Effect of strigolactones and auxins on growth and metabolite content of Sutherlandia frutescens (L.) R. Br. microplants in vitro

The influence of strigolactones as hormones in plants is not fully characterised even though they are known to affect plant architecture, both above ground and in the roots. Using an in vitro system, the effects of the synthetic auxins 1-naphthalene acetic acid and indole-3-butyric acid (NAA and IBA) and synthetic strigolactones (GR24 and Nijmegen-1) were tested on microplant development of Sutherlandia frutescens, a leguminous medicinal plant native to South Africa. Considerable phytochemical variation in wild populations has led to the proposal of using micropropagation for this species. This will assist with domestication and provide plants with a more predictable chemistry for the phytopharmaceuticals industry. Nodal explants with an axillary bud were grown on Murashige and Skoog (Plant Physiol 15:473–497, 1962) medium [0.8 % (m/v) agar (pH 5.8), 3 % (m/v) sucrose and 0.1 g/L myo-inositol] supplemented with NAA, IBA, GR24 and Nijmegen-1, either singly or in combination. The amino acid profile and secondary metabolite pool was monitored using LC–MS-profiling. Treatment with NAA promoted mass shoot production, whilst a combination of NAA and Nijmegen-1 also positively influenced the accumulation of amino acids, flavonoids (sutherlandins) and terpenoids (sutherlandiosides) that S. frutescens produces. Since these compounds represent the presumed active compounds in this species and the biomarkers used in quality control assessment of S. frutescens tissues harvested for the pharmaceutical industry, this treatment holds promise for the commercial production of Sutherlandia extracts and herbal medications.     

SU3, an oxocycloartane diglucoside from Sutherlandia humilis

The structure of a new cycloartane-type triterpene glycoside was determined as 24,25-O-β-d-diglucopyranosyl-6α-hydroxycycloart-3-one (SU3) by spectroscopic methods. This is the first cycloartane diglycoside reported from the genus Sutherlandia (an important South African traditional medicine and general tonic known as cancer bush). It was isolated from a dwarf form of S. frutescens, currently known as Sutherlandia humilis.     

Two important indicators with potential to identify Caragana microphylla in xilin gol grassland from temporal MODIS data

Shrub expansion in arid and semi-arid environment leads to serious issues to earth system regarding net primary production, nutrient cycling, and global carbon balance. Caragana microphylla is a shrub species distributed in Xilin Gol grassland, China, which is expanding into and degrading grassland areas. The current paper proposed two important indicators from Moderate Resolution Imaging Spectroradiometer (MODIS) data that will help potentially distinguish C. microphylla from grass species around it: Difference of Maximum (DM) NDVI/EVI and Difference of Accumulated (DA) NDVI/EVI between wet and dry years. The two indicators were developed based on relative drought resistance of woody vs. grass species. Accuracy assessment was performed using the field survey data collected in Xilin Gol grassland. The results demonstrated that the proposed indicators were effective for identifying C. microphylla, with the overall accuracy of 81.67% and Kappa coefficient of 0.6300. Due to the mixed pixel effect, the performance of the indicators was less desirable when the coverage of C. microphylla was on low levels. In addition, the proposed indicators would be more effective if two years with significant variation in precipitation could be selected. Furthermore, the indicators developed are anticipated to be sensitive to other similar drought resistant shrubs around the world.     

Essential oil variation of cultivated and wild Perilla analyzed by GC/MS

Twenty components extracted from the essential oil in the leaves of 172 samples of Perilla frutescens var. crispa (vegetable crop form), P. frutescens var. frutescens (oil crop form), the wild/weedy form of P. frutescens, and three wild Perilla species, Perilla citriodora, Perilla hirtella and Perilla setoyensis were analyzed using GC/MS. A wide range of essential oil components were found among the wild/weedy form of P. frutescens, whereas distinctive components were detected in each wild Perilla species. Egomaketone, asaron, methyleugenol and 4,6-dimethoxy- or 4,7-dimethoxy-5-(2-propenyl)-1,3-dioxaindan were detected from Perilla for the first time. Limonene derivatives, piperitone and piperitenone, were detected from P. citriodora. Discovery of the limonene derivatives in this Perilla species provides evidence of this wild species being a genome donor of P. frutescens, while limonene synthase has been considered to be a specific enzyme in cultivated P. frutescens. These results will be useful for the evaluation and utilization of Perilla genetic resources.     

