Identification of candidate genes and residues for improving nitrogen use efficiency in the N-sensitive medicinal plant Panax notoginseng.

BACKGROUND: Nitrogen (N) metabolism-related key genes and conserved amino acid sites in key enzymes play a crucial role in improving N use efficiency (NUE) under N stress. However, it is not clearly known about the molecular mechanism of N deficiency-induced improvement of NUE in the N-sensitive rhizomatous medicinal plant Panax notoginseng (Burk.) F. H. Chen. To explore the potential regulatory mechanism, the transcriptome and proteome were analyzed and the three-dimensional (3D) information and molecular docking models of key genes were compared in the roots of P. notoginseng grown under N regimes. RESULTS: Total N uptake and the proportion of N distribution to roots were significantly reduced, but the NUE, N use efficiency in biomass production (NUEb), the recovery of N fertilizer (RNF) and the proportion of N distribution to shoot were increased in the N0-treated (without N addition) plants. The expression of N uptake- and transport-related genes NPF1.2, NRT2.4, NPF8.1, NPF4.6, AVP, proteins AMT and NRT2 were obviously up-regulated in the N0-grown plants. Meanwhile, the expression of CIPK23, PLC2, NLP6, TCP20, and BT1 related to the nitrate signal-sensing and transduction were up-regulated under the N0 condition. Glutamine synthetase (GS) activity was decreased in the N-deficient plants, while the activity of glutamate dehydrogenase (GDH) increased. The expression of genes GS1-1 and GDH1, and proteins GDH1 and GDH2 were up-regulated in the N0-grown plants, there was a significantly positive correlation between the expression of protein GDH1 and of gene GDH1. Glu192, Glu199 and Glu400 in PnGS1 and PnGDH1were the key amino acid residues that affect the NUE and lead to the differences in GDH enzyme activity. The 3D structure, docking model, and residues of Solanum tuberosum and P. notoginseng was similar. CONCLUSIONS: N deficiency might promote the expression of key genes for N uptake (genes NPF8.1, NPF4.6, AMT, AVP and NRT2), transport (NPF1.2 and NRT2.4), assimilation (proteins GS1 and GDH1), signaling and transduction (genes CIPK23, PLC2, NLP6, TCP20, and BT1) to enhance NUE in the rhizomatous species. N deficiency might induce Glu192, Glu199 and Glu400 to improve the biological activity of GS1 and GDH, this has been hypothesized to be the main reason for the enhanced ability of N assimilation in N-deficient rhizomatous species. The key genes and residues involved in improving NUE provide excellent candidates for the breeding of medicinal plants.

Genome-wide identification and characterization of members of the LEA gene family in Panax notoginseng and their transcriptional responses to dehydration of recalcitrant seeds.

BACKGROUND: Late embryogenesis abundant (LEA) proteins play an important role in dehydration process of seed maturation. The seeds of Panax notoginseng (Burkill) F. H. Chen are typically characterized with the recalcitrance and are highly sensitive to dehydration. However, it is not very well known about the role of LEA proteins in response to dehydration stress in P. notoginseng seeds. We will perform a genome-wide analysis of the LEA gene family and their transcriptional responses to dehydration stress in recalcitrant P. notoginseng seeds. RESULTS: In this study, 61 LEA genes were identified from the P. notoginseng genome, and they were renamed as PnoLEA. The PnoLEA genes were classified into seven subfamilies based on the phylogenetic relationships, gene structure and conserved domains. The PnoLEA genes family showed relatively few introns and was highly conserved. Unexpectedly, the LEA_6 subfamily was not found, and the LEA_2 subfamily contained 46 (75.4%) members. Within 19 pairs of fragment duplication events, among them 17 pairs were LEA_2 subfamily. In addition, the expression of the PnoLEA genes was obviously induced under dehydration stress, but the germination rate of P. notoginseng seeds decreased as the dehydration time prolonged. CONCLUSIONS: We found that the lack of the LEA_6 subfamily, the expansion of the LEA_2 subfamily and low transcriptional levels of most PnoLEA genes might be implicated in the recalcitrant formation of P. notoginseng seeds. LEA proteins are essential in the response to dehydration stress in recalcitrant seeds, but the protective effect of LEA protein is not efficient. These results could improve our understanding of the function of LEA proteins in the response of dehydration stress and their contributions to the formation of seed recalcitrance.

