The multidimensionality of plant drought stress: The relative importance of edaphic and atmospheric drought.

Drought threatens plant growth and related ecosystem services. The emergence of plant drought stress under edaphic drought is well studied, whilst the importance of atmospheric drought only recently gained momentum. Yet, little is known about the interaction and relative contribution of edaphic and atmospheric drought on the emergence of plant drought stress. We conducted a gradient experiment, fully crossing gravimetric water content (GWC: maximum water holding capacity-permanent wilting point) and vapour pressure deficit (VPD: 1-2.25 kPa) using five wheat varieties from three species (Triticum monococcum, T. durum & T. aestivum). We quantified the occurrence of plant drought stress on molecular (abscisic acid), cellular (stomatal conductance), organ (leaf water potential) and stand level (evapotranspiration). Plant drought stress increased with decreasing GWC across all organizational levels. This effect was magnified nonlinearly by VPD after passing a critical threshold of soil water availability. At around 20%GWC (soil matric potential 0.012 MPa), plants lost their ability to regulate leaf water potential via stomata regulation, followed by the emergence of hydraulic dysfunction. The emergence of plant drought stress is characterized by changing relative contributions of soil versus atmosphere and their non-linear interaction. This highly non-linear response is likely to abruptly alter plant-related ecosystem services in a drying world.

Inter-laboratory comparison of plant volatile analyses in the light of intra-specific chemodiversity.

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.

Dietary cardenolides enhance growth and change the direction of the fecundity-longevity trade-off in milkweed bugs (Heteroptera: Lygaeinae).

Sequestration, that is, the accumulation of plant toxins into body tissues for defense, was predicted to incur physiological costs and may require resistance traits different from those of non-sequestering insects. Alternatively, sequestering species could experience a cost in the absence of toxins due to selection on physiological homeostasis under permanent exposure of sequestered toxins in body tissues. Milkweed bugs (Heteroptera: Lygaeinae) sequester high amounts of plant-derived cardenolides. Although being potent inhibitors of the ubiquitous animal enzyme Na+/K+-ATPase, milkweed bugs can tolerate cardenolides by means of resistant Na+/K+-ATPases. Both adaptations, resistance and sequestration, are ancestral traits of the Lygaeinae. Using four milkweed bug species (Heteroptera: Lygaeidae: Lygaeinae) and the related European firebug (Heteroptera: Pyrrhocoridae: Pyrrhocoris apterus) showing different combinations of the traits "cardenolide resistance" and "cardenolide sequestration," we tested how the two traits affect larval growth upon exposure to dietary cardenolides in an artificial diet system. While cardenolides impaired the growth of P. apterus nymphs neither possessing a resistant Na+/K+-ATPase nor sequestering cardenolides, growth was not affected in the non-sequestering milkweed bug Arocatus longiceps, which possesses a resistant Na+/K+-ATPase. Remarkably, cardenolides increased growth in the sequestering dietary specialists Caenocoris nerii and Oncopeltus fasciatus but not in the sequestering dietary generalist Spilostethus pandurus, which all possess a resistant Na+/K+-ATPase. We furthermore assessed the effect of dietary cardenolides on additional life history parameters, including developmental speed, longevity of adults, and reproductive success in O. fasciatus. Unexpectedly, nymphs under cardenolide exposure developed substantially faster and lived longer as adults. However, fecundity of adults was reduced when maintained on cardenolide-containing diet for their entire lifetime but not when adults were transferred to non-toxic sunflower seeds. We speculate that the resistant Na+/K+-ATPase of milkweed bugs is selected for working optimally in a "toxic environment," that is, when sequestered cardenolides are stored in the body.

Arabidopsis, tobacco, nightshade and elm take insect eggs as herbivore alarm and show similar transcriptomic alarm responses.