Intergeneric hybridization of marguerite (Argyranthemum frutescens (L.) Sch. Bip.) and Roman chamomile (Chamaemelum nobile (L.) All.) using ovule culture and confirmation of hybridity

To introduce useful characteristics such as fragrance into Argyranthemum frutescens (L.) and to expand the variation, we conducted crosses using A. frutescens as the seed parent and Chamaemelum nobile (L.) All. as the pollen parent. All the tested cross combinations between the three strains of A. frutescens and one strain of C. nobile produced embryos, and healthy plants were obtained by ovule culture. The obtained plantlets had a white ray floret, and the leaf shape was intermediate to those of the parents. The individuals obtained from this cross were subjected to two methods to determine hybridity: flow cytometry analyses and cleaved amplified polymorphic sequence (CAPS) markers. For the CAPS marker, we selected the internal transcribed spacer (ITS) region, which is highly variable among the genera, as the region to be amplified. We selected restriction enzymes BmgT120 I and Afl II, which selectively cut common sequences in the genus Argyranthemum, based on the sequence analysis of one parent strain each of A. frutescens and C. nobile and alignment with known sequences of related species. Flow cytometry analyses and CAPS markers revealed that the individuals obtained from the cross between A. frutescens and C. nobile are intergeneric hybrids. In addition, these established methods were capable of quickly and reliably identifying hybrids between A. frutescens and C. nobile. This report shows for the first time that crossbreeding between A. frutescens (seed parent) and C. nobile (pollen parent) is possible, and further development of Argyranthemum breeding, such as the expansion of variation by intergeneric crosses, is expected.     

Metabolomics and molecular marker analysis to explore pepper (Capsicum sp.) biodiversity

An overview of the metabolic diversity in ripe fruits of a collection of 32 diverse pepper (Capsicum sp.) accessions was obtained by measuring the composition of both semi-polar and volatile metabolites in fruit pericarp, using untargeted LC–MS and headspace GC–MS platforms, respectively. Accessions represented C. annuum, C. chinense, C. frutescens and C. baccatum species, which were selected based on variation in morphological characters, pungency and geographic origin. Genotypic analysis using AFLP markers confirmed the phylogenetic clustering of accessions according to Capsicum species and separated C. baccatum from the C. annuum–C. chinense–C. frutescens complex. Species-specific clustering was also observed when accessions were grouped based on their semi-polar metabolite profiles. In total 88 semi-polar metabolites could be putatively identified. A large proportion of these metabolites represented conjugates of the main pepper flavonoids (quercetin, apigenin and luteolin) decorated with different sugar groups at different positions along the aglycone. In addition, a large group of acyclic diterpenoid glycosides, called capsianosides, was found to be highly abundant in all C. annuum genotypes. In contrast to the variation in semi-polar metabolites, the variation in volatiles corresponded well to the differences in pungency between the accessions. This was particularly true for branched fatty acid esters present in pungent accessions, which may reflect the activity through the acyl branch of the metabolic pathway leading to capsaicinoids. In addition, large genetic variation was observed for many well-established pepper aroma compounds. These profiling data can be used in breeding programs aimed at improving metabolite-based quality traits such as flavour and health-related metabolites in pepper fruits.     