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.

High nitrogen inhibits photosynthetic performance in a shade-tolerant and N-sensitive species Panax notoginseng.

Nitrogen (N) is a primary factor limiting leaf photosynthesis. However, the mechanism of high-N-driven inhibition on photosynthetic efficiency and photoprotection is still unclear in the shade-tolerant and N-sensitive species such as Panax notoginseng. Leaf chlorophyll (Chl) content, Ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) activity and content, N allocation in the photosynthetic apparatus, photosynthetic performance and Chl fluorescence were comparatively analyzed in a shade-tolerant and N-sensitive species P. notoginseng grown under the levels of moderate nitrogen (MN) and high nitrogen (HN). The results showed that Rubisco content, Chl content and specific leaf nitrogen (SLN) were greater in the HN individuals. Rubisco activity, net photosynthetic rate (Anet), photosynthetic N use efficiency (PNUE), maximum carboxylation rate (Vcmax) and maximum electron transport rate (Jmax) were lower when plants were exposed to HN as compared with ones to MN. A large proportion of leaf N was allocated to the carboxylation component under the levels of MN. More N was only served as a form of N storage and not contributed to photosynthesis in HN individuals. Compared with the MN plants, the maximum quantum yield of photosystem II (Fv/Fm), non-photochemical quenching of PSII (NPQ), effective quantum yield and electron transport rate were obviously reduced in the HN plants. Cycle electron flow (CEF) was considerably enhanced in the MN individuals. There was not a significant difference in maximum photo-oxidation P700+ (Pm) between the HN and MN individuals. Most importantly, the HN individuals showed higher K phase in the fast chlorophyll fluorescence induction kinetic curve (OJIP kinetic curve) than the MN ones. The results obtained suggest that photosynthetic capacity might be primarily inhibited by the inactivated Rubisco in the HN individuals, and HN-induced depression of photoprotection might be caused by the photodamage to the donor side of PSII oxygen-evolving complex.

Early Use of Corticosteroid May Prolong SARS-CoV-2 Shedding in Non-Intensive Care Unit Patients with COVID-19 Pneumonia: A Multicenter, Single-Blind, Randomized Control Trial.

BACKGROUND: There is still no clinical evidence available to support or to oppose corticosteroid treatment for coronavirus disease 2019 (COVID-19) pneumonia. OBJECTIVE: To investigate the efficacy and safety of corticosteroid given to the hospitalized patients with COVID-19 pneumonia. METHODS: This was a prospective, multicenter, single-blind, randomized control trial. Adult patients with COVID-19 pneumonia who were admitted to the general ward were randomly assigned to either receive methylprednisolone or not for 7 days. The primary end point was the incidence of clinical deterioration 14 days after randomization. RESULTS: We terminated this trial early because the number of patients with COVID-19 pneumonia in all the centers decreased in late March. Finally, a total of 86 COVID-19 patients underwent randomization. There was no difference of the incidence of clinical deterioration between the methylprednisolone group and control group (4.8 vs. 4.8%, p = 1.000). The duration of throat viral RNA detectability in the methylprednisolone group was 11 days (interquartile range, 6-16 days), which was significantly longer than that in the control group (8 days [2-12 days], p = 0.030). There were no significant differences between the 2 groups in other secondary outcomes. Mass cytometry discovered CD3+ T cells, CD8+ T cells, and NK cells in the methylprednisolone group which were significantly lower than those in the control group after randomization (p < 0.05). CONCLUSIONS: From this prematurely closed trial, we found that the short-term early use of corticosteroid could suppress the immune cells, which may prolong severe acute respiratory syndrome coronavirus 2 shedding in patients with COVID-19 pneumonia. TRIAL REGISTRATION: ClinicalTrials.gov, NCT04273321.

Photosynthetic performance and photosynthesis-related gene expression coordinated in a shade-tolerant species Panax notoginseng under nitrogen regimes.

