Laser capture microdissection transcriptome (LCM RNA-seq) reveals BcDFR is a key gene in anthocyanin synthesis of non-heading Chinese cabbage.

BACKGROUND: Purple non-heading Chinese cabbage [Brassica campestris (syn. Brassica rapa) ssp. chinensis] has become popular because of its richness in anthocyanin. However, anthocyanin only accumulates in the upper epidermis of leaves. Further studies are needed to investigate the molecular mechanisms underlying the specific accumulation of it. RESULTS: In this study, we used the laser capture frozen section method (LCM) to divide purple (ZBC) and green (LBC) non-heading Chinese cabbage leaves into upper and lower epidermis parts (Pup represents the purple upper epidermis, Plow represents the purple lower epidermis, Gup represents the green upper epidermis, Glow represents the green lower epidermis). Through transcriptome sequencing, we found that the DIHYDROFLAVONOL 4-REDUCTASE-encoding gene BcDFR, is strongly expressed in Pup but hardly in others (Plow, Gup, Glow). Further, a deletion and insertion in the promoter of BcDFR in LBC were found, which may interfere with BcDFR expression. Subsequent analysis of gene structure and conserved structural domains showed that BcDFR is highly conserved in Brassica species. The predicted protein-protein interaction network of BcDFR suggests that it interacts with almost all functional proteins in the anthocyanin biosynthesis pathway. Finally, the results of the tobacco transient expression also demonstrated that BcDFR promotes the synthesis and accumulation of anthocyanin. CONCLUSIONS: BcDFR is specifically highly expressed on the upper epidermis of purple non-heading Chinese cabbage leaves and regulates anthocyanin biosynthesis and accumulation. Our study provides new insights into the functional analysis and transcriptional regulatory network of anthocyanin-related genes in purple non-heading Chinese cabbage.

The role of autophagy in insulin resistance and glucolipid metabolism and potential use of autophagy modulating natural products in the treatment of type 2 diabetes mellitus.

Type 2 diabetes mellitus (T2DM) is a severe, long-term condition characterised by disruptions in glucolipid and energy metabolism. Autophagy, a fundamental cellular process, serves as a guardian of cellular health by recycling and renewing cellular components. To gain a comprehensive understanding of the vital role that autophagy plays in T2DM, we conducted an extensive search for high-quality publications across databases such as Web of Science, PubMed, Google Scholar, and SciFinder and used keywords like 'autophagy', 'insulin resistance', and 'type 2 diabetes mellitus', both individually and in combinations. A large body of evidence underscores the significance of activating autophagy in alleviating T2DM symptoms. An enhanced autophagic activity, either by activating the adenosine monophosphate-activated protein kinase and sirtuin-1 signalling pathways or inhibiting the mechanistic target of rapamycin complex 1 signalling pathway, can effectively improve insulin resistance and balance glucolipid metabolism in key tissues like the hypothalamus, skeletal muscle, liver, and adipose tissue. Furthermore, autophagy can increase ß-cell mass and functionality in the pancreas. This review provides a narrative summary of autophagy regulation with an emphasis on the intricate connection between autophagy and T2DM symptoms. It also discusses the therapeutic potentials of natural products with autophagy activation properties for the treatment of T2DM conditions. Our findings suggest that autophagy activation represents an innovative approach of treating T2DM.

The intracellular monoamine oxidase-A inhibitory activity and the protective effect of small hairtail-related peptides in nerve cells (SH-SY5Y).

