Jasmonic acid and heat stress induce high volatile organic compound emissions in Picea abies from needles, but not from roots.

Plants emit diverse volatile organic compounds (VOCs) from their leaves and roots for protection against biotic and abiotic stress. An important signaling cascade activated by aboveground herbivory is the jasmonic acid (JA) pathway that stimulates the production of VOCs. So far it remains unclear if the activation of this pathway also leads to enhanced VOC emissions from conifer roots, and how the interplay of above- and belowground defenses in plants are affected by multiple stressors. Therefore, we simultaneously analyzed needle and root VOC emissions of Picea abies saplings, as well as CO2 and H2O fluxes in response to aboveground JA treatment, heat stress and their interaction in a controlled climate chamber experiment. Continuous online VOC measurements by PTR-TOF-MS showed an inverse pattern of total needle and root VOC emissions, when plants were treated with JA and heat. While needle sesquiterpene emissions increased nine-fold one day after JA application, total root VOC emissions decreased. This was mainly due to reduced emissions of acetone and monoterpenes by roots. In response to aboveground JA treatment, root total carbon emitted as VOCs decreased from 31% to only 4%. While VOC emissions aboveground increased, net CO2 assimilation strongly declined due to JA treatment, resulting in net respiration during the day. Interestingly, root respiration was not affected by aboveground JA application. Under heat the effect of JA on VOC emissions of needles and roots was less pronounced. The buffering effect of heat on VOC emissions following JA treatment points towards an impaired defense reaction of the plants under multiple stress. Our results indicate efficient resource allocation within the plant to protect threatened tissues by a rather local VOC release. Roots may only be affected indirectly by reduced belowground carbon allocation, but are not involved directly in the JA-induced stress response.

Know When You Are Too Many: Density-Dependent Release of Pheromones During Host Colonisation by the European Spruce Bark Beetle, Ips typographus (L.).

Individuals across various animal species communicate their presence to conspecifics. Especially phytophagous and parasitoid insects with their brood developing on limited resources rely on chemical cues, such as host-marking pheromones, to reduce intraspecific competition. Bark beetles are phytophagous insects with some species being economically and ecologically relevant forest pests. Several of them use the volatile compound verbenone to inhibit attraction and reduce intraspecific competition. However, in the Eurasian spruce bark beetle, Ips typographus (L.), temporal emission patterns did so far not quite support the putative function of verbenone as an indicator of densely colonised host trees. More importantly, it is currently unclear how well verbenone emission is actually related to colonisation density and thus intraspecific competition. Here, we inoculated Norway spruce logs with I. typographus at two defined colonisation densities in the greenhouse and measured the emission of verbenone and its precursors α-pinene and verbenol over time. Verbenone emission was 3-7 times greater from colonised logs compared to decaying logs without beetles during the major part of larval development. Furthermore, our data supports the quantitative hypothesis, that the termination of attack on a tree is mediated by a cessation of the release of verbenol and continuous emission of verbenone. The latter is most likely a passively produced host-marking cue reflecting the actual density of conspecifics since per-beetle emission was unaffected by colonisation density. These findings shed new light on the regulation of bark beetle mass aggregations, which are currently causing previously unseen economic damages in temperate forests.

Effects of drought and recovery on soil volatile organic compound fluxes in an experimental rainforest.

Drought can affect the capacity of soils to emit and consume biogenic volatile organic compounds (VOCs). Here we show the impact of prolonged drought followed by rewetting and recovery on soil VOC fluxes in an experimental rainforest. Under wet conditions the rainforest soil acts as a net VOC sink, in particular for isoprenoids, carbonyls and alcohols. The sink capacity progressively decreases during drought, and at soil moistures below ~19%, the soil becomes a source of several VOCs. Position specific 13C-pyruvate labeling experiments reveal that soil microbes are responsible for the emissions and that the VOC production is higher during drought. Soil rewetting induces a rapid and short abiotic emission peak of carbonyl compounds, and a slow and long biotic emission peak of sulfur-containing compounds. Results show that, the extended drought periods predicted for tropical rainforest regions will strongly affect soil VOC fluxes thereby impacting atmospheric chemistry and climate.