BACKGROUND: Nitrogen (N) is an essential component of photosynthetic apparatus. However, the mechanism that photosynthetic capacity is suppressed by N is not completely understood. Photosynthetic capacity and photosynthesis-related genes were comparatively analyzed in a shade-tolerant species Panax notoginseng grown under the levels of low N (LN), moderate N (MN) and high N (HN). RESULTS: Photosynthetic assimilation was significantly suppressed in the LN- and HN-grown plants. Compared with the MN-grown plants, the HN-grown plants showed thicker anatomic structure and larger chloroplast accompanied with decreased ratio of mesophyll conductance (gm) to Rubisco content (gm/Rubisco) and lower Rubisco activity. Meanwhile, LN-grown plants displayed smaller chloroplast and accordingly lower internal conductance (gi). LN- and HN-grown individuals allocated less N to light-harvesting system (NL) and carboxylation system (NC), respectively. N surplus negatively affected the expression of genes in Car biosynthesis (GGPS, DXR, PSY, IPI and DXS). The LN individuals outperformed others with respect to non-photochemical quenching. The expression of genes (FBA, PGK, RAF2, GAPC, CAB, PsbA and PsbH) encoding enzymes of Calvin cycle and structural protein of light reaction were obviously repressed in the LN individuals, accompanying with a reduction in Rubisco content and activity. Correspondingly, the expression of genes encoding RAF2, RPI4, CAB and PetE were repressed in the HN-grown plants. CONCLUSIONS: LN-induced depression of photosynthetic capacity might be caused by the deceleration on Calvin cycle and light reaction of photosynthesis, and HN-induced depression of ones might derive from an increase in the form of inactivated Rubisco.

In vitro evaluation of the antibacterial activities of radezolid and linezolid for Streptococcus agalactiae.

PURPOSE: This study aims to evaluate the antimicrobial activities of linezolid and radezolid against Streptococcus agalactiae in vitro and compared for genetic resistance factors. METHOD: Nonduplicate S. agalactiae clinical isolates (n = 136) were collected and the minimal inhibitory concentrations of antimicrobials were determined by agar dilution methodology. The linezolid-resistant mechanism in the clinical linezolid-non-susceptible S. agalactiae isolates and that induced by linezolid pressure in vitro were analyzed by PCR and sequence alignment. Antimicrobial activities and resistance mechanism distinctions between linezolid and radezolid were further investigated in the clinical linezolid-non-susceptible S. agalactiae isolates and that induced by linezolid pressure in vitro. RESULTS: Our data indicated that 17 (13%) of the 136 clinical S. agalactiae isolates were not susceptible to linezolid. For individual S. agalactiae isolates, including linezolid-nonsusceptible isolates with 23S rRNA V domain mutations, radezolid MIC90 values were generally one-half to one-quarter of the linezolid MIC90 values. Radezolid MICs remained low relative to linezolid MICs among linezolid-resistant S. agalactiae isolates, but exhibited the synchronous increases with the increasing copy numbers of 23S rRNA V domain mutations. Overall, 13 optrA-carrying clinical S. agalactiae isolates were found in this study and their MICs all remained sensitive to both linezolid and radezolid. Clinical S. agalactiae isolates with high radezolid MICs showed clonality clustering to sequence type (ST)10. CONCLUSION: Radezolid exhibits stronger potency against S. agalactiae than linezolid and there is a concerning presence of linezolid-nonsusceptible S. agalactiae in clinical samples.

Comparative transcriptome and metabolome analyses provide new insights into the molecular mechanisms underlying taproot thickening in Panax notoginseng.

BACKGROUND: Taproot thickening is a complex biological process that is dependent on the coordinated expression of genes controlled by both environmental and developmental factors. Panax notoginseng is an important Chinese medicinal herb that is characterized by an enlarged taproot as the main organ of saponin accumulation. However, the molecular mechanisms of taproot enlargement are poorly understood. RESULTS: A total of 29,957 differentially expressed genes (DEGs) were identified during the thickening process in the taproots of P. notoginseng. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment revealed that DEGs associated with "plant hormone signal transduction," "starch and sucrose metabolism," and "phenylpropanoid biosynthesis" were predominantly enriched. Further analysis identified some critical genes (e.g., RNase-like major storage protein, DA1-related protein, and Starch branching enzyme I) and metabolites (e.g., sucrose, glucose, fructose, malate, and arginine) that potentially control taproot thickening. Several aspects including hormone crosstalk, transcriptional regulation, homeostatic regulation between sugar and starch, and cell wall metabolism, were identified as important for the thickening process in the taproot of P. notoginseng. CONCLUSION: The results provide a molecular regulatory network of taproot thickening in P. notoginseng and facilitate the further characterization of the genes responsible for taproot formation in root medicinal plants or crops.

