Integration of transcriptome and metabolome reveals the accumulation of related metabolites and gene regulation networks during quinoa seed development.

Quinoa seeds are gluten- and cholesterol-free, contain all amino acids required by the human body, have a high protein content, provide endocrine regulation, protein supplementation, and cardiovascular protection effects. However, metabolite accumulation and transcriptional regulatory networks in quinoa seed development are not well understood. Four key stages of seed development in Dianli-3260 and Dianli-557 were thus analyzed and 849 metabolites were identified, among which sugars, amino acids, and lipids were key for developmental processes, and their accumulation showed a gradual decrease. Transcriptome analysis identified 40,345 genes, of which 20,917 were differential between the M and F phases, including 8279 and 12,638 up- and down-regulated genes, respectively. Grain development processes were mainly enriched in galactose metabolism, pentose and glucuronate interconversions, the biosynthesis of amino acids, and carbon metabolism pathways, in which raffinose, phosphoenolpyruvate, series and other metabolites are significantly enriched, gene-LOC110689372, Gene-LOC110710556 and gene-LOC110714584 are significantly expressed, and these metabolites and genes play an important role in carbohydrate metabolism, lipid and Amino acid synthesis of quinoa. This study provides a theoretical basis to expand our understanding of the molecular and metabolic development of quinoa grains.

Genetic Diversity and Genome-Wide Association Analysis of the Hulled/Naked Trait in a Barley Collection from Shanghai Agricultural Gene Bank.

Barley is one of the most important cereal crops in the world, and its value as a food is constantly being revealed, so the research into and the use of barley germplasm are very important for global food security. Although a large number of barley germplasm samples have been collected globally, their specific genetic compositions are not well understood, and in many cases their origins are even disputed. In this study, 183 barley germplasm samples from the Shanghai Agricultural Gene Bank were genotyped using genotyping-by-sequencing (GBS) technology, SNPs were identified and their genetic parameters were estimated, principal component analysis (PCA) was preformed, and the phylogenetic tree and population structure of the samples were also analyzed. In addition, a genome-wide association study (GWAS) was carried out for the hulled/naked grain trait, and a KASP marker was developed using an associated SNP. The results showed that a total of 181,906 SNPs were identified, and these barley germplasm samples could be roughly divided into three categories according to the phylogenetic analysis, which was generally consistent with the classification of the traits of row type and hulled/naked grain. Population structure analysis showed that the whole barley population could be divided into four sub-populations (SPs), the main difference from previous classifications being that the two-rowed and the hulled genotypes were sub-divided into two SPs. The GWAS analysis of the hulled/naked trait showed that many associated loci were unrelated to the Nud/nud locus, indicating that there might be new loci controlling the trait. A KASP marker was developed for one exon-type SNP on chromosome 7. Genotyping based on the KASP assay was consistent with that based on SNPs, indicating that the gene of this locus might be associated with the hulled/naked trait. The above work not only lays a good foundation for the future utilization of this barley germplasm population but it provides new loci and candidate genes for the hulled/naked trait.

Transcriptomics integrated with metabolomics reveals the effect of cold stress on rice microspores.

