A Soybean Sucrose Non-Fermenting Protein Kinase 1 Gene, GmSNF1, Positively Regulates Plant Response to Salt and Salt-Alkali Stress in Transgenic Plants.

Soybean is one of the most widely grown oilseed crops worldwide. Several unfavorable factors, including salt and salt-alkali stress caused by soil salinization, affect soybean yield and quality. Therefore, exploring the molecular basis of salt tolerance in plants and developing genetic resources for genetic breeding is important. Sucrose non-fermentable protein kinase 1 (SnRK1) belongs to a class of Ser/Thr protein kinases that are evolutionarily highly conserved direct homologs of yeast SNF1 and animal AMPKs and are involved in various abiotic stresses in plants. The GmPKS4 gene was experimentally shown to be involved with salinity tolerance. First, using the yeast two-hybrid technique and bimolecular fluorescence complementation (BiFC) technique, the GmSNF1 protein was shown to interact with the GmPKS4 protein. Second, the GmSNF1 gene responded positively to salt and salt-alkali stress according to qRT-PCR analysis, and the GmSNF1 protein was localized in the nucleus and cytoplasm using subcellular localization assay. The GmSNF1 gene was then heterologously expressed in yeast, and the GmSNF1 gene was tentatively identified as having salt and salt-alkali tolerance function. Finally, the salt-alkali tolerance function of the GmSNF1 gene was demonstrated by transgenic Arabidopsis thaliana, soybean hairy root complex plants overexpressing GmSNF1 and GmSNF1 gene-silenced soybean using VIGS. These results indicated that GmSNF1 might be useful in genetic engineering to improve plant salt and salt-alkali tolerance.

A soybean calcineurin B-like protein-interacting protein kinase, GmPKS4, regulates plant responses to salt and alkali stresses.

Calcineurin B-like protein-interacting protein kinases (CIPKs) are key elements of plant abiotic stress signaling pathways. CIPKs are SOS2 (Salt Overly Sensitive 2)-like proteins (protein kinase S [PKS] proteins) which all contain a putative FISL motif. It seems that the FISL motif is found only in the SOS2 subfamily of protein kinases. In this study, the full-length cDNA of a soybean CIPK gene (GmPKS4) was isolated and was revealed to have an important role in abiotic stress responses. A qRT-PCR analysis indicated that GmPKS4 expression is upregulated under saline conditions or when exposed to alkali, salt-alkali, drought, or abscisic acid (ABA). A subcellular localization assay revealed the presence of GmPKS4 in the nucleus and cytoplasm. Further studies on the GmPKS4 promoter suggested it affects soybean resistance to various stresses. Transgenic Arabidopsis thaliana and soybean hairy roots overexpressing GmPKS4 had increased proline content as well as high antioxidant enzyme activities but decreased malondialdehyde levels following salt and salt-alkali stress treatments. Additionally, GmPKS4 overexpression activated reactive oxygen species scavenging systems, thereby minimizing damages due to oxidative and osmotic stresses. Moreover, upregulated stress-related gene expression levels were detected in lines overexpressing GmPKS4 under stress conditions. In conclusion, GmPKS4 improves soybean tolerance to salt and salt-alkali stresses. The overexpression of GmPKS4 enhances the scavenging of reactive oxygen species, osmolyte synthesis, and the transcriptional regulation of stress-related genes.

Overexpression of GmCAMTA12 Enhanced Drought Tolerance in Arabidopsis and Soybean.