This study was to investigate the inhibitory activity of small hairtail-related peptides (VFEVFW, LPNSLYQQ, LPNSLYQK, and FADAME) on intracellular monoamine oxidase-A (MAO-A) and their protective effects in a cell model. Specifically, the inhibition activity in SH-SY5Y cells indicated that VFEVFW and LPNSLYQK reduced ∼50% of MAO-A activity in cells, at 0.5 m m. The survival experiment demonstrated that the toxic effect of dexamethasone (DEX) on cells can be significantly alleviated in the presence of peptides, and these peptides can restore (>20%) the mitochondrial membrane potential of SH-SY5Y cells reduced by DEX. Circular dichroism displayed that peptides affected the secondary structure of MAO-A in a concentration-dependent manner. Finally, the real-time quantitative polymerase chain reaction assay revealed that the MAO-A inhibitory activity of the peptides was associated with the upregulation of brain derived neurotrophic factor/cAMP (Cyclic adenosine monophosphate) response element binding protein)/B-cell lymphoma-2 mRNA levels.

Application of Visual Management in Enhancing Work Quality within the Central Sterile Supply Department.

Objective: This study aims to investigate the implementation of visual management in the central sterile supply department, focusing on enhancing work efficiency, improving work quality, reducing errors and accidents, and elevating the satisfaction levels of clinical departments. Methods: Visual flow diagrams were carefully developed and classified by a dedicated team from July to August 2021, led by an established visual management team. Subsequently, department staff underwent organized training sessions to understand the concepts, requirements, and contents of these visual management flow diagrams through a related examination. The application of visual flow diagrams extended to instruments and equipment, infection control, and instrument package management. To assess the impact, a control group comprising 400 pieces of instruments used in the operating room and clinical practice in June 2021 and an observation group with 400 pieces for surgical instruments and clinical use in October 2021 were selected using a convenience sampling method. The study analyzed and compared the qualified rate of instrument cleaning, the qualified rate of instrument packaging, and clinical satisfaction between the two groups. Results: The device qualified rates for instrument cleaning in the observation and control groups were 99% and 95%, respectively; for instrument packaging, they were 96% and 92%. Clinical satisfaction rates were 99% and 90%, respectively. These findings indicate an improved qualified rate for instrument cleaning, instrument packaging, and clinical satisfaction in the observation group compared to the control group before the implementation of visual management, with statistically significant differences (P = .000). Conclusions: The application of visual flow diagrams has a significant positive impact on the work quality in the central sterile supply department and enhances clinical work satisfaction. This approach is deemed suitable for broader promotion.

Licochalcone A alleviates abnormal glucolipid metabolism and restores energy homeostasis in diet-induced diabetic mice.

Licochalcone A (LCA) is a bioactive chalcone compound identified in licorice. This study aimed to investigate the effects of LCA on glucolipid metabolism and energy homeostasis, as well as the underlying mechanisms. Blood glucose levels, oral glucose tolerance, serum parameters, and histopathology were examined in high-fat-high-glucose diet (HFD)-induced diabetic mice, with metformin as a positive control. Additionally, changes in key markers related to glucolipid metabolism and mitochondrial function were analyzed to comprehensively assess LCA's effects on metabolism. The results showed that LCA alleviated metabolic abnormalities in HFD-induced diabetic mice, which were manifested by suppression of lipogenesis, promotion of lipolysis, reduction of hepatic steatosis, increase in hepatic glycogenesis, and decrease in gluconeogenesis. In addition, LCA restored energy homeostasis by promoting mitochondrial biogenesis, enhancing mitophagy, and reducing adenosine triphosphate production. Mechanistically, the metabolic benefits of LCA were associated with the downregulation of mammalian target of rapamycin complex 1 and activation of adenosine monophosphate-activated protein kinase, the two central regulators of metabolism. This study demonstrates that LCA can alleviate abnormal glucolipid metabolism and restore energy balance in diet-induced diabetic mice, highlighting its therapeutical potential for the treatment of diabetes.

BcABF1 Plays a Role in the Feedback Regulation of Abscisic Acid Signaling via the Direct Activation of BcPYL4 Expression in Pakchoi.

