Genome-wide characterization of the bHLH gene family in Gynostemma pentaphyllum reveals its potential role in the regulation of gypenoside biosynthesis.

BACKGROUND: Gynostemma pentaphyllum, an ancient Chinese herbal medicine, serves as a natural source of gypenosides with significant medicinal properties. Basic helix-loop-helix (bHLH) transcription factors play pivotal roles in numerous biological processes, especially in the regulation of secondary metabolism in plants. However, the characteristics and functions of the bHLH genes in G. pentaphyllum remain unexplored, and their regulatory role in gypenoside biosynthesis remains poorly elucidated. RESULTS: This study identified a total of 111 bHLH members in G. pentaphyllum (GpbHLHs), categorizing them into 26 subgroups based on shared conserved motif compositions and gene structures. Collinearity analysis illustrated that segmental duplications predominately lead to the evolution of GpbHLHs, with most duplicated GpbHLH gene pairs undergoing purifying selection. Among the nine gypenoside-related GpbHLH genes, two GpbHLHs (GpbHLH15 and GpbHLH58) were selected for further investigation based on co-expression analysis and functional prediction. The expression of these two selected GpbHLHs was dramatically induced by methyl jasmonate, and their nuclear localization was confirmed. Furthermore, yeast one-hybrid and dual-luciferase assays demonstrated that GpbHLH15 and GpbHLH58 could bind to the promoters of the gypenoside biosynthesis pathway genes, such as GpFPS1, GpSS1, and GpOSC1, and activate their promoter activity to varying degrees. CONCLUSIONS: In conclusion, our findings provide a detailed analysis of the bHLH family and valuable insights into the potential use of GpbHLHs to enhance the accumulation of gypenosides in G. pentaphyllum.

Metabolomics analysis reveals the differences between Abrus cantoniensis Hance and Abrus mollis Hance.

BACKGROUND: Abrus cantoniensis Hance. (Ac) and Abrus mollis (Am), two edible and medicinal plants with economic value in southern China, belong to the Abrus genus. Due to its growth characteristics, Am often replaces Ac in folk medicine. However, the latest National Pharmacopeia of China only recommends Ac. The differences in the metabolite composition of the plants are directly related to the differences in their clinical efficacy. RESULTS: The difference in metabolites were analyzed using an untargeted metabolomic approach based on ultrahigh-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC‒ESI‒MS/MS). The roots (R), stems (S) and leaves (L) of the two varieties were examined, and 635 metabolites belonging to 8 classes were detected. A comparative study revealed clear variations in the metabolic profiles of the two plants, and the AmR group had more active ingredients (flavonoids and terpenoids) than the AcR group. The metabolites classified as flavonoids and triterpene saponins showed considerable variations among the various samples. Both Ac and Am had unique metabolites. Two metabolites (isovitexin-2''-xyloside and soyasaponin V) specifically belong to Ac, and nine metabolites (vitexin-2"-O-galactoside, ethyl salicylate, 6-acetamidohexanoic acid, rhein-8-O-glucoside, hederagenin-3-O-glucuronide-28-O-glucosyl(1,2)-glucoside, methyl dioxindole-3-acetate, veratric acid, isorhamnetin-3-O-sophoroside-7-O-rhamnoside, and isorhamnetin-3-O-sophoroside) specifically belong to Am. CONCLUSIONS: The metabolite differences between Ac and Am cause the differences in their clinical efficacy. Our findings serve as a foundation for further investigation of biosynthesis pathways and associated bioactivities and provide guidance for the clinical application of traditional Chinese medicine.

Alkaloid variations within the genus Stephania (Menispermaceae) in China.

