Additive-Free Anode with High Stability: Nb2CTx MXene Prepared by HCl-LiF Hydrothermal Etching for Lithium-Ion Batteries.

MXenes, represented by Ti3C2Tx, have been widely studied in the electrochemical energy storage fields, including lithium-ion batteries, for their unique two-dimensional structure, tunable surface chemistry, and excellent electrical conductivity. Recently, Nb2CTx, as a new type of MXene, has attracted more and more attention due to its high theoretical specific capacity of 542 mAh g-1. However, the preparation of few-layer Nb2CTx nanosheets with high-quality remains a challenge, which limits their research and application. In this work, high-quality few-layer Nb2CTx nanosheets with a large lateral size and a high conductivity of up to 500 S cm-1 were prepared by a simple HCl-LiF hydrothermal etching method, which is 2 orders of magnitude higher than that of previously reported Nb2CTx. Furthermore, from its aqueous ink, the viscosity-tunable organic few-layer Nb2CTx ink was prepared by HCl-induced flocculation and N-methyl-2-pyrrolidone treatment. When using the organic few-layer Nb2CTx ink as an additive-free anode of lithium-ion batteries, it showed excellent cycling performance with a reversible specific capacity of 524.0 mAh g-1 after 500 cycles at 0.5 A g-1 and 444.0 mAh g-1 after 5000 cycles at 1 A g-1. For rate performance, a specific capacity of 159.8 mAh g-1 was obtained at a high current density of 5 A g-1, and an excellent capacity retention rate of about 95.65% was achieved when the current density returned to 0.5 A g-1. This work presents a simple and scalable process for the preparation of high-quality Nb2CTx and its aqueous/organic ink, which demonstrates important application potential as electrodes for electrochemical energy storage devices.

Magnesium oxide nanoparticles reduce clubroot by regulating plant defense response and rhizosphere microbial community of tumorous stem mustard (Brassica juncea var. tumida).

Clubroot, caused by Plasmodiophora brassicae, is a major disease that significantly impairs the yield of cruciferous crops and causes significant economic losses across the globe. The prevention of clubroot, especially in tumorous stem mustard (without resistant varieties), are is limited and primarily relies on fungicides. Engineered nanoparticles have opened up new avenues for the management of plant diseases, but there is no report on their application in the prevention of clubroot. The results showed that the control efficacy of 500 mg/L MgO NPs against clubroot was 54.92%. However, when the concentration was increased to 1,500 and 2,500 mg/L, there was no significant change in the control effect. Compared with CK, the average fresh and dry weight of the aerial part of plants treated with MgO NPs increased by 392.83 and 240.81%, respectively. Compared with the F1000 treatment, increases were observed in the content of soil available phosphorus (+16.72%), potassium (+9.82%), exchangeable magnesium (+24.20%), and water-soluble magnesium (+20.64%) in the 1,500 mg/L MgO NPs treatment. The enzyme-linked immune sorbent assay (ELISA) results showed that the application of MgO NPs significantly increased soil peroxidase (POD, +52.69%), alkaline protease (AP, +41.21%), alkaline phosphatase (ALP, +79.26%), urease (+52.69%), and sucrase (+56.88%) activities; And also increased plant L-phenylalanine ammonla-lyase (PAL, +70.49%), polyphenol oxidase (PPO, +36.77%), POD (+38.30%), guaiacol peroxidase (POX, +55.46%) activities and salicylic acid (SA, +59.86%) content. However, soil and plant catalase (CAT, -27.22 and - 19.89%, respectively), and plant super oxidase dismutase (SOD, -36.33%) activities were significantly decreased after the application of MgO NPs. The metagenomic sequencing analysis showed that the MgO NPs treatments significantly improved the α-diversity of the rhizosphere soil microbial community. The relative abundance of beneficial bacteria genera in the rhizosphere soil, including Pseudomonas, Sphingopyxis, Acidovorax, Variovorax, and Bosea, was significantly increased. Soil metabolic functions, such as oxidative phosphorylation (ko00190), carbon fixation pathways in prokaryotes (ko00720), indole alkaloid biosynthesis (ko00901), and biosynthesis of various antibiotics (ko00998) were significantly enriched. These results suggested that MgO NPs might control clubroot by promoting the transformation and utilization of soil nutrients, stimulating plant defense responses, and enriching soil beneficial bacteria.

Genetics and metabolic responses of Artemisia annua L to the lake of phosphorus under the sparingly soluble phosphorus fertilizer: evidence from transcriptomics analysis.