Leaf-level metabolic changes in response to drought affect daytime CO2 emission and isoprenoid synthesis pathways.

In the near future, climate change will cause enhanced frequency and/or severity of droughts in terrestrial ecosystems, including tropical forests. Drought responses by tropical trees may affect their carbon use, including production of volatile organic compounds (VOCs), with implications for carbon cycling and atmospheric chemistry that are challenging to predict. It remains unclear how metabolic adjustments by mature tropical trees in response to drought will affect their carbon fluxes associated with daytime CO2 production and VOC emission. To address this gap, we used position-specific 13C-pyruvate labeling to investigate leaf CO2 and VOC fluxes from four tropical species before and during a controlled drought in the enclosed rainforest of Biosphere 2 (B2). Overall, plants that were more drought-sensitive had greater reductions in daytime CO2 production. Although daytime CO2 production was always dominated by non-mitochondrial processes, the relative contribution of CO2 from the tricarboxylic acid cycle tended to increase under drought. A notable exception was the legume tree Clitoria fairchildiana R.A. Howard, which had less anabolic CO2 production than the other species even under pre-drought conditions, perhaps due to more efficient refixation of CO2 and anaplerotic use for amino acid synthesis. The C. fairchildiana was also the only species to allocate detectable amounts of 13C label to VOCs and was a major source of VOCs in B2. In C. fairchildiana leaves, our data indicate that intermediates from the mevalonic acid (MVA) pathway are used to produce the volatile monoterpene trans-ß-ocimene, but not isoprene. This apparent crosstalk between the MVA and methylerythritol phosphate pathways for monoterpene synthesis declined with drought. Finally, although trans-ß-ocimene emissions increased under drought, it was increasingly sourced from stored intermediates and not de novo synthesis. Unique metabolic responses of legumes may play a disproportionate role in the overall changes in daytime CO2 and VOC fluxes in tropical forests experiencing drought.

Drought re-routes soil microbial carbon metabolism towards emission of volatile metabolites in an artificial tropical rainforest.

Drought impacts on microbial activity can alter soil carbon fate and lead to the loss of stored carbon to the atmosphere as CO2 and volatile organic compounds (VOCs). Here we examined drought impacts on carbon allocation by soil microbes in the Biosphere 2 artificial tropical rainforest by tracking 13C from position-specific 13C-pyruvate into CO2 and VOCs in parallel with multi-omics. During drought, efflux of 13C-enriched acetate, acetone and C4H6O2 (diacetyl) increased. These changes represent increased production and buildup of intermediate metabolites driven by decreased carbon cycling efficiency. Simultaneously,13C-CO2 efflux decreased, driven by a decrease in microbial activity. However, the microbial carbon allocation to energy gain relative to biosynthesis was unchanged, signifying maintained energy demand for biosynthesis of VOCs and other drought-stress-induced pathways. Overall, while carbon loss to the atmosphere via CO2 decreased during drought, carbon loss via efflux of VOCs increased, indicating microbially induced shifts in soil carbon fate.

Volatiles of fungal cultivars act as cues for host-selection in the fungus-farming ambrosia beetle Xylosandrus germanus.