The genus Stephania, which is rich in alkaloids, has been used as a traditional medicine or folklore herb against numerous ailments in China. However, the understanding of the variation within the genus Stephania is obscure, which limits the optimal utilization of the genus. An evaluation of the variation within the genus Stephania would help screen the ideal Stephania genotypes for drug utilization. In the present study, alkaloids in the tubers of four commonly cultivated Stephania species in China, i.e., the genotype Stephania kwangsiensis Lo. (SK-guangxi) from Guangxi Province and three Stephania yunnanensis H.S. Lo. genotypes (SY-xueteng, SY-hongteng, and SY-lvteng) sourced from Yunnan Province, were investigated, and the genus variations were compared. The results revealed significant variations in the abundance of alkaloids in tubers within the genus Stephania. The Stephania genotypes SY-xueteng and SY-hongteng showed a relatively high abundance of total alkaloids compared with the Stephania genotypes SK-guangxi and SY-lvteng. Specifically, the Stephania genotype SY-xueteng had a relatively high abundance of palmatine in tubers, and the Stephania genotype SY-hongteng exhibited a high abundance of stephanine in tubers. Our study provides foundations for further utilization of ideal Stephania genotypes by clarifying the variations in the alkaloid contents within the genus in China.

Cultivable endophytic fungal community associated with the karst endemic plant Nervilia fordii and their antimicrobial activity.

Endophytic fungi from medicinal plants with specific pharmacological functions attract much attention to provide the possibility of discovering valuable natural drugs with novel structures and biological activities. Nervilia fordii is a rare and endangered karst endemic plant that is used as medicine and food homology in Guangxi, China. These plants have been reported to have antimicrobial, antitumor, antiviral, and anti-inflammatory activities. However, few studies have focused on the diversity and antibacterial activity of endophytic fungi from N. fordii. In the present study, 184 endophytic fungi were isolated from the healthy tissues of N. fordii, and their molecular diversity and antimicrobial activities were analyzed for the first time. These fungi were categorized into 85 different morphotypes based on the morphological characteristics and the similarity between the target sequence and the reference sequence in the GenBank database. With the exception of 18 unidentified fungi, the fungal isolates belonged to at least 2 phyla, 4 classes, 15 orders, 45 known genera, and 45 different species, which showed high abundance, rich diversity, and obvious tissue specificity. All isolates were employed to screen for their antimicrobial activities via the agar diffusion method against Escherichia coli, Staphylococcus aureus, and Candida tropicalis. Among these endophytes, eight strains (9.41%) displayed inhibitory activity against E. coli, 11 strains (12.94%) against S. aureus, and two strains (2.35%) against C. tropicalis, to some extent. In particular, our study showed for the first time that the fungal agar plugs of Penicillium macrosclerotiorum 1151# exhibited promising antibacterial activity against E. coli and S. aureus. Moreover, the ethyl acetate (EA) extract of P. macrosclerotiorum 1151# had antibacterial effects against E. coli and S. aureus with a minimum inhibitory concentration (MIC) of 0.5 mg ml-1. Further research also confirmed that one of the antimicrobial compounds of P. macrosclerotiorum 1151# was methyl chloroacetate and exhibited excellent antibacterial activity against E. coli and S. aureus up to 1.71-fold and 1.13-fold compared with tetracycline (TET) (5 mg ml-1), respectively. Taken together, the present data suggest that various endophytic fungi of N. fordii could be exploited as sources of novel natural antimicrobial agents.

Integration of transcriptome and targeted metabolome profiling reveals hormone related genes involved in the growth of Bletilla striata.

Bletilla striata (Thunb.) Reichb.f. (BS) is a traditional Chinese medicine with numerous beneficial effects. In our previous study, Aspergillus flavus was isolated from B. striata. To explore the physiological and molecular mechanisms of Aspergillus flavus elicitor (1-G4) that promoted Bletilla striata growth, in this study, we performed the determination of growth indexes and transcriptomics and metabolomics analysis under 5% and 10% 1-G4 conditions. Results showed that 1-G4 elicitor could significantly promote the growth and development of B. striata. With the increasing concentration of 1-G4 elicitor, the contents of SA, ICAld, and ME-IAA significantly increased while the IP and ACC contents decreased dramatically. A total of 1657 DEGs (763 up-regulated and 894 down-regulated) between the control (CK) and 5% elicitor (CK vs G5) and 2415 DEGs (1208 up-regulated and 1207 down-regulated) between the control and 10% elicitor (CK vs G10) were identified. Further, we found that 22, 38, and 2 unigenes were involved in ME-IAA, IP, and ACC, respectively. It was indicated that these unigenes might be involved in B. striata growth. Overall, the current study laid a theoretical foundation for the effective utilization of endophytic fungi and the optimization of germplasm resources of B. striata.

