Integrated physiological, transcriptomics and metabolomics analysis revealed the molecular mechanism of Bupleurum chinense seedlings to drought stress.

Drought stress is a prominent abiotic factor that adversely influences the growth and development of Bupleurum chinense during its seedling stage, negatively impacting biomass and secondary metabolite production, thus affecting yield and quality. To investigate the molecular mechanism underlying the response of B. chinense seedlings under drought stress, this study employed comprehensive physiological, transcriptomic, and metabolomic analyses. The results revealed that under drought stress, the root soluble sugar and free proline content in B. chinense seedlings significantly increased, while the activities of SOD, POD, and CAT increased in the leaves. These findings indicate the presence of distinct response mechanisms in B. chinense to cope with drought stress. Integrated analysis further identified significant correlations between genes and metabolites related to amino acid biosynthesis in the leaves, as well as genes and metabolites associated with acetaldehyde and dicarboxylic acid metabolism. In the roots, genes and metabolites related to plant hormone signaling and the tricarboxylic acid (TCA) cycle showed significant correlations. These findings provide vital views into the molecular-level response mechanisms of B. chinense under drought stress. Moreover, this study establishes the groundwork for identifying drought-tolerant genes and breeding drought-resistant varieties, which could improve the drought tolerance of medicinal plants and have broader implications for agriculture and crop production in water-scarce areas.

GTPase activity of porcine Mx1 plays a dominant role in inhibiting the N-Nsp9 interaction and thus inhibiting PRRSV replication.

Porcine Mx1 is a type of interferon-induced GTPase that inhibits the replication of certain RNA viruses. However, the antiviral effects and the underlying mechanism of porcine Mx1 for porcine reproductive and respiratory syndrome virus (PRRSV) remain unknown. In this study, we demonstrated that porcine Mx1 could significantly inhibit PRRSV replication in MARC-145 cells. By Mx1 segment analysis, it was indicated that the GTPase domain (68-341aa) was the functional area to inhibit PRRSV replication and that Mx1 interacted with the PRRSV-N protein through the GTPase domain (68-341aa) in the cytoplasm. Amino acid residues K295 and K299 in the G domain of Mx1 were the key sites for Mx1-N interaction while mutant proteins Mx1(K295A) and Mx1(K299A) still partially inhibited PRRSV replication. Furthermore, we found that the GTPase activity of Mx1 was dominant for Mx1 to inhibit PRRSV replication but was not essential for Mx1-N interaction. Finally, mechanistic studies demonstrated that the GTPase activity of Mx1 played a dominant role in inhibiting the N-Nsp9 interaction and that the interaction between Mx1 and N partially inhibited the N-Nsp9 interaction. We propose that the complete anti-PRRSV mechanism of porcine Mx1 contains a two-step process: Mx1 binds to the PRRSV-N protein and subsequently disrupts the N-Nsp9 interaction by a process requiring the GTPase activity of Mx1. Taken together, the results of our experiments describe for the first time a novel mechanism by which porcine Mx1 evolves to inhibit PRRSV replication. IMPORTANCE: Mx1 protein is a key mediator of the interferon-induced antiviral response against a wide range of viruses. How porcine Mx1 affects the replication of porcine reproductive and respiratory syndrome virus (PRRSV) and its biological function has not been studied. Here, we show that Mx1 protein inhibits PRRSV replication by interfering with N-Nsp9 interaction. Furthermore, the GTPase activity of porcine Mx1 plays a dominant role and the Mx1-N interaction plays an assistant role in this interference process. This study uncovers a novel mechanism evolved by porcine Mx1 to exert anti-PRRSV activities.

Response of soil microecology to different cropping practice under Bupleurum chinense cultivation.

