Moreover, S. horneri also diminished the protein expression of iNOS, COX-2, and cytokine production, including IL-1ß, TNF-α, and IL-6, on LPS-stimulated microglia activation. S. horneri elicited anti-neuroinflammatory effects by inhibiting phosphorylation of p38 MAPK and NF-κB. In addition, S. horneri inhibited astrocytes and microglia activation in LPS-challenged mice brain. Therefore, these results suggested that S. horneri exerted anti-neuroinflammatory effects on LPS-stimulated microglia cell activation by inhibiting neuroinflammatory factors and NF-κB signaling.

The discovery of novel SNPs associated with group A soyasaponin biosynthesis from Korea soybean core collection.

Soyasaponin is a type of glycoside such as steroids, steroidal alkaloids or triterpenes, which enhance the body immunity. In order to efficiently identify genes and markers related to the soyasaponin, we used a 180K Axiom® SoyaSNP array and whole genome resequencing data from the Korean soybean core collection. As a result of conducting GWAS for group A soyasaponin (Aa and Ab derivatives), 16 significant common markers associated with Aa and Ab derivatives were mapped to chromosome 7, and three candidate genes including Glyma.07g254600 were detected. The functional haplotypes for candidate genes showed that Aa and Ab contents were mainly determined by alleles of AX-90322128, the marker of Glyma.07g254600. In addition, 14 novel SNPs variants closely associated with Aa and Ab derivatives were discovered for Glyma.07g254600. Therefore, the results of this study that identified soyasaponin-associated markers and useful genes utilizing various genomic information could provide insight into functional soybean breeding.

Analysis of Phenotypic Characteristics and Sucrose Metabolism in the Roots of Raphanus sativus L.

The taproot of radish (Raphanus sativus L.) is an important sink organ; it is morphologically diverse and contains large amounts of secondary metabolites. Sucrose metabolism is believed to be important in the development of sink organs. We measured the amounts of glucose, fructose, and sucrose in the roots of sixty three radish accessions and analyzed the association between the sugar content and the root phenotype. Fructose content correlated with the root color and length characteristics, glucose was the most abundant sugar in the roots, and the sucrose content was very low, compared to that of the hexoses in most of the accessions. Expression analysis of the genes involved in sucrose metabolism, transportation, starch synthesis, and cell wall synthesis was performed through RNA sequencing. The genes encoding sucrose synthases (SUSY) and the enzymes involved in the synthesis of cellulose were highly expressed, indicating that SUSY is involved in cell wall synthesis in radish roots. The positive correlation coefficient (R) between the sucrose content and the expression of cell wall invertase and sugar transporter proteins suggest that hexose accumulation could occur through the apoplastic pathway in radish roots. A positive R score was also obtained when comparing the expression of genes encoding SUSY and fructokinase (FK), suggesting that the fructose produced by SUSY is mostly phosphorylated by FK. In addition, we concluded that sucrose was the most metabolized sugar in radish roots.

Effects of a High-Molecular-Weight Polysaccharides Isolated from Korean Persimmon on the Antioxidant, Anti-Inflammatory, and Antiwrinkle Activity.

Persimmon (Diospyros kaki), a familiar and widespread fruit worldwide, is known to exhibit several physiological effects because of the presence of pharmacologically active compounds called phytochemicals. However, its high-molecular-weight compounds, particularly polysaccharides, have not been extensively studied. In this study, D. kaki extract (DK) was fractionated into low- and high-molecular-weight fractions (DK-L and DK-H, respectively) through ethanol fractionation, and their effects on antioxidant, anti-inflammatory, and antiwrinkle activities were investigated by an in vitro system. DK-H contained significantly higher contents of neutral sugar, uronic acid, and polyphenols compared to DK and DK-L. Furthermore, DK-H exhibited significantly improved pharmacological activities, such as antioxidant, anti-inflammatory, and antiwrinkle properties, compared to those of DK and DK-L, demonstrating that DK-H may play an important role in mediating the beneficial effects of persimmon. Sugar composition analysis and molecular characterization indicated that DK-H consisted of a galacturonic acid (GalA)-rich polysaccharide with a molecular weight of >345 kDa that mainly comprised GalA and small amounts of neutral sugar and polyphenol residues. These results suggest that the bioactive fraction DK-H is likely to be a GalA-rich pectic polysaccharide containing a small number of polyphenol residues, which may be a novel candidate in the pharmaceutical and cosmeceutical industries.