BACKGROUND: Microspore culture is one of the important biotechnological tools in plant breeding. The induction of microspore embryogenesis is a critical factor that affects the yield of microspore-derived embryo productions. Cold treatment has been reported to reprogram the gametophytic pathway in various plant species. However, the exact mechanism(s) underlying the effect of cold pre-treatment of floral buds on the efficiency of ME is still not clear. RESULTS: In this study, the effects of cold stress on the microspore totipotency of rice cultivar Zhonghua 11 were investigated. Our results revealed that a 10-day cold treatment is necessary for microspore embryogenesis initiation. During this period, the survival rate of microspores increased and reached a peak at 7 days post treatment (dpt), before decreasing at 10 dpt. RNA-seq analysis showed that the number of DEGs increased from 3 dpt to 10 dpt, with more downregulated DEGs than upregulated ones at the same time point. GO enrichment analysis showed a shift from 'Response to abiotic stimulus' at 3 dpt to 'Metabolic process' at 7 and 10 dpt, with the most significant category in the cellular component being 'cell wall'. KEGG analysis of the pathways revealed changes during cold treatment. Mass spectrometry was used to evaluate the variations in metabolites at 10 dpt compared to 0 dpt, with more downregulated DEMs being determined in both GC-MS and LC-MS modes. These DEMs were classified into 11 categories, Most of the DEMs belonged to 'lipids and lipid-like molecules'. KEGG analysis of DEMs indicates pathways related to amino acid and nucleotide metabolism being upregulated and those related to carbohydrate metabolism being downregulated. An integration analysis of transcriptomics and metabolomics showed that most pathways belonged to 'Amino acid metabolism' and 'Carbohydrate metabolism'. Four DEMs were identified in the interaction network, with stearidonic acid involving in the most correlations, suggesting the potential role in microspore totipotency. CONCLUSIONS: Our findings exhibited the molecular events occurring during stress-induced rice microspore. Pathways related to 'Amino acid metabolism' and 'Carbohydrate metabolism' may play important roles in rice microspore totipotency. Stearidonic acid was identified, which may participate in the initiation of microspore embryogenesis.

Barley Grass Juice Attenuates Hydrodynamic Transfection-Induced HCC Initiation in Mice.

Barley (Hordeum vulgare L.) grass has been recognized as a functional food with a wide spectrum of health-promoting properties. Supplementation with barley grass has the potential to prevent chronic diseases, such as cancer. Here, we investigated whether barley grass could protect against hepatocellular carcinoma (HCC). Our data showed that administration of barley grass juice attenuates tumor development in a hydrodynamic gene delivery-induced model of HCC. The expression levels of the immune cell markers Ptprc and Adgre1 were upregulated in the barley grass juice-treated and normal groups, compared to those in the vehicle group in the HCC model. Immune cells (CD45+, F4/80+, and CLEC4F + iNOS + cells) infiltration in the liver increased following barley grass juice administration. Our results indicate that barley grass could be beneficial for HCC alleviation, partly by regulating immune cell infiltration. The ingredients of barley grass affect immune cell infiltration in HCC, and the detailed mechanism requires further study.

Methylene Blue Reduces Retinal Cell Inflammation, Apoptosis, and Oxidative Stress in a Rat Model of Diabetic Retinopathy via Sirtuin 1 Activation.

Objective: Diabetic retinopathy (DR), characterized by neuronal damage in the retina, is primarily driven by oxidative stress resulting from diabetes (DM). This study investigated the potential effects of methylene blue (MB) on streptozotocin (STZ)-induced DR. Methods: A rat model of DR was established via STZ injection, while a cell model was created using high-glucose (HG) exposure of human retinal microvascular endothelial cells. Evaluation of oxidative stress markers, pro-inflammatory cytokines, and pro-apoptotic proteins was performed based on their expression profiles in human retinal microvascular endothelial cells. Results: MB treatment significantly upregulated the expression of sirtuin 1 (SIRT1), which was found to be downregulated in the retinal tissues of STZ-treated rats and HG-exposed human retinal microvascular endothelial cells, as determined by polymerase chain reaction (PCR). Furthermore, MB therapy effectively suppressed STZ-induced oxidative stress, inflammation, and cell death. Consistent with the in vivo findings, MB activated the expression of SIRT1, thereby protecting HG-treated human retinal microvascular endothelial cells against oxidative stress, inflammation, and apoptosis. Conclusion: These results support the conclusion that MB mitigates DR by activating SIRT1, leading to a reduction of inflammation, apoptosis, and oxidative stress.

Transcriptomic and Metabolomic Analyses Reveal the Importance of Lipid Metabolism and Photosynthesis Regulation in High Salinity Tolerance in Barley (Hordeum vulgare L.) Leaves Derived from Mutagenesis Combined with Microspore Culture.

