WGCNA Reveals Hub Genes and Key Gene Regulatory Pathways of the Response of Soybean to Infection by Soybean mosaic virus.

Soybean mosaic virus (SMV) is one of the main pathogens that can negatively affect soybean production and quality. To study the gene regulatory network of soybeans in response to SMV SC15, the resistant line X149 and susceptible line X97 were subjected to transcriptome analysis at 0, 2, 8, 12, 24, and 48 h post-inoculation (hpi). Differential expression analysis revealed that 10,190 differentially expressed genes (DEGs) responded to SC15 infection. Weighted gene co-expression network analysis (WGCNA) was performed to identify highly related resistance gene modules; in total, eight modules, including 2256 DEGs, were identified. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of 2256 DEGs revealed that the genes significantly clustered into resistance-related pathways, such as the plant-pathogen interaction pathway, mitogen-activated protein kinases (MAPK) signaling pathway, and plant hormone signal transduction pathway. Among these pathways, we found that the flg22, Ca2+, hydrogen peroxide (H2O2), and abscisic acid (ABA) regulatory pathways were fully covered by 36 DEGs. Among the 36 DEGs, the gene Glyma.01G225100 (protein phosphatase 2C, PP2C) in the ABA regulatory pathway, the gene Glyma.16G031900 (WRKY transcription factor 22, WRKY22) in Ca2+ and H2O2 regulatory pathways, and the gene Glyma.04G175300 (calcium-dependent protein kinase, CDPK) in Ca2+ regulatory pathways were highly connected hub genes. These results indicate that the resistance of X149 to SC15 may depend on the positive regulation of flg22, Ca2+, H2O2, and ABA regulatory pathways. Our study further showed that superoxide dismutase (SOD) activity, H2O2 content, and catalase (CAT) and peroxidase (POD) activities were significantly up-regulated in the resistant line X149 compared with those in 0 hpi. This finding indicates that the H2O2 regulatory pathway might be dependent on flg22- and Ca2+-pathway-induced ROS generation. In addition, two hub genes, Glyma.07G190100 (encoding F-box protein) and Glyma.12G185400 (encoding calmodulin-like proteins, CMLs), were also identified and they could positively regulate X149 resistance. This study provides pathways for further investigation of SMV resistance mechanisms in soybean.

Genome-wide analysis of the SCAMPs gene family of soybean and functional identification of GmSCAMP5 in salt tolerance.

The effect of salt damage on plants is mainly caused by the toxic effect of Na+. Studies showed that the secretory carrier membrane proteins were associated with the Na+ transport. However, the salt tolerance mechanism of secretory carrier protein (SCAMP) in soybean was yet to be defined. In this study, ten potential SCAMP genes distributed in seven soybean chromosomes were identified in the soybean genome. The phylogenetic tree of SCAMP domain sequences of several plants can divide SCAMPs into two groups. Most GmSCAMPs genes contained multiple Box4, MYB and MYC cis-elements indicated they may respond to abiotic stresses. We found that GmSCAMP1, GmSCAMP2 and GmSCAMP4 expressed in several tissues and GmSCAMP5 was significantly induced by salt stress. GmSCAMP5 showed the same expression patterns under NaCl treatment in salt-tolerant and salt-sensitive soybean varieties, but the induced time of GmSCAMP5 in salt-tolerant variety was earlier than that of salt-sensitive variety. To further study the effect of GmSCAMP5 on the salt tolerance of soybean plants, compared to GmSCAMP5-RNAi and EV-Control plants, GmSCAMP5-OE had less wilted leave and higher SPAD value. Compared to empty vector control, less trypan blue staining was observed in GmSCAMP5-OE leaves while more staining in GmSCAMP5-RNAi leaves. The Na+ of GmSCAMP5-RNAi plants leaves under NaCl stress were significantly higher than that in EV-Control plants, while significantly lower Na+ in GmSCAMP5-OE plants than in that EV-Control plants. The contents of leaves K+ of GmSCAMP5-RNAi, EV-Control, and GmSCAMP5-OE plants under NaCl stress were opposite to that of leaves Na+ content. Finally, salt stress-related genes NHX1, CLC1, TIP1, SOD1, and SOS1 in transformed hairy root changed significantly compared with the empty control. The research will provide novel information for study the molecular regulation mechanism of soybean salt tolerance.

3-Hydroxy-3-methylglutaryl coenzyme A reductase genes from Glycine max regulate plant growth and isoprenoid biosynthesis.

