Determination of optimal NH4+/K + concentration and corresponding ratio critical for growth of tobacco seedlings in a hydroponic system.

Inherently, ammonium (NH4 +) is critical for plant growth; however, its toxicity suppresses potassium (K+) uptake and vice-versa. Hence, attaining a nutritional balance between these two ions (NH4 + and K+) becomes imperative for the growth of tobacco seedlings. Therefore, we conducted a 15-day experimental study on tobacco seedlings exposed to different concentrations (47 treatments) of NH4 +/K+ at different corresponding 12 ratios simultaneously in a hydroponic system. Our study aimed at establishing the optimal NH4 +-K+ concentration and the corresponding ratio required for optimal growth of different tobacco plant organs during the seedling stage. The controls were the baseline for comparison in this study. Plants with low or excessive NH4 +-K+ concentration had leaf chlorosis or dark greenish colouration, stunted whole plant part biomass, and thin roots. We found that adequate K+ supply is a pragmatic way to mitigate NH4 +-induced toxicity in tobacco plants. The optimal growth for tobacco leaf and root was attained at NH4 +-K+ concentrations 2-2 mM (ratio 1:1), whereas stem growth was optimal at NH4 +-K+ 1-2 mM (1:2). The study provided an insight into the right combination of NH4 +/K+ that could mitigate or prevent NH4 + or K+ stress in the tobacco seedlings.

Projections from the Rostral Zona Incerta to the Thalamic Paraventricular Nucleus Mediate Nociceptive Neurotransmission in Mice.

Zona incerta (ZI) is an integrative subthalamic region in nociceptive neurotransmission. Previous studies demonstrated that the rostral ZI (ZIR) is an important gamma-aminobutyric acid-ergic (GABAergic) source to the thalamic paraventricular nucleus (PVT), but whether the ZIR-PVT pathway participates in nociceptive modulation is still unclear. Therefore, our investigation utilized anatomical tracing, fiber photometry, chemogenetic, optogenetic and local pharmacological approaches to investigate the roles of the ZIRGABA+-PVT pathway in nociceptive neurotransmission in mice. We found that projections from the GABAergic neurons in ZIR to PVT were involved in nociceptive neurotransmission. Furthermore, chemogenetic and optogenetic activation of the ZIRGABA+-PVT pathway alleviates pain, whereas inhibiting the activities of the ZIRGABA+-PVT circuit induces mechanical hypersensitivity and partial heat hyperalgesia. Importantly, in vivo pharmacology combined with optogenetics revealed that the GABA-A receptor (GABAAR) is crucial for GABAergic inhibition from ZIR to PVT. Our data suggest that the ZIRGABA+-PVT pathway acts through GABAAR-expressing glutamatergic neurons in PVT mediates nociceptive neurotransmission.

Glial cells and neurologic autoimmune disorders.

Neurologic autoimmune disorders affect people's physical and mental health seriously. Glial cells, as an important part of the nervous system, play a vital role in the occurrence of neurologic autoimmune disorders. Glial cells can be hyperactivated in the presence of autoantibodies or pathological changes, to influence neurologic autoimmune disorders. This review is mainly focused on the roles of glial cells in neurologic autoimmune disorders and the influence of autoantibodies produced by autoimmune disorders on glial cells. We speculate that the possibility of glial cells might be a novel way for the investigation and therapy of neurologic autoimmune disorders.

Overexpression of NtCBL5A Leads to Necrotic Lesions by Enhancing Na+ Sensitivity of Tobacco Leaves Under Salt Stress.

Many tobacco (Nicotiana tabacum) cultivars are salt-tolerant and thus are potential model plants to study the mechanisms of salt stress tolerance. The CALCINEURIN B-LIKE PROTEIN (CBL) is a vital family of plant calcium sensor proteins that can transmit Ca2+ signals triggered by environmental stimuli including salt stress. Therefore, assessing the potential of NtCBL for genetic improvement of salt stress is valuable. In our studies on NtCBL members, constitutive overexpression of NtCBL5A was found to cause salt supersensitivity with necrotic lesions on leaves. NtCBL5A-overexpressing (OE) leaves tended to curl and accumulated high levels of reactive oxygen species (ROS) under salt stress. The supersensitivity of NtCBL5A-OE leaves was specifically induced by Na+, but not by Cl-, osmotic stress, or drought stress. Ion content measurements indicated that NtCBL5A-OE leaves showed sensitivity to the Na+ accumulation levels that wild-type leaves could tolerate. Furthermore, transcriptome profiling showed that many immune response-related genes are significantly upregulated and photosynthetic machinery-related genes are significantly downregulated in salt-stressed NtCBL5A-OE leaves. In addition, the expression of several cation homeostasis-related genes was also affected in salt-stressed NtCBL5A-OE leaves. In conclusion, the constitutive overexpression of NtCBL5A interferes with the normal salt stress response of tobacco plants and leads to Na+-dependent leaf necrosis by enhancing the sensitivity of transgenic leaves to Na+. This Na+ sensitivity of NtCBL5A-OE leaves might result from the abnormal Na+ compartmentalization, plant photosynthesis, and plant immune response triggered by the constitutive overexpression of NtCBL5A. Identifying genes and pathways involved in this unusual salt stress response can provide new insights into the salt stress response of tobacco plants.

