Marsdenia tenacissima genome reveals calcium adaptation and tenacissoside biosynthesis.

Marsdenia tenacissima is a medicinal plant widely distributed in the calcium-rich karst regions of southwest China. However, the lack of a reference genome has hampered the implementation of molecular techniques in its breeding, pharmacology and domestication. We generated the chromosome-level genome assembly in Apocynaceae using combined SMRT sequencing and Hi-C. The genome length was 381.76 Mb, with 98.9% of it found on 11 chromosomes. The genome contained 222.63 Mb of repetitive sequences and 21 899 predicted gene models, with a contig N50 of 6.57 Mb. Phylogenetic analysis revealed that M. tenacissima diverged from Calotropis gigantea at least 13.43 million years ago. Comparative genomics showed that M. tenacissima underwent ancient shared whole-genome duplication. This event, together with tandem duplication, contributed to 70.71% of gene-family expansion. Both pseudogene analysis and selective pressure calculations suggested calcium-related adaptive evolution in the M. tenacissima genome. Calcium-induced differentially expressed genes (DEGs) were mainly enriched in cell-wall-related processes. Domains (e.g. Fasciclin and Amb_all) and cis-elements (e.g. MYB and MYC) frequently occurred in the coding and promoter regions of cell-wall DEGs, respectively, and the expression levels of these genes correlated significantly with those of calcium-signal-related transcription factors. Moreover, calcium addition increased tenacissoside I, G and H contents. The availability of this high-quality genome provides valuable genomic information for genetic breeding and molecular design, and lends insights into the calcium adaptation of M. tenacissima in karst areas.

Exogenous gibberellic acid shortening after-ripening process and promoting seed germination in a medicinal plant Panax notoginseng.

BACKGROUND: Panax notoginseng (Burk) F.H. Chen is an essential plant in the family of Araliaceae. Its seeds are classified as a type of morphophysiological dormancy (MPD), and are characterized by recalcitrance during the after-ripening process. However, it is not clear about the molecular mechanism on the after-ripening in recalcitrant seeds. RESULTS: In this study, exogenous supply of gibberellic acid (GA3) with different concentrations shortened after-ripening process and promoted the germination of P. notoginseng seeds. Among the identified plant hormone metabolites, exogenous GA3 results in an increased level of endogenous hormone GA3 through permeation. A total of 2971 and 9827 differentially expressed genes (DEGs) were identified in response to 50 mg L-1 GA3 (LG) and 500 mg L-1 GA3 (HG) treatment, respectively, and the plant hormone signal and related metabolic pathways regulated by GA3 was significantly enriched. Weighted gene co-expression network analysis (WGCNA) revealed that GA3 treatment enhances GA biosynthesis and accumulation, while inhibiting the gene expression related to ABA signal transduction. This effect was associated with higher expression of crucial seed embryo development and cell wall loosening genes, Leafy Contyledon1 (LEC1), Late Embryogenesis Abundant (LEA), expansins (EXP) and Pectinesterase (PME). CONCLUSIONS: Exogenous GA3 application promotes germination and shorts the after-ripening process of P. notoginseng seeds by increasing GA3 contents through permeation. Furthermore, the altered ratio of GA and ABA contributes to the development of the embryo, breaks the mechanical constraints of the seed coat and promotes the protrusion of the radicle in recalcitrant P. notoginseng seeds. These findings improve our knowledge of the contribution of GA to regulating the dormancy of MPD seeds during the after-ripening process, and provide new theoretical guidance for the application of recalcitrant seeds in agricultural production and storage.

Illumina-based transcriptomic analysis on recalcitrant seeds of Panax notoginseng for the dormancy release during the after-ripening process.

