Plants have evolved a plethora of different chemical defenses covering nearly all classes of (secondary) metabolites that represent a major barrier to herbivory: Some are constitutive; others are induced after attack. Many compounds act directly on the herbivore, whereas others act indirectly via the attraction of organisms from other trophic levels that, in turn, protect the plant. An enormous diversity of plant (bio)chemicals are toxic, repellent, or antinutritive for herbivores of all types. Examples include cyanogenic glycosides, glucosinolates, alkaloids, and terpenoids; others are macromolecules and comprise latex or proteinase inhibitors. Their modes of action include membrane disruption, inhibition of nutrient and ion transport, inhibition of signal transduction processes, inhibition of metabolism, or disruption of the hormonal control of physiological processes. Recognizing the herbivore challenge and precise timing of plant activities as well as the adaptive modulation of the plants' metabolism is important so that metabolites and energy may be efficiently allocated to defensive activities.

In the interaction between Arabidopsis (Arabidopsis thaliana) and the generalist herbivorous insect Spodoptera littoralis, little is known about early events in defense signaling and their link to downstream phytohormone pathways. S. littoralis oral secretions induced both Ca²⁺ and phytohormone elevation in Arabidopsis. Plant gene expression induced by oral secretions revealed up-regulation of a gene encoding a calmodulin-like protein, CML42. Functional analysis of cml42 plants revealed more resistance to herbivory than in the wild type, because caterpillars gain less weight on the mutant, indicating that CML42 negatively regulates plant defense; cml42 also showed increased aliphatic glucosinolate content and hyperactivated transcript accumulation of the jasmonic acid (JA)-responsive genes VSP2 and Thi2.1 upon herbivory, which might contribute to increased resistance. CML42 up-regulation is negatively regulated by the jasmonate receptor Coronatine Insensitive1 (COI1), as loss of functional COI1 resulted in prolonged CML42 activation. CML42 thus acts as a negative regulator of plant defense by decreasing COI1-mediated JA sensitivity and the expression of JA-responsive genes and is independent of herbivory-induced JA biosynthesis. JA-induced Ca²⁺ elevation and root growth inhibition were more sensitive in cml42, also indicating higher JA perception. Our results indicate that CML42 acts as a crucial signaling component connecting Ca²⁺ and JA signaling. CML42 is localized to cytosol and nucleus. CML42 is also involved in abiotic stress responses, as kaempferol glycosides were down-regulated in cml42, and impaired in ultraviolet B resistance. Under drought stress, the level of abscisic acid accumulation was higher in cml42 plants. Thus, CML42 might serve as a Ca²⁺ sensor having multiple functions in insect herbivory defense and abiotic stress responses.

• Direct and indirect defences against feeding induced by chewing (Spodoptera littoralis) and piercing-sucking (Tetranychus urticae) herbivores, as well as components of signal transduction, were investigated in the model legume Medicago truncatula. • Emitted volatiles, representing a mechanism of indirect defence, were measured and identified by gas chromatography/mass spectrometry (GC-MS). As elements of direct defence, the accumulation of phenolic compounds and of reactive oxygen species (ROS) was assessed using microscopic techniques. Jasmonic acid (JA) and salicylic acid (SA) concentrations were assessed as putative components of signal transduction. • Volatile profiles revealed a sizeable number of different substances emitted, particularly sesquiterpenoids. The qualitative composition clearly differed depending on the type of herbivory. The same held true for JA and SA concentrations. Also, deposition of phenolic compounds and the production of ROS around the wounding sites could be detected. • Conspicuous differences were found in indirect defence and signalling for different types of herbivory. In contrast, no divergence in direct defences was observed; furthermore, the traits investigated exhibited striking similarities to reactions known to occur upon pathogen attack.

Phytohormones are long time known as important components of signalling cascades in plant development and plant responses to various abiotic and biotic challenges. Quantifications of phytohormone levels in plants are typically carried out using GC or LC-MS/MS systems, due to their high sensitivity, specificity, and the fact that not much sample preparation is needed. However, mass spectrometer-based analyses are often affected by the particular sample type (different matrices), extraction procedure, and experimental setups, i.e. the chromatographic separation system and/or mass spectrometer analyser (Triple-quadrupole, Iontrap, TOF, Orbitrap). For these reasons, a validated method is required in order to enable comparison of data that are generated in different laboratories, under different experimental set-ups, and in different matrices. So far, many phytohormone quantification studies were done using either QTRAP or Triple-quadrupole mass spectrometers. None of them was performed under the regime of a fully-validated method. Therefore, we developed and established such validated method for quantification of stress-related phytohormones such as jasmonates, abscisic acid, salicylic acid, IAA, in the model plant Arabidopsis thaliana and the fruit crop Citrus sinensis, using an Iontrap mass spectrometer. All parameters recommended by FDA (US Food and Drug Administration) or EMEA (European Medicines Evaluation Agency) for validation of analytical methods were evaluated: sensitivity, selectivity, repeatability and reproducibility (accuracy and precision).