Exploring Information Exchange between Thesium chinense and Its Host Prunella vulgaris through Joint Transcriptomic and Metabolomic Analysis.

BACKGROUND: Thesium chinense known as the "plant antibiotic" is a facultative root hemi-parasitic herb while Prunella vulgaris can serve as its host. However, the molecular mechanisms underlying the communication between T. chinense and its host remained largely unexplored. The aim of this study was to provide a comprehensive view of transferred metabolites and mobile mRNAs exchanged between T. chinense and P. vulgaris. RESULTS: The wide-target metabolomic and transcriptomic analysis identified 5 transferred metabolites (ethylsalicylate, eriodictyol-7-O-glucoside, aromadendrin-7-O-glucoside, pruvuloside B, 2-ethylpyrazine) and 50 mobile genes between T. chinense and P. vulgaris, as well as haustoria formation related 56 metabolites and 44 genes. There were 4 metabolites (ethylsalicylate, eriodictyol-7-O-glucoside, aromadendrin-7-O-glucoside and pruvuloside B) that are transferred from P. vulgaris to T. chinense, whereas 2-ethylpyrazine was transferred in the opposite direction. Furthermore, we inferred a regulatory network potentially involved in haustoria formation, where three metabolites (N,N'-Dimethylarginine/SDMA, NG,NG-Dimethyl-L-arginine, 2-Acetoxymethyl-anthraquinone) showed significant positive correlations with the majority of haustoria formation-related genes. CONCLUSIONS: These results suggested that there was an extensive exchange of information with P. vulgaris including transferred metabolites and mobile mRNAs, which might facilitate the haustoria formation and parasition of T. chinense.

Authenticating the geographic origins of Atractylodes lancea rhizome chemotypes in China through metabolite marker identification.

Introduction: Atractylodes lancea is widely distributed in East Asia, ranging from Amur to south-central China. The rhizome of A. lancea is commonly used in traditional Chinese medicine, however, the quality of products varies across different regions with different geochemical characteristics. Method: This study aimed to identify the chemotypes of A. lancea from different areas and screen for chemical markers by quantifying volatile organic compounds (VOCs) using a targeted metabolomics approach based on GC-MS/MS. Results: The A. lancea distributed in Hubei, Anhui, Shaanxi, and a region west of Henan province was classified as the Hubei Chemotype (HBA). HBA is characterized by high content of ß-eudesmol and hinesol with lower levels of atractylodin and atractylon. In contrast, the Maoshan Chemotype (MA) from Jiangsu, Shandong, Shanxi, Hebei, Inner Mongolia, and other northern regions, exhibited high levels of atractylodin and atractylon. A total of 15 categories of VOCs metabolites were detected and identified, revealing significant differences in the profiles of terpenoid, heterocyclic compound, ester, and ketone among different areas. Multivariate statistics indicated that 6 compounds and 455 metabolites could serve as candidate markers for differentiating A. lancea obtained from the southern, northern, and Maoshan areas. Discussion: This comprehensive analysis provides a chemical fingerprint of selected A. lancea. Our results highlight the potential of metabolite profiling combined with chemometrics for authenticating the geographical origin of A. lancea.

Drought stress modifies the community structure of root-associated microbes that improve Atractylodes lancea growth and medicinal compound accumulation.