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.

Biofilm formation in erythromycin-resistant Staphylococcus aureus and the relationship with antimicrobial susceptibility and molecular characteristics.

PURPOSE: In this study, we aimed to investigate biofilm formation characteristics in clinical Staphylococcus aureus (S. aureus) isolates with erythromycin (ERY) resistance from China and further analyze their correlations with antimicrobial susceptibility and molecular characteristics. METHODOLOGY: A total of 276 clinical isolates of ERY-resistant S. aureus, including 142 methicillin-resistant S. aureus (MRSA) strains and 134 methicillin-susceptible S. aureus (MSSA) strains, were retrospectively collected in China. Biofilms were determined by crystal violet staining and ERY resistance genes (ermA, ermB and ermC) were detected by polymerase chain reaction. Inducible clindamycin resistance was examined by D test and multilocus sequence typing, and clonal complexes (CCs) based on housekeeping genes were further determined. RESULTS: The frequency of biofilm formation among ERY-resistant S. aureus was 40.9% (113/276) in total and no significant difference was found for the frequency of biofilm formation between ERY-resistant MRSA and ERY-resistant MSSA (44.4% vs 37.3%, P > 0.05). In ERY-resistant MRSA isolates, the frequency of biofilm formation in ermA-positive, gentamicin-resistant and ciprofloxacin-resistant isolates was higher than that in ermA-negative, gentamicin-sensitive and ciprofloxacin-sensitive isolates, respectively (63.9% vs 23.6%, P < 0.01; 60.3% vs 27.5%, P < 0.01; 65.2% vs 26.3%, P < 0.01). In addition, tetracycline resistance facilitated biofilm formation in both ERY-resistant MRSA and MSSA and the frequency of biofilm formation in CC239- or CC7S. aureus isolates with ERY resistance was significantly higher compared with that in CC59S. aureus (both P < 0.01). CONCLUSION: The ermA gene, and gentamicin, ciprofloxacin and tetracycline resistance facilitate biofilm formation in ERY-resistant MRSA isolates and, moreover, ERY-resistant S. aureus isolates with positive biofilm formation exhibited clonality clustering regarding CC239 and CC7.

The Design and Application of Simplified Insole-Based Prototypes with Plantar Pressure Measurement for Fast Screening of Flat-Foot.

This study aimed to find the correlation between conventional Arch Index (AI) measurements and our prototype of a simplified insole-based plantar pressure measurement system and to find out the effective plantar pressure sensor position. Twenty-one subjects participated in this study, which was divided into two groups: 10 subjects with flatfoot and 11 subjects with normal foot. Five force sensitive resistance sensors were used on this prototype using Arduino as the data acquisition device. Two types of trials, namely static and dynamic, were conducted to validate our system against the ink-type AI measurement as a golden standard. The results showed that in the static trial, there was a high linear correlation with the medial arch sensor configuration, while in the dynamic trial, there was a high linear correlation in the medial arch sensor configuration and sensor 5 configuration. This study showed that both static and dynamic tests using the self-developed device could effectively determine most of the flatfoot subjects and suggests that in the future, it can be applied in clinical applications because of its advantages when compared to the expensive-high tech graphic input board and conventional tools, like ink-type based measurements.

[Progress in improvement of continuous monoculture cropping problem in Panax ginseng by controlling soil-borne disease management].

The continuous monoculture cropping problem severely has hindered the land resource of Panax ginseng cultivation and threatened the sustainable development of ginseng industry. There are comprehensive factors causing the continuous monoculture cropping problem, such as deterioration of soil physical and chemical properties, accumulation of allelochemical, increase of pesticide residue and heavy metal, imbalance of rhizospheric micro-ecosystem, and increase of soil-borne diseases. Among soil-borne disease was one of the key factors. More than 40 soil-borne diseases have been reported in the ginseng cultivation, especially, the diseases were more serious in the ginseng replanting land. Here main soil-borne diseases and their prevention way have been summarized, and we try to provide the effective improvement strategy of continuous monoculture cropping problem focusing on the disease control and offer reference for overcoming the ginseng continuous monoculture cropping problem.