Abscisic acid-responsive element-binding factor 1 (ABF1), a key transcription factor in the ABA signal transduction process, regulates the expression of downstream ABA-responsive genes and is involved in modulating plant responses to abiotic stress and developmental processes. However, there is currently limited research on the feedback regulation of ABF1 in ABA signaling. This study delves into the function of BcABF1 in Pakchoi. We observed a marked increase in BcABF1 expression in leaves upon ABA induction. The overexpression of BcABF1 not only spurred Arabidopsis growth but also augmented the levels of endogenous IAA. Furthermore, BcABF1 overexpression in Arabidopsis significantly decreased leaf water loss and enhanced the expression of genes associated with drought tolerance in the ABA pathway. Intriguingly, we found that BcABF1 can directly activate BcPYL4 expression, a critical receptor in the ABA pathway. Similar to BcABF1, the overexpression of BcPYL4 in Arabidopsis also reduces leaf water loss and promotes the expression of drought and other ABA-responsive genes. Finally, our findings suggested a novel feedback regulation mechanism within the ABA signaling pathway, wherein BcABF1 positively amplifies the ABA signal by directly binding to and activating the BcPYL4 promoter.

Photo-Activated Direct Catalytic Oxidation of Gaseous Benzene with a Cu-Connected Serial Heterojunction Array of Co3 O4 /Cux O/Foam Cu Assembled via Layer upon Layer Oxidation.

The foam copper (FCu) has been first used as a promising supporting material to prepare a photo-activated catalyst of Co3 O4 /Cux O/FCu, in which the fine Co3 O4 particles are inlayed on the Cux O nanowires to form a Z-type heterojunction array connected by substrate Cu. The prepared samples have been used as a photo-activated catalyst to directly decompose gaseous benzene and the optimized Co3 O4 /Cux O/FCu demonstrates a 99.5% removal efficiency and 100% mineralizing rate within 15 min in benzene concentration range from 350 to 4000 ppm under simulate solar light irradiation. To track the reactive mechanism, a series of MOx /Cux O/FCu (M = Mn, Fe, Co, Ni, Cu, Zn) is prepared and a novel photo-activated direct catalytic oxidation route is proposed based on the comparative investigation of material properties. Moreover, the approach grew in situ via layer upon layer oxidation on FCu dedicates to the extra lasting reusability and the easy accessibility in the diverse situations. This work provides a novel strategy for the preparation of Cu connected series multidimensional heterojunction array and a promising application for the quick abatement of the high-leveled concentration gaseous benzene and its derivatives from the industrial discharged flow or the accident scene's leakage.

Neutrophil-Mimetic, ROS Responsive, and Oxygen Generating Nanovesicles for Targeted Interventions of Refractory Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is the most prevalent inflammatory joint disease worldwide, leading to irreversible disability and even mortality. Unfortunately, current treatment regimens fail to cure RA due to low therapeutic responses and off-target side effects. Herein, a neutrophil membrane-cloaked, natural anti-arthritic agent leonurine (Leo), and catalase (CAT) co-loaded nanoliposomal system (Leo@CAT@NM-Lipo) is constructed to remodel the hostile microenvironment for RA remission. Due to the inflammation tropism inherited from neutrophils, Leo@CAT@NM-Lipo can target and accumulate in the inflamed joint cavity where high-level ROS can be catalyzed into oxygen by CAT to simultaneously accelerate the drug release and alleviate hypoxia at the lesion site. Besides, the neutrophil membrane camouflaging also enhances the anti-inflammatory potentials of Leo@CAT@NM-Lipo by robustly absorbing pro-arthritogenic cytokines and chemokines. Consequently, Leo@CAT@NM-Lipo successfully alleviated paw swelling, reduced arthritis score, mitigated bone and cartilage damage, and reversed multiple organ dysfunctions in adjuvant-induced arthritis rats (AIA) rats by synergistic effects of macrophage polarization, inflammation resolution, ROS scavenging, and hypoxia relief. Furthermore, Leo@CAT@NM-Lipo manifested excellent biocompatibility both at the cellular and animal levels. Taken together, the study provided a neutrophil-mimetic and ROS responsive nanoplatform for targeted RA therapy and represented a promising paradigm for the treatment of a variety of inflammation-dominated diseases.