Many wood-boring insects use aggregation pheromones during mass colonization of host trees. Bark beetles (Curculionidae: Scolytinae) are a model system, but much less is known about the role of semiochemicals during host selection by ambrosia beetles. As an ecological clade within the bark beetles, ambrosia beetles are obligately dependent on fungal mutualists for their sole source of nutrition. Mass colonization of trees growing in horticultural settings by exotic ambrosia beetles can occur, but aggregation cues have remained enigmatic. To elucidate this mechanism, we first characterized the fungal associates of the exotic, mass-aggregating ambrosia beetle Xylosandrus germanus in Southern Germany. Still-air olfactometer bioassays documented the attraction of X. germanus to its primary nutritional mutualist Ambrosiella grosmanniae and to a lesser extent another common fungal isolate (Acremonium sp.). During two-choice bioassays, X. germanus was preferentially attracted to branch sections (i.e., bolts) that were either pre-colonized by conspecifics or pre-inoculated with A. grosmanniae. Subsequent analyses identified microbial volatile organic compounds (MVOCs) that could potentially function as aggregation pheromones for X. germanus. To our knowledge, this is the first evidence for fungal volatiles as attractive cues during host selection by X. germanus. Adaptive benefits of responding to fungal cues associated with an infestation of conspecifics could be a function of locating a suitable substrate for cultivating fungal symbionts and/or increasing the likelihood of mating opportunities with the flightless males. However, this requires solutions for evolutionary conflict arising due to potential mixing of vertically transmitted and horizontally acquired symbiont strains, which are discussed.

Elucidating Drought-Tolerance Mechanisms in Plant Roots through 1H NMR Metabolomics in Parallel with MALDI-MS, and NanoSIMS Imaging Techniques.

As direct mediators between plants and soil, roots play an important role in metabolic responses to environmental stresses such as drought, yet these responses are vastly uncharacterized on a plant-specific level, especially for co-occurring species. Here, we aim to examine the effects of drought on root metabolic profiles and carbon allocation pathways of three tropical rainforest species by combining cutting-edge metabolomic and imaging technologies in an in situ position-specific 13C-pyruvate root-labeling experiment. Further, washed (rhizosphere-depleted) and unwashed roots were examined to test the impact of microbial presence on root metabolic pathways. Drought had a species-specific impact on the metabolic profiles and spatial distribution in Piper sp. and Hibiscus rosa sinensis roots, signifying different defense mechanisms; Piper sp. enhanced root structural defense via recalcitrant compounds including lignin, while H. rosa sinensis enhanced biochemical defense via secretion of antioxidants and fatty acids. In contrast, Clitoria fairchildiana, a legume tree, was not influenced as much by drought but rather by rhizosphere presence where carbohydrate storage was enhanced, indicating a close association with symbiotic microbes. This study demonstrates how multiple techniques can be combined to identify how plants cope with drought through different drought-tolerance strategies and the consequences of such changes on below-ground organic matter composition.

Ecosystem fluxes during drought and recovery in an experimental forest.

Severe droughts endanger ecosystem functioning worldwide. We investigated how drought affects carbon and water fluxes as well as soil-plant-atmosphere interactions by tracing 13CO2 and deep water 2H2O label pulses and volatile organic compounds (VOCs) in an enclosed experimental rainforest. Ecosystem dynamics were driven by different plant functional group responses to drought. Drought-sensitive canopy trees dominated total fluxes but also exhibited the strongest response to topsoil drying. Although all canopy-forming trees had access to deep water, these reserves were spared until late in the drought. Belowground carbon transport was slowed, yet allocation of fresh carbon to VOCs remained high. Atmospheric VOC composition reflected increasing stress responses and dynamic soil-plant-atmosphere interactions, potentially affecting atmospheric chemistry and climate feedbacks. These interactions and distinct functional group strategies thus modulate drought impacts and ecosystem susceptibility to climate change.

Drought affects carbon partitioning into volatile organic compound biosynthesis in Scots pine needles.

The effect of drought on the interplay of processes controlling carbon partitioning into plant primary and secondary metabolisms, such as respiratory CO2 release and volatile organic compound (VOC) biosynthesis, is not fully understood. To elucidate the effect of drought on the fate of cellular C sources into VOCs vs CO2 , we conducted tracer experiments with 13 CO2 and position-specific 13 C-labelled pyruvate, a key metabolite between primary and secondary metabolisms, in Scots pine seedlings. We determined the stable carbon isotope composition of leaf exchanged CO2 and VOC. Drought reduced the emission of the sesquiterpenes α-farnesene and ß-farnesene but did not affect 13 C-incorporation from 13 C-pyruvate. The labelling patterns suggest that farnesene biosynthesis partially depends on isopentenyl diphosphate crosstalk between chloroplasts and cytosol, and that drought inhibits this process. Contrary to sesquiterpenes, drought did not affect emission of isoprene, monoterpenes and some oxygenated compounds. During the day, pyruvate was used in the TCA cycle to a minor degree but was mainly consumed in pathways of secondary metabolism. Drought partly inhibited such pathways, while allocation into the TCA cycle increased. Drought caused a re-direction of pyruvate consuming pathways, which contributed to maintenance of isoprene and monoterpene production despite strongly inhibited photosynthesis. This underlines the importance of these volatiles for stress tolerance.