Comparative chemical profiling of three TCM drugs in the Paeoniaceae family by UPLC-MS/MS combined with chemometric methods

Paeoniae Radix Rubra (Chi-Shao in Chinese) and Paeoniae Radix Alba (Bai-Shao in Chinese) are two valuable traditional Chinese medicine (TCM) drugs, usually indicated for menstrual disorders and viral infections. Paeonia anomala subsp. anomala (Xinjiang-Shaoyao in Chinese) is taken as Chi-Shao substitute in Xinjiang, China. Due to the diverse growing conditions, there are some differences in chemical compositions of three TCM drugs. An UPLC fingerprint analysis with chemometric methods, including similarity analysis and hierarchical clustering analysis, was applied in the study. By virtue of UPLC-QTOF-MS, 29 components including 18 monoterpene glycosides, 5 galloyl glucoses and 6 phenolic compounds were simultaneously identified. It could be concluded that the UPLC-QTOF-MS combined with chemometric methods could efficiently identify the three TCM drugs, and was a powerful approach for the chemical profiling of three TCM drugs. The developed method provide an available approach for quality control of three TCM drugs.     

小叶锦鸡儿天然居群叶形态性状变异研究

为揭示小叶锦鸡儿(Caragana microphylla)天然居群叶形态性状的变异规律及其生态适应性特征,该研究以10个小叶锦鸡儿天然居群为对象,通过多重比较、巢式方差分析、相关性分析、聚类分析和主成分分析等方法,对7个叶形态性状进行分析。结果表明：(1)小叶锦鸡儿叶形态性状在居群内和居群间均存在极显著差异(P < 0.01),平均变异系数为10.13%,不同性状的变异幅度为6.23%~12.78%;平均叶形态性状的表型分化系数为43.62%,居群内变异(30.09%)大于居群间变异(24.91%),说明居群内是其叶形态性状变异的主要来源。(2)相关性分析表明,环境因子对小叶锦鸡儿的叶形态性状变异有很大的影响,在地理空间上主要呈现出沿海拔梯度的变异模式;主成分分析的结果显示,小叶宽、叶柄宽和叶柄长对小叶锦鸡儿叶形态变异起主导作用;利用欧式距离对小叶锦鸡儿居群进行UPGMA聚类分析结果显示,基于叶形态性状和环境因子可分别将小叶锦鸡儿10个居群分为3类和2类,Mantel检验结果表明,小叶锦鸡儿的叶形态性状变异不存在地理连续性。研究结果为小叶锦鸡儿的适应性进化和开发利用提供了理论依据。     

Single-base cytosine methylation analysis in fruits of three Capsicum species

Single-base cytosine methylation analysis across fruits of Capsicum annuum, C. chinense and C. frutescens showed global average methylation ranging from 82.8–89.1%, 77.6–83.9%, and 22.4–25% at CG, CHG and CHH contexts, respectively. High gene-body methylation at CG and CHG was observed across Capsicum species. The C. annuum showed the highest proportion (>80%) of mCs at different genomic regions compared to C. chinense and C. frutescens. Cytosine methylation for transposable-elements were lower in C. frutescens compared to C. annuum and C. chinense. A total of 510,165 CG, 583112 CHG and 277,897 CHH DMRs were identified across three Capsicum species. The differentially methylated regions (DMRs) distribution analysis revealed C. frutescens as more hypo-methylated compared to C. annuum and C. chinense, and also the presence of more intergenic DMRs in Capsicum genome. At CG and CHG context, gene expression and promoter methylation showed inverse correlations. Furthermore, the observed correlation between methylation and expression of genes suggested the potential role of methylation in Capsicum fruit development/ripening.     

Identification of genetic variations of cultivated and weedy types of Perilla species in Korea and Japan using morphological and SSR markers

To better understand the genetic diversity and relationships of the two cultivated types of Perilla crop and their weedy types in Korea and Japan, we evaluated the genetic variations of 56 accessions by assessing five morphological characteristics and 18 SSR markers. The two cultivated types of var. frutescens and var. crispa were clearly distinguished by seed size, whereas most accessions of cultivated and weedy types of var. crispa cannot be distinguished strictly by seed characteristics. A total of 165 alleles with the SSR analysis were detected with an average number of 9.2 alleles per locus among the 56 Perilla accessions. The number of alleles per locus ranged from two for KWPE-56 and KWPE-39 to 21 for GBPFM-204. Additionally, the genetic diversity of each locus ranged from 0.497 at KWPE-56 and KWPE-39 to 0.959 at GBPFM-204, with an average of 0.692. The average genetic diversity values were 0.549, 0.685, 0.451 and 0.557 for cultivated and weedy types of var. frutescens and for cultivated and weedy types of var. crispa, respectively. The weedy type accessions of var. frutescens and var. crispa evidenced greater variation than the corresponding cultivated type accessions. The accessions of the cultivated and weedy types of var. frutescens and var. crispa from Korea exhibited greater SSR diversity than those of Japan. An UPGMA phylogenetic tree revealed three major groups, which was congruent with their morphological characteristics except for a few odd accessions. SSR markers clarified the genetic relationships between var. frutescens and var. crispa and helped improve our understanding of the genetic diversity of the two cultivated types of P. frutescens and their weedy types in Korea and Japan.     