Panax notoginseng (Burk) F.H. Chen is an economically and medicinally important plant of the family Araliacease, with seed dormancy being a key factor limiting the extended cultivation of P. notoginseng. The seeds belong to the morphophysiological dormancy (MPD) group, and it has also been described as the recalcitrant seed. To date, the molecular mechanism of dormancy release in the recalcitrant seed of P. notoginseng is unknown. In the present study, the transcript profiles of seeds from different after-ripening stages (0, 20, 40 and 60 days) were investigated using Illumina Hiseq 2500 technology. 91 979 946 clean reads were generated, and 81 575 unigenes were annotated in at least one database. In addition, the differentially expressed genes (DEGs) were identified by the pairwise comparisons. We screened out 2483 DEGs by the three key groups of 20 days vs 0 d, 40 d vs 0 d and 60 d vs 0 d. The DEGs were analyzed by gene ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway annotation. Meanwhile, we obtained 78 DEGs related to seeds dormancy release at different after-ripening stages of P. notoginseng, of which 15 DEGs were associated with abscisic acid and gibberellin. 26 DEGs that encode late embryogenesis abundant protein and antioxidant enzyme were correlated with desiccation tolerance in seeds. In summary, the results obtained here showed that PECTINESTERASE-2-LIKE, GA-INSENSITIVE, ENT-KAURENE SYNTHASE, PROTEIN PHOSPHATASE 2C, GIBBERELLIN 2-BETA-DIOXYGENASE, SUPEROXIDE DISMUTASE, L-ASCORBATE PEROXIDASE, CATALASE, LATE EMBRYOGENESIS ABUNDANT PROTEIN DC3 and DEHYDRIN 9 were potentially involved in dormancy release and desiccation sensitivity of P. notoginseng seeds. The data might provide a basis for researches on MPD.

Analysis of the transcriptome of Marsdenia tenacissima discovers putative polyoxypregnane glycoside biosynthetic genes and genetic markers.

Marsdenia tenacissima is a well-known anti-cancer medicinal plant used in traditional Chinese medicine due to bioactive constituents of polyoxypregnane glycosides, such as tenacissosides, marsdenosides and tenacigenosides. Genomic information regarding this plant is very limited, and rare information is available about the biosynthesis of polyoxypregnane glycosides. To facilitate the basic understanding about the polyoxypregnane glycoside biosynthetic pathways, de novo assembling was performed to generate a total of 73,336 contigs and 65,796 unigenes, which represent the first transcriptome of this species. These included 27 unigenes that were involved in steroid biosynthesis and could be related to pregnane backbone biosynthesis. The expression patterns of six unigenes involved in polyoxypregnane biosynthesis were analyzed in leaf and stem tissues by quantitative real time PCR (qRT-PCR) to explore their putative function. Furthermore, a total of 15,295 simple sequence repeats (SSRs) were identified from 11,911 unigenes, of which di-nucleotide motifs were the most abundant.

[Wild resources survey of Marsdenia tenacissima in Honghe, Yunnan].

To ascertain current situation of wild Marsdenia tenacissima resources in Honghe, Yunnan province, the distribution, habitat characteristic and resources reserves of M. tenacissima were surveyed based on interviews and investigation. The results showed that M. tenacissima was found in 7 counties such as Jinping, Mengzi etc, and distributed mainly on the mountainsides from 800 m to 1 200 m. And distribution was affected by many factors, such as light, heat, topography, soil, and vegetation. M. tenacissima grew well in distribution areas. M. tenacissima had averagely a weight of 2.8 kg per plant. Resources reserve of M. tenacissima in Honghe was estimated to 1 300 tons by now but it reduced rapidly in resent years, the wild resources reserve may not meet demand of market. Resources protection and wildlife tending would be conducted to deal with increasing medication requirements.

[Simultaneous determination of chlorogenic acid, scutellarin, 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid in different parts of Erigeron breviscapus by high-performance liquid chromatography].

OBJECTIVE: A high-performance liquid chromatographic (HPLC) method was developed for simultaneous determination of chlorogenic acid, scutellarin, 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid in different parts of Erigerontis Herba. METHOD: The four constituents were measured on an Agilent Zorbax SB-C18 column (4.6 mm x 450 mm, 5 microm) with a gradient elution of acetonitrile (A) -0.3% phosphoric acid solution (B) (0-10 min, 12%-15% A, 10-32 min, 15% A, 32-33 min, 15%-20% A, 33-50 min, 20%-22% A) at wavelength of 335 nm and 327 nm, and a flow rate of 1.0 mL x min(-1) and the column temperature was 30 degrees C. RESULT: Linearity of each standard was established in the concentration range of 0.050 1-1.002 microg for chlorogenic acid, 0.165 9-3.318 microg for chlorogenic acid, 0.049 7-0.994 microg for 3,5-dicaffeoylquinic acid, 0.048 7-0.974 p.g for 4,5-dicaffeoylquinic acid respectively, with correlation coefficient r > 0.999 6. Average recoveries (n = 6) of 4 compounds were 98.53% with a RSD of 0.94%, 99.68% with a RSD of 0.49%, 98.78% with a RSD of 1.1%, 99.06% with a RSD of 0.81%, respectively. CONCLUSION: The developed method is simple, accurate, and precise, it can be used for the quantitative analysis of Erigeron breviscapus.