Barley is the most salt-tolerant cereal crop. However, little attention has been paid to the salt-tolerant doubled haploids of barley derived from mutagenesis combined with isolated microspore culture. In the present study, barley doubled haploid (DH) line 20, which was produced by mutagenesis combined with isolated microspore culture, showed stably and heritably better salt tolerance than the wild type H30 in terms of fresh shoot weight, dry shoot weight, K+/Na+ ratio and photosynthetic characteristics. Transcriptome and metabolome analyses were performed to compare the changes in gene expression and metabolites between DH20 and H30. A total of 462 differentially expressed genes (DEGs) and 152 differentially accumulated metabolites (DAMs) were identified in DH20 compared to H30 under salt stress. Among the DAMs, fatty acids were the most accumulated in DH20 under salt stress. The integration of transcriptome and metabolome analyses revealed that nine key biomarkers, including two metabolites and seven genes, could distinguish DH20 and H30 when exposed to high salt. The pathways of linoleic acid metabolism, alpha-linolenic acid metabolism, glycerolipid metabolism, photosynthesis, and alanine, aspartate and glutamate metabolism were significantly enriched in DH20 with DEGs and DAMs in response to salt stress. These results suggest that DH20 may enhance resilience by promoting lipid metabolism, maintaining energy metabolism and decreasing amino acids metabolism. The study provided novel insights for the rapid generation of homozygous mutant plants by mutagenesis combined with microspore culture technology and also identified candidate genes and metabolites that may enable the mutant plants to cope with salt stress.

Rapid Generation of Barley Homozygous Transgenic Lines Based on Microspore Culture: HvPR1 Overexpression as an Example.

Obtaining homozygous lines from transgenic plants is an important step for phenotypic evaluations, but the selection of homozygous plants is time-consuming and laborious. The process would be significantly shortened if anther or microspore culture could be completed in one generation. In this study, we obtained 24 homozygous doubled haploid (DH) transgenic plants entirely by microspore culture from one T0 transgenic plant overexpressing the gene HvPR1 (pathogenesis-related-1). Nine of the doubled haploids grew to maturity and produced seeds. qRCR (quantitative real-time PCR) validation showed that the HvPR1 gene was expressed differentially even among different DH1 plants (T2) from the same DH0 line (T1). Phenotyping analysis suggested that the overexpression of HvPR1 inhibited nitrogen use efficiency (NUE) only under low nitrogen treatment. The established method of producing homozygous transgenic lines will enable the rapid evaluation of transgenic lines for gene function studies and trait evaluation. As an example, the HvPR1 overexpression of DH lines also could be used for further analysis of NUE-related research in barley.

Transcriptome and Metabolome Analyses Reveal Mechanisms Underlying the Response of Quinoa Seedlings to Nitrogen Fertilizers.

Quinoa (Chenopodium quinoa Willd.) is a dicotyledonous annual amaranth herb that belongs to the family Chenopodiaceae. Quinoa can be cultivated across a wide range of climatic conditions. With regard to its cultivation, nitrogen-based fertilizers have a demonstrable effect on the growth and development of quinoa. How crops respond to the application of nitrogen affects grain quality and yield. Therefore, to explore the regulatory mechanisms that underlie the responses of quinoa seedlings to the application of nitrogen, we selected two varieties (i.e., Dianli-1299 and Dianli-71) of quinoa seedlings and analyzed them using metabolomic and transcriptomic techniques. Specifically, we studied the mechanisms underlying the responses of quinoa seedlings to varying concentrations of nitrogen by analyzing the dynamics of metabolites and genes involved in arginine biosynthesis; carbon fixation; and alanine, aspartate, and glutamate biosynthetic pathways. Overall, we found that differentially expressed genes (DEGs) and differentially expressed metabolites (DEMs) of quinoa are affected by the concentration of nitrogen. We detected 1057 metabolites, and 29,012 genes were annotated for the KEGG. We also found that 15 DEMs and 8 DEGs were key determinants of the differences observed in quinoa seedlings under different nitrogen concentrations. These contribute toward a deeper understanding of the metabolic processes of plants under different nitrogen treatments and provide a theoretical basis for improving the nitrogen use efficiency (NUE) of quinoa.

