Prevention of polycystic ovary syndrome and postmenopausal osteoporosis by inhibiting apoptosis with Shenling Baizhu powder compound.

Objective: Shenling Baizhu powder (SBP) has been shown to reverse the abnormal expression of the aromatic hydrocarbon receptor (AHR) mediated by air pollution. Our study aimed to understand the main ingredient of SBP and investigate its action mechanism in preventing polycystic ovary syndrome (POCS) and postmenopausal osteoporosis (PMO). Methods: The active ingredients of SBP with the highest binding affinity to AHR were screened using a Chinese medicine database, and their binding mechanism was simulated using molecular dynamics simulation (MDS). Rutin was utilized to treat ovarian granulosa cell lines and osteoblast cell lines. The cell lines were treated with a gradient of rutin concentration (0.01 mmol/L, 0.05 mmol/L and 0.1 mmol/L) to find the optimal drug dose. PCR was used to detect AHR and apoptosis-related proteins, and WB to detect the expression of AHR, caspase-3 and cleaved-caspase-3. Finally, the CCK-8 cell proliferation assay detected the proliferation of cells. Results: We obtained Rutin through the Chinese medicine database, and dynamics simulation determined its binding sites. Ovarian granulosa cell lines and osteoblast cell lines were treated with Rutin. RT-PCR and western blotting revealed that the expression of apoptosis-associated protein Bcl-2 was elevated, and the expression of AHR, Bax, caspase-3 and PARP were decreased. CCK-8 results showed accelerated proliferation in both cell types. Conclusion: Rutin, the main ingredient of SBP compound, works by binding to AHR, which can improve POCS and PMO by inhibiting cell apoptosis and by promoting cell proliferation.

CRISPR/Cas9-Based Gene Editing in Soybean.

CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR associated Cas9)-based gene editing is a robust tool for functional genomics research and breeding programs in various crops. In soybean, a number of laboratories have obtained mutants by CRISPR/Cas9 system; however, there has been not yet a detailed method for the CRISPR/Cas9-based gene editing in soybean. Here, we describe the procedures for constructing the CRISPR/Cas9 plasmid suitable for soybean gene editing and the modified protocols for Agrobacterium-mediated soybean transformation and regeneration from cotyledonary node explants containing the Cas9/sgRNA (single guide RNA) transgenes.

CRISPR/Cas9-mediated targeted mutagenesis of GmSPL9 genes alters plant architecture in soybean.

BACKGROUND: The plant architecture has significant effects on grain yield of various crops, including soybean (Glycine max), but the knowledge on optimization of plant architecture in order to increase yield potential is still limited. Recently, CRISPR/Cas9 system has revolutionized genome editing, and has been widely utilized to edit the genomes of a diverse range of crop plants. RESULTS: In the present study, we employed the CRISPR/Cas9 system to mutate four genes encoding SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors of the SPL9 family in soybean. These four GmSPL9 genes are negatively regulated by GmmiR156b, a target for the improvement of soybean plant architecture and yields. The soybean Williams 82 was transformed with the binary CRISPR/Cas9 plasmid, assembled with four sgRNA expression cassettes driven by the Arabidopsis thaliana U3 or U6 promoter, targeting different sites of these four SPL9 genes via Agrobacterium tumefaciens-mediated transformation. A 1-bp deletion was detected in one target site of the GmSPL9a and one target site of the GmSPL9b, respectively, by DNA sequencing analysis of two T0-generation plants. T2-generation spl9a and spl9b homozygous single mutants exhibited no obvious phenotype changes; but the T2 double homozygous mutant spl9a/spl9b possessed shorter plastochron length. In T4 generation, higher-order mutant plants carrying various combinations of mutations showed increased node number on the main stem and branch number, consequently increased total node number per plants at different levels. In addition, the expression levels of the examined GmSPL9 genes were higher in the spl9b-1 single mutant than wild-type plants, which might suggest a feedback regulation on the expression of the investigated GmSPL9 genes in soybean. CONCLUSIONS: Our results showed that CRISPR/Cas9-mediated targeted mutagenesis of four GmSPL9 genes in different combinations altered plant architecture in soybean. The findings demonstrated that GmSPL9a, GmSPL9b, GmSPL9c and GmSPL9 function as redundant transcription factors in regulating plant architecture in soybean.

The CRISPR/Cas9 system and its applications in crop genome editing.

The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein9) system is an RNA-guided genome editing tool that consists of a Cas9 nuclease and a single-guide RNA (sgRNA). By base-pairing with a DNA target sequence, the sgRNA enables Cas9 to recognize and cut a specific target DNA sequence, generating double strand breaks (DSBs) that trigger cell repair mechanisms and mutations at or near the DSBs sites. Since its discovery, the CRISPR/Cas9 system has revolutionized genome editing and is now becoming widely utilized to edit the genomes of a diverse range of crop plants. In this review, we present an overview of the CRISPR/Cas9 system itself, including its mechanism of action, system construction strategies, and the screening methods used to identify mutants containing edited genes. We evaluate recent examples of the use of CRISPR/Cas9 for crop plant improvement, and research into the function(s) of genes involved in determining crop yields, quality, environmental stress tolerance/resistance, regulation of gene transcription and translation, and the construction of mutant libraries and production of transgene-free genome-edited crops. In addition, challenges and future opportunities for the use of the CRISPR/Cas9 system in crop breeding are discussed.

The soybean Rhg1 amino acid transporter gene alters glutamate homeostasis and jasmonic acid-induced resistance to soybean cyst nematode.