Isoprenoids, a large kind of plant natural products, are synthesized by the mevalonate (MVA) pathway in the cytoplasm and the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway in plastids. As one of the rate-limiting enzymes in the MVA pathway of soybean (Glycine max), 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is encoded by eight isogenes (GmHMGR1-GmHMGR8). To begin, we used lovastatin (LOV), a specific inhibitor of GmHMGR, to investigate their role in soybean development. To further investigate, we overexpressed the GmHMGR4 and GmHMGR6 genes in Arabidopsis thaliana. The growth of soybean seedlings, especially the development of lateral roots, was inhibited after LOV treatment, accompanied by a decrease in sterols content and GmHMGR gene expression. After the overexpression of GmHMGR4 and GmHMGR6 in A. thaliana, the primary root length was higher than the wild type, and total sterol and squalene contents were significantly increased. In addition, we detected a significant increase in the product tocopherol from the MEP pathway. These results further support the fact that GmHMGR1-GmHMGR8 play a key role in soybean development and isoprenoid biosynthesis.

A Natural Glucan from Black Bean Inhibits Cancer Cell Proliferation via PI3K-Akt and MAPK Pathway.

A natural α-1,6-glucan named BBWPW was identified from black beans. Cell viability assay showed that BBWPW inhibited the proliferation of different cancer cells, especially HeLa cells. Flow cytometry analysis indicated that BBWPW suppressed the HeLa cell cycle in the G2/M phase. Consistently, RT-PCR experiments displayed that BBWPW significantly impacts the expression of four marker genes related to the G2/M phase, including p21, CDK1, Cyclin B1, and Survivin. To explore the molecular mechanism of BBWPW to induce cell cycle arrest, a transcriptome-based target inference approach was utilized to predict the potential upstream pathways of BBWPW and it was found that the PI3K-Akt and MAPK signal pathways had the potential to mediate the effects of BBWPW on the cell cycle. Further experimental tests confirmed that BBWPW increased the expression of BAD and AKT and decreased the expression of mTOR and MKK3. These results suggested that BBWPW could regulate the PI3K-Akt and MAPK pathways to induce cell cycle arrest and ultimately inhibit the proliferation of HeLa cells, providing the potential of the black bean glucan to be a natural anticancer drug.

Colorimetric Detection of Class A Soybean Saponins by G-Quadruplex-Based Hybridization Chain Reaction.

Soybean saponin is one of the important secondary metabolites in seeds, which has various beneficial physiological functions to human health. GmSg-1 gene is the key enzyme gene for synthesizing class A saponins. It is of great significance to realize the visual and rapid detection of class A saponins at the genetic level. The hybridization chain reaction (HCR) was employed to the visual detection of GmSg-1 gene, which was implemented by changing the length of the target fragment to 92 bp and using the hairpin probes we designed to detect the GmSg-1 a and GmSg-1 b genes. The best condition of HCR reaction is hemin (1.2 µM), Triton X-100 (0.002%), ABTS (3.8 µM), and H2O2 (1.5 mM). It was found that HCR has high specificity for GmSg-1 gene and could be applied to the visual detection of different soybean cultivars containing Aa type, Ab type, and Aa/Ab type saponins, which could provide technical reference and theoretical basis for molecular breeding of soybean and development of functional soybean products.

Colorimetric detection of class A soybean saponins by coupling DNAzyme with the gap ligase chain reaction.

Class A saponins are responsible for the taste of soybean products, and the rapid identification of class A saponins from soybean food is essential for both food safety and cultivar screening. In this study, we propose a colorimetric assay based on the coupling of gap ligase chain reaction (Gap-LCR) with DNAzyme to detect the target GmSg-1 genes of class A soybean saponins with the naked eye, without the involvement of expensive instruments. The limits of detection (LODs) for the GmSg-1a and GmSg-1b genes were determined to be 0.1618 and 0.1625 µM, respectively, with a linear range of 0.2-1.2 µM. The DNAzyme-based Gap LCR assay was successfully employed to identify the target genes from different soybean cultivars, providing a simple means for monitoring the quality of soybean products.

Identification and development of a functional marker from 6-SFT-A2 associated with grain weight in wheat.

As a class of water-soluble, fructose-based oligo- and polysaccharides, fructans are major nonstructural carbohydrates and an important carbon source for grain filling in wheat (Triticum aestivum L.). Four enzymes are involved in fructan synthesis in higher plants, and 6-SFT is a key enzyme in fructan biosynthesis. In this study, thirteen single nucleotide polymorphisms were detected in 6-SFT-A2 in 24 wheat accessions, forming three haplotypes. Two cleaved amplified polymorphic sequence markers developed based on polymorphisms at sites 1870(A-G) and 1951(A-G) distinguished the three haplotypes. 6-SFT-A2 was located on chromosome 4A, between markers P2454.3 and P3465.1 in a doubled haploid (DH) population derived from the cross Hanxuan 10 × Lumai 14. The DH population comprising 150 lines and a historical population consisting of 154 accessions were used in a 6-SFT-A2 marker-trait association analysis. The three haplotypes were significantly associated with thousand-grain weight (TGW) under rainfed conditions. HapIII had a significant positive effect on TGW. There were significant differences between the Hanxuan 10 and Lumai 14 genotypes in both rainfed and irrigated environments. The average TGW of Lumai 14 (HapIII) was higher than that of Hanxuan 10 (HapI). The frequencies of 6-SFT-A2HapIII in cultivars released at different periods showed that it had been strongly positively selected in breeding programs. The preferred HapIII for TGW occurred at higher frequencies in Gansu, Beijing, Shanxi, and Hebei than other regions in northern China.