Induction of Glucoraphasatin Biosynthesis Genes by MYB29 in Radish (Raphanus sativus L.) Roots.

Glucoraphasatin (GRH) is a specific aliphatic glucosinolate (GSL) that is only abundant in radish (Raphanus sativus L.). The gene expression regulating GRH biosynthesis in radish is still poorly understood. We employed a total of 59 radish accessions to analyze GSL profiles and showed that GRH was specific and predominant among the aliphatic GSLs in radish roots. We selected five accessions roots with high, moderate and low GSL biosynthesis, respectively, to conduct a comparative transcriptome analysis and the qRT-PCR of the biosynthesis genes for aliphatic GSLs. In this study, among all the accessions tested, roots with the accession RA157-74 had a high GRH content and showed a significant expression of the aliphatic GSL biosynthesis genes. We defined the genes involved in the GRH biosynthesis process and found that they were regulated by a transcription factor (RSG00789) at the MYB29 locus in radish roots. We found 13 aliphatic GSL biosynthesis genes regulated by the RSG00789 gene in the GRH biosynthesis pathway.

Molecular characterization of glucosinolates and carotenoid biosynthetic genes in Chinese cabbage (Brassica rapa L. ssp. pekinensis).

The present study aimed to investigate the contents of glucosinolates (GSLs) and carotenoids in eleven varieties of Chinese cabbage in relation to the expression level of the important transcription factors. MS and HPLC analysis identified the presence of 13 GSLs (progoitrin, sinigrin, glucoalyssin, gluconapoleiferin, gluconapin, glucocochlearin, glucobrassicanapin, glucoerucin, 4-hydroxyglucobrassicin, glucobrassicin, 4-methoxyglucobrassicin, neoglucobrassicin and gluconasturtiin) and four carotenoids (lutein, zeaxanthin, α-carotene and ß-carotene). GSL contents were varied among the different cabbage varieties. The total GSL content ranged from 2.7 to 57.88 µmol/g DW. The proportion of gluconapin (54%) and glucobrassicanapin (22%) was higher in all the varieties, respectively. Results documented the variation in total and individual carotenoid contents that have also been observed among different varieties; however, the total carotenoid contents ranged from 289.12 to 1001.41 mg kg-1 DW (mean 467.66). Interestingly, the proportion of lutein (66.5) and ß-carotene (25.9) were higher than α-carotene (5.1) and zeaxanthin (2.5%). Consequently, the expression level of the regulatory gene, MYB28 was higher in 'K0648' and was directly proportional to GSL content. Similarly, the expression levels of 1-PSY were higher in 'K0112'; however, the expression levels of 2-ZDS, 3-LCYB, 4-LCYE, 5-CHXB and 7-NCED genes showed no significant difference. In addition, the correlation between GSL and carotenoid contents and gene expression level showed moderate significant difference in each Chinese cabbage.

Expression profiles of BrMYB transcription factors related to glucosinolate biosynthesis and stress response in eight subspecies of Brassica rapa.

Brassica rapa is a polyploid species with phenotypically diverse cultivated subspecies. Glucosinolates (GSLs) are secondary metabolites that contribute to anticarcinogenic activity and plant defense in Brassicaceae. Previously, complete coding sequences of 13 BrMYB transcription factors (TFs) related to GSL biosynthesis were identified in the B. rapa genome. In the present study, we investigated GSL content and expression levels of these BrMYB TFs in 38 accessions belonging to eight subspecies of B. rapa. Twelve identified GSLs were detected and were classified into three chemical groups based on patterns of GSL content and expression profiles of the BrMYB TFs. GSL content and BrMYB TF expression levels differed among genotypes, including B. rapa subspecies pekinensis, chinensis and rapa. BrMYB28.3, BrMYB51.1 and BrMYB122.2 positively regulated GSL content in 38 accessions. Furthermore, expression levels of BrMYB28s and BrMYB34.3 increased under most abiotic and biotic stress treatments. The three BrMYB51 paralogs also showed drastically increased expression levels after infection with Pectobacterium carotovorum. The results of the present study improve our understanding of the functional diversity of these 13 BrMYB TFs during the evolution of polyploid B. rapa.

