Introduction Liverworts are a group of non-vascular plants that possess unique metabolism not found in other plants. Many liverwort metabolites have interesting structural and biochemical characteristics, however the fluctuations of these metabolites in response to stressors is largely unknown. Objectives To investigate the metabolic stress-response of the leafy liverwort Radula complanata. Methods Five phytohormones were applied exogenously to in vitro cultured R. complanata and an untargeted metabolomic analysis was conducted. Compound classification and identification was performed with CANOPUS and SIRIUS while statistical analyses including PCA, ANOVA, and variable selection using BORUTA were conducted to identify metabolic shifts.Results It was found that R. complanata was predominantly composed of carboxylic acids and derivatives, followed by benzene and substituted derivatives, fatty acyls, organooxygen compounds, prenol lipids, and flavonoids. The PCA revealed that samples grouped based on the type of hormone applied, and the variable selection using BORUTA (Random Forest) revealed 71 identified and/or classified features that fluctuated with phytohormone application. The stress-response treatments largely reduced the production of the selected primary metabolites while the growth treatments resulted in increased production of these compounds. 4-(3-Methyl-2-butenyl)-5-phenethylbenzene-1,3-diol was identified as a biomarker for the growth treatments while GDP-hexose was identified as a biomarker for the stress-response treatments. Conclusion Exogenous phytohormone application caused clear metabolic shifts in Radula complanata that deviate from the responses of vascular plants. Further identification of the selected metabolite features can reveal metabolic biomarkers unique to liverworts and provide more insight into liverwort stress responses.

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IntroductionThe demand to develop efficient and reliable analytical methods for the quality control of nutraceuticals is on the rise, together with an increase in the legal requirements for safe and consistent levels of its active principles.ObjectiveTo establish a reliable model for the quality control of widely used Senna preparations used as laxatives and assess its phyto-equivalency.MethodsA comparative metabolomics approach via NMR and MS analyses was employed for the comprehensive measurement of metabolites and analyzed using chemometrics.ResultsUnder optimized conditions, 30 metabolites were simultaneously identified and quantified including anthraquinones, bianthrones, acetophenones, flavonoid conjugates, naphthalenes, phenolics, and fatty acids. Principal component analysis (PCA) was used to define relative metabolite differences among Senna preparations. Furthermore, quantitative 1H NMR (qHNMR) was employed to assess absolute metabolites levels in preparations. Results revealed that 6-hydroxy musizin or tinnevellin were correlated with active metabolites levels, suggesting the use of either of these naphthalene glycosides as markers for official Senna drugs authentication.ConclusionThis study provides the first comparative metabolomics approach utilizing NMR and UPLC–MS to reveal for secondary metabolite compositional differences in Senna preparations that could readily be applied as a reliable quality control model for its analysis.

A crucial feature of plant performance is its strong dependence on the availability of essential mineral nutrients, affecting multiple vital functions. Indeed, mineral-nutrient deficiency is one of the major stress factors affecting plant growth and development. Thereby, nitrogen and potassium represent the most abundant mineral contributors, critical for plant survival. While studying plant responses to nutrient deficiency, one should keep in mind that mineral nutrients, along with their specific metabolic roles, are directly involved in maintaining cell ion homeostasis, which relies on a finely tuned equilibrium between cytosolic and vacuolar ion pools. Therefore, in this chapter we briefly summarize the role of the ion homeostasis system in cell responses to environmental deficiency of nitrate and potassium ions. Special attention is paid to the implementation of plant responses via NO3− and K+ root transport and regulation of ion distribution in cell compartments. These responses are strongly dependent on plant species, as well as severity and duration of nutrient deficiency.

The chapter “Mass Spectrometry Data Processing” focuses on the mass spectrometry data processing workflow. The first step consists of processing the raw MS data using conversion of vendor formats to open standards, followed by feature detection, optionally retention time correction and grouping of features across samples leading to a feature matrix amenable for statistical analysis. The metabolomics community has developed several open source software packages capable of processing large-scale data commonly occurring in metabolomics studies. In the second stage, features of interest are identified, i.e., annotated with names of metabolites, or compound classes. Tandem MS or LC-MS/MS fragmentation data provides structural hints. The MS/MS spectra can be used to search in open and commercial spectral libraries. If no reference spectra are available, in-silico annotation tools or more recently machine learning approaches can be used.

The structure of the microtubule cytoskeleton provides valuable information related to morphogenesis of cells. The cytoskeleton organizes into diverse patterns that vary in cells of different types and tissues, but also within a single tissue. To assess differences in cytoskeleton organization methods are needed that quantify cytoskeleton patterns within a complete cell and which are suitable for large data sets. A major bottleneck in most approaches, however, is a lack of techniques for automatic extraction of cell contours. Here, we present a semi-automatic pipeline for cell segmentation and quantification of microtubule organization. Automatic methods are applied to extract major parts of the contours and a handy image editor is provided to manually add missing information efficiently. Experimental results prove that our approach yields high-quality contour data with minimal user intervention and serves a suitable basis for subsequent quantitative studies.