Rhg1 (resistance to Heterodera glycines 1) is an important locus that contributes to resistance against soybean cyst nematode (SCN; Heterodera glycines Ichinohe), which is the most economically damaging disease of soybean worldwide. Simultaneous overexpression of three genes encoding a predicted amino acid transporter, an α-soluble N-ethylmaleimide-sensitive factor attachment protein (α-SNAP) and a predicted wound-induced protein resulted in resistance to SCN provided by this locus. However, the roles of two of these genes (excluding α-SNAP) remain unknown. Here, we report the functional characterization of Glyma.18G022400, a gene at the Rhg1 locus that encodes the predicted amino acid transporter Rhg1-GmAAT. Although the direct role of Rhg1-GmAAT in glutamate transport was not demonstrated, multiple lines of evidence showed that Rhg1-GmAAT impacts glutamic acid tolerance and glutamate transportation in soybean. Transcriptomic and metabolite profiling indicated that overexpression of Rhg1-GmAAT activated the jasmonic acid (JA) pathway. Treatment with a JA biosynthesis inhibitor reduced the resistance provided by the Rhg1-containing PI88788 to SCN, which suggested that the JA pathway might play a role in Rhg1-mediated resistance to SCN. Our results could be helpful for the clarification of the mechanism of resistance to SCN provided by Rhg1 in soybean.

The Stable Level of Glutamine synthetase 2 Plays an Important Role in Rice Growth and in Carbon-Nitrogen Metabolic Balance.

Glutamine synthetase 2 (GS2) is a key enzyme involved in the ammonium metabolism in plant leaves. In our previous study, we obtained GS2-cosuppressed plants, which displayed a normal growth phenotype at the seedling stage, while at the tillering stage they showed a chlorosis phenotype. In this study, to investigate the chlorosis mechanism, we systematically analyzed the plant growth, carbon-nitrogen metabolism and gene expressions between the GS2-cosuppressed rice and wild-type plants. The results revealed that the GS2-cosuppressed plants exhibited a poor plant growth phenotype and a poor nitrogen transport ability, which led to nitrogen accumulation and a decline in the carbon/nitrogen ratio in the stems. Interestingly, there was a higher concentration of soluble proteins and a lower concentration of carbohydrates in the GS2-cosuppressed plants at the seedling stage, while a contrasting result was displayed at the tillering stage. The analysis of the metabolic profile showed a significant increase of sugars and organic acids. Additionally, gene expression patterns were different in root and leaf of GS2-cosuppressed plants between the seedling and tillering stage. These results indicated the important role of a stable level of GS2 transcription during normal rice development and the importance of the carbon-nitrogen metabolic balance in rice growth.

Overexpressing of OsAMT1-3, a High Affinity Ammonium Transporter Gene, Modifies Rice Growth and Carbon-Nitrogen Metabolic Status.

AMT1-3 encodes the high affinity NH4⁺ transporter in rice roots and is predominantly expressed under nitrogen starvation. In order to evaluate the effect of AMT1-3 gene on rice growth, nitrogen absorption and metabolism, we generated AMT1-3-overexpressing plants and analyzed the growth phenotype, yield, carbon and nitrogen metabolic status, and gene expression profiles. Although AMT1-3 mRNA accumulated in transgenic plants, these plants displayed significant decreases in growth when compared to the wild-type plants. The nitrogen uptake assay using a 15N tracer revealed poor nitrogen uptake ability in AMT1-3-overexpressing plants. We found significant decreases in AMT1-3-overexpressing plant leaf carbon and nitrogen content accompanied with a higher leaf C/N ratio. Significant changes in soluble proteins and carbohydrates were also observed in AMT1-3-overexpressing plants. In addition, metabolite profile analysis demonstrated significant changes in individual sugars, organic acids and free amino acids. Gene expression analysis revealed distinct expression patterns of genes that participate in carbon and nitrogen metabolism. Additionally, the correlation between the metabolites and gene expression patterns was consistent in AMT1-3-overexpressing plants under both low and high nitrogen growth conditions. Therefore, we hypothesized that the carbon and nitrogen metabolic imbalance caused by AMT1-3 overexpressing attributed to the poor growth and yield of transgenic plants.

Accumulated expression level of cytosolic glutamine synthetase 1 gene (OsGS1;1 or OsGS1;2) alter plant development and the carbon-nitrogen metabolic status in rice.

Maintaining an appropriate balance of carbon to nitrogen metabolism is essential for rice growth and yield. Glutamine synthetase is a key enzyme for ammonium assimilation. In this study, we systematically analyzed the growth phenotype, carbon-nitrogen metabolic status and gene expression profiles in GS1;1-, GS1;2-overexpressing rice and wildtype plants. Our results revealed that the GS1;1-, GS1;2-overexpressing plants exhibited a poor plant growth phenotype and yield and decreased carbon/nitrogen ratio in the stem caused by the accumulation of nitrogen in the stem. In addition, the leaf SPAD value and photosynthetic parameters, soluble proteins and carbohydrates varied greatly in the GS1;1-, GS1;2-overexpressing plants. Furthermore, metabolite profile and gene expression analysis demonstrated significant changes in individual sugars, organic acids and free amino acids, and gene expression patterns in GS1;1-, GS1;2-overexpressing plants, which also indicated the distinct roles that these two GS1 genes played in rice nitrogen metabolism, particularly when sufficient nitrogen was applied in the environment. Thus, the unbalanced carbon-nitrogen metabolic status and poor ability of nitrogen transportation from stem to leaf in GS1;1-, GS1;2-overexpressing plants may explain the poor growth and yield.