Understanding of MYB Transcription Factors Involved in Glucosinolate Biosynthesis in Brassicaceae.

Glucosinolates (GSLs) are widely known secondary metabolites that have anticarcinogenic and antioxidative activities in humans and defense roles in plants of the Brassicaceae family. Some R2R3-type MYB (myeloblastosis) transcription factors (TFs) control GSL biosynthesis in Arabidopsis. However, studies on the MYB TFs involved in GSL biosynthesis in Brassica species are limited because of the complexity of the genome, which includes an increased number of paralog genes as a result of genome duplication. The recent completion of the genome sequencing of the Brassica species permits the identification of MYB TFs involved in GSL biosynthesis by comparative genome analysis with A. thaliana. In this review, we describe various findings on the regulation of GSL biosynthesis in Brassicaceae. Furthermore, we identify 63 orthologous copies corresponding to five MYB TFs from Arabidopsis, except MYB76 in Brassica species. Fifty-five MYB TFs from the Brassica species possess a conserved amino acid sequence in their R2R3 MYB DNA-binding domain, and share close evolutionary relationships. Our analysis will provide useful information on the 55 MYB TFs involved in the regulation of GSL biosynthesis in Brassica species, which have a polyploid genome.

Functional analysis of three BrMYB28 transcription factors controlling the biosynthesis of glucosinolates in Brassica rapa.

Glucosinolates (GSLs) are secondary metabolites that have anticarcinogenic activity and play defense roles in plants of the Brassicaceae family. MYB28 is known as a transcription factor that regulates aliphatic GSL biosynthesis in Arabidopsis thaliana. Brassicaceae plants have three orthologous copies of AtMYB28 derived from recent genome triplication. These BrMYB28 genes have a high level of sequence homology, with 81-87% similarities in the coding DNA sequence compared to Arabidopsis. Overexpression of three paralogous BrMYB28 genes in transgenic Chinese cabbage increased the total GSL content in all T1 generation plants and in two inbred lines of homozygous T2 plants. The highest total GSL contents were detected in homozygous T2 lines overexpressing BrMYB28.1, which showed an approximate fivefold increase compared to that of nontransgenic plants. The homozygous T2 lines with overexpressed BrMYB28.1 also showed an increased content of aliphatic, indolic, and aromatic GSLs compared to that of nontransgenic plants. Furthermore, all of the three BrMYB28 genes were identified as negative regulators of BrAOP2 and positive regulators of BrGSL-OH in the homozygous T2 lines. These data indicate the regulatory mechanism of GSL biosynthesis in B. rapa is unlike that in A. thaliana. Our results will provide useful information for elucidating the regulatory mechanism of GSL biosynthesis in polyploid plants.

Cell-autonomous-like silencing of GFP-partitioned transgenic Nicotiana benthamiana.

We previously reported the novel partitioning of regional GFP-silencing on leaves of 35S-GFP transgenic plants, coining the term "partitioned silencing". We set out to delineate the mechanism of partitioned silencing. Here, we report that the partitioned plants were hemizygous for the transgene, possessing two direct-repeat copies of 35S-GFP. The detection of both siRNA expression (21 and 24 nt) and DNA methylation enrichment specifically at silenced regions indicated that both post-transcriptional gene silencing (PTGS) and transcriptional gene silencing (TGS) were involved in the silencing mechanism. Using in vivo agroinfiltration of 35S-GFP/GUS and inoculation of TMV-GFP RNA, we demonstrate that PTGS, not TGS, plays a dominant role in the partitioned silencing, concluding that the underlying mechanism of partitioned silencing is analogous to RNA-directed DNA methylation (RdDM). The initial pattern of partitioned silencing was tightly maintained in a cell-autonomous manner, although partitioned-silenced regions possess a potential for systemic spread. Surprisingly, transcriptome profiling through next-generation sequencing demonstrated that expression levels of most genes involved in the silencing pathway were similar in both GFP-expressing and silenced regions although a diverse set of region-specific transcripts were detected.This suggests that partitioned silencing can be triggered and regulated by genes other than the genes involved in the silencing pathway.

