Identification and Validation of Magnolol Biosynthesis Genes in Magnolia officinalis.

Bacterial infections pose a significant risk to human health. Magnolol, derived from Magnolia officinalis, exhibits potent antibacterial properties. Synthetic biology offers a promising approach to manufacture such natural compounds. However, the plant-based biosynthesis of magnolol remains obscure, and the lack of identification of critical genes hampers its synthetic production. In this study, we have proposed a one-step conversion of magnolol from chavicol using laccase. After leveraging 20 transcriptomes from diverse parts of M. officinalis, transcripts were assembled, enriching genome annotation. Upon integrating this dataset with current genomic information, we could identify 30 laccase enzymes. From two potential gene clusters associated with magnolol production, highly expressed genes were subjected to functional analysis. In vitro experiments confirmed MoLAC14 as a pivotal enzyme in magnolol synthesis. Improvements in the thermal stability of MoLAC14 were achieved through selective mutations, where E345P, G377P, H347F, E346C, and E346F notably enhanced stability. By conducting alanine scanning, the essential residues in MoLAC14 were identified, and the L532A mutation further boosted magnolol production to an unprecedented level of 148.83 mg/L. Our findings not only elucidated the key enzymes for chavicol to magnolol conversion, but also laid the groundwork for synthetic biology-driven magnolol production, thereby providing valuable insights into M. officinalis biology and comparative plant science.

TMT-Based Proteomic Analysis of Continuous Cropping Response in Codonopsis tangshen Oliv.

The growth and development of Codonopsis tangshen, an important herb used in Chinese traditional medicine, have been seriously affected by continuous cropping obstacles. Therefore, understanding the molecular responses of C. tangshen to continuous cropping is imperative to improve its resistance to continuous cropping obstacles. Here, physiological and biochemical results showed that the levels of chlorophyll and malonaldehyde (MDA) were higher in the continuous cropping (LZ) group compared with those of the non-continuous cropping (FLZ) group, while superoxide dismutase (SOD) content was lower in the LZ group than in the FLZ group. Tandem mass tag (TMT)-based proteomic analysis was performed to investigate the response mechanism to continuous cropping obstacles in C. tangshen. A total of 70 differentially expressed proteins (DEPs) were significantly involved in relevant pathways, including photosynthesis, oxidative phosphorylation, ribosome activity, and secondary metabolites. The results suggest that these DEPs in C. tangshen might play a critical role in response to continuous cropping. These findings could provide scientific basis for improving C. tangshen's resistance to continuous cropping obstacles.

Transcriptomic Analysis Provides Novel Insights into the Heat Stress-Induced Response in Codonopsis tangshen.

Codonopsis tangshen Oliv (C. tangshen) is a valuable traditional Chinese medicinal herb with tremendous health benefits. However, the growth and development of C. tangshen are seriously affected by high temperatures. Therefore, understanding the molecular responses of C. tangshen to high-temperature stress is imperative to improve its thermotolerance. Here, RNA-Seq analysis was performed to investigate the genome-wide transcriptional changes in C. tangshen in response to short-term heat stress. Heat stress significantly damages membrane stability and chlorophyll biosynthesis in C. tangshen, as evidenced by pronounced malonaldehyde (MDA), electrolyte leakage (EL), and reduced chlorophyll content. Transcriptome analysis showed that 2691 differentially expressed genes (DEGs) were identified, including 1809 upregulated and 882 downregulated. Functional annotations revealed that the DEGs were mainly related to heat shock proteins (HSPs), ROS-scavenging enzymes, calcium-dependent protein kinases (CDPK), HSP-HSP network, hormone signaling transduction pathway, and transcription factors such as bHLHs, bZIPs, MYBs, WRKYs, and NACs. These heat-responsive candidate genes and TFs could significantly regulate heat stress tolerance in C. tangshen. Overall, this study could provide new insights for understanding the underlying molecular mechanisms of thermotolerance in C. tangshen.

Transcriptome and metabolome analyses reveal novel insights into the seed germination of Michelia chapensis, an endangered species in China.