Metabolic exchange between pathways for isoprenoid synthesis and implications for biosynthetic hydrogen isotope fractionation.

Hydrogen isotope ratios of plant lipids are used for paleoclimate reconstruction, but are influenced by both source water and biosynthetic processes. Measuring 2 H : 1 H ratios of multiple compounds produced by different pathways could allow these effects to be separated, but hydrogen isotope fractionations during isoprenoid biosynthesis remain poorly constrained. To investigate how hydrogen isotope fractionation during isoprenoid biosynthesis is influenced by molecular exchange between the cytosolic and plastidial production pathways, we paired position-specific 13 C-pyruvate labeling with hydrogen isotope measurements of lipids in Pachira aquatica saplings. We find that acetogenic compounds primarily incorporated carbon from 13 C2-pyruvate, whereas isoprenoids incorporated 13 C1- and 13 C2-pyruvate equally. This indicates that cytosolic pyruvate is primarily introduced into plastidial isoprenoids via glyceraldehyde 3-phosphate and that plastidial isoprenoid intermediates are incorporated into cytosolic isoprenoids. Probably as a result of the large differences in hydrogen isotope fractionation between plastidial and cytosolic isoprenoid pathways, sterols from P. aquatica are at least 50‰ less 2 H-enriched relative to phytol than sterols in other plants. These results provide the first experimental evidence that incorporation of plastidial intermediates reduces 2 H : 1 H ratios of sterols. This suggests that relative offsets between the 2 H : 1 H ratios of sterols and phytol can trace exchange between the two isoprenoid synthesis pathways.

Systemic reprogramming of phytohormone profiles and metabolic traits by virulent Diplodia infection in its pine (Pinus sylvestris L.) host.

The widespread ascomycetous fungus Diplodia pinea is a latent, necrotrophic pathogen in Pinus species causing severe damages and world-wide economic losses. However, the interactions between pine hosts and virulent D. pinea are largely not understood. In the present study, systemic defence responses were investigated in non-inoculated, asymptomatic needles and roots of D. pinea infected saplings of two P. sylvestris provenances under controlled greenhouse conditions. Here, we show that D. pinea infection induced a multitude of systemic responses of the phytohormone profiles and metabolic traits. Shared systemic responses of both pine provenances in needles and roots included increased abscisic acid and jasmonic acid levels. Exclusively in the roots of both provenances, enhanced salicylic acid and reduced indole-3-acetic acid levels, structural biomass, and elevated activities of anti-oxidative enzymes were observed. Despite these similarities, the two pine provenances investigated different significantly in the systemic responses of both, phytohormone profiles and metabolic traits in needles and roots. However, the different systemic responses did not prevent subsequent destruction of non-inoculated needles, but rather prevented damage to the roots. Our results provide a detailed view on systemic defence mechanisms of pine hosts that are of particular significance for the selection of provenances with improved defence capacity.

Protein expression plasticity contributes to heat and drought tolerance of date palm.