Optimization of fermentation conditions for surfactin production by B. subtilis YPS-32.

BACKGROUND: Surfactin produced by microbial fermentation has attracted increasing attention because of its low toxicity and excellent antibacterial activity. However, its application is greatly limited by high production costs and low yield. Therefore, it is important to produce surfactin efficiently while reducing the cost. In this study, B. subtilis strain YPS-32 was used as a fermentative strain for the production of surfactin, and the medium and culture conditions for the fermentation of B. subtilis YPS-32 for surfactin production were optimized. RESULTS: First, Landy 1 medium was screened as the basal medium for surfactin production by B. subtilis strain YPS-32. Then, using single-factor optimization, the optimal carbon source for surfactin production by B. subtilis YPS-32 strain was determined to be molasses, nitrogen sources were glutamic acid and soybean meal, and inorganic salts were KCl, K2HPO4, MgSO4, and Fe2(SO4)3. Subsequently, using Plackett-Burman design, MgSO4, time (h) and temperature (°C) were identified as the main effect factors. Finally, Box-Behnken design were performed on the main effect factors to obtain optimal fermentation conditions: temperature of 42.9 °C, time of 42.8 h, MgSO4 = 0.4 g·L- 1. This modified Landy medium was predicted to be an optimal fermentation medium: molasses 20 g·L- 1, glutamic acid 15 g·L- 1, soybean meal 4.5 g·L- 1, KCl 0.375 g·L- 1, K2HPO4 0.5 g·L- 1, Fe2(SO4)3 1.725 mg·L- 1, MgSO4 0.4 g·L- 1. Using the modified Landy medium, the yield of surfactin reached 1.82 g·L- 1 at pH 5.0, 42.9 ℃, and 2% inoculum for 42.8 h, which was 2.27-fold higher than that of the Landy 1 medium in shake flask fermentation. Additionally, under these optimal process conditions, further fermentation was carried out at the 5 L fermenter level by foam reflux method, and at 42.8 h of fermentation, surfactin reached a maximum yield of 2.39 g·L- 1, which was 2.96-fold higher than that of the Landy 1 medium in 5 L fermenter. CONCLUSION: In this study, the fermentation process of surfactin production by B. subtilis YPS-32 was improved by using a combination of single-factor tests and response surface methodology for test optimization, which laid the foundation for its industrial development and application.

In Situ Growth of Cs3Bi2Br9 Quantum Dots on Bi-MOF Nanosheets via Cosharing Bismuth Atoms for CO2 Capture and Photocatalytic Reduction.

Given the global warming caused by excess CO2 accumulation in the atmosphere, it is essential to reduce CO2 by capturing and converting it to chemical feedstock using solar energy. Herein, a novel Cs3Bi2Br9/bismuth-based metal-organic framework (Bi-MOF) composite was prepared via an in situ growth strategy of Cs3Bi2Br9 quantum dots (QDs) on the surface of Bi-MOF nanosheets through coshared bismuth atoms. The prepared Cs3Bi2Br9/Bi-MOF exhibits bifunctional merits for both the high capture and effective conversion of CO2, among which the optimized 3Cs3Bi2Br9/Bi-MOF sample shows a CO2-CO conversion yield as high as 572.24 µmol g-1 h-1 under the irradiation of a 300 W Xe lamp. In addition, the composite shows good stability after five recycles in humid air, and the CO2 photoreduction efficiency does not decrease significantly. The mechanistic investigation uncovers that the intimate atomic-level contact between Cs3Bi2Br9 and Bi-MOF via the coshared atoms not only improves the dispersion of Cs3Bi2Br9 QDs over Bi-MOF nanosheets but also accelerates interfacial charge transfer by forming a strong bonding linkage, which endows it with the best performance of CO2 photoreduction. Our new finding of bismuth-based metal-organic framework/lead-free halide perovskite by cosharing atoms opens a new avenue for a novel preparation strategy of the heterojunction with atomic-level contact and potential applications in capture and photocatalytic conversion of CO2.