Atractylodes lancea is an important medicinal plant in traditional Chinese medicine, its rhizome is rich of volatile secondary metabolites with medicinal values and is largely demanded in modern markets. Currently, supply of high-yield, high-quality A. lancea is mainly achieved via cultivation. Certain soil microbes can benefit plant growth, secondary metabolism and induce resistance to environmental stresses. Hence, studies on the effects of soil microbe communities and isolates microorganisms on A. lancea is extremely meaningful for future application of microbes on cultivation. Here we investigated the effects of the inoculation with an entire soil microbial community on the growth, resistance to drought, and accumulation of major medicinal compounds (hinesol, ß-eudesmol, atractylon and atractylodin) of A. lancea. We analyzed the interaction between A. lancea and the soil microbes at the phylum and genus levels under drought stress of different severities (inflicted by 0%, 10% and 25% PEG6000 treatments). Our results showed that inoculation with soil microbes promoted the growth, root biomass yield, medicinal compound accumulation, and rendered drought-resistant traits of A. lancea, including relatively high root:shoot ratio and high root water content under drought. Moreover, our results suggested drought stress was more powerful than the selectivity of A. lancea in shaping the root-associated microbial communities; also, the fungal communities had a stronger role than the bacterial communities in protecting A. lancea from drought. Specific microbial clades that might have a role in protecting A. lancea from drought stress were identified: at the genus level, the rhizospheric bacteria Bacillus, Dylla and Actinomadura, and rhizospheric fungi Chaetomium, Acrophialophora, Trichoderma and Thielava, the root endophytic bacteria Burkholderia-Caballeronia-Paraburkholderia, Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium, Dylla and Actinomadura, and the root endophytic fungus Fusarium were closely associated with A. lancea under drought stress. Additionally, we acquired several endophytic Paenibacillus, Paraburkholderia and Fusarium strains and verified they had differential promoting effects on the medicinal compound accumulation in A. lancea root. This study reports the interaction between A. lancea and soil microbe communities under drought stress, and provides insights for improving the outcomes in A. lancea farming via applying microbe inoculation.

Maize intercropping enriches plant growth-promoting rhizobacteria and promotes both the growth and volatile oil concentration of Atractylodes lancea.

In the Atractylodes lancea (A. lancea)-maize intercropping system, maize can promote the growth of A. lancea, but it is unclear whether this constitutes an aboveground or belowground process. In this study, we investigated the mechanisms of the root system interaction between A. lancea and maize using three different barrier conditions: no barrier (AI), nylon barrier (AN), and plastic barrier (AP) systems. The biomass, volatile oil concentration, physicochemical properties of the soil, and rhizosphere microorganisms of the A. lancea plant were determined. The results showed that (1) the A. lancea - maize intercropping system could promote the growth of A. lancea and its accumulation of volatile oils; (2) a comparison of the CK, AI, and AP treatments revealed that it was the above-ground effect of maize specifically that promoted the accumulation of both atractylon and atractylodin within the volatile oils of A. lancea, but inhibited the accumulation of hinesol and ß-eudesmol; (3) in comparing the soil physicochemical properties of each treatment group, intercropping maize acidified the root soil of A. lancea, changed its root soil physicochemical properties, and increased the abundance of the acidic rhizosphere microbes of A. lancea at the phylum level; (4) in an analysis of rhizosphere microbial communities of A. lancea under different barrier systems, intercropping was found to promote plant growth-promoting rhizobacteria (PGPR) enrichment, including Streptomyces, Bradyrhizobium, Candidatus Solibacter, Gemmatirosa, and Pseudolabrys, and the biomass of A. lancea was significantly influenced by PGPR. In summary, we found that the rhizosphere soil of A. lancea was acidified in intercropping with maize, causing the accumulation of PGPR, which was beneficial to the growth of A. lancea.

Light and Potassium Improve the Quality of Dendrobium officinale through Optimizing Transcriptomic and Metabolomic Alteration.

BACKGROUND: Dendrobium officinale is a perennial epiphytic herb in Orchidaceae. Cultivated products are the main alternative for clinical application due to the shortage of wild resources. However, the phenotype and quality of D. officinale have changed post-artificial cultivation, and environmental cues such as light, temperature, water, and nutrition supply are the major influencing factors. This study aims to unveil the mechanisms beneath the cultivation-induced variation by analyzing the changes of the metabolome and transcriptome of D. officinale seedlings treated with red- blue LED light and potassium fertilizer. RESULTS: After light- and K-treatment, the D. officinale pseudobulbs turned purple and the anthocyanin content increased significantly. Through wide-target metabolome analysis, compared with pseudobulbs in the control group (P), the proportion of flavonoids in differentially-accumulated metabolites (DAMs) was 22.4% and 33.5% post light- and K-treatment, respectively. The gene modules coupled to flavonoids were obtained through the coexpression analysis of the light- and K-treated D. officinale transcriptome by WGCNA. The KEGG enrichment results of the key modules showed that the DEGs of the D. officinale pseudobulb were enriched in phenylpropane biosynthesis, flavonoid biosynthesis, and jasmonic acid (JA) synthesis post-light- and K-treatment. In addition, anthocyanin accumulation was the main contribution to the purple color of pseudobulbs, and the plant hormone JA induced the accumulation of anthocyanins in D. officinale. CONCLUSIONS: These results suggested that light and potassium affected the accumulation of active compounds in D. officinale, and the gene-flavone network analysis emphasizes the key functional genes and regulatory factors for quality improvement in the cultivation of this medicinal plant.