Climate change is increasing the frequency and intensity of warming and drought periods around the globe, currently representing a threat to many plant species. Understanding the resistance and resilience of plants to climate change is, therefore, urgently needed. As date palm (Phoenix dactylifera) evolved adaptation mechanisms to a xeric environment and can tolerate large diurnal and seasonal temperature fluctuations, we studied the protein expression changes in leaves, volatile organic compound emissions, and photosynthesis in response to variable growth temperatures and soil water deprivation. Plants were grown under controlled environmental conditions of simulated Saudi Arabian summer and winter climates challenged with drought stress. We show that date palm is able to counteract the harsh conditions of the Arabian Peninsula by adjusting the abundances of proteins related to the photosynthetic machinery, abiotic stress and secondary metabolism. Under summer climate and water deprivation, these adjustments included efficient protein expression response mediated by heat shock proteins and the antioxidant system to counteract reactive oxygen species formation. Proteins related to secondary metabolism were downregulated, except for the P. dactylifera isoprene synthase (PdIspS), which was strongly upregulated in response to summer climate and drought. This study reports, for the first time, the identification and functional characterization of the gene encoding for PdIspS, allowing future analysis of isoprene functions in date palm under extreme environments. Overall, the current study shows that reprogramming of the leaf protein profiles confers the date palm heat- and drought tolerance. We conclude that the protein plasticity of date palm is an important mechanism of molecular adaptation to environmental fluctuations.

Heat Waves Change Plant Carbon Allocation Among Primary and Secondary Metabolism Altering CO2 Assimilation, Respiration, and VOC Emissions.

Processes controlling plant carbon allocation among primary and secondary metabolism, i.e., carbon assimilation, respiration, and VOC synthesis are still poorly constrained, particularly regarding their response to stress. To investigate these processes, we simulated a 10-day 38°C heat wave, analysing real-time carbon allocation into primary and secondary metabolism in the Mediterranean shrub Halimium halimifolium L. We traced position-specific 13C-labeled pyruvate into daytime VOC and CO2 emissions and during light-dark transition. Net CO2 assimilation strongly declined under heat, due to three-fold higher respiration rates. Interestingly, day respiration also increased two-fold. Decarboxylation of the C1-atom of pyruvate was the main process driving daytime CO2 release, whereas the C2-moiety was not decarboxylated in the TCA cycle. Heat induced high emissions of methanol, methyl acetate, acetaldehyde as well as mono- and sesquiterpenes, particularly during the first two days. After 10-days of heat a substantial proportion of 13C-labeled pyruvate was allocated into de novo synthesis of VOCs. Thus, during extreme heat waves high respiratory losses and reduced assimilation can shift plants into a negative carbon balance. Still, plants enhanced their investment into de novo VOC synthesis despite associated metabolic CO2 losses. We conclude that heat stress re-directed the proportional flux of key metabolites into pathways of VOC biosynthesis most likely at the expense of reactions of plant primary metabolism, which might highlight their importance for stress protection.

Terpenoids are transported in the xylem sap of Norway spruce.

Norway spruce is a conifer storing large amounts of terpenoids in resin ducts of various tissues. Parts of the terpenoids stored in needles can be emitted together with de novo synthesized terpenoids. Since previous studies provided hints on xylem transported terpenoids as a third emission source, we tested if terpenoids are transported in xylem sap of Norway spruce. We further aimed at understanding if they might contribute to terpenoid emission from needles. We determined terpenoid content and composition in xylem sap, needles, bark, wood and roots of field grown trees, as well as terpenoid emissions from needles. We found considerable amounts of terpenoids-mainly oxygenated compounds-in xylem sap. The terpenoid concentration in xylem sap was relatively low compared with the content in other tissues, where terpenoids are stored in resin ducts. Importantly, the terpenoid composition in the xylem sap greatly differed from the composition in wood, bark or roots, suggesting that an internal transport of terpenoids takes place at the sites of xylem loading. Four terpenoids were identified in xylem sap and emissions, but not within needle tissue, suggesting that these compounds are likely derived from xylem sap. Our work gives hints that plant internal transport of terpenoids exists within conifers; studies on their functions should be a focus of future research.

Heat stress increases the use of cytosolic pyruvate for isoprene biosynthesis.