Transcriptome analysis of Panax vietnamensis var. fuscidicus discovers putative ocotillol-type ginsenosides biosynthesis genes and genetic markers.

BACKGROUND: P. vietnamensis var. fuscidiscus, called "Yesanqi" in Chinese, is a new variety of P. vietnamensis, which was first found in Jinping County, the southern part of Yunnan Province, China. Compared with other Panax plants, this species contains higher content of ocotillol-type saponin, majonoside R2. Despite the pharmacological importance of ocotillol-type saponins, little is known about their biosynthesis in plants. Hence, P. vietnamensis var. fuscidiscus is a suitable medicinal herbal plant species to study biosynthesis of ocotillol-type saponins. In addition, the available genomic information of this important herbal plant is lacking. RESULTS: To investigate the P. vietnamensis var. fuscidiscus transcriptome, Illumina HiSeq™ 2000 sequencing platform was employed. We produced 114,703,210 clean reads, assembled into 126,758 unigenes, with an average length of 1,304 bp and N50 of 2,108 bp. Among these 126,758 unigenes, 85,214 unigenes (67.23%) were annotated based on the information available from the public databases. The transcripts encoding the known enzymes involved in triterpenoid saponins biosynthesis were identified in our Illumina dataset. A full-length cDNA of three Squalene epoxidase (SE) genes were obtained using reverse transcription PCR (RT-PCR) and the expression patterns of ten unigenes were analyzed by reverse transcription quantitative real-time PCR (RT-qPCR). Furthermore, 15 candidate cytochrome P450 genes and 17 candidate UDP-glycosyltransferase genes most likely to involve in triterpenoid saponins biosynthesis pathway were discovered from transcriptome sequencing of P. vietnamensis var. fuscidiscus. We further analyzed the data and found 21,320 simple sequence repeats (SSRs), 30 primer pairs for SSRs were randomly selected for validation of the amplification and polymorphism in 13 P. vietnamensis var. fuscidiscus accessions. Meanwhile, five major triterpene saponins in roots of P. vietnamensis var. fuscidicus were determined using high performance liquid chromatography (HPLC) and evaporative light scattering detector (ELSD). CONCLUSIONS: The genomic resources generated from P. vietnamensis var. fuscidiscus provide new insights into the identification of putative genes involved in triterpenoid saponins biosynthesis pathway. This will facilitate our understanding of the biosynthesis of triterpenoid saponins at molecular level. The SSR markers identified and developed in this study show genetic diversity for this important crop and will contribute to marker-assisted breeding for P. vietnamensis var. fuscidiscus.

[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.

[SSR information in Erigeron breviscapus transcriptome and polymorphism analysis].

OBJECTIVE: The SSR information in the transcriptome of Erigeron breviscapus was analyzed in this study, in order to further develop new functional genes SSR markers laid a solid foundation. METHOD: SSR loci were searched in all of 52,060 unigenes by using est_timmer. Perl program and SSR primers were designed by Primer3. Furthermore, 36 pairs of primers were randomly selected for the polymorphism analysis on 13 Erigeron breviscapus plants collected from different places. RESULT: A total of 3639 SSRs were found in the transcriptome of Erigeron breviscapus, distributed in 3260 unigenes with the distribution frequency of 6.99%. Di-nucleotide repeat was the main type, account for as much as 34.41% of all SSRs, followed by mono-nucleotide (31.41%) and tri-nucleotide repeat motif (28.08%). The di-nucleotide repeat motifs of AT/AT and AC/GT were the predominant repeat types (28.71%). The tri-nucleotide repeat motifs of AAT/AT was the predominant repeat types (7.94%). For validation the availability of those SSR primers, we randomly selected 36 pairs of primers for PCR amplification. Among them, 34 pair primers (94.44%) produced clear and reproductive bands, 19 pair primers showed polymorphism (52.78%), and 13 Erigeron breviscapus plants were divided into 2 groups. CONCLUSION: There are numerous SSRs in Erigeron breviscapus transcriptome with high frequency and various types, this will provide abundant candidate molecular markers for genetic diversity study and genetic map in this plant.