Genome-wide identification of sucrose nonfermenting-1-related protein kinase (SnRK) genes in barley and RNA-seq analyses of their expression in response to abscisic acid treatment.

BACKGROUND: Sucrose nonfermenting-1 (SNF1)-related protein kinases (SnRKs) play important roles in regulating metabolism and stress responses in plants, providing a conduit for crosstalk between metabolic and stress signalling, in some cases involving the stress hormone, abscisic acid (ABA). The burgeoning and divergence of the plant gene family has led to the evolution of three subfamilies, SnRK1, SnRK2 and SnRK3, of which SnRK2 and SnRK3 are unique to plants. Therefore, the study of SnRKs in crops may lead to the development of strategies for breeding crop varieties that are more resilient under stress conditions. In the present study, we describe the SnRK gene family of barley (Hordeum vulgare), the widespread cultivation of which can be attributed to its good adaptation to different environments. RESULTS: The barley HvSnRK gene family was elucidated in its entirety from publicly-available genome data and found to comprise 50 genes. Phylogenetic analyses assigned six of the genes to the HvSnRK1 subfamily, 10 to HvSnRK2 and 34 to HvSnRK3. The search was validated by applying it to Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) genome data, identifying 50 SnRK genes in rice (four OsSnRK1, 11 OsSnRK2 and 35 OsSnRK3) and 39 in Arabidopsis (three AtSnRK1, 10 AtSnRK2 and 26 AtSnRK3). Specific motifs were identified in the encoded barley proteins, and multiple putative regulatory elements were found in the gene promoters, with light-regulated elements (LRE), ABA response elements (ABRE) and methyl jasmonate response elements (MeJa) the most common. RNA-seq analysis showed that many of the HvSnRK genes responded to ABA, some positively, some negatively and some with complex time-dependent responses. CONCLUSIONS: The barley HvSnRK gene family is large, comprising 50 members, subdivided into HvSnRK1 (6 members), HvSnRK2 (10 members) and HvSnRK3 (34 members), showing differential positive and negative responses to ABA.

Comparative transcriptome analysis reveals compatible and recalcitrant genotypic response of barley microspore-derived embryogenic callus toward Agrobacterium infection.

BACKGROUND: The Agrobacterium mediated transformation has been routinely used in lots of plant species as a powerful tool to deliver genes of interest into a host plant. However, the transformation of elite and commercially valuable cultivar is still limited by the genotype-dependency, and the efficiency of Agrobacterium infection efficiency is crucial for the success of transformation. RESULTS: In this study, the microspore-derived embryogenic calli (MDEC) of barley elite cultivars and breeding lines were employed as unique subjects to characterize the genotypic response during Agrobacterium infection process. Our results identified compatible barley genotypes (GanPi 6 and L07, assigned as GP6-L07 group) and one recalcitrant genotype (Hong 99, assigned as H99) for the Agrobacterium strain LBA4404 infection using GUS assay. The accumulation trend of reactive oxygen species (ROS) was similar among genotypes across the time course. The results of RNA-seq depicted that the average expressional intensity of whole genomic genes was similar among barley genotypes during Agrobacterium infection. However, the numbers of differentially expressed genes (DEGs) exhibited significant expressional variation between GP6-L07 and H99 groups from 6 to 12 h post-inoculation (hpi). Gene ontology (GO) enrichment analysis revealed different regulation patterns for the predicted biological processes between the early (up-regulated DEGs overrepresented at 2 hpi) and late stages (down-regulated DEGs overrepresented from 6 to 24 hpi) of infection. KEGG analysis predicted 12 pathways during Agrobacterium infection. Among which one pathway related to pyruvate metabolism was enriched in GP6 and L07 at 6 hpi. Two pathways related to plant hormone signal transduction and DNA replication showed expressional variation between GP6-L07 and H99 at 24 hpi. It was further validated by qRT-PCR assay for seven candidate genes (Aldehyde dehydrogenase, SAUR, SAUR50, ARG7, Replication protein A, DNA helicase and DNA replication licensing factor) involved in the three pathways, which are all up-regulated in compatible while down-regulated in recalcitrant genotypes, suggesting the potential compatibility achieved at later stage for the growth of Agrobacterium infected cells. CONCLUSIONS: Our findings demonstrated the similarity and difference between compatible and recalcitrant genotypes of barley MDEC upon Agrobacterium infection. Seven candidate genes involved in pyruvate metabolism, hormonal signal transduction and DNA replication were identified, which advocates the genotypic dependency during Agrobacterium infection process.

