BpGRP1 acts downstream of BpmiR396c/BpGRF3 to confer salt tolerance in Betula platyphylla.

Glycine-rich RNA-binding proteins (GRPs) have been implicated in the responses of plants to environmental stresses, but the function of GRP genes involved in salt stress and the underlying mechanism remain unclear. In this study, we identified BpGRP1 (glycine-rich RNA-binding protein), a Betula platyphylla gene that is induced under salt stress. The physiological and molecular responses to salt tolerance were investigated in both BpGRP1-overexpressing and suppressed conditions. BpGRF3 (growth-regulating factor 3) was identified as a regulatory factor upstream of BpGRP1. We demonstrated that overexpression of BpGRF3 significantly increased the salt tolerance of birch, whereas the grf3-1 mutant exhibited the opposite effect. Further analysis revealed that BpGRF3 and its interaction partner, BpSHMT, function upstream of BpGRP1. We demonstrated that BpmiR396c, as an upstream regulator of BpGRF3, could negatively regulate salt tolerance in birch. Furthermore, we uncovered evidence showing that the BpmiR396c/BpGRF3 regulatory module functions in mediating the salt response by regulating the associated physiological pathways. Our results indicate that BpmiR396c regulates the expression of BpGRF3, which plays a role in salt tolerance by targeting BpGRP1.

Targeted trace ingredients coupled with chemometric analysis for consistency evaluation of Panax notoginseng saponins injectable formulations.

Evaluating the consistency of herb injectable formulations could improve their product quality and clinical safety, particularly concerning the composition and content levels of trace ingredients. Panax notoginseng Saponins Injection (PNSI), widely used in China for treating acute cardiovascular diseases, contains low-abundance (10%-25%) and trace saponins in addition to its five main constituents (notoginsenoside R1, ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, and ginsenoside Rd). This study aimed to establish a robust analytical method and assess the variability in trace saponin levels within PNSI from different vendors and formulation types. To achieve this, a liquid chromatography-triple quadrupole mass spectrometry (LC-MS/MS) method employing multiple ions monitoring (MIM) was developed. A "post-column valve switching" strategy was implemented to eliminate highly abundant peaks (NR1, Rg1, and Re) at 26 min. A total of 51 saponins in PNSI were quantified or relatively quantified using 18 saponin standards, with digoxin as the internal standard. This study evaluated 119 batches of PNSI from seven vendors, revealing significant variability in trace saponin levels among different vendors and formulation types. These findings highlight the importance of consistent content in low-abundance and trace saponins to ensure product control and clinical safety. Standardization of these ingredients is crucial for maintaining the quality and effectiveness of PNSI in treating acute cardiovascular diseases.

Mass spectrometry imaging: new eyes on natural products for drug research and development.

Natural products (NPs) and their structural analogs represent a major source of novel drug development for disease prevention and treatment. The development of new drugs from NPs includes two crucial aspects. One is the discovery of NPs from medicinal plants/microorganisms, and the other is the evaluation of the NPs in vivo at various physiological and pathological states. The heterogeneous spatial distribution of NPs in medicinal plants/microorganisms or in vivo can provide valuable information for drug development. However, few molecular imaging technologies can detect thousands of compounds simultaneously on a label-free basis. Over the last two decades, mass spectrometry imaging (MSI) methods have progressively improved and diversified, thereby allowing for the development of various applications of NPs in plants/microorganisms and in vivo NP research. Because MSI allows for the spatial mapping of the production and distribution of numerous molecules in situ without labeling, it provides a visualization tool for NP research. Therefore, we have focused this mini-review on summarizing the applications of MSI technology in discovering NPs from medicinal plants and evaluating NPs in preclinical studies from the perspective of new drug research and development (R&D). Additionally, we briefly reviewed the factors that should be carefully considered to obtain the desired MSI results. Finally, the future development of MSI in new drug R&D is proposed.

Calculating Relative Correction Factors for Quantitative Analysis with HILIC-HPLC-ELSD Method: Eight Fructooligosaccharides of Morinda Officinalis as a Case Study.