[SSR information in transcriptome of Pinellia ternata].

OBJECTIVE: In order to establish new molecular markers and to design SSR primers,the SSR information was analyzed in the transcriptome of Pinellia ternata. METHODS: The unigenes in transcriptome were filtered and then redundant sequences were removed by the tool of EST-trimmer and cross-match, respectively. Meanwhile, the non-redundant unigenes were examined by the tool of MISA. RESULTS: 14,468 SSRs were found, and these SSRs were distributed in 12 000 unigenes in the examined sequences. Frequency of occurrences for SSRs was 16.24%, and density of distribution was on average 1/4.33 kb in the present study. Di-nucleotide and tri-nucleotide were the major repeated types,accounting for 51.15% and 41.50%, respectively. AG/CT and CCG/CGG were most frequent motifs in di-nucleotide and tri-nucleotide, accounting for 41.54% and 11. 84%, respectively. 87.14% motifs in the length ranged from 12 bp to 20 bp. CONCLUSION: The SSR site in the transcriptome of Pinellia ternata have the characteristics of high frequency of occurrences, type richness, high density and high potential of polymorphism, and these characteristics might be applied in the study on genetic diversity, marker-assisted breeding,and genetic map of Pinellia ternata.

Integrated metabolome and transcriptome analysis reveals the regulatory mechanism of low nitrogen-driven biosynthesis of saponins and flavonoids in Panax notoginseng.

BACKGROUND: Nitrogen (N) is an important macronutrient involved in the biosynthesis of primary and secondary metabolites in plants. However, the metabolic regulatory mechanism of low-N-induced triterpenoid saponin and flavonoid accumulation in rhizomatous medicinal Panax notoginseng (Burk.) F. H. Chen remains unclear. METHODS: To explore the potential regulatory mechanism and metabolic basis controlling the response of P. notoginseng to N deficiency, the transcriptome and metabolome were analysed in the roots. RESULTS: The N content was significantly reduced in roots of N0-treated P. notoginseng (0 kg·N·667 m-2). The C/N ratio was enhanced in the N-deficient P. notoginseng. N deficiency promotes the accumulation of amino acids (L-proline, L-leucine, L-isoleucine, L-norleucine, L-arginine, and L-citrulline) and sugar (arabinose, xylose, glucose, fructose, and mannose), thus providing precursor metabolites for the biosynthesis of flavonoids and triterpenoid saponins. Downregulation of key structural genes (PAL, PAL3, ACC1, CHS2, PPO, CHI3, F3H, DFR, and FGT), in particular with the key genes of F3H, involved in the flavonoid biosynthesis pathway possibly induced the decrease in flavonoid content with increased N supply. Notoginsenoside R1, ginsenoside Re, Rg1, Rd, F1, R1 + Rg1 + Rb1 and total triterpenoid saponins were enhanced in the N0 groups than in the N15 (15 kg·N·667 m-2) plants. Higher phosphoenolpyruvate (an intermediate of glycolyticwith pathway metabolism) and serine (an intermediate of photorespiration) levels induced by N deficiency possibly promote saponin biosynthesis through mevalonic acid (MVA) and methylerythritol (MEP) pathways. Genes (MVD2, HMGS, HMGR1, HMGR2, DXR, and HMGR1) encoding the primary enzymes HMGS, HMGR, DXR, and MVD in the MVA and MEP pathways were significantly upregulated in the N0-treated P. notoginseng. The saponin biosynthesis genes DDS, DDS, CYP716A52, CYP716A47, UGT74AE2, and FPS were upregulated in the N-deficient plants. Upregulation of genes involved in saponin biosynthesis promotes the accumulation of triterpenoid saponins in the N0-grown P. notoginseng. CONCLUSIONS: N deficiency enhances primary metabolisms, such as amino acids and sugar accumulation, laying the foundation for the synthesis of flavonoids and triterpenoid saponins in P. notoginseng. F3H, DDS, FPS, HMGR, HMGS and UGT74AE2 can be considered as candidates for functional characterisation of the N-regulated accumulation of triterpenoid saponins and flavonoids in future.