The effects of cropping practices on the rhizosphere soil physical properties and microbial communities of Bupleurum chinense have not been studied in detail. The chemical properties and the microbiome of rhizosphere soil of B. chinense were assessed in the field trial with three cropping practices (continuous monocropping, Bupleurum-corn intercropping and Bupleurum-corn rotation). The results showed cropping practices changed the chemical properties of the rhizosphere soil and composition, structure and diversity of the rhizosphere microbial communities. Continuous monocropping of B. chinense not only decreased soil pH and the contents of NO3--N and available K, but also decreased the alpha diversity of bacteria and beneficial microorganisms. However, Bupleurum-corn rotation improved soil chemical properties and reduced the abundance of harmful microorganisms. Soil chemical properties, especially the contents of NH4+-N, soil organic matter (SOM) and available K, were the key factors affecting the structure and composition of microbial communities in the rhizosphere soil. These findings could provide a new basis for overcoming problems associated with continuous cropping and promote development of B. chinense planting industry by improving soil microbial communities.

Chromosome-Level Genome Assembly of Bupleurum chinense DC Provides Insights Into the Saikosaponin Biosynthesis.

Bupleurum chinense DC is a plant widely used in Chinese traditional medicine. Saikosaponins are the major bioactive constituents of B. chinense DC. Saikosaponins biosynthesis in Bupleurum has been more intensively studied than any other metabolic processes or bioactive constituents. However, whole-genome sequencing and chromosome-level assembly for Bupleurum genus have not been reported yet. Here, we report a high-quality chromosome-level genome of B. chinense DC. through the integration of PacBio long-read sequencing, Illumina short-read sequencing, and Hi-C sequencing. The genome was phased into haplotype 0 (621.27 Mb with a contig N50 of 16.86 Mb and a scaffold N50 of 92.25 Mb) and haplotype 1 (600.48 Mb with a contig N50 of 23.90 Mb and a scaffold N50 of 102.68 Mb). A total of 45,909 and 35,805 protein-coding genes were predicted in haplotypes 0 and 1, respectively. The enrichment analyses suggested that the gene families that expanded during the evolution of B. chinense DC are involved in the biosynthesis of isoquinoline alkaloid, tyrosine, and anthocyanin. Furthermore, we analyzed the genes involved in saikosaponin biosynthesis and determined the candidate P450 and UGT genes in the third stage of saikosaponins biosynthetic, which provided new insight into the saikosaponins biosynthetic. The genomic data provide a valuable resource for future investigations of the molecular mechanisms, biological functions, and evolutionary adaptations of B. chinense DC.

Ceramide-Induced Cell Death Depends on Calcium and Caspase-Like Activity in Rice.

Ceramide sphingolipids are major components of membranes. C2 and C6 ceramides induce programmed cell death (PCD) in animals and plants, and we previously showed that C2 and C6 ceramides induce PCD in rice (Oryza sativa) protoplasts. However, the mechanistic link between sphingolipids and PCD in rice remains unclear. Here, we observed that calcium levels increased rapidly after ceramide treatment. Moreover, the calcium channel inhibitor LaCl3 and the intracellular calcium chelator acetoxymethyl-1, 2-bis (2-aminophenoxy) ethic acid (BAPTA-AM) inhibited this calcium increase and prevented ceramide-induced PCD. Moreover, caspase-3-like protease activity increased significantly in C6 ceramide-treated protoplasts, and a caspase-specific inhibitor prevented C6 ceramide-induced cell death. We also detected the other typical PCD events including ATP loss. DIDS (4, 49-diisothiocyanatostilbene- 2, 29-disulfonic acid), an inhibitor of voltage-dependent anion channels (VDACs), decreased C6 ceramide-induced cell death. Together, this evidence suggests that mitochondria played an important role in C6 ceramide-induced PCD.

Association between interleukin-8 gene -251 A/T polymorphism and the risk of coronary artery disease: A meta-analysis.