Objective: Because the response of evaporating light scattering detector (ELSD) being in a nonlinear mode, there is no consensus on the method of calculating its relative correction factors (RCF), which limits the application of the quantitative analysis for multi-components by a single marker (QAMS) with LC-ELSD. Methods: Using eight fructooligosaccharides of Morinda officinalis as a case study, the nystose (GF3) as a single standard was adopted to develop a QAMS method to simultaneously determine the other seven fructooligosaccharides with HILIC-HPLC-ELSD method. Six calculation methods of RCF were investigated to select the most reasonable method. The relative error of content between the QAMS and the external standard method (ESM) obtained from 30 batches of samples was used as an indicator to evaluate the six methods. Finally, a chemometrics analysis was performed to find the differential components among MO and its three processing products. Results: It was first reported that only one calculation method was scientific for calculating RCF for the LC-ELSD method. The RCFs of GF3 to the other seven fructooligosaccharides (GF1-GF8) were obtained as 0.86, 0.91, 0.93, 1.05, 1.15, 1.12, and 1.18, respectively. The QAMS of eight fructooligosaccharides of Morinda officinalis was validated with good linearity (R 2 > 0.9998) and accepted the accuracy of 95-105% (RSD < 1.81%) based on nystose. Finally, Morinda officinalis and its three processed products were distinguished and could be differed based on the content of the eight fructooligosaccharides. Conclusion: The scientific calculation method of RCF would be of great significance for developing the QAMS method in Pharmacopoeia when performing the LC-ELSD method.

Authentication of Shenqi Fuzheng Injection via UPLC-Coupled Ion Mobility-Mass Spectrometry and Chemometrics with Kendrick Mass Defect Filter Data Mining.

Nearly 5% of the Shenqi Fuzheng Injection's dry weight comes from the secondary metabolites of Radix codonopsis and Radix astragali. However, the chemical composition of these metabolites is still vague, which hinders the authentication of Shenqi Fuzheng Injection (SFI). Ultra-high performance liquid chromatography with a charged aerosol detector was used to achieve the profiling of these secondary metabolites in SFI in a single chromatogram. The chemical information in the chromatographic profile was characterized by ion mobility and high-resolution mass spectrometry. Polygonal mass defect filtering (PMDF) combined with Kendrick mass defect filtering (KMDF) was performed to screen potential secondary metabolites. A total of 223 secondary metabolites were characterized from the SFI fingerprints, including 58 flavonoids, 71 saponins, 50 alkaloids, 30 polyene and polycynes, and 14 other compounds. Among them, 106 components, mainly flavonoids and saponins, are contributed by Radix astragali, while 54 components, mainly alkaloids and polyene and polycynes, are contributed by Radix codonopsis, with 33 components coming from both herbs. There were 64 components characterized using the KMDF method, which increased the number of characterized components in SFI by 28.70%. This study provides a solid foundation for the authentification of SFIs and the analysis of its chemical composition.

Transcriptomic Analysis of Mature Transgenic Poplar Expressing the Transcription Factor JERF36 Gene in Two Different Environments.

During the last several decades, a number of transgenic or genetically modified tree varieties with enhanced characteristics and new traits have been produced. These trees have become associated with generally unsubstantiated concerns over health and environmental safety. We conducted transcriptome sequencing of transgenic Populus alba × P. berolinensis expressing the transcription factor JERF36 gene (ABJ01) and the non-transgenic progenitor line (9#) to compare the transcriptional changes in the apical buds. We found that 0.77% and 1.31% of the total expressed genes were significant differentially expressed in ABJ01 at the Daqing and Qiqihar sites, respectively. Among them, 30%-50% of the DEGs contained cis-elements recognized by JERF36. Approximately 5% of the total number of expressed genes showed significant differential expression between Daqing and Qiqihar in both ABJ01 and 9#. 10 DEGs resulting from foreign gene introduction, 394 DEGs that resulted solely from the environmental differences, and 47 DEGs that resulted from the combination of foreign gene introduction and the environment were identified. The number of DEGs resulting from environmental factors was significantly greater than that resulting from foreign gene introduction, and the combined effect of the environmental effects with foreign gene introduction was significantly greater than resulting from the introduction of JERF36 alone. GO and KEGG annotation showed that the DEGs mainly participate in the photosynthesis, oxidative phosphorylation, plant hormone signaling, ribosome, endocytosis, and plant-pathogen interaction pathways, which play important roles in the responses to biotic and abiotic stresses ins plant. To enhance its adaptability to salt-alkali stress, the transgenic poplar line may regulate the expression of genes that participate in the photosynthesis, oxidative phosphorylation, MAPK, and plant hormone signaling pathways. The crosstalk between biotic and abiotic stress responses by plant hormones may improve the ability of both transgenic and non-transgenic poplars to defend against pathogens. The results of our study provide a basis for further studies on the molecular mechanisms behind improved stress resistance and the unexpected effects of transgenic gene expression in poplars, which will be significant for improving the biosafety evaluation of transgenic trees and accelerating the breeding of new varieties of forest trees resistant to environmental stresses.