Michelia chapensis Dandy, a well-known medicinal woody plant endemic to China, is endangered and seriously constricted by seed dormancy-induced low-regeneration in natural conditions. Cold stratification can effectively reduce seed dormancy and promote the seed germination of M. chapensis. However, the molecular events and systematic changes that occurred during seed germination in M. chapensis remain largely unknown. In this study, we carried out transcriptomic and metabolomic analyses to elucidate the potential molecular mechanisms underlying seed germination in M. chapensis under cold stratification. The results showed that the embryo cells became bigger and looser with increasing stratification time. Moreover, the endosperm appeared reduced due to the consumption of nutrients. Seventeen phytohormones were examined by the metabolome targeted for hormones. Compared with the ES (no stratification), the levels of indole-3-acetic acid (IAA) and gibberellin A3 (GA3) were increased in the MS (stratification for 45 days), while the abscisic acid (ABA) was downregulated in both MS and LS (stratification for 90 days). The transcriptome profiling identified 24975 differentially expressed genes (DEGs) in the seeds during germination. The seed germination of M. chapensis was mainly regulated by the biological pathways of plant hormone signal transduction, energy supply, secondary metabolite biosynthesis, photosynthesis-related metabolism, and transcriptional regulation. This study reveals the biological evidence of seed germination at the transcriptional level and provides a foundation for unraveling molecular mechanisms regulating the seed germination of M. chapensis.

Transcriptome analysis provides novel insights into the soil amendments induced response in continuously cropped Codonopsis tangshen.

Codonopsis tangshen Oliv (C. tangshen) is an important Chinese traditional medicinal plant with various health benefits. However, the growth of C. tangshen are seriously affected by continuous cropping, which led to the decrease of the yield and quality. A field experiment was conducted to learn the effects of soil amendments on the growth of C. tangshen under continuous cropping condition, and the biological events which occurred at molecular level were investigated. The results indicated that the content of chlorophyll a (Chl a), chlorophyll b (Chl b), and carotenoid (Car) was significantly higher in SCPM (silicon-calcium-potassium-magnesium fertilizer), SCPMA (SCPM combined with azoxystrobin) and SCPMAOM (SCPM combined with azoxystrobin and organic manure) treatments. Moreover, the yield and the levels of alkaloid, polysaccharide, flavone and total protein in the treatments of SCPM, SCPMA and SCPMAOM were significantly higher than those in the control, and these indexes were all highest in the SCPMAOM treatment. RNA-sequencing (RNA-Seq) is an economical and efficient method to obtain genetic information for species with or without available genome data. In this study, RNA-Seq was performed to understand how continuously cropped C. tangshen responded to the soil amendments at the transcriptome level. The number of differentially expressed genes (DEGs) were as follows: CK vs. SCPM (719 up- and 1456 down-), CK vs. SCPMA (1302 up- and 1748 down-), CK vs. SCPMAOM (1274 up- and 1678 down-). The soil amendments affected the growth of C. tangshen mainly by regulating the genes involved in pathways of 'photosynthesis,' 'plant hormone signal transduction,' 'biosynthesis of unsaturated fatty acids,' 'phenylpropanoid biosynthesis,' and 'starch and sucrose metabolism,' etc. qRT-PCR was performed to validate the expressions of 10 target genes such as CP26, PsaF, and POX, etc., which verified the reliability of RNA-Seq results. Overall, this study revealed the roles and underlying mechanisms of the soil amendments in regulating the growth of continuously cropped C. tangshen at transcriptome level. These findings are beneficial for improving the continuous cropping tolerance and may be valuable for future genetic improvement of C. tangshen.

Effects of foliar selenium application on growth and rhizospheric soil micro-ecological environment of Atractylodes macrocephala Koidz.

Atractylodes macrocephala (A. macrocephala), a famous medicinal herb in China, is widely cultivated and consumed in China with various beneficial effects. Numerous studies have shown that selenium (Se) plays an important role in promoting plant growth, although Se has not been considered an essential element for higher plants. The objectives of this research were to determine the effects of foliar Se application (0, 2.5, 5.0, 10.0 and 20.0 mg m-2 Se in sodium selenite, sprayed monthly from May to August) on the growth and rhizospheric soil micro-ecological environment of A. macrocephala, and explore the possible mechanisms underlying plant response to foliar Se application through a field experiment. The results were: The foliar application of 5.0 mg m-2 Se significantly increased the survival rate of A. macrocephala compared to the control. The yield of A. macrocephala was increased when the Se level maintained belowed 10.0 mg m-2 but decreased when Se level reached 20.0 mg m-2. The Se content in the rhizome of A. macrocephala showed a significant positive correlation with the Se level, while the insect attack rate was significantly negatively correlated with the Se level. However, foliar Se application hardly affected the concentration of bioactive compound atractylenolide in the rhizome of A. macrocephala. Notably, the application of foliar Se changed the content of partial soil nutrients, microbial diversity and composition in the rhizosphere soil of A. macrocephala. Bacterial diversity was positively correlated with A. macrocephala growth whereas fungal diversity was negatively correlated, suggesting that microbial diversity in the rhizosphere soils is closely related to plant growth. Moreover, correlation analysis showed that available potassium, Burkholderia and Cupriavidus in rhizospheric soil might be critical factors for promoting the growth of A. macrocephala. Overall, the foliar application of Se at moderate concentration was beneficial for the growth of A. macrocephala, and 5.0-10.0 mg m-2 Se level was the optimum. Our findings revealed novel insights into the response of A. macrocephala to foliar Se application from plant growth, rhizospheric soil nutrient and microbial community composition .