Rapid Visual Detection Method for Barley Yellow Mosaic Virus Using Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP).

Barley yellow mosaic disease, caused mainly by Barley yellow mosaic virus (BaYMV) and Barley mild mosaic virus, is a devastating disease of barley and is a threat to Eurasian barley production. Early detection is essential for effective management of the pathogens and to assure food security. In this study, a simple, rapid, specific, sensitive, and visual method was developed to detect BaYMV using loop-mediated isothermal amplification (LAMP). Two pairs of oligonucleotide primers (inner and outer primers) were designed to amplify the gene encoding the coat protein of BaYMV. The optimal conditions for the LAMP method were determined, and a one-step reverse transcription (RT)-LAMP method was also developed. Subsequently, the fastest processing time for RT-LAMP was determined. Among eight plant viruses examined using the LAMP method, only BaYMV was detectable, suggesting that the assay was highly specific. The RT-LAMP method was 10 times more sensitive than the RT-PCR method in the sensitivity test. To further shorten the virus detection process, a dye was added to the RT-LAMP products, and positive reactions were simply read by the naked eye via a color change (from orange to light green) under visible light. Barley samples from the middle and lower reaches of the Yangtze River basin, where BaYMV broke out very seriously in 1970s, were detected by the newly established RT-LAMP method. The results showed that all samples were positive for BaYMV, indicating the potential risk of the virus in these areas. This newly established LAMP/RT-LAMP method could be a promising tool for barley protection and food security control.

Novel low-nitrogen stress-responsive long non-coding RNAs (lncRNA) in barley landrace B968 (Liuzhutouzidamai) at seedling stage.

BACKGROUND: Reducing the dependence of crop production on chemical fertilizer with its associated costs, carbon footprint and other environmental problems is a challenge for agriculture. New solutions are required to solve this problem, and crop breeding for high nitrogen use efficiency or tolerance of low nitrogen availability has been widely considered to be a promising approach. However, the molecular mechanisms of high nitrogen use efficiency or low-nitrogen tolerance in crop plants are still to be elucidated, including the role of long non-coding RNAs (lncRNAs). RESULTS: In this study, we identified 498 lncRNAs in barley (Hordeum vulgare) landrace B968 (Liuzhutouzidamai), of which 487 were novel, and characterised 56 that were responsive to low-nitrogen stress. For functional analysis of differentially-expressed lncRNAs, the gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment of co-expressed and co-located protein-coding genes were analyzed, and interactions with annotated co-expressed protein coding genes or micro RNAs (miRNAs) were further predicted. Target mimicry prediction between differentially-expressed lncRNAs and miRNAs identified 40 putative target mimics of lncRNAs and 58 target miRNAs. Six differentially-expressed lncRNAs were further validated by qPCR, and one in particular showed consistent differential expression using both techniques. Expression levels of most of the lncRNAs were found to be very low, and this may be the reason for the apparent inconsistency between RNA-seq and qPCR data. CONCLUSIONS: The analysis of lncRNAs that are differentially-expressed under low-nitrogen stress, as well as their co-expressed or co-located protein coding genes and target mimics, could elucidate complex and hitherto uncharacterised mechanisms involved in the adaptation to low-nitrogen stress in barley and other crop plants.