Chromosome-level genome assembly of Gynostemma pentaphyllum provides insights into gypenoside biosynthesis.

Gynostemma pentaphyllum (Thunb.) Makino is an economically valuable medicinal plant belonging to the Cucurbitaceae family that produces the bioactive compound gypenoside. Despite several transcriptomes having been generated for G. pentaphyllum, a reference genome is still unavailable, which has limited the understanding of the gypenoside biosynthesis and regulatory mechanism. Here, we report a high-quality G. pentaphyllum genome with a total length of 582 Mb comprising 1,232 contigs and a scaffold N50 of 50.78 Mb. The G. pentaphyllum genome comprised 59.14% repetitive sequences and 25,285 protein-coding genes. Comparative genome analysis revealed that G. pentaphyllum was related to Siraitia grosvenorii, with an estimated divergence time dating to the Paleogene (∼48 million years ago). By combining transcriptome data from seven tissues, we reconstructed the gypenoside biosynthetic pathway and potential regulatory network using tissue-specific gene co-expression network analysis. Four UDP-glucuronosyltransferases (UGTs), belonging to the UGT85 subfamily and forming a gene cluster, were involved in catalyzing glycosylation in leaf-specific gypenoside biosynthesis. Furthermore, candidate biosynthetic genes and transcription factors involved in the gypenoside regulatory network were identified. The genetic information obtained in this study provides insights into gypenoside biosynthesis and lays the foundation for further exploration of the gypenoside regulatory mechanism.

Identification of Isoflavonoid Biosynthesis-Related R2R3-MYB Transcription Factors in Callerya speciosa (Champ. ex Benth.) Schot Using Transcriptome-Based Gene Coexpression Analysis.

The R2R3-MYB family is one of the largest plant transcription factor (TF) families playing vital roles in defense, plant growth, and secondary metabolism biosynthesis. Although this gene family has been studied in many species, isoflavonoid biosynthesis-related R2R3-MYB TFs in Callerya speciosa (Champ. ex Benth.) Schot, a traditional Chinese medicinal herb, are poorly understood. Here, a total of 101 R2R3-MYB TFs were identified from C. speciosa transcriptome dataset. 25 clades divided into five functional groups were clustered based on the sequence similarity and phylogenetic tree. Conserved motifs and domain distribution, expression patterns, and coexpression networks were also employed to identify the potential R2R3-MYB TFs in the regulation of isoflavonoid biosynthesis. In silico evaluation showed that the deduced R2R3-CsMYB proteins contain highly conserved R2R3 repeat domain at the N-terminal region, that is the signature motif of R2R3-type MYB TFs. Eight potential TFs (CsMYB17, CsMYB36, CsMYB41, CsMYB44, CsMYB45, CsMYB46, CsMYB72, and CsMYB81) had high degrees of coexpression with four key isoflavonoid biosynthetic genes (CsIFS, CsCHS7, CsHID-1, and CsCHI3), in which CsMYB36 as a potential regulator possessed the highest degree. HPLC analysis showed that formononetin and maackiain contents were significantly increased during the development of tuberous roots, which might be controlled by both related R2R3-CsMYBs and structural genes involved in the isoflavonoid biosynthesis pathway. The transcriptome data were further validated by reverse transcription real-time PCR (RT-qPCR) analysis, and similar expression profiles between TFs and key structural genes were identified. This study was the first step toward the understanding of the R2R3-MYB TFs regulating isoflavonoid biosynthesis in C. speciosa. The results will provide information for further functional analysis and quality improvement through genetic manipulation of these potential R2R3-CsMYB genes in C. speciosa.