BACKGROUND: The association between interleukin-8 (IL-8) gene polymorphism -251 A>T and susceptibility to coronary artery disease (CAD) has been investigated previously; however, results remain controversial. Thus, a meta-analysis was conducted to reassess the effects of this polymorphism on CAD risks. METHODS: The PubMed, Cochrane Library, China National Knowledge Infrastructure, and Wanfang databases were searched for relevant studies published up to December, 2018. The pooled odds ratios (OR) were calculated using STATA 13.0 software for allelic (A vs T) as well as homozygote (AA vs TT), heterozygote (AT vs TT), recessive (AA vs AT + TT), and dominant (AA + AT vs TT) genotype models, respectively. RESULTS: Ten case-control studies (3744 cases and 3660 controls) were included. Overall, a significant association of IL-8 gene -251 A > T polymorphism with an increased risk of CAD was only observed in the dominant genotype model (OR = 1.48), but not others. In the subgroup analysis, significantly increased risks were also found for Chinese (OR = 1.64), polymerase chain reaction-restriction fragment length polymorphism genotyping (OR = 1.61), acute coronary syndrome (ACS) type (OR = 1.92 for 3 datasets; OR = 1.88 for 4 datasets), high quality (OR = 1.64), and age/gender matching status (OR = 1.55) under the dominant model. Furthermore, significantly increased risks were also found for ACS type under allelic (OR = 1.32 for 3 datasets; OR = 127 for 4 datasets), homozygote (OR = 1.64 for 3 datasets; OR = 1.50 for 4 datasets), heterozygote (OR = 1.32 for 3 datasets; OR = 1.30 for 4 datasets), and recessive (OR = 1.40 for 3 datasets; OR = 1.28 for 4 datasets) models. CONCLUSION: This meta-analysis suggests that Chinese patients carrying -251A allele of IL-8 may have an increased risk for the development of CAD, especially ACS.

Application of high-throughput amplicon sequencing-based SSR genotyping in genetic background screening.

BACKGROUND: Host genetic backgrounds affect gene functions. The genetic backgrounds of genetically engineered organisms must be identified to confirm their genetic backgrounds identity with those of recipients. Marker-assisted backcrossing (MAB), transgenesis and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) editing are three commonly used genetic engineering techniques. However, methods for genetic background screening between genetically engineered organisms and corresponding recipients suffer from low efficiency, low accuracy or high cost. RESULTS: Here, we improved our previously reported AmpSeq-SSR method, an amplicon sequencing-based simple sequence repeat (SSR) genotyping method, by selecting SSR loci with high polymorphism among varieties. Ultimately, a set of 396 SSRs was generated and applied to evaluate the genetic backgrounds identity between rice lines developed through MAB, transgenesis, and CRISPR/Cas9 editing and the respective recipient rice. We discovered that the percentage of different SSRs between the MAB-developed rice line and its recipient was as high as 23.5%. In contrast, only 0.8% of SSRs were different between the CRISPR/Cas9-system-mediated rice line and its recipient, while no SSRs showed different genotypes between the transgenic rice line and its recipient. Furthermore, most differential SSRs induced by MAB technology were located in non-coding regions (62.9%), followed by untranslated regions (21.0%) and coding regions (16.1%). Trinucleotide repeats were the most prevalent type of altered SSR. Most importantly, all altered SSRs located in coding regions were trinucleotide repeats. CONCLUSIONS: This method is not only useful for the background evaluation of genetic resources but also expands our understanding of the unintended effects of different genetic engineering techniques. While the work we present focused on rice, this method can be readily extended to other organisms.

[A real-time fluorescence quantitative PCR method for source identification of Crocus sativus and Carthamus tinctorius].

The specific PCR primer was designed base on ITS2 sequence in GenBank, and we developed a SYBRGreen real-time fluorescence quantitative PCR system for identification of Crocus sativus and Carthamus tinctorius source. Compared with Chinese herbal medicine DNA barcode technique, this method showed characteristics of shorter time, higher specificity and sensitivity. Using this method to detect 15 samples, 4 were C. sativus, 8 were C. tinctorius, and the other 3 samples were none of them. The result was in accordance with Chinese herbal medicine DNA barcode. This study lays the foundation for identification of related Chinese medical materials.

Early Rehabilitation Therapy Is Beneficial for Patients With Prolonged Mechanical Ventilation After Coronary Artery Bypass Surgery.