Information Entropy-Based Strategy for the Quantitative Evaluation of Extensive Hyperspectral Images to Better Unveil Spatial Heterogeneity in Mass Spectrometry Imaging.

Hyperspectral images can be generated from mass spectrometry imaging (MSI) data for the intuitive data visualization purpose. However, hundreds of HSIs can be generated by different dimensionality reduction methods, which poses great challenges in selecting the high-quality images with the best intuitive visualization results of the MSI data. Here, we presented a novel approach that objectively evaluates the image quality of the hyperspectral images. The applicability of this method was demonstrated by analyzing the MSI data acquired from human prostate cancer biopsy samples and mouse brain tissue section, which harbored an intrinsic tissue heterogeneity. Our method was based on the information entropy and contrast measured from image information content and image definition, respectively. The heterogeneity of the MSI data from high-dimensional space was reduced to three-dimensional embeddings and thoroughly evaluated to achieve satisfactory visualization results. The application of information entropy and contrast can be used to choose the optimized visualization results rapidly and objectively from an extensive number of hyperspectral images and be adopted to evaluate and optimize different dimensionality reduction algorithms and their hyperparameter combinations. In conclusion, the information entropy-based strategy could be a bridge between chemometrician and biologists.

Morphological, physiological, and transcriptional responses to low nitrogen stress in Populus deltoides Marsh. clones with contrasting nitrogen use efficiency.

BACKGROUND: Nitrogen (N) is one of the main factors limiting the wood yield in poplar cultivation. Understanding the molecular mechanism of N utilization could play a guiding role in improving the nitrogen use efficiency (NUE) of poplar. RESULTS: In this study, three N-efficient genotypes (A1-A3) and three N-inefficient genotypes (C1-C3) of Populus deltoides were cultured under low N stress (5 µM NH4NO3) and normal N supply (750 µM NH4NO3). The dry matter mass, leaf morphology, and chlorophyll content of both genotypes decreased under N starvation. The low nitrogen adaptation coefficients of the leaves and stems biomass of group A were significantly higher than those of group C (p < 0.05). Interestingly, N starvation induced fine root growth in group A, but not in group C. Next, a detailed time-course analysis of enzyme activities and gene expression in leaves identified 2062 specifically differentially expressed genes (DEGs) in group A and 1118 in group C. Moreover, the sensitivity to N starvation of group A was weak, and DEGs related to hormone signal transduction and stimulus response played an important role in the low N response this group. Weighted gene co-expression network analysis identified genes related to membranes, catalytic activity, enzymatic activity, and response to stresses that might be critical for poplar's adaption to N starvation and these genes participated in the negative regulation of various biological processes. Finally, ten influential hub genes and twelve transcription factors were identified in the response to N starvation. Among them, four hub genes were related to programmed cell death and the defense response, and PodelWRKY18, with high connectivity, was involved in plant signal transduction. The expression of hub genes increased gradually with the extension of low N stress time, and the expression changes in group A were more obvious than those in group C. CONCLUSIONS: Under N starvation, group A showed stronger adaptability and better NUE than group C in terms of morphology and physiology. The discovery of hub genes and transcription factors might provide new information for the analysis of the molecular mechanism of NUE and its improvement in poplar.

Overexpression of ThMYB8 mediates salt stress tolerance by directly activating stress-responsive gene expression.