Integrative analyses of targeted metabolome and transcriptome of Isatidis Radix autotetraploids highlighted key polyploidization-responsive regulators.

BACKGROUND: Isatidis Radix, the root of Isatis indigotica Fort. (Chinese woad) can produce a variety of efficacious compound with medicinal properties. The tetraploid I. indigotica plants exhibit superior phenotypic traits, such as greater yield, higher bioactive compounds accumulation and enhanced stress tolerance. In this study, a comparative transcriptomic and metabolomic study on Isatidis Radix autotetraploid and its progenitor was performed. RESULTS: Through the targeted metabolic profiling, 283 metabolites were identified in Isatidis Radix, and 70 polyploidization-altered metabolites were obtained. Moreover, the production of lignans was significantly increased post polyploidization, which implied that polyploidization-modulated changes in lignan biosynthesis. Regarding the transcriptomic shift, 2065 differentially expressed genes (DEGs) were identified as being polyploidy-responsive genes, and the polyploidization-altered DEGs were enriched in phenylpropanoid biosynthesis and plant hormone signal transduction. The further integrative analysis of polyploidy-responsive metabolome and transcriptome showed that 1584 DEGs were highly correlated with the 70 polyploidization-altered metabolites, and the transcriptional factors TFs-lignans network highlighted 10 polyploidy-altered TFs and 17 fluctuated phenylpropanoid pathway compounds. CONCLUSIONS: These results collectively indicated that polyploidization contributed to the high content of active compounds in autotetraploid roots, and the gene-lignan pathway network analysis highlighted polyploidy-responsive key functional genes and regulators.

Detection and Biological Characteristics of Alternaria alternata Resistant to Difenoconazole from Paris polyphylla var. chinensis, an Indigenous Medicinal Herb.

Black spot caused by Alternaria alternata (BSAA) is one of the most common diseases of Paris polyphylla var. chinensis, causing yield losses in China. Demethylation inhibitors (DMIs) have been used to control this disease in China for decades. Some farmers have complained about the decreased efficacy of DMIs against BSAA. The objective of this study was to detect and characterize the resistance of A. alternata against difenoconazole from P. polyphylla var. chinensis during 2018. Of the 22 isolates of A. alternata obtained from Sichuan Province in the southwest of China, 20 were resistant to difenoconazole. Mycelial growth rates and sporulation of the difenoconazole-resistant (DfnR) isolates were not different from those of the difenoconazole-sensitive (DfnS) isolates. No cross resistance between difenoconazole and tebuconazole or propiconazole was observed. Mutations were identified at gene AaCYP51 of DfnR isolates based on the sequence alignment of the DfnR and DfnS isolates. All of the mutations could be divided into three resistant genotypes, I (K715R + Y781C), II (K715R + D1140G + T1628A), and III (no mutation). The docking total score of the DfnS isolates was 5.6020, higher than the resistant genotype I (4.4599) or the resistant genotype II (3.8651), suggesting that the DMI resistance of A. alternata may be caused by the decreased affinity between AaCYP51 and difenoconazole.

Detection and Fitness of Dicarboximide-Resistant Isolates of Alternaria alternata from Dendrobium officinale, a Chinese Indigenous Medicinal Herb.