We investigated the effects of early rehabilitation therapy on prolonged mechanically ventilated patients after coronary artery bypass surgery (CABG).A total of 106 patients who underwent CABG between June 2012 and May 2015 were enrolled and randomly assigned into an early rehabilitation group (53 cases) and a control group (53 cases). The rehabilitation therapy consisted of 6 steps including head up, transferring from supination to sitting, sitting on the edge of bed, sitting in a chair, transferring from sitting to standing, and walking along a bed. The patients received rehabilitation therapy in the intensive care unit (ICU) after CABG in the early rehabilitation group. The control group patients received rehabilitation therapy after leaving the ICU.The results showed that the early rehabilitation therapy could significantly decrease the duration of mechanical ventilation (early rehabilitation group: 8.1 ± 3.3 days; control group: 13.9 ± 4.1 days, P < 0.01), hospital stay (early rehabilitation group: 22.0 ± 3.8 days; control group: 29.1 ± 4.6 days, P < 0.01), and ICU stay (early rehabilitation group: 11.7 ± 3.2 days; control group: 18.3 ± 4.2 days, P < 0.01) for patients requiring more than 72 hours prolonged mechanical ventilation. The results of Kaplan-Meier analysis showed that the proportions of patients remaining on mechanical ventilation in the early rehabilitation group were larger than that in the control group after 7 days of rehabilitation therapy (logrank test: P < 0.01). The results provide evidence for supporting the application of early rehabilitation therapy in patients requiring prolonged mechanical ventilation after CABG.

Evolutionary Comparison of Two Combinatorial Regulators of SBP-Box Genes, MiR156 and MiR529, in Plants.

A complete picture of the evolution of miRNA combinatorial regulation requires the synthesis of information on all miRNAs and their targets. MiR156 and miR529 are two combinatorial regulators of squamosa promoter binding protein-like (SBP-box) genes. Previous studies have clarified the evolutionary dynamics of their targets; however, there have been no reports on the evolutionary patterns of two miRNA regulators themselves to date. In this study, we investigated the evolutionary differences between these two miRNA families in extant land plants. Our work found that miR529 precursor, especially of its mature miRNA sequence, has a higher evolutionary rate. Such accelerating evolution of miR529 has significantly effects on its structural stability, and sequence conservation against existence of itself. By contrast, miR156 evolves more rapidly in loop region of the stable secondary structure, which may contribute to its functional diversity. Moreover, miR156 and miR529 genes have distinct rates of loss after identical duplication events. MiR529 genes have a higher average loss rate and asymmetric loss rate in duplicated gene pairs, indicating preferred miR529 gene losses become another predominant mode of inactivation, that are implicated in the contraction of this family. On the contrary, duplicated miR156 genes have a low loss rate, and could serve as another new source for functional diversity. Taken together, these results provide better insight into understanding the evolutionary divergence of miR156 and miR529 family in miRNA combinational regulation network.

Elevated lipoprotein-associated phospholipase A2 is associated with progression of nonculprit lesions after percutaneous coronary intervention.

Lipoprotein-associated phospholipase A2 (Lp-PLA2) is an enzyme that hydrolyzes oxidized phospholipids to generate bioactive proatherogenic products. Nonculprit lesions have been assumed to contribute to the pathogenesis of recurrent acute coronary syndrome (ACS). The role of LP-PLA2 in the progression of nonculprit coronary lesions after successful percutaneous coronary intervention (PCI) remains unclear. Our study included 123 patients with ACS who underwent initial PCI and a long-term follow-up (mean interval, one year) with coronary angiography. Among them, 19 patients were diagnosed as the progression of nonculprit lesions, based on the presence of at least one of the following factors: (1) ≥ 10% reduction in the diameter of a preexisting ≥ 50% stenosis; (2) ≥ 30% reduction in the diameter of a < 50% stenosis; and (3) early-onset stenosis with ≥ 30% reduction in the diameter of a segment that was normal on the primary angiogram. Blood sampling was drawn from all patients at 12-14 hours after PCI. The ACS patients with progression had higher total cholesterol (4.47 ± 1.02 mmol/L vs. 3.59 ± 0.57 mmol/L, P < 0.05), higher levels of Lp-PLA2 activity (14.39 ± 6.13 nmol/min/ml vs. 8.86 ± 3.14 nmol/min/ml, P < 0.001) and a higher proportion of multi-vessel disease than those without progression. Multivariate logistic regression analysis showed that Lp-PLA2 activity (ß = 0.024, P = 0.005) was an independent predictor for rapid progression of nonculprit coronary lesions. In conclusion, elevated Lp-PLA2 activity is associated with rapid progression of nonculprit coronary lesions in ACS patients who underwent PCI.