Salinity induced changes in the chloroplast proteome of the aquatic pteridophyte <Emphasis Type="Italic">Azolla microphylla</Emphasis>

The growth of the nitrogen fixing aquatic pteridophyte Azolla microphylla is severely affected by salinity. Salinity exposure (0.5%) resulted in significant reduction in chlorophyll a and b content, altered chl a/b ratio and photosynthetic efficiency (Fv/Fm). Chloroplasts maintain photosynthesis but are highly sensitive to salinity stress. Chloroplast proteins extracted from A. microphylla was separated by two-dimensional electrophoresis (2DE) and approximately 200 proteins were observed on each gel. Forty two differentially expressed protein spots were detected and out of this 17 could be identified through MALDI-TOF-MS/MS analysis. Out of the 17 identified proteins, 15 were found to be down regulated and 2 proteins were up regulated. Most of the down regulated proteins were associated with Calvin cycle, ATP synthesis, oxygen evolution, photosystem I and ROS scavenging. The results show changes in proteome dynamics of the chloroplasts of A. microphylla and such changes may lead to reduction in growth and metabolism. The primary target of salinity in A. microphylla is photosynthesis and the changes in the proteome dynamics of the chloroplasts lead to reduced growth.     

A chemotaxonomic assessment of four indigenous South African Lippia species using GC–MS and vibrational spectroscopy of the essential oils

Lippia species are widely used as traditional remedies by many South African communities. The identification of Lippia spp. indigenous to South Africa is currently based on the morphology and geographical origin of the specimen. However, morphological similarities are evident and due to sympatric distributions, different species may co-occur in the same natural environment, thereby presenting difficulties in the taxonomic delimitation of these taxa. In this paper, gas chromatography–mass spectrometry (GC–MS) and vibrational spectroscopic analyses were used to investigate the chemotaxonomic differentiation of four Lippia species (L. javanica, L. scaberrima, L. rehmannii and L. wilmsii), based on the essential oil composition. The results demonstrate that multivariate classification of chromatographic data enabled separation of Lippia oils into three distinct clusters representing L. scaberrima, L. rehmannii and L. javanica. With the aid of chemometric algorithms, it was also possible to use both mid infrared (MIR) and near infrared (NIR) spectroscopy for a clear distinction of the Lippia spp., with results comparable to those obtained by GC–MS. Orthogonal projections to latent structures (OPLS) presented a better classification algorithm than principal component analysis (PCA), in both GC–MS and spectroscopy analyses. Chemical variations within the Lippia spp. complex were evident, particularly for L. javanica and L. scaberrima, which exhibited significant intra-species variation.     

Two New Alkaloids from Fuzi and Their Metabolites Study

Former study suggests alkaloids from herbs of Aconitum genus plants possess excellent bioactivities, which exert great value for related deeper chemical constituent investigation. Herein, chemical isolation was performed and four alkaloids were isolated from Fuzi, of which two were new ones, and the other two were reported NMR data for the first time. Their chemical structures were identified by NMR data, high resolution MS, UV and IR analysis. Additionally, the MS fragmentation patterns were explored, formerly, that of hetisane alkaloid was rarely reported, and fragmentation mechanism of the diagnostic ion was proposed. Based on these fragment pathway, metabolites and metabolic pathways of four compounds were investigated in rat liver microsomes using UPLC−Q/TOF−MS, and dehydrogenation product was firstly found from metabolites of hetisane alkaloid.     