Terpenes isolated from Polyalthia simiarum and their cytotoxic activities.

Two new C31 triterpenes, polysimiaric acid A (1) and B (2) as well as one new clerodane diterpenoid, 16,16-dimethoxy-cleroda-3,13Z-dien-15-oic acid (3), together with six known compounds were isolated from Polyalthia simiarum. Their structures were determined by analysis of 1D and 2D NMR data. Three new compounds were tested for their cytotoxicity against five human tumour cell lines. Compound 3 showed cytotoxic activities against SMMC-7721 with the IC50 value of 22.43 µM.

Transcriptomic analysis of short-term heat stress response in Pinellia ternata provided novel insights into the improved thermotolerance by spermidine and melatonin.

Heat stress has been a major environmental factor limiting the growth and development of Pinellia ternata which is an important Chinese traditional medicine. It has been reported that spermidine (SPD) and melatonin (MLT) play pivotal roles in modulating heat stress response (HSR). However, the roles of SPD and MLT in HSR of P. ternata, and the potential mechanism is still unknown. Here, exogenous SPD and MLT treatments alleviated heat-induced damages in P. ternata, which was supported by the increased chlorophyll content, OJIP curve, and relative water content, and the decreased malondialdehyde and electrolyte leakage. Then, RNA sequencing between CK (control) and Heat (1 h of heat treatment) was conducted to analyze how genes were in response to short-term heat stress in P. ternata. A total of 14,243 (7870 up- and 6373 down-regulated) unigenes were differentially expressed after 1 h of heat treatment. Bioinformatics analysis revealed heat-responsive genes mainly included heat shock proteins (HSPs), ribosomal proteins, ROS-scavenging enzymes, genes involved in calcium signaling, hormone signaling transduction, photosynthesis, pathogen resistance, and transcription factors such as heat stress transcription factors (HSFs), NACs, WRKYs, and bZIPs. Among them, PtABI5, PtNAC042, PtZIP17, PtSOD1, PtHSF30, PtHSFB2b, PtERF095, PtWRKY75, PtGST1, PtHSP23.2, PtHSP70, and PtLHC1 were significantly regulated by SPD or MLT treatment with same or different trends under heat stress condition, indicating that exogenous application of MLT and SPD might enhance heat tolerance in P. ternata through regulating these genes but may with different regulatory patterns. These findings contributed to the identification of potential genes involved in short-term HSR and the improved thermotolerance by MLT and SPD in P. ternata, which provided important clues for improving thermotolerance of P. ternata.

Transcriptome analysis reveals novel insights into the continuous cropping induced response in Codonopsis tangshen, a medicinal herb.

Codonopsis tangshen Oliv. (C. tangshen Oliv.), a famous medicinal herb in China, is seriously affected by continuous cropping (C-cro). The physiological and biochemical results indicated that C-cro significantly affected the malonaldehyde (MDA) and chlorophyll content, as well as activities of catalase (CAT) and superoxide dismutase (SOD) when compared with the non-continuous cropping (NC-cro) group. Transcriptome profiling found 762 differentially expressed genes, including 430 up-regulated and 332 down-regulated genes by C-cro. In addition, pathway enrichment analysis revealed that genes related to 'Tyrosine degradation I', 'Glycogen synthesis' and 'Phenylalanine and tyrosine catabolism' were up-regulated, and genes associated with 'Signal transduction', 'Immune system', etc. were down-regulated by C-cro. The expression of target genes was further validated by Q-PCR. In this study, we demonstrated the effects of C-cro on C. tangshen at the transcriptome level, and found possible C-cro responsive candidate genes. These findings could be further beneficial for improving the continuous cropping tolerance.