Plants respond to insect eggs with transcriptional changes, resulting in enhanced defence against hatching larvae. However, it is unknown whether phylogenetically distant plant species show conserved transcriptomic responses to insect eggs and subsequent larval feeding. We used Generally Applicable Gene set Enrichment (GAGE) on gene ontology terms to answer this question and analysed transcriptome data from Arabidopsis thaliana, wild tobacco (Nicotiana attenuata), bittersweet nightshade (Solanum dulcamara) and elm trees (Ulmus minor) infested by different insect species. The different plant-insect species combinations showed considerable overlap in their transcriptomic responses to both eggs and larval feeding. Within these conformable responses across the plant-insect combinations, the responses to eggs and feeding were largely analogous, and about one-fifth of these analogous responses were further enhanced when egg deposition preceded larval feeding. This conserved transcriptomic response to eggs and larval feeding comprised gene sets related to several phytohormones and to the phenylpropanoid biosynthesis pathway, of which specific branches were activated in different plant-insect combinations. Since insect eggs and larval feeding activate conserved sets of biological processes in different plant species, we conclude that plants with different lifestyles share common transcriptomic alarm responses to insect eggs, which likely enhance their defence against hatching larvae.

Slug Feeding Triggers Dynamic Metabolomic and Transcriptomic Responses Leading to Induced Resistance in Solanum dulcamara.

Induced plant responses to insect herbivores are well studied, but we know very little about responses to gastropod feeding. We aim to identify the temporal dynamics of signaling- and defense-related plant responses after slug feeding in relation to induced resistance. We exposed Solanum dulcamara plants to feeding by the gray field slug (GFS; Deroceras reticulatum) for different periods and tested disks of local and systemic leaves in preference assays. Induced responses were analyzed using metabolomics and transcriptomics. GFS feeding induced local and systemic responses. Slug feeding for 72 h more strongly affected the plant metabolome than 24 h feeding. It increased the levels of a glycoalkaloid (solasonine), phenolamides, anthocyanins, and trypsin protease inhibitors as well as polyphenol oxidase activity. Phytohormone and transcriptome analyses revealed that jasmonic acid, abscisic acid and salicylic acid signaling were activated. GFS feeding upregulated more genes than that it downregulated. The response directly after feeding was more than five times higher than after an additional 24 h without feeding. Our research showed that GFS, like most chewing insects, triggers anti-herbivore defenses by activating defense signaling pathways, resulting in increased resistance to further slug feeding. Slug herbivory may therefore impact other herbivores in the community.

Insect egg deposition renders plant defence against hatching larvae more effective in a salicylic acid-dependent manner.

Plants can improve their antiherbivore defence by taking insect egg deposition as cue of impending feeding damage. Previous studies showed that Pieris brassicae larvae feeding upon egg-deposited Brassicaceae perform worse and gain less weight than larvae on egg-free plants. We investigated how P. brassicae oviposition on Arabidopsis thaliana affects the plant's molecular and chemical responses to larvae. A transcriptome comparison of feeding-damaged leaves without and with prior oviposition revealed about 200 differently expressed genes, including enhanced expression of PR5, which is involved in salicylic acid (SA)-signalling. SA levels were induced by larval feeding to a slightly greater extent in egg-deposited than egg-free plants. The adverse effect of egg-deposited wild-type (WT) plants on larval weight was absent in an egg-deposited PR5-deficient mutant or other mutants impaired in SA-mediated signalling, that is, sid2/ics1, ald1, and pad4. In contrast, the adverse effect of egg-deposited WT plants on larvae was retained in egg-deposited npr1 and wrky70 mutants impaired further downstream in SA-signalling. Oviposition induced accumulation of flavonols in WT plants with and without feeding damage, but not in the PR5-deficient mutant. We demonstrated that egg-mediated improvement of A. thaliana's antiherbivore defence involves SA-signalling in an NPR1-independent manner and is associated with accumulation of flavonols.

Interactive Responses of Solanum Dulcamara to Drought and Insect Feeding are Herbivore Species-Specific.