A conserved cysteine motif is critical for rice ceramide kinase activity and function.

BACKGROUND: Ceramide kinase (CERK) is a key regulator of cell survival in dicotyledonous plants and animals. Much less is known about the roles of CERK and ceramides in mediating cellular processes in monocot plants. Here, we report the characterization of a ceramide kinase, OsCERK, from rice (Oryza sativa spp. Japonica cv. Nipponbare) and investigate the effects of ceramides on rice cell viability. PRINCIPAL FINDINGS: OsCERK can complement the Arabidopsis CERK mutant acd5. Recombinant OsCERK has ceramide kinase activity with Michaelis-Menten kinetics and optimal activity at 7.0 pH and 40°C. Mg2+ activates OsCERK in a concentration-dependent manner. Importantly, a CXXXCXXC motif, conserved in all ceramide kinases and important for the activity of the human enzyme, is critical for OsCERK enzyme activity and in planta function. In a rice protoplast system, inhibition of CERK leads to cell death and the ratio of added ceramide and ceramide-1-phosphate, CERK's substrate and product, respectively, influences cell survival. Ceramide-induced rice cell death has apoptotic features and is an active process that requires both de novo protein synthesis and phosphorylation, respectively. Finally, mitochondria membrane potential loss previously associated with ceramide-induced cell death in Arabidopsis was also found in rice, but it occurred with different timing. CONCLUSIONS: OsCERK is a bona fide ceramide kinase with a functionally and evolutionarily conserved Cys-rich motif that plays an important role in modulating cell fate in plants. The vital function of the conserved motif in both human and rice CERKs suggests that the biochemical mechanism of CERKs is similar in animals and plants. Furthermore, ceramides induce cell death with similar features in monocot and dicot plants.

Antidiabetic effect and mechanism of chitooligosaccharides.

The aim of this study was to observe the antidiabetic effect and mechanism of chitooligosaccharides (COS). Type 2 diabetic rats were fed a high-energy diet together with an injection of streptozotocin (STZ). After 8 weeks of COS treatment, the changes in glycometabolism, insulin sensitivity, serum hepatic marker enzyme levels, liver glycogen content, expressions of glucose transporter GLUT-4, malonaldehyde content, superoxide dismutase activity and morphology of the pancreas were observed. The results showed that COS significantly reduced fasting blood glucose (FBG), fasting insulin (FINS), increased the insulin sensitivity index (ISI) and improved oral glucose tolerance. COS increased liver glucokinase activity and glycogen content and upregulated the expressions of GLUT-4 mRNA in adipose and soleus muscle. They also raised the superoxide dismutase activity and reduced the malonaldehyde content in pancreas homogenate. Pancreas hematoxylin/eosin (HE) staining of the diabetic rats showed ruptured islet, but changes of pancreatic islet in the animals were minimized by administration of COS. The effect of COS on pancreatic beta cell (INS-1) in vitro was also examined. It was found that COS played important roles in INS-1 cells by promoting proliferation, increasing glucose stimulated insulin release, upregulating the expressions of GLUT-2 mRNA and protecting against STZ-induced apoptosis. The results from the present study indicate COS have protective effect for type 2 diabetes by ameliorating insulin resistance, promoting the proliferation of beta cells, increasing insulin secretion and protecting beta cells.

Induction of programmed cell death in Arabidopsis and rice by single-wall carbon nanotubes.

PREMISE OF THE STUDY: Single-walled carbon nanotubes (SWCNTs) have many unique structural and mechanical properties. Their potential applications, especially in biomedical engineering and medical chemistry, have been increasing in recent years, but the toxicological impact of nanoparticles has rarely been studied in plants. • METHODS: We exposed Arabidopsis and rice leaf protoplasts to SWCNTs and examined cell viability, DNA damage, reactive oxygen species generation, and related gene expression. We also tested the effects of nanoparticles on Arabidopsis leaves after injecting a SWCNT solution. EM-TUNEL (electron-microscopic terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling) and a cerium chloride staining method were used. • KEY RESULTS: SWCNTs caused adverse cellular responses including cell aggregation, chromatin condensation along with a TUNEL-positive reaction, plasma membrane deposition, and H(2)O(2) accumulation. The effect of SWCNTs on the survival of cells was dose dependent, with 25 µg/mL inducing 25% cell death in 6 h. In contrast, activated carbon, which is not a nano-sized carbon particle, did not induce cell death even 24 h after treatments. The data indicated that the nano-size of the particle is a critical factor for toxicity. Moreover, endocytosis-like structures with cerium chloride deposits formed after SWCNT treatment, suggesting a possible pathway for nanoparticles to traverse the cell membrane. • CONCLUSIONS: Consequently, SWCNTs have an adverse effect on protoplasts and leaves through oxidative stress, leading to a certain amount of programmed cell death. Although nanomaterials have great advantages in many respects, the benefits and side effects still need to be assessed carefully.