Apoptotic vesicles activate autophagy in recipient cells to induce angiogenesis and dental pulp regeneration.

Extracellular vesicles (EVs) derived from living cells play important roles in donor cell-induced recipient tissue regeneration. Although numerous studies have found that cells undergo apoptosis after implantation in an ischemic-hypoxic environment, the roles played by the EVs released by apoptotic cells are largely unknown. In this study, we obtained apoptotic vesicles (apoVs) derived from human deciduous pulp stem cells and explored their effects on the dental pulp regeneration process. Our work showed that apoVs were ingested by endothelial cells (ECs) and elevated the expression of angiogenesis-related genes, leading to pulp revascularization and tissue regeneration. Furthermore, we found that, at the molecular level, apoV-carried mitochondrial Tu translation elongation factor was transported and regulated the angiogenic activation of ECs via the transcription factor EB-autophagy pathway. In a beagle model of dental pulp regeneration in situ, apoVs recruited endogenous ECs and facilitated the formation of dental-pulp-like tissue rich in blood vessels. These findings revealed the significance of apoptosis in tissue regeneration and demonstrated the potential of using apoVs to promote angiogenesis in clinical applications.

Figure Correction: Using Social Media to Help Understand Patient-Reported Health Outcomes of Post-COVID-19 Condition: Natural Language Processing Approach.

[This corrects the article DOI: 10.2196/45767.].

Using Social Media to Help Understand Patient-Reported Health Outcomes of Post-COVID-19 Condition: Natural Language Processing Approach.

BACKGROUND: While scientific knowledge of post-COVID-19 condition (PCC) is growing, there remains significant uncertainty in the definition of the disease, its expected clinical course, and its impact on daily functioning. Social media platforms can generate valuable insights into patient-reported health outcomes as the content is produced at high resolution by patients and caregivers, representing experiences that may be unavailable to most clinicians. OBJECTIVE: In this study, we aimed to determine the validity and effectiveness of advanced natural language processing approaches built to derive insight into PCC-related patient-reported health outcomes from social media platforms Twitter and Reddit. We extracted PCC-related terms, including symptoms and conditions, and measured their occurrence frequency. We compared the outputs with human annotations and clinical outcomes and tracked symptom and condition term occurrences over time and locations to explore the pipeline's potential as a surveillance tool. METHODS: We used bidirectional encoder representations from transformers (BERT) models to extract and normalize PCC symptom and condition terms from English posts on Twitter and Reddit. We compared 2 named entity recognition models and implemented a 2-step normalization task to map extracted terms to unique concepts in standardized terminology. The normalization steps were done using a semantic search approach with BERT biencoders. We evaluated the effectiveness of BERT models in extracting the terms using a human-annotated corpus and a proximity-based score. We also compared the validity and reliability of the extracted and normalized terms to a web-based survey with more than 3000 participants from several countries. RESULTS: UmlsBERT-Clinical had the highest accuracy in predicting entities closest to those extracted by human annotators. Based on our findings, the top 3 most commonly occurring groups of PCC symptom and condition terms were systemic (such as fatigue), neuropsychiatric (such as anxiety and brain fog), and respiratory (such as shortness of breath). In addition, we also found novel symptom and condition terms that had not been categorized in previous studies, such as infection and pain. Regarding the co-occurring symptoms, the pair of fatigue and headaches was among the most co-occurring term pairs across both platforms. Based on the temporal analysis, the neuropsychiatric terms were the most prevalent, followed by the systemic category, on both social media platforms. Our spatial analysis concluded that 42% (10,938/26,247) of the analyzed terms included location information, with the majority coming from the United States, United Kingdom, and Canada. CONCLUSIONS: The outcome of our social media-derived pipeline is comparable with the results of peer-reviewed articles relevant to PCC symptoms. Overall, this study provides unique insights into patient-reported health outcomes of PCC and valuable information about the patient's journey that can help health care providers anticipate future needs. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): RR2-10.1101/2022.12.14.22283419.