Exogenous abscisic acid prolongs the dormancy of recalcitrant seed of Panax notoginseng.

The seeds of Panax notoginseng (Burk.) F. H. Chen are typically characterized by their recalcitrance and after-ripening process and exhibit a high water content at harvest as well as a high susceptibility to dehydration. Storage difficulty and the low germination of recalcitrant seeds of P. notoginseng are known to cause an obstacle to agricultural production. In this study, the ratio of embryo to endosperm (Em/En) in abscisic acid (ABA) treatments (1 mg·l-1 and 10 mg·l-1, LA and HA) was 53.64% and 52.34%, respectively, which were lower than those in control check (CK) (61.98%) at 30 days of the after-ripening process (DAR). A total of 83.67% of seeds germinated in the CK, 49% of seeds germinated in the LA treatment, and 37.33% of seeds germinated in the HA treatment at 60 DAR. The ABA, gibberellin (GA), and auxin (IAA) levels were increased in the HA treatment at 0 DAR, while the jasmonic acid (JA) levels were decreased. ABA, IAA, and JA were increased, but GA was decreased with HA treatment at 30 DAR. A total of 4,742, 16,531, and 890 differentially expressed genes (DEGs) were identified between the HA-treated and CK groups, respectively, along with obvious enrichment in the ABA-regulated plant hormone pathway and the mitogen-activated protein kinase (MAPK) signaling pathway. The expression of pyracbactin resistance-like (PYL) and SNF1-related protein kinase subfamily 2 (SnRK2s) increased in the ABA-treated groups, whereas the expression of type 2C protein phosphatase (PP2C) decreased, both of which are related to the ABA signaling pathway. As a result of the changes in expression of these genes, increased ABA signaling and suppressed GA signaling could inhibit the growth of the embryo and the expansion of developmental space. Furthermore, our results demonstrated that MAPK signaling cascades might be involved in the amplification of hormone signaling. Meanwhile, our study uncovered that the exogenous hormone ABA could inhibit embryonic development, promote dormancy, and delay germination in recalcitrant seeds. These findings reveal the critical role of ABA in regulating the dormancy of recalcitrant seeds, and thereby provide a new insight into recalcitrant seeds in agricultural production and storage.

[Study on HPLC characteristic fingerprint of Yunnan Dipsacus asper].

OBJECTIVE: To study the HPLC fingerprint of yunnan Dipsacus asper and provide a reliable method for sciencetific evaluation and quality control. METHODS: 16 batches of Dipsacus asper were collected from 11 counties (cities, districts) of 5 prefectures or municipals in Yunnan provinice, which were the mainly distribution region of Dipsacus asper. Samples were analyzed on an Aglient Zorbax SB-18 (4.6 mm x 250 mm, 5 microm) eluted with methanol (A) and water (B) as mobile phase in gradient clution. The flow rate was 1 ml/min, and the column temperature was set at 38 degrees C. The detector wavelength was 220 nm. RESULTS: A HPLC fingerprint method was established, 14 common peaks were selected and the similarity ranged from 0.674 to 0.965, cluster analysis could classified these 16 batches of Dipsacus asper into 2 groups. CONCLUSION: Dipsacus japonicus is found for the first time in yunnan. The fingerprint of Dipsacus asper in different origin have a extremely high similarity;The method is accurate and reliable.

[Chemical constituents of Dipsacus asper].