Medicago truncatula, owing to its small diploid genome (∼500 Mbp), short life cycle, and high natural diversity makes it a good model plant and has opened the door of opportunities for scientists interested in studying legume biology. But over the years, challenges are also being faced for genetic manipulation of this plant. Many genetic manipulation protocols have been published involving Agrobacterium tumefaciens, a pathogen causing tumor disease in plants. These protocols apart from being difficult to achieve, are also time consuming. Nowadays, an easy, less time consuming and highly reproducible Agrobacterium rhizogenes based method is in use by many research groups. This method generates composite plants having transformed roots on a wild‐type shoot. Here, stable transformed lines that can be propagated over time are not achieved by this method, but for root‐development or root–microbe interaction studies this method has proven to be a useful tool for the community. In addition, transformed roots can be propagated by root organ cultures (ROCs), wherein transformed roots are propagated on sucrose containing media without any shoot part. Occasionally, even stable transgenic plants can be regenerated from transgenic roots. In this chapter, developments and improvements of various transformation protocols are discussed. The suitability of composite plants is highlighted by a study on mycorrhization of transformed and non‐transformed roots, which did not show differences in the mycorrhization rate and developmental stages of the arbuscular mycorrhizal (AM) fungus inside the roots as well as in transcript accumulation and metabolite levels of roots. Finally, applications of the A. rhizogenes based transformation method are discussed.

Mass spectrometry coupled with LC (liquid chromatography) separation has developed into a technique routinely applied for targeted as well as for nontargeted analysis of complex biological samples, not only in plant biochemistry. Earlier on, LC‐MS (liquid chromatography–mass spectrometry) was mostly part of the efforts for identification of one or few unknown metabolites of interest as part of a phytochemical study. As a major strategy, unknown compounds had to be purified in sufficient quantities. The purified fractions were then subjected to LC‐MS/MS as part of the structural elucidation, mostly complemented by NMR (nuclear magnetic resonance) analysis. With the advance of mass spectrometry instrumentation, LC‐MS is now widely applied for analysis of crude plant extracts and large numbers (100s to 1000s) of samples. It has become an essential part of metabolomic studies (see Metabolomics), aiming at the comprehensive coverage of the metabolite profiles of cells, tissues, or organs. Owing to the huge chemical diversity of small molecules, conditions for the extraction will restrict the subfraction of the metabolome, which can be actually analyzed. The conditions for LC have to be adjusted to allow good separation of the particular metabolites from the respective extract. Major consideration will be the selection of an appropriate column and suitable eluents, the establishment of gradient profiles, temperature conditions, and so on.

IntroductionThe production of marine drugs in its normal habitats is often low and depends greatly on ecological conditions. Chemical synthesis of marine drugs is not economically feasible owing to their complex structures. Biotechnology application via elicitation represents a promising tool to enhance metabolites yield that has yet to be explored in soft corals.ObjectivesStudy the elicitation impact of salicylic acid (SA) and six analogues in addition to a systemic acquired resistance inducer on secondary metabolites accumulation in the soft coral Sarcophyton ehrenbergi along with the symbiont zooxanthellae and if SA elicitation effect is extended to other coral species S. glaucum and Lobophyton pauciliforum.MethodsPost elicitation in the three corals and zooxanthella, metabolites were extracted and analyzed via UHPLC-MS coupled with chemometric tools.ResultsMultivariate data analysis of the UHPLC-MS data set revealed clear segregation of SA, amino-SA, and acetyl-SA elicited samples. An increased level ca. 6- and 8-fold of the diterpenes viz., sarcophytonolide I, sarcophine and a C28-sterol, was observed in SA and amino-SA groups, respectively. Post elicitation, the level of diepoxy-cembratriene increased 1.5-fold and 2.4-fold in 1 mM SA, and acetyl-SA (aspirin) treatment groups, respectively. S. glaucum and Lobophyton pauciliforum showed a 2-fold increase of diepoxy-cembratriene levels.ConclusionThese results suggest that SA could function as a general and somewhat selective diterpene inducing signaling molecule in soft corals. Structural consideration reveals initial structure–activity relationship (SAR) in SA derivatives that seem important for efficient diterpene and sterol elicitation.

Plant glandular trichomes are epidermal differentiations that are dedicated to the production of specialized metabolites, which constitute a first line of defense against pathogens and herbivores. The secretions of these metabolic factories are chemically very diverse, including of terpenoid, fatty acid, or phenylpropanoid origins. They find uses in various industrial areas, for example as pharmaceutical, flavor, or fragrance ingredients or as insecticides. Recent progress in the elucidation of biosynthesis pathways for these compounds has opened up novel opportunities for metabolic engineering in microorganisms as well as in plants.