In nature, plants are frequently subjected to multiple biotic and abiotic stresses, resulting in a convergence of adaptive responses. We hypothesised that hormonal signalling regulating defences to different herbivores may interact with drought responses, causing distinct resistance phenotypes. To test this, we studied the hormonal and transcriptomic responses of Solanum dulcamara subjected to drought and herbivory by the generalist Spodoptera exigua (beet armyworm; BAW) or the specialist Leptinotarsa decemlineata (Colorado potato beetle; CPB). Bioassays showed that the performance of BAW, but not CPB, decreased on plants under drought compared to controls. While drought did not alter BAW-induced hormonal responses, it enhanced the CPB-induced accumulation of jasmonic acid and salicylic acid (SA), and suppressed ethylene (ET) emission. Microarray analyses showed that under drought, BAW herbivory enhanced several herbivore-induced responses, including cell-wall remodelling and the metabolism of carbohydrates, lipids, and secondary metabolites. In contrast, CPB herbivory enhanced several photosynthesis-related and pathogen responses in drought-stressed plants. This may divert resources away from defence production and increase leaf nutritive value. In conclusion, while BAW suffers from the drought-enhanced defences, CPB may benefit from the effects of enhanced SA and reduced ET signalling. This suggests that the fine-tuned interaction between the plant and its specialist herbivore is sustained under drought.

Oviposition by Spodoptera exigua on Solanum dulcamara Alters the Plant's Response to Herbivory and Impairs Larval Performance.

Plant resistance traits against insect herbivores are extremely plastic. Plants respond not only to the herbivory itself, but also to oviposition by herbivorous insects. How prior oviposition affects plant responses to larval herbivory is largely unknown. Combining bioassays and defense protein activity assays with microarray analyses and metabolite profiling, we investigated the impact of preceding oviposition on the interaction of Solanum dulcamara with the generalist lepidopteran herbivore Spodoptera exigua at the levels of the plant's resistance, transcriptome and metabolome. We found that oviposition increased plant resistance to the subsequent feeding larvae. While constitutive and feeding-induced levels of defensive protease inhibitor activity remained unaffected, pre-exposure to eggs altered S. dulcamara's transcriptional and metabolic response to larval feeding in leaves local and systemic to oviposition. In particular, genes involved in phenylpropanoid metabolism were more strongly expressed in previously oviposited plants, which was reflected by reciprocal changes of primary metabolites upstream and within these pathways. Our data highlight that plants integrate signals from non-threatening life stages of their natural enemies to optimize their response when they become actually attacked. The observed transcriptional and metabolic reshaping of S. dulcamara's response to S. exigua herbivory suggests a role of phenylpropanoids in oviposition-primed plant resistance.

Moth oviposition shapes the species-specific transcriptional and phytohormonal response of Nicotiana attenuata to larval feeding.

Oviposition by lepidopteran herbivores on Nicotiana attenuata primes plant defence responses that are induced by the feeding larvae. While oviposition by both the generalist Spodoptera exigua and the specialist Manduca sexta primes the production of defensive phenylpropanoids, their larvae are differentially affected. We investigate here the impact of prior oviposition on the transcriptome and phytohormone levels of plants that were later attacked by larvae to find regulatory signals of this priming. In a full-factorial design, we evaluated the effects of oviposition and herbivory by both species. Oviposition alone had only subtle effects at the transcriptional level. Laval feeding alone induced species-specific plant responses. Larvae of the generalist regulated phytohormones and gene expression stronger than larvae of the specialist. A day after larvae started to feed, we detected no significant alterations of the plant's response to larval feeding due to prior oviposition by conspecific moths. Yet, oviposition by each of the species profoundly influenced the plant's transcriptional and phytohormonal response to feeding larvae of the other species. Remarkably, the species-specific plant responses to larval feeding shifted towards the response normally elicited by larvae of the ovipositing species. Thus, plants may already recognise an insect's identity upon its oviposition.

The Plant Immunity Regulating F-Box Protein CPR1 Supports Plastid Function in Absence of Pathogens.