The medicinal herb Artemisia annua L. is prized for its capacity to generate artemisinin, which is used to cure malaria. Potentially influencing the biomass and secondary metabolite synthesis of A. annua is plant nutrition, particularly phosphorus (P). However, most soil P exist as insoluble inorganic and organic phosphates, which results to low P availability limiting plant growth and development. Although plants have developed several adaptation strategies to low P levels, genetics and metabolic responses to P status remain largely unknown. In a controlled greenhouse experiment, the sparingly soluble P form, hydroxyapatite (Ca5OH(PO4)3/CaP) was used to simulate calcareous soils with low P availability. In contrast, the soluble P form KH2PO4/KP was used as a control. A. annua's morphological traits, growth, and artemisinin concentration were determined, and RNA sequencing was used to identify the differentially expressed genes (DEGs) under two different P forms. Total biomass, plant height, leaf number, and stem diameter, as well as leaf area, decreased by 64.83%, 27.49%, 30.47%, 38.70%, and 54.64% in CaP compared to KP; however, LC-MS tests showed an outstanding 37.97% rise in artemisinin content per unit biomass in CaP contrary to KP. Transcriptome analysis showed 2015 DEGs (1084 up-regulated and 931 down-regulated) between two P forms, including 39 transcription factor (TF) families. Further analysis showed that DEGs were mainly enriched in carbohydrate metabolism, secondary metabolites biosynthesis, enzyme catalytic activity, signal transduction, and so on, such as tricarboxylic acid (TCA) cycle, glycolysis, starch and sucrose metabolism, flavonoid biosynthesis, P metabolism, and plant hormone signal transduction. Meanwhile, several artemisinin biosynthesis genes were up-regulated, including DXS, GPPS, GGPS, MVD, and ALDH, potentially increasing artemisinin accumulation. Furthermore, 21 TF families, including WRKY, MYB, bHLH, and ERF, were up-regulated in reaction to CaP, confirming their importance in P absorption, internal P cycling, and artemisinin biosynthesis regulation. Our results will enable us to comprehend how low P availability impacts the parallel transcriptional control of plant development, growth, and artemisinin production in A. annua. This study could lay the groundwork for future research into the molecular mechanisms underlying A. annua's low P adaptation.

Evaluation of Reference Genes for Normalizing RT-qPCR and Analysis of the Expression Patterns of WRKY1 Transcription Factor and Rhynchophylline Biosynthesis-Related Genes in Uncaria rhynchophylla.

Uncaria rhynchophylla (Miq.) Miq. ex Havil, a traditional medicinal herb, is enriched with several pharmacologically active terpenoid indole alkaloids (TIAs). At present, no method has been reported that can comprehensively select and evaluate the appropriate reference genes for gene expression analysis, especially the transcription factors and key enzyme genes involved in the biosynthesis pathway of TIAs in U. rhynchophylla. Reverse transcription quantitative PCR (RT-qPCR) is currently the most common method for detecting gene expression levels due to its high sensitivity, specificity, reproducibility, and ease of use. However, this methodology is dependent on selecting an optimal reference gene to accurately normalize the RT-qPCR results. Ten candidate reference genes, which are homologues of genes used in other plant species and are common reference genes, were used to evaluate the expression stability under three stress-related experimental treatments (methyl jasmonate, ethylene, and low temperature) using multiple stability analysis methodologies. The results showed that, among the candidate reference genes, S-adenosylmethionine decarboxylase (SAM) exhibited a higher expression stability under the experimental conditions tested. Using SAM as a reference gene, the expression profiles of 14 genes for key TIA enzymes and a WRKY1 transcription factor were examined under three experimental stress treatments that affect the accumulation of TIAs in U. rhynchophylla. The expression pattern of WRKY1 was similar to that of tryptophan decarboxylase (TDC) under ETH treatment. This research is the first to report the stability of reference genes in U. rhynchophylla and provides an important foundation for future gene expression analyses in U. rhynchophylla. The RT-qPCR results indicate that the expression of WRKY1 is similar to that of TDC under ETH treatment. It may coordinate the expression of TDC, providing a possible method to enhance alkaloid production in the future through synthetic biology.

Feasibility and Clinical Outcome Predictors of Mechanical Thrombectomy in Distal Arterial Occlusion Causing Acute Ischemic Stroke: A Monocentric Retrospective Study.