Black spot, caused by Alternaria alternata, poses a severe threat to the industry of Dendrobium officinale, a Chinese indigenous medicinal herb. Dicarboximide fungicides (DCFs) have been intensively used to control this disease for decades in China, and offer excellent efficacy. The resistance of phytopathogenic pathogens against DCFs are reportedly selected in fields; however, the DCF resistance of A. alternata from D. officinale is not well understood. The isolates of A. alternata with low procymidone resistance (ProLR) were detected in the commercial orchards of D. officinale in China in 2018 and biochemically characterized in this study. The result showed that the ProLR isolates were selected in the commercial orchards with a resistance frequency of 100%, and no significant difference in mycelial growth, sporulation, and virulence was observed among the ProLR and procymidone-sensitive (ProS) isolates. A positive cross-resistance pattern was exhibited between procymidone and iprodione. Results of amino acid sequence alignment of AaOS-1 from the tested isolates showed that all of the ProLR genotypes could be categorized into two groups, including group I (mutations at AaOs-1) and group II (no mutation). Under procymidone (5.0 µg/ml) treatment conditions, the AaOs-1 expression levels increased in the ProS isolates and ranged from approximately 2.94- to 3.69-fold higher than those under procymidone-free conditions, while the AaOs-1 expressions of the ProLR isolates were significantly lower than those in the ProS isolates under the same conditions. The data indicated that the mutations at AaOs-1 are involved in the DCF resistance of A. alternata selected in the D. officinale orchards.

[Transcriptome analysis of seed embryo in dormancy and dormancy release state of Thesium chinense].

We used exogenous GA_3 to break the seed dormancy of Thesium chinense. We used high-throughput sequencing technology was used to sequence the transcriptome of dormant seed embryos and dormancy breaking seed embryos of Th. chinense, and the data was analyzed bioinformatically and systematically. The results showed that exogenous GA_3 could effectively break the seed dormancy of Th. chinense; 73 794 up-regulated genes and 42 776 down regulated genes were obtained by transcriptome sequencing; 116 570 diffe-rential genes were annotated by GO function to GO items such as metabolism process, cell process, cell, cell component, binding and catalytic activity. A total of 133 metabolic pathways were found by Pathway analysis of 26 508 differentially expressed genes. In the process of dormancy release, DEGs were mainly enriched in translation, carbohydrate metabolism, folding, classification, degradation and amino acid metabolism. Based on the annotation results in KEGG database, 20 metabolic pathways related to dormancy release were found. Dormancy release of Th. chinense seeds is a complex biological process, including cell morphology construction, secondary metabolite synthesis, sugar metabolism and plant signal transduction, among which plant hormone signal transduction is one of the key factors to regulate dormancy release. The results of qRT-PCR showed that the sequencing results were consistent with the actual results.

[Analysis of differential genes related to dormancy release of Paris polyphylla var. chinensis seeds by transcriptome sequencing].

The study aims at exploring the expression of differential genes and related metabolic pathways in the process of seed dormancy release. The dormant embryo and the dormant released embryo of Paris polyphylla var. chinensis were used as the test materials, a new generation high-throughput sequencing methods to sequence the transcriptome of the samples was used to carry out systematic bioinformatics analysis. We obtained 62 882 650 and 62 263 366 clean reads from the DNA libraries of the samples before and after dormancy breaking. A total of 69 248 differentially expressed genes(DEGs) were obtained, 56 426 up-regulated genes and 12 822 down-regulated genes. There are 138 267 differentially expressed genes in the process of embryo dormancy release, which were annotated by GO function to 58 subclasses of biological processes, molecular functions and cell components. The annotated differentially expressed genes were closely related to metabolic processes, biological regulation, cell component synthesis and enzyme catalytic activity. We found 139 metabolic pathways through pathway analysis of 58 722 differentially expressed genes. Before and after dormancy, DEGs were mainly enriched in carbon metabolism, secondary metabolite biosynthesis and polysaccharide metabolism. Based on the annotation results in KEGG database, we found 16 metabolic pathways related to the dormancy release of P. polyhoylla var. chinensis. A large number of differentially expressed genes were involved in embryo morphogenesis, polysaccharide decomposition and protein synthesis during seed development and dormancy release. It involves the interaction of multiple metabolic pathways and constitutes a complex regulation network for dormancy relief.

[Comparative analysis of active components and transcriptome between autotetraploid and diploid of Dendrobium huoshanense].