The CBL-Interacting Protein Kinase NtCIPK23 Positively Regulates Seed Germination and Early Seedling Development in Tobacco (Nicotiana tabacum L.).

CBL-interacting protein kinase (CIPK) family is a unique group of serine/threonine protein kinase family identified in plants. Among this family, AtCIPK23 and its homologs in some plants are taken as a notable group for their importance in ions transport and stress responses. However, there are limited reports on their roles in seedling growth and development, especially in Solanaceae plants. In this study, NtCIPK23, a homolog of AtCIPK23 was cloned from Nicotiana tabacum. Expression analysis showed that NtCIPK23 is mainly expressed in the radicle, hypocotyl, and cotyledons of young tobacco seedlings. The transcriptional level of NtCIPK23 changes rapidly and spatiotemporally during seed germination and early seedling growth. To study the biological function of NtCIPK23 at these stages, the overexpressing and CRISPR/Cas9-mediated knock-out (ntcipk23) tobacco lines were generated. Phenotype analysis indicated that knock-out of NtCIPK23 significantly delays seed germination and the appearance of green cotyledon of young tobacco seedling. Overexpression of NtCIPK23 promotes cotyledon expansion and hypocotyl elongation of young tobacco seedlings. The expression of NtCIPK23 in hypocotyl is strongly upregulated by darkness and inhibited under light, suggesting that a regulatory mechanism of light might underlie. Consistently, a more obvious difference in hypocotyl length among different tobacco materials was observed in the dark, compared to that under the light, indicating that the upregulation of NtCIPK23 contributes greatly to the hypocotyl elongation. Taken together, NtCIPK23 not only enhances tobacco seed germination, but also accelerate early seedling growth by promoting cotyledon greening rate, cotyledon expansion and hypocotyl elongation of young tobacco seedlings.

NH4+ Toxicity, Which Is Mainly Determined by the High NH4+/K+ Ratio, Is Alleviated by CIPK23 in Arabidopsis.

Ammonium (NH4+) toxicity is always accompanied by ion imbalances, and NH4+ and potassium (K+) exhibit a competitive correlation in their uptake and transport processes. In Arabidopsis thaliana, the typical leaf chlorosis phenotype in the knockout mutant of calcineurin B-like interacting protein kinase 23 (CIPK23) is high-NH4+-dependent under low-K+ condition. However, the correlation of K+ and NH4+ in the occurrence of leaf chlorosis in the cipk23 mutant has not been deeply elucidated. Here, a modified hydroponic experimental system with different gradients of NH4+ and K+ was applied. Comparative treatments showed that NH4+ toxicity, which is triggered mainly by the high ratio of NH4+ to K+ (NH4+/K+ ≥ 10:1 for cipk23) but not by the absolute concentrations of the ions, results in leaf chlorosis. Under high NH4+/K+ ratios, CIPK23 is upregulated abundantly in leaves and roots, which efficiently reduces the leaf chlorosis by regulating the contents of NH4+ and K+ in plant shoots, while promoting the elongation of primary and lateral roots. Physiological data were obtained to further confirm the role CIPK23 in alleviating NH4+ toxicity. Taken all together, CIPK23 might function in different tissues to reduce stress-induced NH4+ toxicity associated with high NH4+/K+ ratios by regulating the NH4+-K+ balance in Arabidopsis.

The Arabidopsis Calcium-Dependent Protein Kinases (CDPKs) and Their Roles in Plant Growth Regulation and Abiotic Stress Responses.

As a ubiquitous secondary messenger in plant signaling systems, calcium ions (Ca2+) play essential roles in plant growth and development. Within the cellular signaling network, the accurate decoding of diverse Ca2+ signal is a fundamental molecular event. Calcium-dependent protein kinases (CDPKs), identified commonly in plants, are a kind of vital regulatory protein deciphering calcium signals triggered by various developmental and environmental stimuli. This review chiefly introduces Ca2+ distribution in plant cells, the classification of Arabidopsis thaliana CDPKs (AtCDPKs), the identification of the Ca2+-AtCDPK signal transduction mechanism and AtCDPKs' functions involved in plant growth regulation and abiotic stress responses. The review presents a comprehensive overview of AtCDPKs and may contribute to the research of CDPKs in other plants.