Optimization of saponin extraction from the leaves of Panax notoginseng and Panax quinquefolium and evaluation of their antioxidant, antihypertensive, hypoglycemic and anti-inflammatory activities.

Panax notoginseng and Panax quinquefolium are important economic plants that utilize dried roots for medicinal and food dual purposes; there is still insufficient research of their stems and leaves, which also contain triterpenoid saponins. The extraction process was developed with a total saponin content of 12.30 ± 0.34% and 12.19 ± 0.64% for P. notoginseng leaves (PNL) and P. quinquefolium leaves (PQL) extracts, respectively. PNL and PQL saponin extracts showed good antioxidant, antihypertensive, hypoglycemic, and anti-inflammatory properties in vitro and RAW264.7 cells. A total of 699 metabolites were identified in PNL and PQL saponin extracts, with the majority being triterpenoid saponins, flavonoids and amino acids. Fourteen ginsenosides, 18 flavonoids or alkaloids, and 16 amino acids were enriched in both saponin extracts. Overall, the utilization of saponins from medicinal plants PNL and PQL has been developed to facilitate systematic research in the functional food and natural product industries.

[Phylogeny and functional divergence of ω-6 and ω-3 fatty acid desaturase families].

The deduced amino acid sequences characteristics, phylogeny, and functional diverge of ω-6 and ω-3 fatty acid desaturase families were analyzed by using Bioinformatics methods. The results showed that all the deduced amino acid sequences shared three highly conserved histidine rich motifs (Hisbox). All the plastidial ω-6 and ω-3 fatty acid desaturases possessed putative N-terminal signal peptide with different amino acids. A relatively conserved hydrophobic region composed of 10 amino-acid residues was found in the middle of signal peptides, which is presumed to be the functional region of the signal peptide of these enzymes. Most of the plant microsomal ω-6 and ω-3 fatty acid desaturases (FAD2 and FAD3) contained a KKXX-like motif of endoplasmic reticulum (ER) retention signal at the C-terminus. However, no such motif was detected in safflower CtFAD2-3, CtFAD2-4, CtFAD2-5, CtFAD2-6, and CtFAD2-7, while an aromatic aa enriched signal (YKNK) was found at their C-terminus which has been reported to be both necessary and sufficient for maintaining localization of the enzymes in the ER. All the amino acid sequences were divided into four categories through phylogenetic analysis. It was suggested that ω-3 fatty acid desaturase originates in a prokaryotic lineage from ω-6 fatty acid desaturase. Both plastidial and microsomal ω-3 fatty acid desaturases could be divided into dicotyledonous and monocotyledonous subgroups, which inferred that functional differentiation of plastidial and microsomal ω-3 fatty acid desaturases had been formed before the divergence of dicotyledonous and monocotyledonous plants. Seed type and housekeeping type FAD2 diverged after the formation of dicotyledonous plants. Except for plant FAD3/plant FAD2, posterior probability values over 0.80 amino acid sites were identified among the functional differentiation subsets, which were mainly distributed at the front and back end of Hisbox I, and the front end of Hisbox II. This indicated that the variations of these amino acid sites played an important role in the size and conformation of protein functional domains and subfamily functional divergence.

[Effect of different plant growth regulators on yield and quality of Angelica dahurica var. formosana development].

OBJECTIVE: To investigate the effect of plant growth regulators on the growth and quality of Angelica dahurica var. formosana. METHOD: Five plant growth regulators: chlormequat chloride (CCC), Mepiquat chloride (PIX), Gibberellic acid (GA3), Paclobutrazol (PP333) and Maleic Hydrazide (MH) were sprayed in rosette stage, the effects of these plant growth regulators (PGRs) on the growth, yield and quality of A. dahurica var. formosanaw were observed. The biological traits were first measured and then imperatorin and isoimperatorin contents in roots were determined by HPLC. RESULT: Low concentration GA3 increased the yield while not influenced the premature bolting rate and the coumarin content. CONCLUSION: Spraying of GA3 (30 mg x L(-1)) could guarantee the growth and development of A. dahurica var. formosana to have a higher yield and maintain the active ingredients content in the root as well.