Chemical profiling with cytokine stimulating investigations of Sutherlandia frutescens L. R. (Br.) (Fabaceae)

Sutherlandia frutescens, one of the medicinal plants of southern Africa, has been widely used to boost the immune system by various ethnic groups. This study aims to provide initial scientific evidence for in vitro immune modulating activities of extracts of S. frutescens on cytokines, including interleukins 4, 6, 8, 10, 12p70 and TNF produced by the HL60 cell lines, as well as to identify possible compounds present therein. This will assist in guiding further studies to an active compound. The chemical profile of extracts, fractions and compounds was determined using high performance liquid chromatography coupled to mass spectrometry. Results from the initial in vitro experiments conducted indicated that extracts from S. frutescens possessed immune modulating as well as anti-inflammatory activities. This in vitro study showed that an ethanolic extract appeared to recruit the various inflammatory cytokines to the site of infection upon stimulation with phorbol 12-myristate 13-acetate, where essentially the non-polar compounds present in the ethanol extract contributed to most of the activity observed for this extract.     

Molecular-networking-guided discovery of species-specific markers for discriminating five medicinal Paris herbs

BackgroundParidis Rhizoma (PR) is a famous traditional herbal medicine. Apart from two officially recorded species, viz. Paris polyphylla Smith var. yunnanensis (Franch.) Hand. - Mazz. (PPY) and P. polyphylla Smith var. chinensis (Franch.) Hara (PPC), there are still many other species used as folk medicine. It is necessary to understand the metabolic differences among Paris species.PurposeTo establish a strategy that can discover species-specific steroidal saponin markers to distinguish closely-related Paris herbs for quality and safety control.MethodsA new strategy of molecular-networking-guided discovery of species-specific markers was proposed. Firstly, the ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS) was applied to obtain the MS and MS/MS data of all samples. Then, molecular networking (MN) was created using MS/MS data to prescreen the steroidal saponins for subsequent analysis. Next, the principal component analysis (PCA) and orthogonal partial least square discriminant analysis (OPLS-DA) models were established to discover potential markers. Finally, the verification, identification and distribution of chemical markers were performed.ResultsA total of 126 steroidal saponins were screened out from five species using MN. Five species were classified successfully by OPLS-DA model, and 18 species-specific markers were discovered combining the variable importance in the projection (VIP) value, P value (one-way ANOVA) and their relative abundance. These markers could predict the species of Paris herbs correctly.ConclusionThese results revealed that this new strategy could be an efficient way for chemical discrimination of medicinal herbs with close genetic relationship.     

Comparative analysis of plastomes in Oxalidaceae: Phylogenetic relationships and potential molecular markers

The wood sorrel family, Oxalidaceae, is mainly composed of annual or perennial herbs, a few shrubs, and trees distributed from temperate to tropical zones. Members of Oxalidaceae are of high medicinal, ornamental, and economic value. Despite the rich diversity and value of Oxalidaceae, few molecular markers or plastomes are available for phylogenetic analysis of the family. Here, we reported four new whole plastomes of Oxalidaceae and compared them with plastomes of three species in the family, as well as the plastome of Rourea microphylla in the closely related family Connaraceae. The eight plastomes ranged in length from 150,673 bp (Biophytum sensitivum) to 156,609 bp (R. microphylla). Genome annotations revealed a total of 129–131 genes, including 83–84 protein-coding genes, eight rRNA genes, 37 tRNA genes, and two to three pseudogenes. Comparative analyses showed that the plastomes of these species have minor variations at the gene level. The smaller plastomes of herbs B. sensitivum and three Oxalis species are associated with variations in IR region sizes, intergenic region variation, and gene or intron loss. We identified sequences with high variation that may serve as molecular markers in taxonomic studies of Oxalidaceae. The phylogenetic trees of selected superrosid representatives based on 76 protein-coding genes corroborated the Oxalidaceae position in Oxalidales and supported it as a sister to Connaraceae. Our research also supported the monophyly of the COM (Celastrales, Oxalidales, and Malpighiales) clade.     

