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.

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.

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.

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.

Molecular Identification and Targeted Quantitative Analysis of Medicinal Materials from Uncaria Species by DNA Barcoding and LC-MS/MS.

The genus Uncaria is an important source of traditional Chinese medicines with multiple therapeutic effects. The identification of the correct species and accurate determination of the contents of bioactive constituents are important for quality control of Uncaria medicinal materials. Here, an integrated evaluation system based on DNA barcoding for species identification and quantitative analysis by LC-MS/MS has been established. DNA barcoding based on the ITS2 barcode region showed sufficient discriminatory power to precisely identify 24 samples from seven Uncaria species. The length of the 24 ITS2 sequences of Uncaria samples is 227 bp, and 17 variation sites were detected. Additionally, the results of qualitative and quantitative chemical analyses by LC-MS/MS indicated that the chemical compositions of all Uncaria samples were similar; while their contents of targeted alkaloids in samples from different species and origin areas were different. The contents of rhynchophylline (RC) and isorhynchophylline (IRC) were 2.9⁻1612 mg/kg and 2.60⁻1299 mg/kg in all tested samples, respectively. This study concludes that DNA barcoding should be used as the first screening step for Uncaria medicinal materials. Then, integration of DNA barcoding with chemical analyses should be applied in quality control of Uncaria medicinal materials in the medicinal industry.

The effects of genetic variation and environmental factors on rhynchophylline and isorhynchophylline in Uncaria macrophylla Wall. from different populations in China.

Uncaria macrophylla Wall. is an important Chinese medicinal herb. Rhynchophylline (RIN) and isorhynchophylline (IRN) are its major active compounds. We investigated the influence of genetic differentiation and environmental factors on the RIN and IRN to find the main influencing factors of their contents and lay the foundation for the following cultivation and breeding. We used inter-simple sequence repeat (ISSR) markers to investigate the genetic diversity, and high-performance liquid chromatography (HPLC) to measure the contents of RIN and IRN in 200 samples of U. macrophylla obtained from nine natural populations, and then to analyze the correlation between genetic differentiation, environmental factors of sampling sites and the contents of RIN and IRN. We found that High intra-population (80.05%) and low inter-population (19.95%) genetic diversity existed in the samples of U. macrophylla. To some extent, genetic differentiation and the contents of RIN and IRN had correlation in individual populations (such as JH, MH, XM, and ML). The RIN and IRN contents were significant negatively correlated with the precipitation in May (RIRN = -0.771, p = 0.015) and June (RRIN = -0.814, p = 0.008; RIRN = -0.921, p = 0.000), indicating that precipitation was the main affecting factor of their contents. Interestingly, the analysis results showed that the RIN content had a significant positive correlation (r = 0.585, p = 0.000) with the IRN content (they are isomers); the proportion of RIN had a significant negative correlation with the sum of the two (r = -0.390, p<0.0001), while the proportion of IRN had a significant positive correlation (r = 0.390, p<0.0001). It meant that, with the total quantity of the two compounds increased, the proportion of RIN decreased and the proportion of IRN increased, illustrating that their conversion exist some regularity. Moreover, the content ratio of RIN and IRN was significant positively correlated with the January precipitation (r = 0.716, p = 0.030), implying that January may be the key period for the mutual transformation of RIN and IRN.

Transcriptome Analysis of Taxillusi chinensis (DC.) Danser Seeds in Response to Water Loss.

BACKGROUND: Taxillus chinensis (DC.) Danser, the official species of parasitic loranthus that grows by parasitizing other plants, is used in various traditional Chinese medicine prescriptions. ABA-dependent and ABA-independent pathways are two major pathways in response to drought stress for plants and some genes have been reported to play a key role during the dehydration including dehydration-responsive protein RD22, late embryogenesis abundant (LEA) proteins, and various transcription factors (TFs) like MYB and WRKY. However, genes responding to dehydration are still unknown in loranthus. METHODS AND RESULTS: Initially, loranthus seeds were characterized as recalcitrant seeds. Then, biological replicates of fresh loranthus seeds (CK), and seeds after being dehydrated for 16 hours (Tac-16) and 36 hours (Tac-36) were sequenced by RNA-Seq, generating 386,542,846 high quality reads. A total of 164,546 transcripts corresponding to 114,971 genes were assembled by Trinity and annotated by mapping them to NCBI non-redundant (NR), UniProt, GO, KEGG pathway and COG databases. Transcriptome profiling identified 60,695, 56,027 and 66,389 transcripts (>1 FPKM) in CK, Tac-16 and Tac-36, respectively. Compared to CK, we obtained 2,102 up-regulated and 1,344 down-regulated transcripts in Tac-16 and 1,649 up-regulated and 2,135 down-regulated transcripts in Tac-36 by using edgeR. Among them some have been reported to function in dehydration process, such as RD22, heat shock proteins (HSP) and various TFs (MYB, WRKY and ethylene-responsive transcription factors). Interestingly, transcripts encoding ribosomal proteins peaked in Tac-16. It is indicated that HSPs and ribosomal proteins may function in early response to drought stress. Raw sequencing data can be accessed in NCBI SRA platform under the accession number SRA309567. CONCLUSIONS: This is the first time to profile transcriptome globally in loranthus seeds. Our findings provide insights into the gene regulations of loranthus seeds in response to water loss and expand our current understanding of drought tolerance and germination of seeds.

[Effects of controlled atmosphere storage on fruit quality of Siraitia grosvenorii during storage].

UNLABELLED: To explore the condition of CA storage for fruit of Siraitia grosvenorii. METHOD: The CA storage of 5% CO2, 15% CO2, 30% O2 under normal temperature was applied in the experiments, and the results were compared. RESULT: The results showed that CA storage condition could alleviate the reduction of weight loss and water content, the best CA storage condition were 5% CO2, which alleviates the reduction of firmness and increases of soluble solid content during the 45 day period.

[Inspection on result of artemisinin content rank distribution in Guangxi].

OBJECTIVE: In order to enhance the yield of artemisinin, makes out the Artemisia annua adaptive area regional assignment in Guangxi. To ensure the nicety in study, on the base of literature study and experience on the spot, the article inspect the division result. METHOD: By document analysis and colleted data of A. annua, make out sample collect proceed and inspect the result of artemisinin content rank distribution in Guangxi. RESULT AND CONCLUSION: Result of A. annua regional assignment is checked out in the article, the result passes the check by AQL (32, 4). The conclusions insure subsequence study and the A. annua sample collect. The result of artemisinin content rank distribution in Guangxi can be used in artemisinin production.

[Evaluation on germplasm resources of main production area of Artemisia annua in China].

OBJECTIVE: To evaluate and select excellent germplasm resources of Artemisia annua L., providing basic data for data base and breeding of A. annua. METHOD: Seventy-two germplasms of A. annua, which were collected from main production areas, were planted in germplasm resources garden in Jingxi and Nanning under the same conditions. The samples were gathered in the squaring stage. Artemisinin was extracted by supersonic wave and determined by ultraviolet spectrophotometry. RESULT: The leaf's yield and the content of artemisinin in the samples of different germplasms were varied significantly. Effect of environment on artemisinin content was more than that of hereditary factors. The content of artemisinin was different when the same material of A. annua grew in different place. The content of artemisinin in next year's plant was decreasing. CONCLUSIONS: Seven germplasms from south of China have been selected, their content of artemisinin was above 0.9% and calculated yield was more than 2250 kg x hm(-2). The content of artemisinin has been affected by hereditary factors and variation of the growth environment.