BACKGROUND: The feasibility and clinical outcome predictors of mechanical thrombectomy (MT) for strokes caused by distal arterial occlusion (DAO) remain the subject of debate. METHODS: A retrospective analysis was conducted of patients with consecutive acute ischemic stroke treated using MT. Clinical and procedural-associated factors were studied to compare the efficacy, safety, and short-term and long-term outcomes of MT between the proximal arterial occlusion (PAO) and DAO groups. The predictors of a good functional outcome in the DAO group were also identified. RESULTS: A total of 116 patients were included in this study, of whom 23 (19.8%) underwent MT for DAO. A higher complete recanalization rate was independently associated with PAO in adjusted models [adjusted odds ratio, 0.596; 95% CI, 0.377-0.941]. The measures of safety and clinical outcome showed no significant differences between the DAO and PAO groups. The National Institute of Health stroke scale (NIHSS) score on admission, hybrid technique use, and complete recanalization rate emerged as independent predictors of a good functional outcome in the DAO group. CONCLUSIONS: The efficacy, safety, and short-term and long-term outcomes of DAO thrombectomy were similar to those of PAO thrombectomy. The good functional outcome predictors of MT in DAO included NIHSS on admission, hybrid technique use, and complete recanalization. Overall, the findings lead us to propose that MT may be considered a feasible option for treating DAO after a careful risk-benefit analysis.

In situconstruction of N-doped Ti3C2Txconfined worm-like Fe2O3nanoparticles by Fe-O-Ti bonding for LIBs anode with superior cycle performance.

The development of Fe2O3as lithium-ion batteries (LIBs) anode is greatly restricted by its poor electronic conductivity and structural stability. To solve these issues, this work presentsin situconstruction of three-dimensional crumpled Fe2O3@N-Ti3C2Txcomposite by solvothermal-freeze-drying process, in which wormlike Fe2O3nanoparticles (10-50 nm)in situnucleated and grew on the surface of N-doped Ti3C2Txnanosheets with Fe-O-Ti bonding. As a conductive matrix, N-doping endows Ti3C2Txwith more active sites and higher electron transfer efficiency. Meanwhile, Fe-O-Ti bonding enhances the stability of the Fe2O3/N-Ti3C2Txinterface and also acts as a pathway for electron transmission. With a large specific surface area (114.72 m2g-1), the three-dimensional crumpled structure of Fe2O3@N-Ti3C2Txfacilitates the charge diffusion kinetics and enables easier exposure of the active sites. Consequently, Fe2O3@N-Ti3C2Txcomposite exhibits outstanding electrochemical performance as anode for LIBs, a reversible capacity of 870.2 mAh g-1after 500 cycles at 0.5 A g-1, 1129 mAh g-1after 280 cycles at 0.2 A g-1and 777.6 mAh g-1after 330 cycles at 1 A g-1.

Selection of Reference Genes in Evodia rutaecarpa var. officinalis and Expression Patterns of Genes Involved in Its Limonin Biosynthesis.

E. rutaecarpa var. officinalis is a traditional Chinese medicinal plant known for its therapeutic effects, which encompass the promotion of digestion, the dispelling of cold, the alleviation of pain, and the exhibition of anti-inflammatory and antibacterial properties. The principal active component of this plant, limonin, is a potent triterpene compound with notable pharmacological activities. Despite its significance, the complete biosynthesis pathway of limonin in E. rutaecarpa var. officinalis remains incompletely understood, and the underlying molecular mechanisms remain unexplored. The main purpose of this study was to screen the reference genes suitable for expression analysis in E. rutaecarpa var. officinalis, calculate the expression patterns of the genes in the limonin biosynthesis pathway, and identify the relevant enzyme genes related to limonin biosynthesis. The reference genes play a pivotal role in establishing reliable reference standards for normalizing the gene expression data, thereby ensuring precision and credibility in the biological research outcomes. In order to identify the optimal reference genes and gene expression patterns across the diverse tissues (e.g., roots, stems, leaves, and flower buds) and developmental stages (i.e., 17 July, 24 August, 1 September, and 24 October) of E. rutaecarpa var. officinalis, LC-MS was used to analyze the limonin contents in distinct tissue samples and developmental stages, and qRT-PCR technology was employed to investigate the expression patterns of the ten reference genes and eighteen genes involved in limonin biosynthesis. Utilizing a comprehensive analysis that integrated three software tools (GeNorm ver. 3.5, NormFinder ver. 0.953 and BestKeeper ver. 1.0) and Delta Ct method alongside the RefFinder website, the best reference genes were selected. Through the research, we determined that Act1 and UBQ served as the preferred reference genes for normalizing gene expression during various fruit developmental stages, while Act1 and His3 were optimal for different tissues. Using Act1 and UBQ as the reference genes, and based on the different fruit developmental stages, qRT-PCR analysis was performed on the pathway genes selected from the "full-length transcriptome + expression profile + metabolome" data in the limonin biosynthesis pathway of E. rutaecarpa var. officinalis. The findings indicated that there were consistent expression patterns of HMGCR, SQE, and CYP450 with fluctuations in the limonin contents, suggesting their potential involvement in the limonin biosynthesis of E. rutaecarpa var. officinalis. This study lays the foundation for further research on the metabolic pathway of limonin in E. rutaecarpa var. officinalis and provides reliable reference genes for other researchers to use for conducting expression analyses.