In this study, the roots, stems and leaves of diploid and autotetraploid Dendrobium huoshanense were used as materials to compare their contents of polysaccharides and alkaloids, and the transcriptome sequencing analysis was carried out. The results showed that the contents of polysaccharides and alkaloids in the roots, stems and leaves of tetraploid were 7.6%, 34.5%, 17.2%, 0.01%, 0.024% and 0.035% higher than those of diploid D. huoshanense, respectively. The contents of active components in different tissues were significantly different. There were 3 687 differentially expressed genes in diploid and tetraploid D. huoshanense, of which 2 346 genes were up-regulated and 1 341 down regulated. Go functional analysis showed that these genes were mainly involved in growth and development, stress resistance and other related functions. KEGG pathway analysis showed that most of the differential genes were concentrated in the processes of carbon metabolism, signal transduction, carbohydrate metabolism, amino acid metabolism and energy metabolism. The differential expression of key genes involved in the metabolism of polysaccharides, terpenes and polyketones, amino acid metabolism, hormone synthesis and signal transduction in diploid and tetraploid plants may be the main reason for the high energy content, the increase of active components and the growth potential of tetraploid plants.

[Identification and differential expression of miRNA related to seed dormancy of Paris polyphylla var. chinensis].

The purpose of this study was to explore the expression pattern of miRNA in the process of embryo dormancy and provide a reference for the mechanism of regulating seed dormancy and germination by miRNA. We used high-throughput sequencing technology, bioinformatics analysis and real-time fluorescent quantitative PCR(qPCR) technology to sequence, screen and identify miRNAs of dormant and dormant embryos. The results showed that there were 23 811 977, 24 276 695, 20 611 876 and 20 601 811 unique sequences in the four sample libraries during the period of dormancy and dormancy release. MiRNAs are mainly distributed between 21 and 24 nt, among which the length of 24 nt occurred most frequently. A total of 31 known miRNAs were identified, belonging to 13 different families. 93 new miRNAs were predicted by bioinformatics software. Ten miRNAs(mir156 a-5 p, mir160 a-5 p, mir160 h-1, mir169 a-5 p, mir157 d, mir159 a-1, mir395-3, mir156 f-5 p, mir156-2 and mir171 a-3 p) were screened out. In this study, 10 miRNAs related to seed dormancy release were identified. The target genes mainly involved carbohydrate metabolism, plant hormone signal transduction, cell division and growth. The results of qRT-PCR showed that the sequencing results were consistent with the actual results.

A comparative morphological and transcriptomic study on autotetraploid Stevia rebaudiana (bertoni) and its diploid.

Stevia rebaudiana is an important medical plant for producing steviol glycosides (SGs) or stevioside. Autotetraploids (4x = 44) show an increasing level of morphology, physiology and tolerances comparing to diploids (2x = 22). However, little information regarded on the comparative transcriptome analysis between diploid and autotetraploid S. rebaudiana was found. In this study, synthetic autotetraploid was induced and morphological features were confirmed. A comprehensive transcriptome of stevia leaf, stem and root from the diploids and autotetraploids was constructed based on RNA-seq, yielded 1,000,892,422 raw reads and subsequently assembled into 251,455 transcripts, corresponded to 146,130 genes. Pairwise comparisons of the six leaf libraries between the diploids and autotetraploids revealed 4114 differentially expression genes (DEGs), in which 2105 (51.17%) were up-regulated in autotetraploids and associated with SGs biosynthesis, plant growth and secondary metabolism. Moreover, weighted gene co-expression network analysis showed co-expressed genes of fifteen genes of SG biosynthesis pathway were enriched in photosynthesis, flavonoid and secondary metabolic process, plant growth and morphogenesis. A hundred of DEGs related to plant resistance were identified by interviewing PlantPReS database. This study has highlighted molecular changes related to SGs metabolism of polyploidy, and advanced our understanding in plant resistance responsible for phenotypic change of autotetraploids.

[In vitro autotetraploid induction and analysis on DNA methylation diversity of Platycodon grandiflorum].

