# The Chemistry of Stress: Understanding the 'Cry for Help' of Plant Roots
## 5 Challenges, Opportunities, and Future Directions  
New developments in metabolomics offer a great potential to gain new insights into the mechanisms underlying stress-induced belowground chemical interactions as it allows for the discovery of novel compounds or combinations of compounds that directly or indirectly alleviate (a) biotic stresses. In general, detection of plant metabolites is extremely challenging, as there is no single-instrument platform available to effectively measure the overall coverage of plant metabolites and standards for many root exudates are currently lacking. To date, mass spectrometry-based metabolomic techniques (such as LC-MS and GC-MS) are the most sensitive for simultaneous analysis of a large number of soluble and volatile compounds [72]. Especially mass spectrometry imaging (MSI) has emerged as a valuable tool, with numerous applications in the field of plant metabolomics [73]. MSI was used for spatio-temporal metabolite and elemental profiling of salt stressed barley during initial stages of plant germination [74]. MSI analytical techniques enable high-resolution spatial mapping of a large variety of biomolecules, providing qualitative and quantitative chemical information, in a single experiment. MSI can help to spatially elucidate the metabolite composition of the intact roots with minimal to no sample preparation [75]. Yet, the precise identification of the relevant plant metabolites and the assessment of their exact function remain a difficult and time-consuming process. In contrast to genes and proteins, metabolites have much greater structural diversity: they are not simply combinations of 4-20 letters of the gene or protein alphabet. The developments and combinations of novel metabolomics approaches and bioinformatics pipelines to search multiple databases for the identification of compounds in a metabolomics profile are crucial and urgently needed (e.g., [76; 77]). Most studies, so far, are focused on plant metabolites induced by individual stress under controlled conditions, but in nature, plants are typically subjected to a multiple stress factors at the same time. To date, little is known about plant metabolic responses to multiple stresses that occur either simultaneously or sequentially. Although several stresses have similarities (i.e., damaging plant tissue), each stress can lead to specific metabolic responses. Furthermore, the chemical responses to stress can depend on stress severity and duration [78]. For example, mild and short time aboveground stress seldom elicits release of stress VOCs, whereas severe stresses lead to major qualitative and quantitative changes in VOCs emission [79]. While responses to stresses are studied predominantly with young plants very little is learned about how mature plants perceive stresses and recruit or activate beneficial members of the root microbiome through chemical cues. Root exudates have been shown to change significantly during plant development and having an impact on microbiome composition [80]. In the case of 18 grass species, drought had a conserved effect on the plant microbiome in younger plants while microbiome of the drought stressed mature plants were more species-specific yet diverse members of Actinobacteria were enriched in both cases [81]. Whether these observations relate to different qualities and/or quantities of plant exudates or due to structural changes related to plant development remains unknown. The majority of studies on stress-induced plant metabolites are based on experiments with domesticated plants and hence, we know very little about the responses of the wild relatives in natural ecosystems. Both in agricultural and natural ecosystems, plants are subject to multiple biotic and abiotic stresses. Even if they are resilient to a single stress, plants are often compromised to tolerate multiple stresses occurring at the same time.