Transcriptome Analysis Identifies Candidate Genes and Functional Pathways Controlling the Response of Two Contrasting Barley Varieties to Powdery Mildew Infection.

Powdery mildew caused by Blumeria graminis f. sp. hordei (Bgh) is one of the most serious diseases in barley. The numerous barley varieties across China provide valuable genetic resources to screen the resistant germplasm and to discover the primary genes of resistance to powdery mildew. In this study, Chinese barley variety Feng 7 was identified as a highly resistant genotype which limited Bgh colonization by cell apoptosis using leaf staining assay, while another variety Hua 30 showed high susceptibility. The performance of high resistance to Bgh in F1 plants from the two varieties suggested dominant gene(s) controlled the resistance to powdery mildew in Feng 7. To understand the host transcriptional response to Bgh infection, these two barley varieties Feng 7 and Hua 30 were inoculated with Bgh, and their transcriptional profiling using RNA sequencing (RNA-seq) at four time points (12 h post-inoculation (hpi), 24 hpi, 48 hpi, and 72 hpi) were compared. 4318 differentially expressed genes (DEGs), including 2244 upregulated and 2074 downregulated genes, were detected in Feng 7, compared with Hua 30 at 12 hpi. 4907 DEGs (2488 upregulated and 2419 downregulated) were detected at 24 hpi. 4758 DEGs (2295 upregulated and 2463 downregulated) were detected at 48 hpi. 3817 DEGs (2036 upregulated and 1781 downregulated) were detected at 72 hpi. The results showed the number of DEGs between two varieties peaked at 24 hpi (for the upregulated) or 48 hpi (for the downregulated), which is matched with the processing of Bgh infection. In addition, the number of upregulated DEGs involved in the functional pathways of plant defense (mitogen-activated protein kinase (MAPK) pathway and plant hormone signal transduction) is elevated remarkably at 24 hpi. Six candidate genes (PR13, glutaredoxin, alcohol dehydrogenase, and cytochrome P450) were identified in Feng 7. All of them present continuous expression at higher levels upon Bgh infection, compared with the performance in Hua 30, which revealed the potential contribution to Feng 7 mediate resistance to Bgh. In conclusion, the candidate genes and relevant pathways provided key information towards understanding the defense of barley to Bgh attack and the molecular mechanisms of different genetic resistance to powdery mildew.

Transient Overexpression of HvSERK2 Improves Barley Resistance to Powdery Mildew.

Somatic embryogenesis receptor-like kinases (SERKs) play an essential role in plant response to pathogen infection. Here we identified three SERK genes (HvSERK1/2/3) from barley, and aimed to determine their implication in defense responses to barley powdery mildew (Bgh). Although HvSERK1/2/3 share the characteristic domains of the SERK family, only HvSERK2 was significantly induced in barley leaves during Bgh infection. The expression of HvSERK2 was rapidly induced by hydrogen peroxide (H2O2) treatment, but not by treatment with salicylic acid (SA), methyl jasmonate (MeJA), ethephon (ETH), or abscisic acid (ABA). Bioinformatics analysis of the cloned HvSERK2 promoter revealed that it contains several elements responsible for defense responses against pathogens. Promoter functional analysis showed that the HvSERK2 promoter was induced by Bgh and H2O2. Subcellular localization analysis of HvSERK2 indicated that it is mainly located on the plasma membrane. Transient overexpression of HvSERK2 in epidermal cells of the susceptible barley cultivar Hua 30 reduced the Bgh haustorium index from 58.6% to 43.2%. This study suggests that the HvSERK2 gene plays a positive role in the improvement of barley resistance to powdery mildew, and provides new insight into the function of SERK genes in the biotic stress response of plants.