[Rapid establishment of suspension cell lines in japonica rice].

With Jingkang No.5 (PiA), calli of the PiA induced for 10-15 days were transferred into amino acid liquid culture medium, to establish excellent rice suspension cell lines successfully in a relative short time. The growth characteristics and differentiation conditions of suspension cells were measured at different phases. Results revealed that the optimal subculture time was 7-10 days, and the cells cultured for 30-120 days had the best differentiation ability (57.1%) and regeneration ability (20%). This study is promising in further using the suspension cell for genetic transformation and protoplasm isolation.

Phenotypic characterization, genetic analysis and gene-mapping for a brittle mutant in rice.

Plant mechanical strength is an important agronomic trait of rice. An ethyl methane sulfonate (EMS)-induced rice mutant, fragile plant 2 (fp2), showed morphological changes and reduced mechanical strength. Genetic analysis indicated that the brittle of fp2 was controlled by a recessive gene. The fp2 gene was mapped on chromosome 10. Anatomical analyses showed that the fp2 mutation caused the reduction of cell length and cell wall thickness, increasing of cell width, and the alteration of cell wall structure as well as the vessel elements. The consequence was a global alteration in plant morphology. Chemical analyses indicated that the contents of cellulose and lignin decreased, and hemicelluloses and silicon increased in fp2. These results were different from the other mutants reported in rice. Thus, fp2 might affect the deposition and patterning of microfibrils, the biosynthesis and deposition of cell wall components, which influences the formation of primary and secondary cell walls, the thickness of cell walls, cell elongation and expansion, plant morphology and plant strength in rice.

Characterization and identification of a novel mutant fon(t) on floral organ number and floral organ identity in rice.

The floral-organ-number mutant fon(t) was firstly discovered in the progeny of a cross between a diploid (Chunjiang 683) and a haploid (SARIV-620-A) rice cultivar. The fon(t) mutant showed normal vegetative development and produced normal inflorescence structures. Difference between the mutant and the wild type was observed when the stamen primordia began to form. The mature flowers of fon(t) mutant showed open-hull phenotypes, which resulted in the exposure of stamens and stigmas. Normally, a single fon(t) floret consisted of six to nine stamens and one or two pistils. In addition, stamen/pistil-like structures and bulged tissues near ovaries were also observed in a few fon(t) florets. But homeotic transformation of lodicules into palea/lemma-like organs was observed almost in all the open-hull florets. The phenotypes of fon(t) flowers also suggested that fon(t) gene might affect flower organ identity in the inner whorls. Genetic analysis showed that the fon(t) mutant was controlled by a single recessive gene.

Morphological, anatomical and genetic analysis for a rice mutant with abnormal hull.

A mutant with abnormal hull was first discovered from a twin-seedling strain W2555 in rice (Oryza sativa L.). The mutant had sparse branches and decreased number of florets from the base to the peak. Frequently, the florets at the top of the panicle did not develop completely. The underdeveloped florets often showed slender and white in their life cycle. Genetic analysis indicated that the mutant traits were controlled by a single recessive gene (temporarily designated as ah). ah gene controlled the development of inflorescence meristem and the flower organ. The florets of mutant showed degenerated lemma and palea. Stamens and lodicules were homeoticly transformed into pistils and palea/lemma-like structures, respectively. It seemed that ah mutant phenotypes of the homeotic conversions in lodicules and stamens were very similar to that of the B loss-of-function spw1 gene reported previously in rice.