In order to investigate the epigenetic variations between diploid and autotetraploid of Platycodon grandiflorus. The diploid buds of P. grandiflorus were soaked in the mixture of different concentration colchicines and 0.002 g•mL ⁻¹ dimethyl sulphoxide (DMSO)．The identification of autotetraploid plants were based on morphological characteristics, chromosome number and flow cytometry. And then the level and pattern of DNA methylation explored by using the technology of methylation sensitive amplified polymorphism (MSAP)．The result demonstrated that the buds soaked in 0.2% colchicines and 0.002 g•mL ⁻¹ DMSO solution for 12 h was ideal conditions to induce autotetraploid of P. grandiflorus, with induction rate of 32.0%.The diploid and tetraploid plants existed distinctly differences in morphological indexes.Totally,1 586 bands were amplified by 20 pairs of selective primers, of which 764 and 822 bands were detected in diploid and autotetraploid respectively. The total methylation ratio,full methylation ratio and hemimethylated ratio were 91.25%,61.25% and 30.65% in diploid of P. grandiflorus,respectively.However,the total methylation ratio,full methylation ratio and hemimethylated ratio of autotetraploid of P. grandiflorus were 86.13%,54.38% and 31.75%, respectively. Compared with diploid, the genomic DNA total methylate ratio and full methylation ratio of autotetration plants decreased by 6.02% and 7.14%.But the hemimethylated ratio of autotetraploid was higher than that of diploid, which more than 1.6%. All this results indicated that DNA methylation patterns have adjusted during the polyploidy process．.

[Morphology and AFLP analysis of tetraploid plantlets of Atractylodes macrocephala].

In order to investigate the genetic basis of morphological variation of tetraploid plantlets of Atractylodes macrocephala, diploid plantlets were taken as experimental material, sterile filtration colchicine was used to soak 0.5-1.0 cm long buds. The difference between morphology and stomatal of diploid and tetraploid of A. macrocephala was compared, and genome polymorphism was explored by AFLP. The results showed that the buds dipped in 0.1% colchicine solution for 36 h was optimal conditions to induce tetraploid of A. macrocephala with induction rate of 32.0%. Morphological indexes such as leaf area index, leaf length and width, the density of stomas and the number of chloroplast of tetraploid were distinctly different from diploid. Four hundred and fifty-one bands ranging with 80-500 bp were amplified with 24 pairs of primers, the rate of polymorphism was 32.59%. These amplification sites of diploid were different from tetraploid of A. macrocephala, and the differences in morphology of them were reflected in the DNA polymorphism.

[Residuals of organochlorine pesticides and heavy metals in soil and water of planting base and Liriope muscari].

OBJECTIVE: To analyze the residuals of organochlorine pesticides and heavy metals in soil, water and Radix Liriopes from planting bases in Quanzhou city, and evaluate the quality of the herb. METHOD: GC method was applied to determine residuals of organochlorine pesticides, the contents of Pb, Cd, Cu, Cr, and As were determined by IPC. RESULT: The contents of residuals of organochlorine pesticides and heavy metals varied among the samples of soil, water and Radix liriopes, but all of the residuals met requirement of the national standard. CONCLUSION: Residuals of organochlorine pesticides and heavy metals in soil, water and Radix liriopes from planting bases in Quanzhou city were conformed to GAP.

[Application and study of HPLC fingerprint of Lonicera japonica for identification of geo-authenticity].

OBJECTIVE: To establish a chromatographic fingerprint of Lonicera japonica and apply it to identify geo-authentic of L. japonica. METHOD: The HPLC was applied in chromatographic separation and data were analysed by "computed aided similarity evaluation" software. RESULT: There exited distinct difference of chemical components group between and non-authentic samples. CONCLUSION: The established HPLC fingerprint can be used for the identification of geo-authentic of L. japonica.

[Determination of 18 organophosphate pesticide residues in Flos Lonicerae].

OBJECTIVE: To establish an analysical method for the quantitative determination of 18 kinds of organophosphate pesticide residues in Flos Lonicerae. METHOD: The pesticides in samples were extracted with acetone by Ultrasonic wave Assistant Extraction (UAE), purified by Solid-Phase Extraction (SPE). Using PFPD detecter gas chromatography with HP-1701 column. RESULT AND CONCLUSION: All 18 pesticides were separated well in 30 minutes. The detection limits was 4.0-18.0 microg x kg(-1). The range of recovery and RSD were 83.64%-88.65% and 2.8%-6.0%, respectively. And it met the requirements of pesticide multi-residues analysis.