Hormonal and transcriptional analyses provides new insights into the molecular mechanisms underlying root thickening and isoflavonoid biosynthesis in Callerya speciosa (Champ. ex Benth.) Schot.

Callerya speciosa (Champ. ex Benth.) Schot is a traditional Chinese medicine characterized by tuberous roots as the main organ of isoflavonoid accumulation. Root thickening and isoflavonoid accumulation are two major factors for yield and quality of C. speciosa. However, the underlying mechanisms of root thickening and isoflavonoid biosynthesis have not yet been elucidated. Here, integrated morphological, hormonal and transcriptomic analyses of C. speciosa tuberous roots at four different ages (6, 12, 18, 30 months after germination) were performed. The growth cycle of C. speciosa could be divided into three stages: initiation, rapid-thickening and stable-thickening stage, which cued by the activity of vascular cambia. Endogenous changes in phytohormones were associated with developmental changes during root thickening. Jasmonic acid might be linked to the initial development of tuberous roots. Abscisic acid seemed to be essential for tuber maturation, whereas IAA, cis-zeatin and gibberellin 3 were considered essential for rapid thickening of tuberous roots. A total of 4337 differentially expressed genes (DEGs) were identified during root thickening, including 15 DEGs participated in isoflavonoid biosynthesis, and 153 DEGs involved in starch/sucrose metabolism, hormonal signaling, transcriptional regulation and cell wall metabolism. A hypothetical model of genetic regulation associated with root thickening and isoflavonoid biosynthesis in C. speciosa is proposed, which will help in understanding the underlying mechanisms of tuberous root formation and isoflavonoid biosynthesis.

Genome-Wide Identification of R2R3-MYB Transcription Factors: Discovery of a "Dual-Function" Regulator of Gypenoside and Flavonol Biosynthesis in Gynostemma pentaphyllum.

The R2R3-MYB gene family participates in several plant physiological processes, especially the regulation of the biosynthesis of secondary metabolites. However, little is known about the functions of R2R3-MYB genes in Gynostemma pentaphyllum (G. pentaphyllum), a traditional Chinese medicinal herb that is an excellent source of gypenosides (a class of triterpenoid saponins) and flavonoids. In this study, a systematic genome-wide analysis of the R2R3-MYB gene family was performed using the recently sequenced G. pentaphyllum genome. In total, 87 R2R3-GpMYB genes were identified and subsequently divided into 32 subgroups based on phylogenetic analysis. The analysis was based on conserved exon-intron structures and motif compositions within the same subgroup. Collinearity analysis demonstrated that segmental duplication events were majorly responsible for the expansion of the R2R3-GpMYB gene family, and Ka/Ks analysis indicated that the majority of the duplicated R2R3-GpMYB genes underwent purifying selection. A combination of transcriptome analysis and quantitative reverse transcriptase-PCR (qRT-PCR) confirmed that Gynostemma pentaphyllum myeloblastosis 81 (GpMYB81) along with genes encoding gypenoside and flavonol biosynthetic enzymes exhibited similar expression patterns in different tissues and responses to methyl jasmonate (MeJA). Moreover, GpMYB81 could bind to the promoters of Gynostemma pentaphyllum farnesyl pyrophosphate synthase 1 (GpFPS1) and Gynostemma pentaphyllum chalcone synthase (GpCHS), the key structural genes of gypenoside and flavonol biosynthesis, respectively, and activate their expression. Altogether, this study highlights a novel transcriptional regulatory mechanism that suggests that GpMYB81 acts as a "dual-function" regulator of gypenoside and flavonol biosynthesis in G. pentaphyllum.

Transcriptome-wide analysis of the AP2/ERF transcription factor gene family involved in the regulation of gypenoside biosynthesis in Gynostemma pentaphyllum.