Transcriptome analysis reveals translational regulation in barley microspore-derived embryogenic callus under salt stress.

KEY MESSAGE: Transcriptome analysis of barley embryogenic callus from isolated microspore culture under salt stress uncovered a role of translation inhibition and selective activation of stress-specific proteins in cellular defense. Soil salinity is one of the major abiotic stresses which constrains the plant growth and reduces the productivity of field crops. In this study, it was observed that the salt stress in barley isolated microspore culture impacted not only on the quantity of embryogenic callus but also on the quality for later differentiation. The barley microspore-derived embryogenic callus, a transient intermediate form linked cells and plants, was employed for a global transcriptome analysis by RNA sequencing to provide new insights into the cellular adaptation or acclimation to stress. A total of 596 differentially expressed genes (DEGs) were identified, in which 123 DEGs were up-regulated and 473 DEGs were down-regulated in the embryogenic callus produced from microspore culture under salt stress as compared to the control conditions. KEGG pathway analysis identified 'translation' (27 DEGs; 12.56 %) as the largest group and followed by 'folding, sorting and degradation' (25 DEGs; 11.63 %) in 215 mapped metabolic pathways. The results of RNA-Seq data and quantitative real-time polymerase chain reaction validation showed that the genes related to translation regulation (such as eIF1A, RPLP0, RPLP2, VARS) were down-regulated to control general protein synthesis, and the genes related to endoplasmic reticulum stress response (such as small heat shock protein genes) were selectively up-regulated against protein denaturing during microspore embryogenesis under continuous salt stress. These transcriptional remodeling might affect the essential protein synthesis for the cell development to fulfill totipotency under salt stress.

Key phytochemicals contributing to the bitterness of quinoa.

Despite its nutritional components and potential health benefits, the bitterness of quinoa seed limits its utilization in the food industry. Saponins are believed to be the main cause of the bitterness, but it is still uncertain which specific compound is responsible. This study aimed to isolate the main components contributing to the bitterness in quinoa seed by solvent extraction and various column chromatography techniques guided by sensory evaluation. Five compounds were identified by mass spectrometry and nuclear magnetic resonance analyses, with the dose-over-threshold factors from 29.03 to 198.89. The results confirmed that triterpenoids are responsible for the bitter taste in quinoa seed, with phytolaccagenic acid derivatives being the primary contributor. Additionally, kaempferol 3-O-(2″, 6″-di-O-α-rhamnopyranosyl)-ß-galactopyranoside (namely mauritianin), was demonstrated for the first time to be associated with the bitterness of quinoa. This study could provide new insight into the bitter compound identification in quinoa.

TMF suppresses chondrocyte hypertrophy in osteoarthritic cartilage by mediating the FOXO3a/BMPER pathway.

Osteoarthritis (OA) is a disease of the joints, characterized by chronic inflammation, cartilage destruction and extracellular matrix (ECM) remodeling. Aberrant chondrocyte hypertrophy promotes cartilage destruction and OA development. Collagen X, the biomarker of chondrocyte hypertrophy, is upregulated by runt-related transcription factor 2 (Runx2), which is mediated by the bone morphogenetic protein 4 (BMP4)/Smad1 signaling pathway. BMP binding endothelial regulator (BMPER), a secreted glycoprotein, acts as an agonist of BMP4. 5,7,3',4'-tetramethoxyflavone (TMF) is a natural flavonoid derived from Murraya exotica L. Results of our previous study demonstrated that TMF exhibits chondroprotective effects against OA development through the activation of Forkhead box protein O3a (FOXO3a) expression. However, whether TMF suppresses chondrocyte hypertrophy through activation of FOXO3a expression and inhibition of BMPER/BMP4/Smad1 signaling remains unknown. Results of the present study revealed that TMF inhibited collagen X and Runx2 expression, inhibited BMPER/BMP4/Smad1 signaling, and activated FOXO3a expression; thus, protecting against chondrocyte hypertrophy and OA development. However, BMPER overexpression and FOXO3a knockdown impacted the protective effects of TMF. Thus, TMF inhibited chondrocyte hypertrophy in OA cartilage through mediating the FOXO3a/BMPER signaling pathway.

