The growing interest in the efficacy of phytomedicines and herbal supplements but also the increase in legal requirements for safety and reliable contents of active principles drive the development of analytical methods for the quality control of complex, multicomponent mixtures as found in plant extracts of value for the pharmaceutical industry. Here, we describe an ultra-performance liquid chromatography method (UPLC) coupled with quadrupole time of flight mass spectrometry (qTOF-MS) measurements for the large scale analysis of H. perforatum plant material and its commercial preparations. Under optimized conditions, we were able to simultaneously quantify and identify 21 metabolites including 4 hyperforins, 3 catechins, 3 naphthodianthrones, 5 flavonoids, 3 fatty acids, and a phenolic acid. Principal component analysis (PCA) was used to ensure good analytical rigorousness and define both similarities and differences among Hypericum samples. A selection of batches from 9 commercially available H. perforatum products available on the German and Egyptian markets showed variable quality, particularly in hyperforins and fatty acid content. PCA analysis was able to discriminate between various preparations according to their global composition, including differentiation between various batches from the same supplier. To the best of our knowledge, this study provides the first approach utilizing UPLC-MS-based metabolic fingerprinting to reveal secondary metabolite compositional differences in Hypericum extract.

Hop (Humulus lupulus L. Cannabaceae) is an economically important crop. In addition to its role in beer brewing, its pharmaceutical applications have been of increasing importance in recent years. Bitter acids (prenylated polyketides), prenylflavonoids and essential oils, are the primary phytochemical components that account for hop medicinal value. An integrated approach utilizing nuclear magnetic resonance (NMR) and mass spectrometry (MS) techniques was used for the first large-scale metabolite profiling in Humulus lupulus. Resins and extracts prepared from 13 hop cultivars were analysed using NMR, liquid chromatography (LC)-MS and fourier transform ion cyclotron resonance (FTICR)-MS in parallel and subjected to principal component analysis (PCA). A one pot extraction method, compatible with both MS and NMR measurement was developed to help rule out effects due to differences in extraction protocols. Under optimised conditions, we were able to simultaneously quantify and identify 46 metabolites including 18 bitter acids, 12 flavonoids, 3 terpenes, 3 fatty acids and 2 sugars. Cultivars segregation in PCA plots generated from both LC-MS and NMR data were found comparable and mostly influenced by differences in bitter acids composition among cultivars. FTICR-MS showed inconsistent PCA loading plot results which are likely due to preferential ionisation and also point to the presence of novel isoprenylated metabolites in hop. This comparative metabolomic approach provided new insights for the complementariness and coincidence for these different technology platform applications in hop and similar plant metabolomics projects.

Glycyrrhiza glabra, commonly known as licorice, is a popular herbal supplement used for the treatment of chronic inflammatory conditions and possesses anticancer and antiviral activities. This species contains a plethora of phytochemicals including terpenoids, saponins, flavonoids, polyamines and polysaccharides. The full complement of bioactive compounds has yet to be elucidated, a step necessary in order to explain its medicinal use. There are over 30 species in the Glycyrrhiza genus world-wide, most of which have been little characterized in terms of phytochemical or pharmacological properties. Here, large scale multi-targeted metabolic profiling and fingerprinting techniques were utilized to help gain a broader insight into Glycyrrhiza species chemical composition. UV, MS and NMR spectra of extracted components were connected with NMR, MS, and multivariate analyses data from Glycyrrhiza glabra, Glycyrrhiza uralensis, Glycyrrhiza inflata and Glycyrrhiza echinata. Major peaks in 1H NMR and MS spectra contributing to the discrimination among species were assigned as those of glycyrrhizin, 4-hydroxyphenyl acetic acid, and glycosidic conjugates of liquiritigenin/isoliquiritigenin. Primary metabolites profiling using GC–MS revealed the presence of cadaverine, an amino acid, exclusively found in G. inflata roots. Both LC–MS and NMR were found effective techniques in sample classification based on genetic and or geographical origin as revealed from derived PCA analysis.

Recognition of pathogen attack or elicitation with pathogen-associated molecular patterns (PAMPs) leads to defense signaling that includes activation of the three mitogen-activated protein kinases (MPKs), MPK3, MPK4 and MPK6 in Arabidopsis. Recently, we demonstrated the activation of a fourth MPK, MPK11, after treatment with flg22, a 22 amino acid PAMP derived from bacterial flagellin. Here, we extended the study by examining elicitation with two other PAMPs, elf18 (derived from bacterial elongation factor EF-Tu) and ch8 (N-acetylchitooctaose derived from fungal chitin). Both PAMPs led to rapid MPK11 transcript accumulation and increased MPK11 kinase activity, suggesting that multiple PAMPs (or stresses) can activate MPK11. However, probably due to functional redundancies, bacteria-induced phytoalexin accumulation does not absolutely require MPK11.