MYB transcription factors are important in abiotic stress responses; however, the detailed mechanisms are unclear. Tamarix hispida contains multiple MYB genes. The present study characterized T. hispida MYB8 (ThMYB8) during salt stress using transgenic T. hispida and Arabidopsis assays. ThMYB8 overexpression and ThMYB8 RNAi analysis demonstrated that ThMYB8 enhanced the salt stress tolerance. Transgenic Arabidopsis ectopic expression of ThMYB8 significantly increased root growth, fresh weight, and seed germination rate compared with that of the wild-type under salt stress. Physiological parameters analysis in T. hispida and Arabidopsis showed that ThMYB8 overexpressing plants had the lowest levels of O2, H2O2, cell death, malondialdehyde, and electrolyte leakage. Overexpression of ThMYB8 regulated Na+ and K+ concentrations in plant tissues while maintaining K+/Na+ homeostasis. Analysis using qRT-PCR and ChIP-PCR identified possible downstream ThMYB8-regulated genes. ThMYB8 regulated the expression of ThCYP450-2 (cytochrome p450-2), Thltk (leucine-rich repeat transmembrane protein kinase), and ThTIP (aquaporin TIP) by binding to the MBSI motif ('CAACTG') in their promoters. The results indicated that ThMYB8 enhanced salt stress tolerance in T. hispida by regulating gene expression related to the activation of stress-associated physiological changes, such as enhanced reactive oxygen species scavenging capability, maintaining K+/Na+ homeostasis, and decreasing the malondialdehyde content and lipid peroxidation cell membranes.

Comprehensive Analysis of MYB Gene Family and Their Expressions Under Abiotic Stresses and Hormone Treatments in Tamarix hispida.

The MYB transcription factors (TFs) is a plant TF families, which involves in hormone signal transduction, and abiotic stress tolerance, etc. However, there are few studies on the MYB TFs family and its regulatory mechanism in Tamarix hispida. In this study, 14 MYB genes (named ThMYB1 - ThMYB14) were cloned and characterized from T. hispida. The transcription profiles of ThMYBs in T. hispida under different abiotic stress conditions were monitored using qRT-PCR. Most of studied ThMYBs were significantly downregulated and/or upregulated by salt and osmotic stress, ABA, GA3 and JA treatments in at least one organ. Especially, ThMYB13 was induced in the leaves and roots of T. hispida when exposed to NaCl treatment at all study periods, indicating that it may involve in salt stress. To further study ThMYB13 function, ThMYB13 overexpression and knock-down plants and control plants transformed with an empty pROKII were obtained using a transient transformation system. Overexpression of ThMYB13 in T. hispida displayed the lowest O2-, H2O2 and MDA accumulation, minimal cell death, the most stable K+/Na+ ratio and the lowest electrolyte leakage rate among the three kinds of transient expression in T. hispida. Conversely, the RNAi-silencing, transiently transformed plants displayed the opposite physiological changes. Therefore, ThMYB13 might play a role in salt stress tolerance in transgenic T. hispida plants.

Three new cardiac glycosides obtained from the roots of Streblus asper Lour. and their cytotoxic and melanogenesis-inhibitory activities.

Three new cardiac glycosides strophanthidin-3-O-α-l-rhamnopyranosyl-(1→4)-6-deoxy-ß-d-allopyranoside (1), 5ßH-16ß-acetylkamaloside (2), and mansonin-19-carboxylic acid (3) along with seven known steroids including five cardiac glycosides were isolated from the methanol extracts of Streblus asper Lour. roots. The structures of these compounds were established by spectroscopic analyses. The cytotoxicities of crude extracts and all the isolated compounds were evaluated against four human cancer cell lines (HL60, A549, AZ521, and SKBR3). Furthermore, the selective index (SI) of each compound was measured by the ratio of cytotoxic effect on a normal cell line (WI38) to the cytotoxic effect on cancer cell line (A549). The results suggested that cardiac glycosides (2, 4, and 6-8) exhibited significant cytotoxicities with IC50 values from 0.01 to 3.77 µM as well as high selective index for WI38/A549 (SI 1.50-24.26), and they displayed superior selectivities when compared with the reference cisplatin (SI 1.09). Preliminary structure-activity relationships (SARs) were also discussed regarding the type of C-10 group in the cardiac glycosides being a crucial factor in determining the cytotoxic activities and regarding the sugar moieties having much less of an active role than the type of C-10 group. In addition, the melanogenesis-inhibitory abilities of these compounds were also evaluated. Cardiac glycosides (3 and 6-8) displayed moderate inhibition effects on melanogenesis with melanin content (MC) of 26.22-74.90% at a concentration of 100 µM, thus showing high cell viability (CV: 77.94-111.70%) compared with that of the reference arbutin (MC: 82.50% and CV: 107.60%). Furthermore, western blot analysis of melanogenesis-related proteins suggested that 3 could inhibit melanogenesis by suppressing the protein expressions of TRP-2 and tyrosinase.