Fifteen transcription factors in the CAMTA (calmodulin binding transcription activator) family of soybean were reported to differentially regulate in multiple stresses; however, their functional analyses had not yet been attempted. To characterize their role in stresses, we first comprehensively analyzed the GmCAMTA family in silico and thereafter determined their expression pattern under drought. The bioinformatics analysis revealed multiple stress-related cis-regulatory elements including ABRE, SARE, G-box and W-box, 10 unique miRNA (microRNA) targets in GmCAMTA transcripts and 48 proteins in GmCAMTAs' interaction network. We then cloned the 2769 bp CDS (coding sequence) of GmCAMTA12 in an expression vector and overexpressed in soybean and Arabidopsis through Agrobacterium-mediated transformation. The T3 (Transgenic generation 3) stably transformed homozygous lines of Arabidopsis exhibited enhanced tolerance to drought in soil as well as on MS (Murashige and Skoog) media containing mannitol. In their drought assay, the average survival rate of transgenic Arabidopsis lines OE5 and OE12 (Overexpression Line 5 and Line 12) was 83.66% and 87.87%, respectively, which was ~30% higher than that of wild type. In addition, the germination and root length assays as well as physiological indexes such as proline and malondialdehyde contents, catalase activity and leakage of electrolytes affirmed the better performance of OE lines. Similarly, GmCAMTA12 overexpression in soybean promoted drought-efficient hairy roots in OE chimeric plants as compare to that of VC (Vector control). In parallel, the improved growth performance of OE in Hoagland-PEG (polyethylene glycol) and on MS-mannitol was revealed by their phenotypic, physiological and molecular measures. Furthermore, with the overexpression of GmCAMTA12, the downstream genes including AtAnnexin5, AtCaMHSP, At2G433110 and AtWRKY14 were upregulated in Arabidopsis. Likewise, in soybean hairy roots, GmELO, GmNAB and GmPLA1-IId were significantly upregulated as a result of GmCAMTA12 overexpression and majority of these upregulated genes in both plants possess CAMTA binding CGCG/CGTG motif in their promoters. Taken together, we report that GmCAMTA12 plays substantial role in tolerance of soybean against drought stress and could prove to be a novel candidate for engineering soybean and other plants against drought stress. Some research gaps were also identified for future studies to extend our comprehension of Ca-CaM-CAMTA-mediated stress regulatory mechanisms.

Comprehensive Genomic Analysis and Expression Profiling of Diacylglycerol Kinase (DGK) Gene Family in Soybean (Glycine max) under Abiotic Stresses.

Diacylglycerol kinase (DGK) is an enzyme that plays a pivotal role in abiotic and biotic stress responses in plants by transforming the diacylglycerol into phosphatidic acid. However, there is no report on the characterization of soybean DGK genes in spite of the availability of the soybean genome sequence. In this study, we performed genome-wide analysis and expression profiling of the DGK gene family in the soybean genome. We identified 12 DGK genes (namely GmDGK1-12) which all contained conserved catalytic domains with protein lengths and molecular weights ranging from 436 to 727 amino acids (aa) and 48.62 to 80.93 kDa, respectively. Phylogenetic analyses grouped GmDGK genes into three clusters-cluster I, cluster II, and cluster III-which had three, four, and five genes, respectively. The qRT-PCR analysis revealed significant GmDGK gene expression levels in both leaves and roots coping with polyethylene glycol (PEG), salt, alkali, and salt/alkali treatments. This work provides the first characterization of the DGK gene family in soybean and suggests their importance in soybean response to abiotic stress. These results can serve as a guide for future studies on the understanding and functional characterization of this gene family.

Protective effects of ginsenoside Rg1 against hydrogen peroxide-induced injury in human neuroblastoma cells.

The active ingredient of ginseng, ginsenosides Rg1, has been shown to scavenge free radicals and improve antioxidant capacity. This study hypothesized that ginsenosides Rg1 has a protective role in human neuroblastoma cells injured by H2O2. Ginsenosides Rg1 at different concentrations (50 and 100 µM) was used to treat H2O2 (150 µM)-injured SH-SY5Y cells. Results demonstrated that ginsenoside Rg1 elevated the survival rate of SH-SY5Y cells injured by H2O2, diminished the amount of leaked lactate dehydrogenase, and increased superoxide dismutase activity. Ginsenoside Rg1 effectively suppressed caspase-3 immunoreactivity, and contributed to heat shock protein 70 gene expression, in a dose-dependent manner. These results indicate that ginsenoside Rg1 has protective effects on SH-SY5Y cells injured by H2O2 and that its mechanism of action is associated with anti-oxidation and the inhibition of apoptosis.

The AaDREB1 Transcription Factor from the Cold-Tolerant Plant Adonis amurensis Enhances Abiotic Stress Tolerance in Transgenic Plant.