The redox imbalanced 6 mutant (rimb6) of Arabidopsis thaliana was isolated in a genetic screening approach for mutants with defects in chloroplast-to-nucleus redox signaling. It has an atypically low activation status of the 2-Cys peroxiredoxin-A promoter in the seedling stage. rimb6 shows wildtype-like germination, seedling development and greening, but slower growth and reduced biomass in the rosette stage. Mapping of the casual mutation revealed that rimb6 carries a single nucleotide polymorphism in the gene encoding CONSTITUTIVE EXPRESSER OF PATHOGENESIS RELATED (PR) GENES 1, CPR1 (At4g12560), leading to a premature stop codon. CPR1 is known as a repressor of pathogen signaling and regulator of microtubule organization. Allelism of rimb6 and cpr1 revealed a function of CPR1 in chloroplast stress protection. Expression studies in pathogen signaling mutants demonstrated that CPR1-mediated activation of genes for photosynthesis and chloroplast antioxidant protection is, in contrast to activation of pathogen responses, regulated independently from PAD4-controlled salicylic acid (SA) accumulation. We conclude that the support of plastid function is a basic, SA-independent function of CPR1.

NaMYB8 regulates distinct, optimally distributed herbivore defense traits.

When herbivores attack, plants specifically reconfigure their metabolism. Herbivory on the wild tobacco Nicotiana attenuata strongly induces the R2R3 MYB transcriptional activator MYB8, which was reported to specifically regulate the accumulation of phenolamides (PAs). We discovered that transcriptional regulation of trypsin protease inhibitors (TPIs) and a threonine deaminase (TD) also depend on MYB8 expression. Induced distributions of PAs, TD and TPIs all meet predictions of optimal defense theory: their leaf concentrations increase with the fitness value and the probability of attack of the tissue. Therefore, we suggest that these defensive compounds have evolved to be co-regulated by MYB8.

Solanum dulcamara's response to eggs of an insect herbivore comprises ovicidal hydrogen peroxide production.

Plants can respond to insect oviposition, but little is known about which responses directly target the insect eggs and how. Here, we reveal a mechanism by which the bittersweet nightshade Solanum dulcamara kills the eggs of a generalist noctuid herbivore. The plant responded at the site of oviposition by Spodoptera exigua with formation of neoplasms and chlorotic tissue, accumulation of reactive oxygen species and induction of defence genes and proteins. Transcriptome analysis revealed that these responses were reflected in the transcriptional reprogramming of the egg-laden leaf. The plant-mediated egg mortality on S. dulcamara was not present on a genotype lacking chlorotic leaf tissue at the oviposition sites on which the eggs are exposed to less hydrogen peroxide. As exposure to hydrogen peroxide increased egg mortality, while catalase supplementation prevented the plants from killing the eggs, our results suggest that reactive oxygen species formation directly acts as an ovicidal plant response of S. dulcamara.

Transcriptomic responses of Solanum dulcamara to natural and simulated herbivory.

Plants are attacked by diverse herbivores and respond with manifold defence responses. To study transcriptional and other early regulation events of these plant responses, herbivory is often simulated to standardize the temporal and spatial dynamics that vary tremendously for natural herbivory. Yet, to what extent such simulations of herbivory are able to elicit the same plant response as real herbivory remains largely undetermined. We examined the transcriptional response of a wild model plant to herbivory by lepidopteran larvae and to a commonly used herbivory simulation by applying the larvae's oral secretions to standardized wounds. We designed a microarray for Solanum dulcamara and showed that the transcriptional responses to real and to simulated herbivory by Spodoptera exigua overlapped moderately by about 40%. Interestingly, certain responses were mimicked better than others; 60% of the genes upregulated but not even a quarter of the genes downregulated by herbivory were similarly affected by application of oral secretions to wounds. While the regulation of genes involved in signalling, defence and water stress was mimicked well by the simulated herbivory, most of the genes related to photosynthesis, carbohydrate- and lipid metabolism were exclusively regulated by real herbivory. Thus, wounding and application of oral secretions decently mimics herbivory-induced defence responses but likely not the reallocation of primary metabolites induced by real herbivory.