[Expression of yeast acyl-delta9 desaturase for fatty acid biosynthesis in tobacco].

Palmitoleic acid (16:1delta9), an unusual monounsaturated fatty acid, is highly valued for human nutrition, medication and industry. Plant oils containing large amounts of palmitoleic acid are the ideal resource for biodiesel production. To increase accumulation of palmitoleic acid in plant tissues, we used a yeast (Saccharomyees cerevisiae) acyl-CoA-delta9 desaturase (Scdelta9D) for cytosol- and plastid-targeting expression in tobacco (Nicotiana tabacum L.). By doing this, we also studied the effects of the subcellular-targeted expression of this enzyme on lipid synthesis and metabolism in plant system. Compared to the wild type and vector control plants, the contents of monounsaturated palmitoleic (16:1delta9) and cis-vaccenic (18:1delta11) were significantly enhanced in the Scdelta9D-transgenic leaves whereas the levels of saturated palmitic acid (16:0) and polyunsaturated linoleic (18:2) and linolenic (18:3) acids were reduced in the transgenics. Notably, the contents of 16:1delta9 and 18:1delta11 in the Scdelta9D plastidal-expressed leaves were 2.7 and 1.9 folds of that in the cytosolic-expressed tissues. Statistical analysis appeared a negative correlation coefficient between 16:0 and 16:1delta9 levels. Our data indicate that yeast cytosolic acyl-CoA-delta9 desaturase can convert palmitic (16:0) into palmitoleic acid (16:1delta9) in high plant cells. Moreover, this effect of the enzyme is stronger with the plastid-targeted expression than the cytosol-target expression. The present study developed a new strategy for high accumulation of omega-7 fatty acids (16:1delta9 andl8:1delta11) in plant tissues by protein engineering of acyl-CoA-delta9 desaturase. The findings would particularly benefit the metabolic assembly of the lipid biosynthesis pathway in the large-biomass vegetative organs such as tobacco leaves for the production of high-quality biodiesel.

Expression of yeast acyl-CoA-∆9 desaturase leads to accumulation of unusual monounsaturated fatty acids in soybean seeds.

An acyl-CoA-Δ9 desaturase from Saccharomyces cerevisiae was expressed by subcellular-targeting in soybean (Glycine max) seeds with the goal of increasing palmitoleic acid (16:1Δ9), a high-valued fatty acid (FA), and simultaneously decreasing saturated FA in oil. The expression resulted in the conversion of palmitic acid (16:0) to 16:1Δ9 in soybean seeds. 16:1Δ9 and its elongation product cis-vaccenic acid (18:1Δ11) were increased to 17 % of the total fatty acids by plastid-targeted expression of the enzyme. Other lipid changes include the decrease of polyunsaturated FA and saturated FA, suggesting that a mechanism exists downstream in oil biosynthesis to compensate the FA alternation. This is the first time a cytosolic acyl-CoA-∆9 desaturase is functionally expressed in plastid and stronger activity was achieved than its cytosolic expression. The present study provides a new strategy for converting 16:0 to 16:1Δ9 by engineering acyl-CoA-Δ9 desaturase in commercialized oilseeds.

[Metabolic engineering of edible plant oils].

Plant seed oil is the major source of many fatty acids for human nutrition, and also one of industrial feedstocks. Recent advances in understanding of the basic biochemistry of seed oil biosynthesis, coupled with cloning of the genes encoding the enzymes involved in fatty acid modification and oil accumulation, have set the stage for the metabolic engineering of oilseed crops that produce "designer" plant seed oils with the improved nutritional values for human being. In this review we provide an overview of seed oil biosynthesis/regulation and highlight the key enzymatic steps that are targets for gene manipulation. The strategies of metabolic engineering of fatty acids in oilseeds, including overexpression or suppression of genes encoding single or multi-step biosynthetic pathways and assembling the complete pathway for the synthesis of long-chain polyunsaturated fatty acids (e.g. arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid) are described in detail. The current "bottlenecks" in using common oilseeds as "bioreactors" for commercial production of high-value fatty acids are analyzed. It is also discussed that the future research focuses of oilseed metabolic engineering and the prospects in creating renewable sources and promoting the sustainable development of human society and economy.