Dehydration-responsive element binding (DREB) transcription factors (TFs) play important roles in the regulation of plant resistance to environmental stresses and can specifically bind to dehydration-responsive element/C-repeat element (DRE/CRT) proteins (G/ACCGAC) and activate expression of many stress-inducible genes. Here, we cloned and characterized a novel gene (AaDREB1) encoding the DREB1 transcription factor from the cold-tolerant plant Adonis amurensis. Quantitative real-time (qRT)-PCR results indicated that AaDREB1 expression was induced by salt, drought, cold stress, and abscisic acid application. A yeast one-hybrid assay demonstrated that AaDREB1 encodes a transcription activator and specifically binds to DRE/CRT. Furthermore, transgenic Arabidopsis and rice harboring AaDREB1 showed enhanced tolerance to salt, drought, and low temperature. These results indicated that AaDREB1 might be useful in genetic engineering to improve plant stress tolerance.

Gene expression profiling of soybean leaves and roots under salt, saline-alkali and drought stress by high-throughput Illumina sequencing.

Salt, saline-alkali and drought stresses are major environmental constraints for the production and yield of soybean worldwide. To identify genes responsible for stress tolerance, the transcriptional profiles of genes in leaves and roots of seedlings (two-leaf stage) of the soybean inbred line HJ-1 were examined after 48 h under various stress conditions; salt (120 mM NaCl), saline-alkali (70 mM NaCl and 50mM NaHCO(3)) and drought (2% PEG 8000). Gene expression at the transcriptional level was investigated using high-throughput Illumina sequencing technology and bioinformatics tools. Under salt, saline-alkali and drought stress, 874, 1897, and 535 genes, respectively, were up-regulated in leaves, and 1822, 1731 and 1690 genes, respectively, were up-regulated in roots, compared with expression in the corresponding organ in control plants. Comparisons among salt, saline-alkali and drought stress yielded similar results in terms of the percentage of genes classified into each GO category. Moreover, 69 genes differentially expressed in both organs with similar expression patterns clustered together in the taxonomic tree across all conditions. Furthermore, comparison of gene expression among salt, saline-alkali and drought treated plants revealed that genes associated with calcium-signaling and nucleic acid pathways were up-regulated in the responses to all three stresses, indicating a degree of cross-talk among these pathways. These results could provide new insights into the stress tolerance mechanisms of soybean.

Sequence mining and transcript profiling to explore differentially expressed genes associated with lipid biosynthesis during soybean seed development.

BACKGROUND: Soybean (Glycine max L.) is one of the most important oil crops in the world. It is desirable to increase oil yields from soybean, and so this has been a major goal of oilseed engineering. However, it is still uncertain how many genes and which genes are involved in lipid biosynthesis. RESULTS: Here, we evaluated changes in gene expression over the course of seed development using Illumina (formerly Solexa) RNA-sequencing. Tissues at 15 days after flowering (DAF) served as the control, and a total of 11592, 16594, and 16255 differentially expressed unigenes were identified at 35, 55, and 65 DAF, respectively. Gene Ontology analyses detected 113 co-expressed unigenes associated with lipid biosynthesis. Of these, 15 showed significant changes in expression levels (log2fold values ≥ 1) during seed development. Pathway analysis revealed 24 co-expressed transcripts involved in lipid biosynthesis and fatty acid biosynthesis pathways. We selected 12 differentially expressed genes and analyzed their expressions using qRT-PCR. The results were consistent with those obtained from Solexa sequencing. CONCLUSION: These results provide a comprehensive molecular biology background for research on soybean seed development, particularly with respect to the process of oil accumulation. All of the genes identified in our research have significance for breeding soybeans with increased oil contents.

Clinical research on comprehensive treatment of senile vascular dementia.

OBJECTIVE: To observe the curative effect of combined Chinese medicine, acupuncture and rehabilitation therapy on vascular dementia (VD), and to compare the Western medicine piracetam. METHODS: Total 134 VD patients screened in reference to the standard for VD diagnosis in DSM-IV were randomly divided into a Chinese medicine plus rehabilitation group (32 cases), a Chinese medicine plus acupuncture group (33 cases), a Chinese medicine and acupuncture plus rehabilitation group (37 cases) and a Western medicine piracetam group (32 cases). Mini Mental State Examination (MMSE) and Bless Behavior Scale (BBS) were used to assess changes in intelligence disorder before treatment and after 12-week treatment. RESULTS: BBS score, living ability and daily habit were enhanced after treatment in all groups. Cognitive function and behavioral ability were improved with similar total curative effects in all the 4 groups. Directional ability and short-term memory ability were significantly enhanced after treatment (P < 0.05) in both the Chinese medicine plus rehabilitation group and the Chinese medicine plus acupuncture group groups. Living ability was significantly improved after treatment in the Chinese medicine and acupuncture plus rehabilitation group (P < 0.01). However, no obvious difference was shown before and after treatment in the piracetam group. CONCLUSION: The improvement of living ability in the comprehensive treatment group is better than that in the other groups. Chinese medicine and acupuncture plus rehabilitation treatment can improve intelligence and living ability of senile VD patients.