Induced plant defences in biological control of arthropod pests: a double-edged sword.

Biological control is an important ecosystem service delivered by natural enemies. Together with breeding for plant defence, it constitutes one of the most promising alternatives to pesticides for controlling herbivores in sustainable crop production. Especially induced plant defences may be promising targets in plant breeding for resistance against arthropod pests. Because they are activated upon herbivore damage, costs are only incurred when defence is needed. Moreover, they can be more specific than constitutive defences. Nevertheless, inducible defence traits that are harming plant pest organisms may interfere with biological control agents, such as predators and parasitoids. Despite the vast fundamental knowledge on plant defence mechanisms and their effects on natural enemies, our understanding of the feasibility of combining biological control with induced plant defence in practice is relatively poor. In this review, we focus on arthropod pest control and present the most important features of biological control with natural enemies and of induced plant defence. Furthermore, we show potential synergies and conflicts among them and, finally, identify gaps and list opportunities for their combined use in crop protection. We suggest that breeders should focus on inducible resistance traits that are compatible with the natural enemies of arthropod pests, specifically traits that help communities of natural enemies to build up. © 2017 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Sequential above- and belowground herbivory modifies plant responses depending on herbivore identity.

BACKGROUND: Herbivore-induced changes in plant traits can cause indirect interactions between spatially and/or temporally separated herbivores that share the same host plant. Feeding modes of the herbivores is one of the major factors that influence the outcome of such interactions. Here, we tested whether the effects of transient aboveground herbivory for seven days by herbivores of different feeding guilds on tomato plants (Solanum lycopersicum) alters their interaction with spatially as well as temporally separated belowground herbivores. RESULTS: The transient aboveground herbivory by both chewing caterpillars (Spodoptera exigua) and sucking aphids (Myzus persicae) had significant impacts on plant traits such as plant growth, resource allocation and phytohormone contents. While the changes in plant traits did not affect the overall performance of the root-knot nematodes (Meloidogyne incognita) in terms of total number of galls, we found that the consequences of aboveground herbivory for the plants can be altered by the subsequent nematode herbivory. For example, plants that had hosted aphids showed compensatory growth when they were later challenged by nematodes, which was not apparent in plants that had hosted only aphids. In contrast, plants that had been fed by S. exigua larvae did not show such compensatory growth even when challenged by nematodes. CONCLUSION: The results suggest that the earlier aboveground herbivory can modify plant responses to subsequent herbivores, and such modifications may depend upon identity and/or feeding modes of the aboveground herbivores.

Jasmonate signalling in plants shapes plant-insect interaction ecology.

The phytohormone jasmonic acid (JA) regulates the induction of direct and indirect defences against herbivores. By now, the biochemical pathway of JA-signalling has been well resolved, allowing the use of an interdisciplinary toolbox and spurring the mechanistic investigation of plant-insect interactions. Recent advances show that JA-mediated plant responses are involved in the competitive and trophic interactions between various organisms throughout at least four trophic levels and therefore likely shape natural communities. Moreover, JA-mediated responses can be primed or suppressed by various environmental factors that are related to herbivory or not. Yet, to integrate the complex interactions at the physiological and ecological levels into community ecology, an examination of the often onetime discoveries for general rules and new bioinformatic approaches are required.

Extrafloral nectar secretion from wounds of Solanum dulcamara.