Gynostemma pentaphyllum is a traditional Chinese medicinal herb, serving as natural source of gypenosides (triterpene saponins). The APETALA2/ethylene response factor (AP2/ERF) transcription factors, playing essential regulation roles in plant biotic and abiotic stress responses and secondary metabolism biosynthesis. However, the regulation roles of AP2/ERF transcription factors in gypenosides biosynthesis in G. pentaphyllum remains little understood. In the present study, 125 AP2/ERF genes were identified from G. pentaphyllum transcriptome datasets. Phylogenetic, conserved motifs and expression pattern were employed to comprehensively analyze the 125 GpAP2/ERF genes. Based on the sequence similarity and phylogeny tree, the 125 GpAP2/ERF genes can be classified into 10 groups. Moreover, the distribution of conserved motifs among GpAP2/ERF proteins in phylogenetic trees was consistent with previous studies, thus supporting the classification. Expression profiling indicated that the 125 GpAP2/ERF genes exhibited distinct tissue-specific expression patterns. As confirmed by qRT-PCR, the four candidate GpAP2/ERF genes and gypenoside biosynthetic genes were highly expressed in leaves and/or flowers, and show similar expression patterns in response to MeJA. Base on the expression patterns and phylogenetic relationships, two GpAP2/ERF genes were considered as potential regulatory genes for gypenoside biosynthesis. Our study enhances understanding roles of GpAP2/ERF genes in regulation of gypenosides biosynthesis.

Novel Substituted Thiophenes and Sulf-Polyacetylene Ester from Echinops ritro L.

Three new substituted bithiophenes (1⁻3), and one new sulf-polyacetylene ester, ritroyne A (16) were isolated from the whole plant of Echinops ritro together with twelve known substituted thiophenes. The structures were elucidated on the basis of extensive spectroscopic analysis including 1D and 2D NMR as well as MS. Furthermore, the absolute configuration of ritroyne A (16) was established by computational methods. In bioscreening experiments, four compounds (2, 4, 12, 14) showed similar antibacterial activity against Staphylococcus aureus ATCC 2592 with levofloxacin (8 µg/mL). Five compounds (2, 4, 9, 12, 14) exhibited antibacterial activities against Escherichia coli ATCC 25922, with minimum inhibitory concentration (MIC) values of 32⁻64 µg/mL. Three compounds (2, 4, 12) exhibited antifungal activities against Candida albicans ATCC 2002 with MIC values of 32⁻64 µg/mL. However, compound 16 did not exhibit antimicrobial activities against three microorganisms.

[Comparative study on the infrared fingerprint of Abrus cantoniensis based on the methods of sequential analysis of dual-indexes and cluster analysis].

The methods of sequential analysis of dual-indexes and cluster analysis were utilized to investigate the infrared fingerprints of A. cantoniensis planted in different years and different places in Guangxi, China. The results showed that 6 samples were able to be completely separated only through 13 point smoothing, when the dual-indexes analysis was applied in the present research, and the accurate relationship between these samples could be inspected and expressed by quantitative relationships under 6-dimensional spaces; however, the effect of cluster was bad only through 13 point smoothing of raw spectra, and it was very difficult to find out the regular sequences while the cluster analysis was applied. Furthermore, the 6 samples were able to be completely separated if raw spectra were dealed with 1st derivative after 13 point smoothing, and the clustering effects were more obvious and 6 samples of A. cantoniensis were completely separated. The above two methods could be used to evaluate the quality of Chinese medicinal materials easily when the sample was not excessive quantitatively, but the method of dual-indexes analysis was more difficult than the clustering analysis if the sample size was too large, since a mass of data such as common peak ratio and variation peak ratio of the IR fingerprint spectra were processed and analyzed statistically, while this method could accurately find out the closest relationship between any samples through comparing the quantitative relationships of common peak ratio and variation peak ratio of each sample under 6-dimensional space; the precision of cluster analysis was less than dual-indexes analysis, but it was more convenient than dual-indexes analysis when large sample data were analysed. Finally the above two methods all showed that the chemical composition of the A. cantoniensis was similar in the same cultivated area, but the difference in chemical composition of A. cantoniensis in different years was distinct even they were in the same place.