[Use of traditional Chinese medicine during the Red Army period in Chinese history].

In this paper, the authors make an analysis of the historical literature during the Red Army period of the agrarian revolution war, dealing with the situation of traditional Chinese medicine in the Red Army. During that time the Red Army had created revolutionary medical hospitals, gathering herbal medicine, growing herbal plants and producing Chinese medicines. At the same time the Red Army paid great attention to enriching Chinese medicine, cultivating practitioners and treating and preventing diseases using traditional Chinese medicine. During the Red Army period there was an extreme lack of medical facilities; traditional Chinese medicine played an important role in ensuring the fighting capabilities of the Red Army units. Looking back at the Red Army period, the development of our tradition can be seen, which enables future development of traditional Chinese medicine, as well as integrated medicine.

[Effect of Shuyusan on neuropeptide Y and serotonin expression in the hippocampal neurons of rats with chronic mild unpredictable stress-induced depression].

OBJECTIVE: To investigate the effect of Shuyusan decoction on neuropeptide Y (NPY) and serotonin (5-HT) expression in the hippocampus and plasma of rats with chronic mild unpredictable stressors depression. METHODS: Fifty Wistar rats were randomly divided into 5 groups, namely the normal control group, model group, fluoxetine group, and high- and low-dose Shuyusan groups. Except for those in the normal control group, all the rats were subjected to chronic mild unpredicted stress for 21 consecutive days with corresponding treatments. Open-field test was used to assess the behavioral changes of the rats. The content of NPY in the hippocampus and plasma was detected by competitive enzyme-linked immunosorbent assay, and immunocytochemistry was used to determine the expression of 5-HT in the hippocampus. RESULTS: NPY levels in the hippocampus and plasma was significantly decreased in the model group as compared with that in the normal control group (P<0.05). Treatments with fluoxetine and high-dose Shuyusan both significantly increased NPY levels in the hippocampus and plasma in the depressive rats (P<0.05), resulting also in significantly increased 5-HT-immunoreactive neurons in the cerebral cortex and hippocampus and the average optical density (P<0.05). CONCLUSION: Shuyusan decoction can effectively increase plasma and hippocampus NPY levels and the number of 5-HT-positive neurons in the cerebral cortex and happocampus of rats with chronic mild unpredictable stress-induced depression.

Dwarf apple MbDREB1 enhances plant tolerance to low temperature, drought, and salt stress via both ABA-dependent and ABA-independent pathways.

In higher plants, DREB1/CBF-type transcription factors play an important role in tolerance to low temperatures, drought, and high-salt stress. These transcription factors bind to CRT/DRE elements in promoter regions of target genes, regulating their expression. In this study, we cloned and characterized a novel gene encoding a DREB1 transcription factor from dwarf apple, Malus baccata (GenBank accession number: EF582842). Expression of MbDREB1 was induced by cold, drought, and salt stress, and also in response to exogenous ABA. Subcellular localization analyses revealed that MbDREB1 localizes in the nucleus. A yeast activity assay demonstrated that the MbDREB1 gene encodes a transcription activator, which specifically binds to DRE/CRT elements. Compared with wild-type plants, transgenic Arabidopsis overexpressing MbDREB1 showed increased tolerance to low temperature, drought, and salt stresses. Analysis of the MbDREB1 promoter revealed an ABA-responsive element (ABRE), an inducer of CBF expression 1 (ICE1)-like binding site, two MYB recognition sites, and three stress-inducible GT-1 boxes. GUS activities driven by the MbDREB1 promoter in transgenic Arabidopsis increased in response to ABA, cold temperature, drought, and salt treatments. Interestingly, the expression of both ABA-independent and ABA-dependent stress-induced genes (COR15a and rd29B, respectively) was activated under normal growth conditions in Arabidopsis overexpressing MbDREB1. These results suggest that MbDREB1 functions as a transcription factor and increases plant tolerance to low temperature, drought, and salt stress via both ABA-dependent and ABA-independent pathways.