Biodegradation of lignin and nicotine with white rot fungi for the delignification and detoxification of tobacco stalk.

BACKGROUND: Tobacco stalk is one kind of abundant crop residues in China. The high lignification of tobacco stalk increases its reusing cost and the existing of nicotine will cause serious pollution. The biodegradation of lignocellulosic biomass has been demonstrated to be an environmental and economical approach for the utilization of plant stalk. Meanwhile, many nicotine-degrading microorganisms were found in nature. However, microorganisms which could degraded both nicotine and lignin haven't been reported. Therefore, it's imperative to find some suitable microorganisms to break down lignin and simultaneously remove nicotine in tobacco stalk. RESULTS: The nicotine in tobacco stalk could be degraded effectively by Trametes versicolor, Trametes hirsute and Phanerochaete chrysosporium. The nicotine content in tobacco stalk was lowered to below 500 mg/kg (a safe concentration to environment) after 10 days of fermentation with Phanerochaete chrysosporium and Trametes versicolor, and 15 days with Trametes hirsute. The degradation rate of lignin in the fermented tobacco stalk was 37.70, 51.56 and 53.75% with Trametes versicolor, Trametes hirsute and Phanerochaete chrysosporium, respectively. Meanwhile, 24.28% hemicellulose was degraded by Phanerochaete chrysosporium and 28.19% cellulose was removed by Trametes hirsute. Through the enzyme activity analysis, the main and highest ligninolytic enzymes produced by Phanerochaete chrysosporium, Trametes hirsute and Trametes versicolor were lignin peroxidase (88.62 U · L-1), manganese peroxidase (100.95 U · L-1) and laccase (745.65 U · L-1). Meanwhile, relatively high and stable cellulase activity was also detected during the fermentation with Phanerochaete chrysosporium, and the highest endoglucanase, exoglucanase and filter paper enzyme activities were 0.38 U · mL-1, 0.45 U · mL-1 and 0.35U · mL-1, respectively. Moreover, the products in the fermentation of tobacco stalk with P. chrysosporium were identified with GC-MS, besides the chemicals produced in the degradation of lignin and nicotine, some small molecular valuable chemicals and fatty acid were also detected. CONCLUSIONS: Our study developed a new method for the degradation and detoxification of tobacco stalk by fermentation with white rot fungi Phanerochaete chrysosporium and Trametes hirsute. The different oxidative enzymes and chemical products detected during the degradation indicated a possible pathway for the utilization of tobacco stalk.

Mechanisms and Physiological Roles of the CBL-CIPK Networking System in Arabidopsis thaliana.

Calcineurin B-like protein (CBL)-CBL-interacting protein kinase (CIPK) network is one of the vital regulatory mechanisms which decode calcium signals triggered by environmental stresses. Although the complicated regulation mechanisms and some novel functions of CBL-CIPK signaling network in plants need to be further elucidated, numerous advances have been made in its roles involved in the abiotic stresses. This review chiefly introduces the progresses about protein interaction, classification and expression pattern of different CBLs and CIPKs in Arabidopsis thaliana, summarizes the physiological roles of CBL-CIPK pathway while pointing out some new research ideas in the future, and finally presents some unique perspectives for the further study. The review might provide new insights into the functional characterization of CBL-CIPK pathway in Arabidopsis, and contribute to a deeper understanding of CBL-CIPK network in other plants or stresses.

The Calcium Sensor CBL-CIPK Is Involved in Plant's Response to Abiotic Stresses.

Abiotic stress halts the physiological and developmental process of plant. During stress condition, CBL-CIPK complex is identified as a primary element of calcium sensor to perceive environmental signals. Recent studies established that this complex regulates downstream targets like ion channels and transporters in adverse stages conditions. Crosstalks between the CBL-CIPK complex and different abiotic stresses can extend our research area, which can improve and increase the production of genetically modified crops in response to abiotic stresses. How this complex links with environmental signals and creates adjustable circumstances under unfavorable conditions is now one of the burning issues. Diverse studies are already underway to delineate this signalling mechanism underlying different interactions. Therefore, up to date experimental results should be concisely published, thus paving the way for further research. The present review will concisely recapitulate the recent and ongoing research progress of positive ions (Mg(2+), Na(+), and K(+)), negative ions (NO3 (-), PO4 (-)), and hormonal signalling, which are evolving from accumulating results of analyses of CBL and CIPK loss- or gain-of-function experiments in different species along with some progress and perspectives of our works. In a word, this review will give one step forward direction for more functional studies in this area.

Nicotiana sylvestris calcineurin B-like protein NsylCBL10 enhances salt tolerance in transgenic Arabidopsis.