Identification and mapping of a novel dominant resistance gene, TuRB07 to Turnip mosaic virus in Brassica rapa.

KEY MESSAGE: A novel dominant resistance gene, TuRB07, was found to confer resistance to an isolate of TuMV strain C4 in B. rapa line VC1 and mapped on the top of chromosome A06. The inheritance of resistance to Turnip mosaic virus in Brassica rapa was investigated by crossing the resistant line, VC1 with the susceptible line, SR5, and genotyping and phenotyping diverse progenies derived from this cross. Both a doubled haploid population, VCS3M-DH, an F2 and two BC1 (F1 × VC1 and F1 × SR5) populations were created. Population tests revealed that the resistance to the TuMV C4 isolate in B. rapa is controlled by a single dominant gene. This resistance gene, TuRB07 was positioned on the top of linkage group A06 of the B. rapa genome through bulk segregation analysis and fine mapping recombinants in three doubled haploid- and one backcross population using microsatellite markers developed from BAC end sequences. Within the region between the two closely linked markers flanking TuRB07, H132A24-s1, and KS10960, in the Chiifu reference genome, two genes encoding nucleotide-binding site and leucine-rich repeat proteins with a coiled-coil motif (CC-NBS-LRR), Bra018862 and Bra018863 were identified as candidate resistance genes. The gene Bra018862 is truncated, but the gene Bra018863 has all the domains to function. Furthermore, the analysis of structural variation using resequencing data of VC1 and SR5 revealed that Bra018863 might be a functional gene because the gene has no structural variation in the resistant line VC1 when compared with Chiifu, whereas at the other NBS-LRR genes large deletions were identified in the resistant line. Allelic differences of Bra018863 were found between VC1 and SR5, supporting the notion that this gene is a putative candidate gene for the virus resistance.

Mapping quantitative trait loci for tissue culture response in VCS3M-DH population of Brassica rapa.

KEY MESSAGE: Quantitative trait loci (QTL) controlling callus induction and plant regeneration were identified in the VCS3M-DH population of Brassica rapa. Quantitative trait loci (QTL) controlling callus induction and plant regeneration were identified in the VCS3M-DH population of Brassica rapa. The VCS3M-DH population showed wide and continuous variation in callus induction and shoot regeneration. Significant coefficient correlations were detected between these two parameters. Broad-sense heritability (h (2)) for the two traits was around 0.7, indicating genetic regulation of regeneration ability in this population. In the composite interval mapping analysis, two QTLs for callus induction ability, qCi2 and qCi7, were mapped on chromosome A02 and A07, explaining 28.6 % of phenotypic variation. For plant regeneration, four QTLs, qPr6-1 qPr6-2, qPr7, and qPr9 were identified on chromosome A06, A07, and A09, which in total explained 50.1 % of phenotypic variation. Furthermore, 15 putative candidate genes were found on the interval of the six QTLs, which were related to various plant hormones, MADS-box genes, and serine/threonine related genes. These results provide important information to identify genes related to tissue culture ability in B. rapa.

Identification and characterization of stress resistance related genes of Brassica rapa.

Two biotic stress resistance related genes from the full-length cDNA library of Brassica rapa cv. Osome were identified from EST analysis and determined to be pathogenesis-related (PR) 12 Brassica defensin-like family protein (BrDLFP) and PR-10 Brassica Betv1 allergen family protein (BrBetv1AFP) after sequence analysis and homology study with other stress resistance related same family genes. In the expression analysis, both genes expressed in different organs and during all developmental growth stages in healthy plants. Expression of BrDLFP significantly increased and BrBetv1AFP gradually decreased after infection with Pectobacterium carotovorum subsp. carotovorum in Chinese cabbage. Expression of these two genes significantly changed after cold, salt, drought and ABA stress treatments. These two PR genes may therefore be involved in the plant resistance against biotic and abiotic stresses.