OBJECTIVE: To study the chemical constituents of Dipsacus asper. METHODS: Column chromatography on Silica gel and RP-C18 were applied for isolation and purification of the constituents. Their structures were identified by spectral and chemical methods. RESULTS: From the crude MeOH fraction of Dipsacus asper, 12 compounds were isolated and identified as Sucrose (1), beta-sitosterol (2), Oleanic acid (3), Triplostoside A (4), Loganin (5), Loganin acid (6), Sweroside (7), Epi-vogeloside ( 8), Vogeloside (9), Akebiasaponin D(10), Cauloside A(11),7-Deoxyloganic acid (12). CONCLUSION: Compounds 8, 9, 12 are isolated from this plant for the first time.

iTRAQ and RNA-seq analyses provide an insight into mechanisms of recalcitrance in a medicinal plant Panax notoginseng seeds during the after-ripening process.

Panax notoginseng (Burk) F.H. Chen is an important economic and medicinal plant from the family of Araliaceae, and its seed is characterised by the recalcitrance and after-ripening process. However, the molecular mechanism on the dehydration sensitivity is not clear in recalcitrant seeds. In the present study, isobaric tag for relative and absolute quantification (iTRAQ) and RNA-seq were used to analyse the proteomic and transcriptomic changes in seeds of P. notoginseng in days after-ripening (DAR). A total of 454 differentially expressed proteins (DEPs) and 12000 differentially expressed genes (DEGs) were obtained. The activity of enzymes related to antioxidant system were significantly increased, and the late embryogenesis abundant (LEA) protein family and most members of glutathione metabolism enzymes have been downregulated during the after-ripening process. The lack or inadequate accumulation of LEA proteins in the embryo and the low activity of antioxidant defense in glutathione metabolism might be the key factors leading to the dehydration sensitivity in recalcitrant seeds of P. notoginseng. In addition, the increased activity of elycolysis (EMP), citric acid cycle (TCA) and pentose phosphate pathway (PPP) pathways might be one of important signals to complete the after-ripening process. Overall, our study might provide a new insight into the molecular mechanism on dehydration sensitivity of recalcitrant seeds.

Unigene-based RNA-seq provides insights on drought stress responses in Marsdenia tenacissima.

Marsdenia tenacissima is a well-known anti-cancer medicinal plant used in traditional Chinese medicine, which often grows on the karst landform and the water conservation capacity of land is very poorly and drought occurrences frequently. We found M. tenacissima has strong drought resistance because of continuousdrought16 d, the leaves of M. tenacissima were fully curly and dying. But the leaves were fully almost recovering after re-watering 24h. The activity of SOD and POD were almost doubled under drought stress. The content of osmotic regulating substance proline and soluble sugar were three times than control group. But after re-watering, these indexes were declined rapidly. Three cDNA libraries of control, drought stress, and re-watering treatments were constructed. There were 43,129,228, 47,116,844, and 42,815,454 clean reads with Q20 values of 98.06, 98.04, and 97.88respectively.SRA accession number of raw data was PRJNA498187 on NCBI. A total of 8672, 6043, and 6537 differentially expressed genes (DEGs) were identified in control vs drought stress, control vs re-watering, and drought stress vs re-watering, respectively. In addition, 1039, 1016, and 980 transcription factors (TFs) were identified, respectively. Among them, 363, 267, and 299 TFs were identified as DEGs in drought stress, re-watering, and drought stress and re-watering, respectively. These differentially expressed TFs mainly belonged to the bHLH, bZIP, C2H2, ERF, MYB, MYB-related, and NAC families. A comparative analysis found that 1174 genes were up-regulated and 2344 were down-regulated under drought stress and this pattern was the opposite to that found after re-watering. Among the up-regulated genes, 64 genes were homologous to known functional genes that directly protect plants against drought stress. Furthermore, 44 protein kinases and 38 TFs with opposite expression patterns under drought stress and re-watering were identified, which are possibly candidate regulators for drought stress resistance in M. tenacissima. Our study is the first to characterize the M. tenacissima transcriptome in response to drought stress, and will serve as a useful resource for future studies on the functions of candidate protein kinases and TFs involved in M. tenacissima drought stress resistance.

Geographical traceability of Marsdenia tenacissima by Fourier transform infrared spectroscopy and chemometrics.