Network pharmacology-based research on the active component and mechanism of the antihepatoma effect of Rubia cordifolia L.

Rubia cordifolia L. is widely used in Asia and its antihepatoma effect has been proved by in vitro and in vivo experiments. However, there are few studies on its specific mechanism. In the present study, the network pharmacology method was used to construct the component/target/pathway molecular regulatory network for the antihepatoma effect of Rubia cordifolia L. to explore the effective components of Rubia cordifolia L. and its potential mechanism. The chemical components of Rubia cordifolia L. were identified through literature and databases, and the components were evaluated and screened by drug likeness and pharmacokinetic characteristics (ADMET). The targets of active components were predicted according to the reverse pharmacophore matching model. The hepatic carcinoma-related genes were found in databases, and antihepatoma-related gene targets were selected through comparison. The functions of target genes and related pathways were analyzed and screened using the Database for Annotation, Visualization and Integrated Discovery, and the component/target/pathways network of antihepatoma effect of Rubia cordifolia L. was constructed using Cytoscape software. Finally, 16 active compounds were screened from Rubia cordifolia L., and 39 gene targets, including AKT1, mitogen-activated protein kinase 1, and epidermal growth factor receptor, were involved. Rubia cordifolia L. also affected the hepatitis B, phosphoinositide-3-kinase-protein kinase B, and mitogen-activated protein kinase signaling pathways. Many direct-acting tumor-related signaling pathways and indirect-acting hepatitis pathways inhibit the generation of liver cancer. The present study provided a scientific basis for further elucidating the mechanism of Rubia cordifolia L. against liver cancer.

[Absorption and Distribution of Nitrogen,Phosphorus and Potassium in Atractylodes macrocephala].

Objective: To investigate the absorption and distribution of nitrogen（ N ), phosphorus（ P） and potassium（ K） in Atractylodes macrocephala by field experiment. Methods: The samples of Atractylodes macrocephala were carried out in different growing periods,and the amount of dry matter and the contents of N,P and K in different parts of the plant were determined,respectively. Results: The results showed that one-year-old Atractylodes macrocephala of budding stage and rhizome swelling stage were the key period of rapid nutrient absorption, and the absorbed nutrient element mainly allocated to the aerial part. For the two-year-old Atractylodes macrocephala, flowering stage was the key period of rapid nutrient absorption, and the absorbed nutrient element mainly allocated to the rhizome. The average N,P2O5 and K2O absorption per 100 kg rhizome product in two-year-old Atractylodes macrocephala were 2. 65,0. 91 and 2. 16 kg, respectively. Conclusion: In addition to the appropriate base fertilizer, one-year-old Atractylodes macrocephala cultivation should pay attention to the topdressing during the budding stage and the rhizome swelling stage, while the two-year-old Atractylodes macrocephala cultivation should pay attention to the topdressing during the flowering stage.

Effective protein extraction protocol for proteomics studies of Jerusalem artichoke leaves.

Protein extraction is a crucial step for proteomics studies. To establish an effective protein extraction protocol suitable for two-dimensional electrophoresis (2DE) analysis in Jerusalem artichoke (Helianthus tuberosus L.), three different protein extraction methods-trichloroacetic acid/acetone, Mg/NP-40, and phenol/ammonium acetate-were evaluated using Jerusalem artichoke leaves as source materials. Of the three methods, trichloroacetic acid/acetone yielded the best protein separation pattern and highest number of protein spots in 2DE analysis. Proteins highly abundant in leaves, such as Rubisco, are typically problematic during leaf 2DE analysis, however, and this disadvantage was evident using trichloroacetic acid/acetone. To reduce the influence of abundant proteins on the detection of low-abundance proteins, we optimized the trichloroacetic acid/acetone method by incorporating a PEG fractionation approach. After optimization, 363 additional (36.2%) protein spots were detected on the 2DE gel. Our results suggest that trichloroacetic acid/acetone method is a better protein extraction technique than Mg/NP-40 and phenol/ammonium acetate in Jerusalem artichoke leaf 2DE analysis, and that trichloroacetic acid/acetone method combined with PEG fractionation procedure is the most effective approach for leaf 2DE analysis of Jerusalem artichoke.