Differences and biocontrol potential of haustorial endophytic fungi from Taxillus Chinensis on different host plants.

BACKGROUND: To explore the community composition and diversity of the endophytic fungi in Taxillus chinensis, samples of the parasites growing on seven different hosts, Morus alba, Prunus salicina, Phellodendron chinense, Bauhinia purpurea, Dalbergia odorifera, Diospyros kaki and Dimocarpus longan, were isolated. The strains were identified by their morphological characteristics and their internal transcribed spacer (ITS) sequences. RESULTS: 150 different endophytic fungi were isolated from the haustorial roots of the seven hosts with a total isolation rate of 61.24%. These endophytic fungi were found to belong to 1 phylum, 2 classes, 7 orders, 9 families, 11 genera and 8 species. Among of them, Pestalotiopsis, Neopestalotiopsis and Diaporthe were the dominant genera, accounting for 26.67, 17.33 and 31.33% of the total number of strains, respectively. Diversity and similarity analyses showed that the endophytic fungi isolated from D. longan (H'=1.60) had the highest diversity index. The highest richness indexes were found in M. alba and D. odorifera (both 2.23). The evenness index of D. longan was the highest (0.82). The similarity coefficient of D. odorifera was the most similar to D. longan and M. alba (33.33%), while the similarity coefficient of P. chinense was the lowest (7.69%) with M. alba and D. odorifera. Nine strains showed antimicrobial activities. Among them, Pestalotiopsis sp., N. parvum and H. investiens showed significant antifungal activity against three fungal phytopathogens of medicinal plants. At the same time, the crude extracts from the metabolites of the three endophytic fungi had strong inhibitory effects on the three pathogens. Pestalotiopsis sp., N. parvum and H. investiens had the strongest inhibitory effects of S. cucurbitacearum, with inhibitory rates of 100%, 100% and 81.51%, respectively. In addition, N. parvum had a strong inhibitory effect on D. glomerata and C. cassicola, with inhibitory rates of 82.35% and 72.80%, respectively. CONCLUSIONS: These results indicate that the species composition and diversity of endophytic fungi in the branches of T. chinensis were varied in the different hosts and showed good antimicrobial potential in the control of plant pathogens.

RNA sequencing in Artemisia annua L explored the genetic and metabolic responses to hardly soluble aluminum phosphate treatment.

Artemisia annua L. is a medicinal plant valued for its ability to produce artemisinin, a molecule used to treat malaria. Plant nutrients, especially phosphorus (P), can potentially influence plant biomass and secondary metabolite production. Our work aimed to explore the genetic and metabolic response of A. annua to hardly soluble aluminum phosphate (AlPO4, AlP), using soluble monopotassium phosphate (KH2PO4, KP) as a control. Liquid chromatography-mass spectrometry (LC-MS) was used to analyze artemisinin. RNA sequencing, gene ontology (GO), and the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were applied to analyze the differentially expressed genes (DEGs) under poor P conditions. Results showed a significant reduction in plant growth parameters, such as plant height, stem diameter, number of leaves, leaf areas, and total biomass of A. annua. Conversely, LC-MS analysis revealed a significant increase in artemisinin concentration under the AlP compared to the KP. Transcriptome analysis revealed 762 differentially expressed genes (DEGs) between the AlP and the KP. GH3, SAUR, CRE1, and PYL, all involved in plant hormone signal transduction, showed differential expression. Furthermore, despite the downregulation of HMGR in the artemisinin biosynthesis pathway, the majority of genes (ACAT, FPS, CYP71AV1, and ALDH1) were upregulated, resulting in increased artemisinin accumulation in the AlP. In addition, 12 transcription factors, including GATA and MYB, were upregulated in response to AlP, confirming their importance in regulating artemisinin biosynthesis. Overall, our findings could contribute to a better understanding the parallel transcriptional regulation of plant hormone transduction and artemisinin biosynthesis in A. annua L. in response to hardly soluble phosphorus fertilizer.