Identification and expression analysis of chitinase genes related to biotic stress resistance in Brassica.

Brassica is a very important vegetable group because of its contribution to human nutrition and consequent economic benefits. However, biotic stress is a major concern for these crops and molecular biology techniques offer the most efficient of approaches to address this concern. Chitinase is an important biotic stress resistance-related gene. We identified three genes designated as Brassica chitinase like protein (BrCLP1), BrCLP2 and BrCLP3 from a full-length cDNA library of Brassica rapa cv. Osome. Sequence analysis of these genes confirmed that BrCLP1 was a class IV chitinase, and BrCLP2 and BrCLP3 were class VII chitinases. Also, these genes showed a high degree of homology with other biotic stress resistance-related plant chitinases. In expression analysis, organ-specific expression of all three genes was high except BrCLP1 in all the organs tested and BrCLP2 showed the highest expression compared to the other genes in flower buds. All these genes also showed expression during all developmental growth stages of Chinese cabbage. In addition, BrCLP1 was up-regulated with certain time of infection by Pectobacterium carotovorum subsp. carotovorum in Chinese cabbage plants during microarray expression analysis. On the other hand, expression of BrCLP2 and BrCLP3 were increased after 6 h post inoculation (hpi) but decreased from 12 hpi. All these data suggest that these three chitinase genes may be involved in plant resistance against biotic stresses.

Sequence and structure of Brassica rapa chromosome A3.

BACKGROUND: The species Brassica rapa includes important vegetable and oil crops. It also serves as an excellent model system to study polyploidy-related genome evolution because of its paleohexaploid ancestry and its close evolutionary relationships with Arabidopsis thaliana and other Brassica species with larger genomes. Therefore, its genome sequence will be used to accelerate both basic research on genome evolution and applied research across the cultivated Brassica species. RESULTS: We have determined and analyzed the sequence of B. rapa chromosome A3. We obtained 31.9 Mb of sequences, organized into nine contigs, which incorporated 348 overlapping BAC clones. Annotation revealed 7,058 protein-coding genes, with an average gene density of 4.6 kb per gene. Analysis of chromosome collinearity with the A. thaliana genome identified conserved synteny blocks encompassing the whole of the B. rapa chromosome A3 and sections of four A. thaliana chromosomes. The frequency of tandem duplication of genes differed between the conserved genome segments in B. rapa and A. thaliana, indicating differential rates of occurrence/retention of such duplicate copies of genes. Analysis of 'ancestral karyotype' genome building blocks enabled the development of a hypothetical model for the derivation of the B. rapa chromosome A3. CONCLUSIONS: We report the near-complete chromosome sequence from a dicotyledonous crop species. This provides an example of the complexity of genome evolution following polyploidy. The high degree of contiguity afforded by the clone-by-clone approach provides a benchmark for the performance of whole genome shotgun approaches presently being applied in B. rapa and other species with complex genomes.

A multi-residue method for the determination of 203 pesticides in rice paddies using gas chromatography/mass spectrometry.

This work reports a rapid, specific and sensitive multi-residue method based on the Quick Easy Cheap Effective Rugged and Safe (QuEChERS) sample preparation method and gas chromatography with mass spectrometric detection in the selected ion monitoring mode (GC-MS-SIM) using one quantification ion and two identification ions for the routine analysis of 203 pesticides in rice paddies. Analyses of fortified rice paddy samples were performed at different levels (0.05, 0.20 and 0.50 mg kg(-1)). Mean recoveries from five replicates ranged from 75% to 115%, with coefficients of variation lower than 17%. The limit of quantification was in the range of 0.002-0.05 mg kg(-1) for the pesticides. 1040 rice paddy samples were analyzed for method application.