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INTRODUCTION: Assessing intraspecific variation in plant volatile organic compounds (VOCs) involves pitfalls that may bias biological interpretation, particularly when several laboratories collaborate on joint projects. Comparative, inter-laboratory ring trials can inform on the reproducibility of such analyses. OBJECTIVES: In a ring trial involving five laboratories, we investigated the reproducibility of VOC collections with polydimethylsiloxane (PDMS) and analyses by thermal desorption-gas chromatography-mass spectrometry (TD-GC-MS). As model plant we used Tanacetum vulgare, which shows a remarkable diversity in terpenoids, forming so-called chemotypes. We performed our ring-trial with two chemotypes to examine the sources of technical variation in plant VOC measurements during pre-analytical, analytical, and post-analytical steps. METHODS: Monoclonal root cuttings were generated in one laboratory and distributed to five laboratories, in which plants were grown under laboratory-specific conditions. VOCs were collected on PDMS tubes from all plants before and after a jasmonic acid (JA) treatment. Thereafter, each laboratory (donors) sent a subset of tubes to four of the other laboratories (recipients), which performed TD-GC-MS with their own established procedures. RESULTS: Chemotype-specific differences in VOC profiles were detected but with an overall high variation both across donor and recipient laboratories. JA-induced changes in VOC profiles were not reproducible. Laboratory-specific growth conditions led to phenotypic variation that affected the resulting VOC profiles. CONCLUSION: Our ring trial shows that despite large efforts to standardise each VOC measurement step, the outcomes differed both qualitatively and quantitatively. Our results reveal sources of variation in plant VOC research and may help to avoid systematic errors in similar experiments.
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Compuestos Orgánicos Volátiles , Compuestos Orgánicos Volátiles/análisis , Reproducibilidad de los Resultados , Metabolómica , Terpenos/análisis , Cromatografía de Gases y Espectrometría de Masas/métodos , PlantasRESUMEN
MAIN CONCLUSION: Solanum dulcamara primary and adventitious roots showed qualitative and quantitative differences in their steroidal glycosides profile. This opened new venues to evaluate the bioactivity of these molecules in belowground ecosystems. The Solanum genus is characterized by the presence of steroidal glycosides (SGs) that confer herbivore resistance and serve as drug precursors in the pharmaceutical industry. Solanum dulcamara is a self-compatible, sexually reproducing species that produces seeds after buzz-pollination. In addition, primordia on the stem facilitate clonal propagation via adventitious root (AR) formation. ARs contain aerenchyma being developmentally and morphologically different from primary roots (PRs). Therefore, we hypothesized that ARs and PRs have different SG profiles. Aiming to assess differences in SGs profiles in S. dulcamara roots in relation to their origins and morphologies, we used liquid chromatography coupled to electron spray ionization quadruple time of flight mass spectrometry (LC-ESI-qToF-MS) to profile SGs from PRs and ARs of seven S. dulcamara individuals. Mass fragmentation pattern analysis indicated the presence of 31 SG-type structures, including those with spirostans and furostans moieties. We assigned the 31 structures to 9 classes of steroidal aglycons (SAgls) that differ in hydroxylation and degree of unsaturation. We found that SAgls were conjugated with di-, tri- and tetra saccharides whereby one compound contained a malonylated sugar. Principle component analysis showed that SG profiles of PRs and ARs separated on the first principal component, supporting our hypothesis. Specifically, PRs contain higher number of SGs than ARs with some compounds exclusively present in PRs. Our results reveal a high level of novel chemodiversity in PRs and ARs of Solanum dulcamara. The knowledge gained will deepen our understanding of SGs biosynthesis and their functional role in plant-environment interactions.