Optimization of Isolation and Transformation of Protoplasts from Uncaria rhynchophylla and Its Application to Transient Gene Expression Analysis.

Protoplast-based engineering has become an important tool for basic plant molecular biology research and developing genome-edited crops. Uncaria rhynchophylla is a traditional Chinese medicinal plant with a variety of pharmaceutically important indole alkaloids. In this study, an optimized protocol for U. rhynchophylla protoplast isolation, purification, and transient gene expression was developed. The best protoplast separation protocol was found to be 0.8 M D-mannitol, 1.25% Cellulase R-10, and 0.6% Macerozyme R-10 enzymolysis for 5 h at 26 °C in the dark with constant oscillation at 40 rpm/min. The protoplast yield was as high as 1.5 × 107 protoplasts/g fresh weight, and the survival rate of protoplasts was greater than 90%. Furthermore, polyethylene glycol (PEG)-mediated transient transformation of U. rhynchophylla protoplasts was investigated by optimizing different crucial factors affecting transfection efficiency, including plasmid DNA amount, PEG concentration, and transfection duration. The U. rhynchophylla protoplast transfection rate was highest (71%) when protoplasts were transfected overnight at 24 °C with the 40 µg of plasmid DNA for 40 min in a solution containing 40% PEG. This highly efficient protoplast-based transient expression system was used for subcellular localization of transcription factor UrWRKY37. Finally, a dual-luciferase assay was used to detect a transcription factor promoter interaction by co-expressing UrWRKY37 with a UrTDC-promoter reporter plasmid. Taken together, our optimized protocols provide a foundation for future molecular studies of gene function and expression in U. rhynchophylla.

Integrated metabolome and transcriptome analysis identifies candidate genes involved in triterpenoid saponin biosynthesis in leaves of Centella asiatica (L.) Urban.

Centella asiatica (L.) Urban is a well-known medicinal plant which has multiple pharmacological properties. Notably, the leaves of C. asiatica contain large amounts of triterpenoid saponins. However, there have only been a few studies systematically elucidating the metabolic dynamics and transcriptional differences regarding triterpenoid saponin biosynthesis during the leaf development stages of C. asiatica. Here, we performed a comprehensive analysis of the metabolome and transcriptome to reveal the dynamic patterns of triterpenoid saponin accumulation and identified the key candidate genes associated with their biosynthesis in C. asiatica leaves. In this study, we found that the key precursors in the synthesis of terpenoids, including DMAPP, IPP and ß-amyrin, as well as 22 triterpenes and eight triterpenoid saponins were considered as differentially accumulated metabolites. The concentrations of DMAPP, IPP and ß-amyrin showed significant increases during the entire stage of leaf development. The levels of 12 triterpenes decreased only during the later stages of leaf development, but five triterpenoid saponins rapidly accumulated at the early stages, and later decreased to a constant level. Furthermore, 48 genes involved in the MVA, MEP and 2, 3-oxidosqualene biosynthetic pathways were selected following gene annotation. Then, 17 CYP450s and 26 UGTs, which are respectively responsible for backbone modifications, were used for phylogenetic-tree construction and time-specific expression analysis. From these data, by integrating metabolomics and transcriptomics analyses, we identified CaHDR1 and CaIDI2 as the candidate genes associated with DMAPP and IPP synthesis, respectively, and CaßAS1 as the one regulating ß-amyrin synthesis. Two genes from the CYP716 family were confirmed as CaCYP716A83 and CaCYP716C11. We also selected two UGT73 families as candidate genes, associated with glycosylation of the terpenoid backbone at C-3 in C. asiatica. These findings will pave the way for further research on the molecular mechanisms associated with triterpenoid saponin biosynthesis in C. asiatica.

Comparative Proteomic Analysis Provides New Insights into the Development of Haustorium in Taxillus chinensis (DC.) Danser.

Taxillus chinensis is an important medicinal and parasitic plant that attacks other plants for living. The development of haustorium is a critical process, imperative for successful parasitic invasion. To reveal the mechanisms underlying haustorium development, we performed an iTRAQ-based proteomics analysis which led to the identification of several differentially abundant proteins (DAPs) in fresh seeds (CK), baby (FB), and adult haustoria (FD). A total of 563 and 785 DAPs were identified and quantified in the early and later developmental stages, respectively. Pathway enrichment analysis revealed that the DAPs are mainly associated with metabolic pathways, ribosome, phenylpropanoid biosynthesis, and photosynthesis. In addition, DAPs associated with the phytohormone signaling pathway changed markedly. Furthermore, we evaluated the content of various phytohormones during different stages of haustoria development. These results indicated that phytohormones are very important for haustorium development. qRT-PCR results validated that the mRNA expression levels were consistent with the expression of proteins, suggesting that our results are reliable. This is the first report on haustoria proteomes in the parasitic plant, Taxillus chinensis, to the best of our knowledge. Our findings will enhance our understanding of the molecular mechanism of haustoria development.