The increasing occurrence of heatwaves has intensified temperature stress on terrestrial vegetation. Here, we investigate how two contrasting isoprene-emitting tropical species, Ficus benjamina and Pachira aquatica, cope with heat stress and assess the role of internal plant carbon sources for isoprene biosynthesis in relation to thermotolerance. To our knowledge, this is the first study to report isoprene emissions from P. aquatica. We exposed plants to two levels of heat stress and determined the temperature response curves for isoprene and photosynthesis. To assess the use of internal C sources in isoprene biosynthesis, plants were fed with 13C position-labelled pyruvate. F. benjamina was more heat tolerant with higher constitutive isoprene emissions and stronger acclimation to higher temperatures than P. aquatica, which showed higher induced isoprene emissions at elevated temperatures. Under heat stress, both isoprene emissions and the proportion of cytosolic pyruvate allocated into isoprene synthesis increased. This represents a mechanism that P. aquatica, and to a lesser extent F. benjamina, has adopted as an immediate response to sudden increase in heat stress. However, in the long run under prolonged heat, the species with constitutive emissions (F. benjamina) was better adapted, indicating that plants that invest more carbon into protective emissions of biogenic volatile organic compounds tend to suffer less from heat stress.

Differences in isoprenoid-mediated energy dissipation pathways between coastal and interior Douglas-fir seedlings in response to drought.

Plants have evolved energy dissipation pathways to reduce photooxidative damage under drought when photosynthesis is hampered. Non-volatile and volatile isoprenoids are involved in non-photochemical quenching of excess light energy and scavenging of reactive oxygen species. A better understanding of trees' ability to cope with and withstand drought stress will contribute to mitigate the negative effects of prolonged drought periods expected under future climate conditions. Therefore we investigated if Douglas-fir (Pseudotsuga menziesii(Mirb.)) provenances from habitats with contrasting water availability reveal intraspecific variation in isoprenoid-mediated energy dissipation pathways. In a controlled drought experiment with 1-year-old seedlings of an interior and a coastal Douglas-fir provenance, we assessed the photosynthetic capacity, pool sizes of non-volatile isoprenoids associated with the photosynthetic apparatus, as well as pool sizes and emission of volatile isoprenoids. We observed variation in the amount and composition of non-volatile and volatile isoprenoids among provenances, which could be linked to variation in photosynthetic capacity under drought. The coastal provenance exhibited an enhanced biosynthesis and emission of volatile isoprenoids, which is likely sustained by generally higher assimilation rates under drought. In contrast, the interior provenance showed an enhanced photoprotection of the photosynthetic apparatus by generally higher amounts of non-volatile isoprenoids and increased amounts of xanthophyll cycle pigments under drought. Our results demonstrate that there is intraspecific variation in isoprenoid-mediated energy dissipation pathways among Douglas-fir provenances, which may be important traits when selecting provenances suitable to grow under future climate conditions.

Optimization of photosynthesis and stomatal conductance in the date palm Phoenix dactylifera during acclimation to heat and drought.

We studied acclimation of leaf gas exchange to differing seasonal climate and soil water availability in slow-growing date palm (Phoenix dactylifera) seedlings. We used an extended Arrhenius equation to describe instantaneous temperature responses of leaf net photosynthesis (A) and stomatal conductance (G), and derived physiological parameters suitable for characterization of acclimation (Topt , Aopt and Tequ ). Optimum temperature of A (Topt ) ranged between 20-33°C in winter and 28-45°C in summer. Growth temperature (Tgrowth ) explained c. 50% of the variation in Topt , which additionally depended on leaf water status at the time of measurement. During water stress, light-saturated rates of A at Topt (i.e. Aopt ) were reduced to 30-80% of control levels, albeit not limited by CO2 supply per se. Equilibrium temperature (Tequ ), around which A/G and substomatal [CO2 ] are constant, remained tightly coupled with Topt . Our results suggest that acclimatory shifts in Topt and Aopt reflect a balance between maximization of photosynthesis and minimization of the risk of metabolic perturbations caused by imbalances in cellular [CO2 ]. This novel perspective on acclimation of leaf gas exchange is compatible with optimization theory, and might help to elucidate other acclimation and growth strategies in species adapted to differing climates.