Efficient isolated microspore culture protocol for callus induction and plantlet regeneration in japonica rice (Oryza sativa L.).

BACKGROUND: Isolated microspore culture is a useful biotechnological technique applied in modern plant breeding programs as it can produce doubled haploid (DH) plants and accelerate the development of new varieties. Furthermore, as a single-cell culture technique, the isolated microspore culture provides an excellent platform for studying microspore embryogenesis. However, the reports on isolated microspore culture are rather limited in rice due to the low callus induction rate, poor regeneration capability, and high genotypic dependency. The present study developed an effective isolated microspore culture protocol for high-frequency androgenesis in four japonica rice genotypes. Several factors affecting the isolated microspore culture were studied to evaluate their effects on callus induction and plantlet regeneration. RESULTS: Low-temperature pre-treatment at 4 â„ƒ for 10-15 days could effectively promote microspore embryogenesis in japonica rice. A simple and efficient method was proposed for identifying the microspore developmental stage. The anthers in yellow-green florets located on the second type of primary branch on the rice panicle were found to be the optimal stage for isolated microspore culture. The most effective induction media for callus induction were IM2 and IM3, depending on the genotype. The optimal concentration of 2, 4-D in the medium for callus induction was 1 mg/L. Callus induction was negatively affected by a high concentration of KT over 1.5 mg/L. The differentiation medium suitable for japonica rice microspore callus comprised 1/2 MS, 2 mg/L 6-BA, 0.5 mg/L NAA, 30 g/L sucrose, and 6 g/L agar. The regeneration frequency of the four genotypes ranged from 61-211 green plantlets per 100 mg calli, with Chongxiangjing showing the highest regeneration frequency. CONCLUSIONS: This study presented an efficient protocol for improved callus induction and green plantlet regeneration in japonica rice via isolated microspore culture, which could provide valuable support for rice breeding and genetic research.

Real-Time Quantitative PCR: Primer Design, Reference Gene Selection, Calculations and Statistics.

Real-time quantitative PCR is a technique that can measure the content of the target nucleic acid sequence of interest in a given sample. It is mainly divided into absolute and relative quantitative methods. The relative quantification is mainly used in gene expressions for functional genomic and transcriptome studies. However, to use this technology accurately, there are some key points to master. First, specific primers need to be designed to ensure amplification of the gene of interest (GOI). Second, the appropriate reference gene or reference gene combination has to be selected. Finally, scientific gene expression level calculations and statistics are required to obtain accurate results. Therefore, this work proposes a workflow for relative quantitative PCR and introduces the relevant points so that beginners can better understand and use this technology.

Why Do Bystanders Become Cyberbullies? The Effect of Perceived Reasonableness of Online Messages on Bystanders' Aggressive Tendency and Its Underlying Mechanism.

The objectives of this study were to examine the influence of bystanders' perceived reasonableness of online messages on their aggressive tendency toward victims and to examine the mediating role of bystanders' attribution of responsibility to victims on their aggressive tendency toward the victims. Our study involved two parts: In Study 1, 295 Taiwanese undergraduates were recruited, and questionnaires were distributed to them to measure their perceived reasonableness of cyberbullying attacks. In Study 2, a total of 78 university students were recruited. The participants' perceived reasonableness of cyberbullying attacks was reduced through experimental manipulation. Subsequently, they were randomly assigned to a group with relatively low reasonableness or a control group. The results of both studies revealed that the participants' aggressive tendency toward the victim was influenced by their perceived reasonableness of cyberbully messages. The relation between perceived reasonableness and aggressive tendency was mediated by the participants' attribution of responsibility to the victim.