Plants usually close wounds rapidly to prevent infections and the loss of valuable resources such as assimilates(1). However, herbivore-inflicted wounds on the bittersweet nightshade Solanum dulcamara appear not to close completely and produce sugary wound secretions visible as droplets. Many plants across the plant kingdom secrete sugary nectar from extrafloral nectaries(2) to attract natural enemies of herbivores for indirect defence(3,4). As ants forage on wound edges of S. dulcamara in the field, we hypothesized that wound secretions are a form of extrafloral nectar (EFN). We show that, unlike EFN from known nectaries, wound secretions are neither associated with any specific structure nor restricted to certain locations. However, similar to EFN, they are jasmonate-inducible and the plant controls their chemical composition. Wound secretions are attractive for ants, and application of wound secretion mimics increases ant attraction and reduces herbivory on S. dulcamara plants in a natural population. In greenhouse experiments, we reveal that ants can defend S. dulcamara from two of its native herbivores, slugs and flea beetle larvae. Since nectar is defined by its ecological function as a sugary secretion involved in interactions with animals(5), such 'plant bleeding' could be a primitive mode of nectar secretion exemplifying an evolutionary origin of structured extrafloral nectaries.

The role of phytophagy by predators in shaping plant interactions with their pests.

Zoophytophagy is common among predacious arthropods, but research on their role in plant-herbivore interactions is generally focused on predation effects whereas their phytophagy is largely neglected. Our recent study revealed the ability of zoophytophagous predators to induce defense related traits and to affect herbivore performance apart from predation through the plant. Additionally, we show here that predator-exposed plants suffer less damage compared to unexposed plants. Thus, zoophytophagous organisms likely shape community structure by both their predation on herbivores and their phytophagy. Here, we consider zoophytophagous predators as plant vaccination factors and outline how their dual role in affecting herbivores may impact their use in biological pest control. Because plant responses to phytophagy and phytopathogens are known to interact, zoophytophagous predators may also affect plant-pathogen interactions. When we consider these indirect interactions with different plant pest organisms, we will likely better understand the ecology of the complex relationships among plants, herbivores and predators. Moreover, a comprehensive knowledge on the effects of the phytophagy of predators in these ecological interactions will potentially allow us to enhance sustainability in pest control.

Drought and flooding have distinct effects on herbivore-induced responses and resistance in Solanum dulcamara.

In the field, biotic and abiotic stresses frequently co-occur. As a consequence, common molecular signalling pathways governing adaptive responses to individual stresses can interact, resulting in compromised phenotypes. How plant signalling pathways interact under combined stresses is poorly understood. To assess this, we studied the consequence of drought and soil flooding on resistance of Solanum dulcamara to Spodoptera exigua and their effects on hormonal and transcriptomic profiles. The results showed that S. exigua larvae performed less well on drought-stressed plants than on well-watered and flooded plants. Both drought and insect feeding increased abscisic acid and jasmonic acid (JA) levels, whereas flooding did not induce JA accumulation. RNA sequencing analyses corroborated this pattern: drought and herbivory induced many biological processes that were repressed by flooding. When applied in combination, drought and herbivory had an additive effect on specific processes involved in secondary metabolism and defence responses, including protease inhibitor activity. In conclusion, drought and flooding have distinct effects on herbivore-induced responses and resistance. Especially, the interaction between abscisic acid and JA signalling may be important to optimize plant responses to combined drought and insect herbivory, making drought-stressed plants more resistant to insects than well-watered and flooded plants.

Priming and memory of stress responses in organisms lacking a nervous system.

Experience and memory of environmental stimuli that indicate future stress can prepare (prime) organismic stress responses even in species lacking a nervous system. The process through which such organisms prepare their phenotype for an improved response to future stress has been termed 'priming'. However, other terms are also used for this phenomenon, especially when considering priming in different types of organisms and when referring to different stressors. Here we propose a conceptual framework for priming of stress responses in bacteria, fungi and plants which allows comparison of priming with other terms, e.g. adaptation, acclimation, induction, acquired resistance and cross protection. We address spatial and temporal aspects of priming and highlight current knowledge about the mechanisms necessary for information storage which range from epigenetic marks to the accumulation of (dormant) signalling molecules. Furthermore, we outline possible patterns of primed stress responses. Finally, we link the ability of organisms to become primed for stress responses (their 'primability') with evolutionary ecology aspects and discuss which properties of an organism and its environment may favour the evolution of priming of stress responses.