A combination of Fourier transform infrared spectroscopy with chemometrics tools provided an approach for studying Marsdenia tenacissima according to its geographical origin. A total of 128 M. tenacissima samples from four provinces in China were analyzed with FTIR spectroscopy. Six pattern recognition methods were used to construct the discrimination models: support vector machine-genetic algorithms, support vector machine-particle swarm optimization, K-nearest neighbors, radial basis function neural network, random forest and support vector machine-grid search. Experimental results showed that K-nearest neighbors was superior to other mathematical algorithms after data were preprocessed with wavelet de-noising, with a discrimination rate of 100% in both the training and prediction sets. This study demonstrated that FTIR spectroscopy coupled with K-nearest neighbors could be successfully applied to determine the geographical origins of M. tenacissima samples, thereby providing reliable authentication in a rapid, cheap and noninvasive way.

Photosynthesis, light energy partitioning, and photoprotection in the shade-demanding species Panax notoginseng under high and low level of growth irradiance.

Partitioning of light energy into several pathways and its relation to photosynthesis were examined in a shade-demanding species Panax notoginseng (Burkill) F.H.Chen ex C.Y.Wu & K.M.Feng grown along a light gradient. In fully light-induced leaves, the actual efficiency of PSII photochemistry (ΔF/Fm'), electron transport rate (ETR), non-photochemical quenching (NPQ) and photochemical quenching (qP) were lower in low-light-grown plants; this was also the case in fully dark-adapted leaves under a simulated sunfleck. In response to varied light intensity, high-light-grown plants showed greater quantum yields of light-dependent non-photochemical quenching (ΦNPQ) and PSII photochemistry (ΦPSII) and smaller quantum yields of fluorescence and constitutive thermal dissipation (Φf,d). Under the simulated sunfleck, high-light-grown plants showed greater ΦPSII and smaller Φf,d. There were positive relationships between net photosynthesis (Anet) and ΦNPQ+f,d and negative relationships between Anet and ΦPSII in fully light-induced leaves; negative correlations of Anet with ΦNPQ+f,d and positive correlations of Anet with ΦPSII were observed in fully dark-adapted leaves. In addition, more nitrogen was partitioned to light-harvesting components in low-light-grown plants, whereas leaf morphology and anatomy facilitate reducing light capture in high-light-grown plants. The pool of xanthophyll pigments and the de-epoxidation state was greater in high-light-grown plants. Antioxidant defence was elevated by increased growth irradiance. Overall, the evidences from P. notoginseng suggest that in high-light-grown shade-demanding plants irradiated by high light more electrons were consumed by non-net carboxylative processes that activate the component of NPQ, that low-light-grown plants correspondingly protect the photosynthetic apparatus against photodamage by reducing the efficiency of PSII photochemistry under high light illumination, and that during the photosynthetic induction, the ΔpH-dependent (qE) component of NPQ might dominate photoprotection, but the NPQ also depresses the enhancement of photosynthesis via competition for light energy.

Determination of multiple elements in samples of the medicinal plant Marsdenia tenacissima and estimation of geographic origin via pattern recognition techniques.

Multi-element analysis of the medicinal plant Marsdenia tenacissima was used to develop a reliable method of tracing the geographical source of the samples. The concentrations of 27 elements in 128 samples from 4 provinces in China were analyzed by inductively coupled plasma-atomic emission spectroscopy. Pattern recognition techniques, viz. principal component analysis (PCA), cluster analysis (CA), stepwise linear discriminant analysis (SLDA) and k-nearest neighbor analysis (KNN), were used for this purpose. It was verified that 21 elements in the M. tenacissima samples from different regions showed significant differences (P < 0.05). The PCA explained 87.36 % of the variance with the first seven principal component variables, and a score plot produced from the largest three principal components showed that the source area of most samples could be correctly distinguished. The CA showed that samples were separated into three clusters. The SLDA produced an overall correct classification rate of 87.5 % and a cross-validation rate of 85.2 %. The KNN analysis performed ideally, with an average identification rate of 100 % for the training set and 93.33 % for the test set. These results laid the foundation for the application of multi-element analysis combined with pattern recognition techniques for tracing the geographical origin of samples of medicinal plants.