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Vesicle fusion processes in plants are important for both development and stress responses. Transgenic potato plants with reduced expression of SYNTAXIN-RELATED1 (StSYR1), a gene encoding the potato homolog of Arabidopsis PENETRATION1 (AtPEN1), display spontaneous necrosis and chlorosis at later stages of development. In accordance with this developmental defect, tuber number, weight and overall yield are significantly reduced in StSYR1-RNAi lines. Enhanced resistance of StSYR1-RNAi plants to Phytophthora infestans, the causal agent of late blight disease of potato, correlates with enhanced levels of salicylic acid, whereas levels of 12-oxophytodienoic acid and jasmonic acid are unaltered. Cultured cells of StSYR1-RNAi lines secrete at least two compounds which are not detectable in the supernatant of control cells, suggesting an involvement of StSYR1 in secretion processes to the apoplast.

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• The oomycete Phytophthora infestans is the causal agent of late blight, the most devastating disease of potato. The importance of vesicle fusion processes and callose deposition for defense of potato against Phytophthora infestans was analyzed.• Transgenic plants were generated, which express RNA interference constructs targeted against plasma membrane‐localized SYNTAXIN‐RELATED 1 (StSYR1) and SOLUBLE N‐ETHYLMALEIMIDE‐SENSITIVE FACTOR ADAPTOR PROTEIN 33 (StSNAP33), the potato homologs of Arabidopsis AtSYP121 and AtSNAP33, respectively.• Phenotypically, transgenic plants grew normally, but showed spontaneous necrosis and chlorosis formation at later stages. In response to infection with Phytophthora infestans, increased resistance of StSYR1‐RNAi plants, but not StSNAP33‐RNAi plants, was observed. This increased resistance correlated with the constitutive accumulation of salicylic acid and PR1 transcripts. Aberrant callose deposition in Phytophthora infestans‐infected StSYR1‐RNAi plants coincided with decreased papilla formation at penetration sites. Resistance against the necrotrophic fungus Botrytis cinerea was not significantly altered. Infiltration experiments with bacterial solutions of Agrobacterium tumefaciens and Escherichia coli revealed a hypersensitive phenotype of both types of RNAi lines.• The enhanced defense status and the reduced growth of Phytophthora infestans on StSYR1‐RNAi plants suggest an involvement of syntaxins in secretory defense responses of potato and, in particular, in the formation of callose‐containing papillae.

Targeted proteomics via selected reaction monitoring is a powerful mass spectrometric technique affording higher dynamic range, increased specificity and lower limits of detection than other shotgun mass spectrometry methods when applied to proteome analyses. However, it involves selective measurement of predetermined analytes, which requires more preparation in the form of selecting appropriate signatures for the proteins and peptides that are to be targeted. There is a growing number of software programs and resources for selecting optimal transitions and the instrument settings used for the detection and quantification of the targeted peptides, but the exchange of this information is hindered by a lack of a standard format. We have developed a new standardized format, called TraML, for encoding transition lists and associated metadata. In addition to introducing the TraML format, we demonstrate several implementations across the community, and provide semantic validators, extensive documentation, and multiple example instances to demonstrate correctly written documents. Widespread use of TraML will facilitate the exchange of transitions, reduce time spent handling incompatible list formats, increase the reusability of previously optimized transitions, and thus accelerate the widespread adoption of targeted proteomics via selected reaction monitoring.

The Lotus japonicus SYMBIOSIS RECEPTOR-LIKE KINASE (SYMRK) is required for symbiotic signal transduction upon stimulation of root cells by microbial signaling molecules. Here, we identified members of the SEVEN IN ABSENTIA (SINA) E3 ubiquitin-ligase family as SYMRK interactors and confirmed their predicted ubiquitin-ligase activity. In Nicotiana benthamiana leaves, SYMRK–yellow fluorescent protein was localized at the plasma membrane, and interaction with SINAs, as determined by bimolecular fluorescence complementation, was observed in small punctae at the cytosolic interface of the plasma membrane. Moreover, fluorescence-tagged SINA4 partially colocalized with SYMRK and caused SYMRK relocalization as well as disappearance of SYMRK from the plasma membrane. Neither the localization nor the abundance of Nod-factor receptor1 was altered by the presence of SINA4. SINA4 was transcriptionally upregulated during root symbiosis, and rhizobia inoculated roots ectopically expressing SINA4 showed reduced SYMRK protein levels. In accordance with a negative regulatory role in symbiosis, infection thread development was impaired upon ectopic expression of SINA4. Our results implicate SINA4 E3 ubiquitin ligase in the turnover of SYMRK and provide a conceptual mechanism for its symbiosis-appropriate spatio-temporal containment.