A De Novo Transcriptome Analysis Identifies Cold-Responsive Genes in the Seeds of Taxillus chinensis (DC.) Danser.

Taxillus chinensis (DC.) Danser, a parasitic plant of the Loranthaceae family, grows by attacking other plants. It has a long history of being used in Chinese medicine to treat multiple chronic diseases. We previously observed that T. chinensis seeds are sensitive to cold. In this study, we performed transcriptome sequencing for T. chinensis seeds treated by cold (0°C) for 0 h, 12 h, 24 h, and 36 h. TRINITY assembled 257,870 transcripts from 223,512 genes. The GC content and N50 were calculated as 42.29% and 1,368, respectively. Then, we identified 42,183 CDSs and 35,268 likely proteins in the assembled transcriptome, which contained 1,622 signal peptides and 6,795 transmembrane domains. Next, we identified 17,217 genes (FPKM > 5) and 2,333 differentially expressed genes (DEGs) in T. chinensis seeds under cold stress. The MAPK pathway, as an early cold response, was significantly enriched by the DEGs in the T. chinensis seeds after 24 h of cold treatment. Known cold-responsive genes encoding abscisic acid-associated, aquaporin, C-repeat binding factor (CBF), cold-regulated protein, heat shock protein, protein kinase, ribosomal protein, transcription factor (TF), zinc finger protein, and ubiquitin were deregulated in the T. chinensis seeds under cold stress. Notably, the upregulation of CBF gene might be the consequences of the downregulation of MYB and GATA TFs. Additionally, we identified that genes encoding CDC20, YLS9, EXORDIUM, and AUX1 and wound-responsive family protein might be related to novel mechanisms of T. chinensis seeds exposed to cold. This study is first to report the differential transcriptional induction in T. chinensis seeds under cold stress. It will improve our understanding of parasitic plants in response to cold and provide a valuable resource for future studies.

Effects of endophytic fungi on parasitic process of Taxillus chinensis.

Taxillus chinensis (DC.) Danser is an extensively used medicinal shrub in the traditional as well as modern systems of medicines. It is a perennial hemiparasitic plant, which is difficult to propagate artificially because of its low parasitic rate. Successful parasitism of parasitic plants is to fuse their tissues and connect their vasculature to the host vasculature building a physiological bridge, which can efficiently withdraw water, sugars and nutrients from their host plants. It is reported that endophytic fungi play an important role in cell wall degradation and fusion, which is the key forming process of the physiological bridge. Therefore, in this study, the endophytic fungi from T. chinensis of different hosts were isolated, and then the organisms that could degrade the main components of the cell walls were screened out using a medium consisting of guaihuol and cellulose degradation capacity. The results showed that five strains were screened out from 72 endophytic fungi of T. chinensis which with high enzyme activities for lignocellulosic degradation. The laccase and cellulase activities of five strains reached their peaks at day 7, and the highest enzyme activities of these two enzymes were found in strain P6, which was 117.66 and 1.66 U/mL, respectively. Manganese peroxidase of strain 4 and lignin peroxidase of strain N6 also reached their peaks at day 7 and were the highest among the 5 strains, with enzyme activities of 11.61 and 6.64 U/mL, respectively. Strains 4, 15, 31, N6 and P6 were identified as Colletotrichum sp., Nigerrospora sphaerica, Exserohilum sp., Diaporthe phaseolorum and Pestalotiopsis sp., respectively, according to their morphological and molecular biology properties. The endophytic fungi may secrete efficient cell wall degradation enzymes, which promote the dissolution and relaxation of the cell wall between T. chinensis and host, thus contributing to the parasitism of T. chinensis.

Chromosome-Level Genome Assembly of the Hemiparasitic Taxillus chinensis (DC.) Danser.