Catalytic Electrophilic Halogenation of Arenes with Electron-Withdrawing Substituents.

The electrophilic halogenation of arenes is perhaps the simplest method to prepare aryl halides, which are important structural motifs in agrochemicals, materials, and pharmaceuticals. However, the nucleophilicity of arenes is weakened by the electron-withdrawing substituents, whose electrophilic halogenation reactions usually require harsh conditions and lead to limited substrate scopes and applications. Therefore, the halogenation of arenes containing electron-withdrawing groups (EWGs) and complex bioactive compounds under mild conditions has been a long-standing challenge. Herein, we describe Brønsted acid-catalyzed halogenation of arenes with electron-withdrawing substituents under mild conditions, providing an efficient protocol for aryl halides. The hydrogen bonding of Brønsted acid with the protic solvent 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) enables this transformation and thus solves this long-standing problem.

Cloning and characterization of the Cry79Aa1 gene from a lepidopteran active strain of Bacillus thuringiensis.

Bacillus thuringiensis (Bt) has been used globally as a biopesticide for effective and environmentally friendly pest control. Research has intensified following the development of resistance by lepidopteran species to Bt insecticidal crystal proteins. Discovering new Bt strains with novel toxin properties which can overcome resistance is one of the strategies to improve pesticide sustainability. The genome of the Bacillus thuringiensis LTS290 strain was sequenced and assembled in 252 contigs containing a total of 6,391,328 bp. The novel cry79Aa1 gene from this strain was identified and cloned. Cry79Aa1 contains 729 amino acid residues and a molecular mass of 84.8 kDa by SDS-PAGE analysis. Cry79Aa1 was found to be active against the lepidopteran larvae of Spodoptera exigua, Helicoverpa armigera, and Plutella xylostella with LC50 values of 13.627 µg/mL, 42.8 µg/mL, and 38.086 µg/mL, respectively. However, Cry79Aa1 protein showed almost no insecticidal activity against Leguminivora glycinivorella, although some degree of growth retardation was observed.

SHED promote angiogenesis in stem cell-mediated dental pulp regeneration.

Dental pulp, plays an indispensable role in maintaining homeostasis of the tooth. Pulp necrosis always causes tooth nutrition deficiency and abnormal root development, which leads to tooth discoloration, fracture or even loss. Our previous study showed implantation of autologous SHED could regenerate functional dental pulp. However, the detailed mechanism of the implanted SHED participating in dental pulp regeneration remains unknown. In this study, we implanted SHED in a porcine dental pulp regeneration model to evaluate the regenerative effect and identify whether SHED promoted angiogenesis in regenerated dental pulp. Firstly we verified that xenogenous SHED had the ability to regenerated pulp tissue of host in vivo. Then we found the vasculature in regenerated pulp originated from implanted SHED. In addition, stem cells were isolated from regenerated dental pulp, which exhibited good multi-differentiation properties and promoted angiogenesis in pulp regeneration process and these results demonstrated that SHED promoted angiogenesis in stem cell-mediated dental pulp regeneration.

Discovery of dipeptidyl peptidase 4 inhibitory peptides from Largemouth bass (Micropterus salmoides) by a comprehensive approach.