Successful Wide Hybridization and Introgression Breeding in a Diverse Set of Common Peppers (Capsicum annuum) Using Different Cultivated Ají (C. baccatum) Accessions as Donor Parents

Capsicum baccatum, commonly known as ají, has been reported as a source of variation for many different traits to improve common pepper (C. annuum), one of the most important vegetables in the world. However, strong interspecific hybridization barriers exist between them. A comparative study of two wide hybridization approaches for introgressing C. baccatum genes into C. annuum was performed: i) genetic bridge (GB) using C. chinense and C. frutescens as bridge species; and, ii) direct cross between C. annuum and C. baccatum combined with in vitro embryo rescue (ER). A diverse and representative collection of 18 accessions from four cultivated species of Capsicum was used, including C. annuum (12), C. baccatum (3), C. chinense (2), and C. frutescens (1). More than 5000 crosses were made and over 1000 embryos were rescued in the present study. C. chinense performed as a good bridge species between C. annuum and C. baccatum, with the best results being obtained with the cross combination [C. baccatum (♀) × C. chinense (♂)] (♀) × C. annuum (♂), while C. frutescens gave poor results as bridge species due to strong prezygotic and postzygotic barriers. Virus-like-syndrome or dwarfism was observed in F₁ hybrids when both C. chinense and C. frutescens were used as female parents. Regarding the ER strategy, the best response was found in C. annuum (♀) × C. baccatum (♂) crosses. First backcrosses to C. annuum (BC₁s) were obtained according to the crossing scheme [C. annuum (♀) × C. baccatum (♂)] (♀) × C. annuum (♂) using ER. Advantages and disadvantages of each strategy are discussed in relation to their application to breeding programmes. These results provide breeders with useful practical information for the regular utilization of the C. baccatum gene pool in C. annuum breeding.     

Genetic variability and differentiation of <Emphasis Type="Italic">Caragana microphylla</Emphasis> populations as revealed by RAPD markers

Genetic variability in random amplified polymorphic DNA (RAPD) was studied in 90 individuals of Caragana microphylla, an outcrossing perennial shrub species, from five natural populations sampled in Inner Mongolia steppe of China on a small scale. Nineteen selected primers were used to amplify DNA samples, and totally 225 bands were detected. The percentage of polymorphic bands within populations ranged form 58.22% to 63.56%, with an average of 60% at the population level and 71.11% at the species level, indicating relatively high genetic variations in C. microphylla species. Shannon’s information index (l) and Nei’s gene diversity (h) showed the similar trend with each other. According to the analysis of Nei’s gene diversity, the percentage of genetic variation among populations was 7.13%, indicating a low level of genetic differentiation among populations. There existed a strong gene flow (N _m = 3.26) among populations. Although AMOVA analysis also revealed most variation was within populations (Φ_ST = 4.1%), a significant proportion was observed among populations (P < 0.001) in the present study, suggesting genetic differentiation occurred among populations at a certain extent. Based on Mantel’s tests and the results of previous studies, the genetic structure pattern of C. microphylla accorded with the isolation-by-distance model on a very large scale, however, on a small scale, the significant genetic differentiation among populations might be enhanced by the micro-environmental divergence among the sampling sites, rather than by geographic factors. Analysis of the genetic variations of C. microphylla populations provided useful information for the adaptive strategy of Caragana species.     

Volatile constituents of four Inula species of Bulgarian origin

In this study the volatile components of four Inula species (Inula germanica L., I. bifrons (L.) L., I. ensifolia L., and I. salicina L.) were analysed by GC-FID/MS. A total of 141 chemical compounds were identified. A distinct volatile chemical profiles with high variation in the type of compounds was observed. Inula germanica was rich in oxygenated monoterpenes (54.7%), with cis-carvyl acetate (20.7%) and 1,8-cineole (14.6%) as main components. Sesquiterpenoids dominated in I. bifrons (60.3%), while the relatively high percentage of fatty acids characterized the other two species I. ensifolia and I. salicina (44.1 and 39.8%, respectively). Principal component analysis (PCA) and cluster analysis (CA) were used to investigate the variations in the volatiles of different Inula species.