The hemiparasitic Taxillus chinensis (DC.) Danser is a root-parasitizing medicinal plant with photosynthetic ability, which is lost in other parasitic plants. However, the cultivation and medical application of the species are limited by the recalcitrant seeds of the species, and even though the molecular mechanisms underlying this recalcitrance have been investigated using transcriptomic and proteomic methods, genome resources for T. chinensis have yet to be reported. Accordingly, the aim of the present study was to use nanopore, short-read, and high-throughput chromosome conformation capture sequencing to construct a chromosome-level assembly of the T. chinensis genome. The final genome assembly was 521.90 Mb in length, and 496.43 Mb (95.12%) could be grouped into nine chromosomes with contig and scaffold N50 values of 3.80 and 56.90 Mb, respectively. In addition, a total of 33,894 protein-coding genes were predicted, and gene family clustering identified 11 photosystem-related gene families, thereby indicating photosynthetic ability, which is a characteristic of hemiparasitic plants. This chromosome-level genome assembly of T. chinensis provides a valuable genomic resource for elucidating the genetic basis underlying the recalcitrant characteristics of T. chinensis seeds and the evolution of photosynthesis loss in parasitic plants.

Identification of MicroRNAs in Taxillus chinensis (DC.) Danser Seeds under Cold Stress.

Taxillus chinensis (DC.) Danser, a parasitic plant that belongs to the Loranthaceae family, has a long history of being used in the Chinese medicine. We observed that the loranthus seeds were sensitive to temperature and could lose viability below 0°C quickly. Thus, we performed small RNA sequencing to study the microRNA (miRNA) regulation in the loranthus seeds under cold stress. In total, we identified 600 miRNAs, for the first time, in the loranthus seeds under cold stress. Then, we detected 224, 229, and 223 miRNAs (TPM > 1) in A0 (control), A1 (cold treatment for 12 h at 0°C), and A2 (cold treatment for 36 h at 0°C), respectively. We next identified 103 differentially expressed miRNAs (DEmiRs) in the loranthus seeds in response to cold. Notably, miR408 was upregulated during the cold treatment, which can regulate genes encoding phytocyanin family proteins and phytophenol oxidases. Some DEmiRs were specific to A1 and may function in early response to cold, such as gma-miR393b-3p, miR946, ath-miR779.2-3p, miR398, and miR9662. It is interesting that ICE3, IAA13, and multiple transcription factors (e.g., WRKY and CRF4 and TCP4) regulated by the DEmiRs have been reported to respond cold in other plants. We further identified 4, 3, and 4 DEmiRs involved in the pathways "responding to cold," "responding to abiotic stimulus," and "seed development/germination," respectively. qRT-PCR was used to confirm the expression changes of DEmiRs and their targets in the loranthus seeds during the cold treatment. This is the first time to study cold-responsive miRNAs in loranthus, and our findings provide a valuable resource for future studies.

Physiological change alters endophytic bacterial community in clubroot of tumorous stem mustard infected by Plasmodiophora brassicae.

BACKGROUND: Endophytic bacteria are considered as symbionts living within plants and are influenced by abiotic and biotic environments. Pathogen cause biotic stress, which may change physiology of plants and may affect the endophytic bacterial communiy. Here, we reveal how endophytic bacteria in tumorous stem mustard (Brassica juncea var. tumida) are affected by plant physiological changes caused by Plasmodiophora brassicae using 16S rRNA high-throughput sequencing. RESULTS: The results showed that Proteobacteria was the dominant group in both healthy roots and clubroots, but their abundance differed. At the genus level, Pseudomonas was dominant in clubroots, whereas Rhodanobacter was the dominant in healthy roots. Hierarchical clustering, UniFrac-weighted principal component analysis (PCA), non-metric multidimensional scaling (NMDS) and analysis of similarities (ANOSIM) indicated significant differences between the endophytic bacterial communities in healthy roots and clubroots. The physiological properties including soluble sugar, soluble protein, methanol, peroxidase (POD) and superoxide dismutase (SOD) significantly differed between healthy roots and clubroots. The distance-based redundancy analysis (db-RDA) and two-factor correlation network showed that soluble sugar, soluble protein and methanol were strongly related to the endophytic bacterial community in clubroots, whereas POD and SOD correlated with the endophytic bacterial community in healthy roots. CONCLUSIONS: Our results illustrate that physiologcial changes caused by P. brassicae infection may alter the endophytic bacterial community in clubroots of tumorous stem mustard.

Isolation, Screening, and Identification of Actinomycetes with Antifungal and Enzyme Activity Assays against Colletotrichum dematium of Sarcandra glabra.