Dipeptidyl peptidase-4 (DPP4) is the prime target for glycemic control by inactivating glucagon-like peptide and decreasing postprandial glucose levels. Food protein-derived peptides have been considered to be capable of inhibiting DPP4. In this study, a novel strategy was developed by coupling in silico gastrointestinal digestion, pharmacophore and three-dimensional quantitative structure-activity relationships (3D-QSAR) analysis to discover DPP4 inhibitory peptide, and in vitro assay was confirmed. Specifically, the simulated gastrointestinal hydrolysis was firstly performed on Largemouth bass (Micropterus salmoides) proteins, the generated peptides were used to establish peptide library. Secondly, 60 DPP4 inhibitors were selected and pharmacophore model was generated; moreover, 40 DPP4 inhibitory tripeptides were collected to construct 3D-QSAR model. Thirdly, the pharmacophore and 3D-QSAR models were employed to screen the above peptide library. Lastly, the in vitro activity assay was performed, which showed that the six tripeptides (VSM, ISW, VSW, ICY, ISD and ISE) exhibited inhibitory activities on DPP4, and ICY was the most active tripeptide with the IC50 value of 0.73 mM. This is the first identification of Largemouth bass protein-derived peptides as DPP4 inhibitor, which is good for the development of food protein-derived peptides with glucose lowering activity.

Discovery of monoamine oxidase A inhibitory peptides from hairtail (Trichiurus japonicus) using in vitro simulated gastrointestinal digestion and in silico studies.

The aim of this study was to effectively obtain monoamine oxidase A (MAO-A) inhibitory peptides from in vitro simulated gastrointestinal digestion and to assess the correspondences between in silico prediction and in vitro confirmation. Fractions (<3 kDa) from ultrafiltration of pepsin and simulated gastrointestinal enzymes hydrolysates exhibited the highest MAO-A inhibitory activity with IC50 values of 0.61 and 2.54 mg/mL, respectively. After sequencing and then screening by HPEPDOCK, 11 high-score peptides and 2 low-score peptides were selected for further synthesis. Remarkable correlation was found between (-)docking scores and MAO-A inhibitory activity of the synthesized peptides, and among which VVFEVFW showed the highest MAO-A inhibitory activity (IC50 = 0.405 mM). Current research suggested that in silico is an effective method to screen MAO-A inhibitory peptides from hairtail protein hydrolysates, and these peptides can be used as functional ingredients for MAO-A inhibition or potential alternatives for antidepressant.

Alfalfa MsCBL4 enhances calcium metabolism but not sodium transport in transgenic tobacco under salt and saline-alkali stress.

KEY MESSAGE: MsCBL4 expression in tobacco enhanced its salt and saline-alkali stress tolerance by regulating calcium accumulation in roots, indicating the important role of calcium metabolism in plant saline-alkali stress tolerance The calcineurin B-like (CBL) family of proteins play important roles in plant abiotic stress tolerance and signal transduction. CBL4 is known to participate in the Salt Overly Sensitive pathway; however, little is currently known regarding the mechanisms underlying the response of CBL4 to saline-alkali stress. In this study, we cloned and characterized the alfalfa MsCBL4 gene. We found that MsCBL4 showed the highest expression in root tissues and was induced by salt and saline-alkali stress, with the latter causing higher induction. Overexpression of MsCBL4 in tobacco enhanced salt and saline-alkali stress tolerance and reduced the Na+/K+ ratio in roots of transgenic lines. Salt (30 and 300 mM NaCl) and saline-alkali (30 mM NaHCO3) stress assays performed for MsCBL4 transgenic tobacco lines revealed a substantial influx of sodium ions in roots under saline-alkali stress and indicated that the expression of MsCBL4 had little influence on sodium ion content reduction. In contrast, in roots subjected to saline-alkali stress, calcium accumulation occurred and was significantly enhanced by the overexpression of MsCBL4. Physiological and biochemical analyses indicated that MsCBL4 plays an important role in saline-alkali stress tolerance via its influence on the regulation of calcium transport and accumulation. These results provide novel insights into the saline-alkali stress tolerance mechanisms of plants.