[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.

[Determination of total flavonoids in Abrus cantoniensis and its dynamic changes].

OBJECTIVE: To develop a method for ultrasonic extraction and determination of total flavonoids in Abrus cantoniensis, and to analyze its dynamic changes. METHOD: The optimized condition of extraction of total flavonoids was studied with orthogonal design. The contents of total flavonoids in different organs and of different growth stages were determined by UV-visible spectrophotometer. RESULT: The ethanol volume and extraction times were the main factors impacting the effects of ultrasonic extraction. The content of total flavonoids in stems were higher than in roots and the lowest in leaves. The dynamic changes of total flavonoids contents in roots and stems of A. cantoniensis were in similar trends. Its total flavonoids content in the two parts of plant increased gradually with the growth and reached the maximum in October, and the content decreased significantly in Feburay of next year. The content of total flavonoids in leaves reached also to the highest value before leaves fell off. CONCLUSION: The optimized extraction method of total flavonoids in Abrus contoniensis was obtained with three times with 80% ethanol at 20 times of volume for 30 min. The results implied that the best yield and quality may be obtained before leaves fall.

[Isolation and biological characteristics of rhizobia strains from Abrus cantoniensis].

OBJECTIVE: The research aimed at studying the biological characteristics of rhizobia isolated from Abrus cantoniensis. METHOD: The rhizobia strains, isolated from different environments in Guangxi, were studied for their growing characters and the generation time. They were also compared for survival capabilities under stresses caused by NaCl, pH, temperature, and different kinds and concentration of antibiotics. RESULT: The strains obtained from A. cantoniensis in subtropical zone produced alkali in YMA medium, the average generation time was 14.8 hours, and thus they belong to slow-growing rhizobia. Rhizobia strains differed greatly in respect to tolerance of high temperature, adaptability of acidic environment and sensitivity to four antibiotics, but they had the same abilities of using different carbon and nitrogen sources. After 70 days from inoculated strains, the seedling formed nodules on the root (85.0%), and the dry matter of vine was increased by 51.1%. CONCLUSION: The rhizobia strains isolated from different ecological environments are good germplasm resources of tolerances to high temperature and acidic environment. The research will greatly help utilize the rhizobia resources and enhance the quality of crude drugs of medicinal leguminosae.

[Effects of different nitrogenous compounds on growth and nodulation of Abrus cantoniensis].

OBJECTIVE: The research aimed at the effects of different nitrogenous compounds on growth and nodulation of Abrus cantoniensis. METHOD: After the seedlings of the herb were inoculated with rhizobia in potted culture, they were supplied with nutrition solutions which contained the three nitrogenous compounds, KNO3, NH4NO3, (NH4)2SO4 of different nitrogen concentration. The growth and nodulation of seedlings was determined after 70 days. RESULT: Different nitrogenous compounds were able to enhance the vegetable growth of seedlings variously. The effect of (NH4)2SO4 and NH4NO3 on growth was better than that of KNO3. Seedlings nodulation was obviously inhibited by these nitrogenous compounds. Their inhibitory effects ranked NH4NO3 > (NH4)2SO4 > KNO3. The treatments of KNO3 and the lower concentration treatments of NH4NO3 and (NH4)2 SO4 didn't inhibit the nodulation of seedlings, but the higher concentration treatment of NH4NO3 and (NH4)2SO4 severely inhibited nodulation or even made a no formation of nodule. CONCLUSION: The results showed that ammonium nitrogen the higher inhibitory ability to the nodulation of seedlings of A. cantoniensis than nitrate nitrogen. Therefore, the application of ammonium nitrogen fertilizer should be controlled in culture of the herb, which is in favor of increasing the function of biological nitrogen fixation and the quality of the medicinal materials of A. cantoniensis.