A serious leaf disease caused by Colletotrichum dematium was found during the cultivation of Sarcandra glabra in Jingxi, Rong'an, and Donglan Counties in Guangxi Province, which inflicted huge losses to plant productivity. Biological control gradually became an effective control method for plant pathogens. Many studies showed that the application of actinomycetes in biological control has been effective. Therefore, it may be of great significance to study the application of actinomycetes on controlling the diseases caused by S. glabra. Strains of antifungal actinomycetes capable of inhibiting C. dematium were identified, isolated and screened from healthy plants tissues and the rhizospheres in soils containing S. glabra. In this study, 15 actinomycetes strains were isolated and among these, strains JT-2F, DT-3F, and JJ-3F, appeared to show antagonistic effects against anthracnose of S. glabra. The strains JT-2F and DT-3F were isolated from soil, while JJ-3F was isolated from plant stems. The antagonism rate of strain JT-2F was 86.75%, which was the highest value among the three strains. Additionally, the JT-2F strain also had the strongest antagonistic activity when the antagonistic activities were tested against seven plant pathogens. Strain JT-2F is able to produce proteases and cellulase to degrade the protein and cellulose components of cell walls of C. dematium, respectively. This results in mycelia damage which leads to inhibition of the growth of C. dematium. Strain JT-2F was identified as Streptomyces tsukiyonensis based on morphological traits and 16S rDNA sequence analysis.

De novo Transcriptome Reveals Gene Changes in the Development of the Endosperm Chalazal Haustorium in Taxillus chinensis (DC.) Danser.

Loranthus (Taxillus chinensis) is a facultative, hemiparasite and stem parasitic plant that attacks other plants for living. Transcriptome sequencing and bioinformatics analysis were applied in this study to identify the gene expression profiles of fresh seeds (CK), baby (FB), and adult haustoria tissues (FD). We assembled 160,571 loranthus genes, of which 64,926, 35,417, and 47,249 were aligned to NR, GO, and KEGG pathway databases, respectively. We identified 14,295, 15,921, and 16,402 genes in CK, FB, and FD, respectively. We next identified 5,480 differentially expressed genes (DEGs) in the process, of which 258, 174, 81, and 94 were encoding ribosomal proteins (RP), transcription factors (TF), ubiquitin, and disease resistance proteins, respectively. Some DEGs were identified to be upregulated along with the haustoria development (e.g., 68 RP and 26 ubiquitin genes). Notably, 36 RP DEGs peak at FB; 10 ER, 5 WRKY, 6 bHLH, and 4 MYB TF genes upregulated only in FD. Further, we identified 4 out of 32 microRNA genes dysregulated in the loranthus haustoria development. This is the first haustoria transcriptome of loranthus, and our findings will improve our understanding of the molecular mechanism of haustoria.

Rapid Screening of Forskolin-Type Diterpenoids of Blumea aromatica DC Using Ultra-High-Performance Liquid Chromatography Tandem Quadrupole Time-of-Flight Mass Spectrometry Based on the Mass Defect Filtering Approach.

The discovery of new active compounds of natural products tends to be increasingly more challenging due to chemical complexity and unpredictable matrices. Forskolin is an active natural labdane-type diterpenoid ingredient widely used worldwide for the treatment of glaucoma, heart failure, hypertension, diabetes, and asthma, and is expected to be a promising anticancer, anti-inflammation, and anti-HIV agent. In recent years, demand for forskolin in the medicine market has increased dramatically. However, natural forskolin originates exclusively from traditional Indian herb medicine Coleus forskohlii (Willd.) Briq. In a previous study, we isolated a series of diterpenoids including an 8,13-epoxy-14ene labdane carbon skeleton from Blumea aromatica DC. In order to identify alternative plant resources, a novel and effective strategy was proposed for the screening of potential forskolin-type diterpenoids (FSKD) compounds obtained from B. aromatica, using the mass defect filtering (MDF) strategy via ultra-high-performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry (UHPLC-QTOF/MS) approach. Within a narrow, well-defined mass defect range, the strategy developed could significantly improve the detection efficiency of selected FSKD compounds by filtering out certain major or moderate interference compounds. Additionally, the MS/MS cleavage behavior and the characteristic diagnostic ions of the FSKD compounds were proposed to be used in aiding structural identification of the filtration compounds. As a result, a total of 38 FSKD of B. aromatica were filtered out and tentatively identified. To the best of our knowledge, it was the first time that these forskolin-type diterpenoids were identified in B. aromatica, which significantly expands our understanding of the chemical constituents of Blumea species, and allows B. aromatica to be used as a potential alternative plant resource that contains these forskolin-type active compounds. The strategy proposed was proven efficient and reliable for the discovery of novel compounds of herbal extracts.