KEY MESSAGE: Nicotiana sylvestris calcineurin B-like protein NsylCBL10 improves tolerance to high-salt stress through better maintenance of Na (+) balance. The calcineurin B-like (CBL) proteins represent a unique group of plant calcium sensors and play an important role in regulating the response of a plant cell to the stress. Although many studies have been made in Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa) and poplar (Populus trichocarpa), the characterization and elucidation of the functions of CBLs in tobacco have not yet been reported. In this study, NsylCBL10, a CBL gene showing higher similarities to other CBL10 genes, was cloned from Nicotiana sylvestris. NsylCBL10 is expressed in most of the tobacco tissues, and the protein targets to the plasma membrane specifically. Over-expression of NsylCBL10 enhanced the salt tolerance of Arabidopsis wild type plants greatly, and rescued the high-salt-sensitive phenotype of Arabidopsis cbl10 mutant. The analysis of ion content indicated that over-expressing NsylCBL10 in plants is able to maintain a lower Na(+)/K(+) ratio in roots and higher Na(+)/K(+) ratio in shoots, compared with cbl10 mutant. The results suggest that NsylCBL10 might play an important role in response to high salinity stress in N. sylvestris, by keeping a better ionic homeostasis to reduce the damage of toxic ion to the plant cell.

[Development and assessment of a chemiluminescence assay kit for detecting anti-HEV IgG].

OBJECTIVE: To develop an anti-hepatitis E virus (anti-HEV) IgG chemiluminescence assay kit and assess its clinical application. METHODS: The HEV recombinant antigen was used as coating antigen, horseradish peroxidase (HRP)-conjugated monoclonal anti-human IgG as the secondary antibody, and the luminol chemiluminescent reaction system as a substrate. The sensitivity, specificity, precision and other technical indicators of the kit were evaluated using the HEV national reference product, and a contrast experiment was conducted on 1012 serum samples by the kit developed in this research and a commercialized anti-HEV IgG chemiluminescence assay kit. RESULTS: The sensitivity, specificity, precision and stability of all the three batches of kit reached national standard. In the detection of 1012 clinical serum samples, the positive coincidence rate of both kits was 97.4%, the negative coincidence rate was 99.4%, and the total coincidence rate reached 98.4%. CONCLUSION: An anti-HEV IgG chemiluminescence assay kit has been successfully developed. The kit is of high sensitivity and specificity, easy to operate. It is applicable to the clinical diagnosis and epidemiological survey of HEV infection.

[Isolation of microorganisms producing enzyme capable of degrading tobacco straw and nicotine].

OBJECTIVE: The aim of this study was to screen tobacco straw and nicotine degrading microorganism. METHODS: The bacterium was isolated from tobacco field soil using medium containing tobacco straw as the sole carbon and nitrogen source. We identified the bacterium through morphological and physiological characterization combined with the result of 16S rRNA gene sequence and data analysis. We also studied the lignocelluloses degradation and enzyme activities related to the degradation of lignin and cellulose in liquid state fermentation of tobacco stalk. RESULTS: The bacterium was identified as Bacillus megaterium and we had demonstrated that it has a good ability to degrade lignin in tobacco straw when fermented in liquid state. It showed the highest laccase production of 418. 52 U/L while the highest lignin peroxides and manganese peroxides activity was 19. 71 U/L and 64. 71 U/L. On the other hand, we also found that nicotine in tobacco stem was totally degraded 20 d after inoculation. CONCLUSION: to the isolated Bacillus megaterium is capable of degrading tobacco straw partially and nicotine totally.

[Establishment and evaluation of the diagnostic kit for anti-HIV1/2 antibody and P24 antigen].

OBJECTIVE: To establish and evaluate an Enzyme Immunoassay diagnostic kit combined with anti-HIV1/2 antibody and P24 antigen for shortening the examination window period of HIV infection in HIV laboratory diagnosis. METHODS: The enzyme-linked reaction plates was coated by anti-HIV P24 monoclonal antibody and HIV 1/2 antigen. Labeling HIV1/2 antigen and anti-HIV P24 polyclonal antibody with horseradish peroxidase, setup an integrated ELISA kit for detecting anti-HIV-1/2 antibody and HIV P24 antigen, and evaluate the specificity and sensitivity of this kit. RESULTS: The sensitivity of testing P24 antigen was up to 0.2 ng/ml. 78 serum samples of patients with AIDS, 85 serum samples of healthy people were compared with Abbott EIA kit, the coincidence was 100%. 12 051 sera from normal persons and patients were examined, the sensitivity of 100 %and specificity of 99.62 %, respectively. CONCLUSION: The anti-HIV1/2 antibody and HIV P24 antigen can be measured at the same time using this EIA kit, while the examination window period of HIV infection is shortened. Thus, the method is suitable for laboratory diagnosis and epidemiological investigation.