Cowpea volatiles induced by beet armyworm or fall armyworm differentially prime maize plants.

Exposure to herbivore-induced plant volatiles (HIPVs) is known to enhance the defense responses in plants. This so-called priming effect has only been marginally studied in intercropping systems. We tested whether HIPVs from cowpea, which often serves as an intercrop alongside maize, can prime herbivore-induced volatile emissions in maize. Conventional volatile collection assays and real-time mass spectrometry revealed that maize plants that were exposed to HIPVs from cowpea infested with Spodoptera exigua caterpillars emitted more than control plants when they themselves were subsequently damaged by the same pest. The enhanced emission was only evident on the first day after infestation. Maize plants that were exposed to HIPVs from cowpea infested by S. frugiperda larvae showed no priming effect and released considerably less upon S. frugiperda infestation than upon S. exigua infestation. The latter may be explained by the fact that S. frugiperda is particularly well adapted to feed on maize and is known to suppress maize HIPV emissions. Our results imply that HIPVs from cowpea, depending on the inducing insect herbivore, may strongly prime maize plants. This deserves further investigation, also in other intercropping systems, as it can have important consequences for tritrophic interactions and crop protection.

Influence of Brevibacterium linens RS16 on foliage photosynthetic and volatile emission characteristics upon heat stress in Eucalyptus grandis.

Heat stress induces secondary metabolic changes in plants, channeling photosynthetic carbon and energy, away from primary metabolic processes, including, growth. Use of ACC (1-aminocyclopropane-1-carboxylate) deaminase containing plant growth promoting bacteria (PGPB) in conferring heat resistance in plants and the role of PGPB, in altering net carbon assimilation, constitutive and stress volatile emissions has not been studied yet. We exposed leaves of Eucalyptus grandis inoculated and non-inoculated with PGPB Brevibacterium linens RS16 to two levels of heat stress (37 °C and 41 °C for 5 min) and quantified temporal changes in foliage photosynthetic characteristics and volatile emission rates at 0.5 h, day 1 and day 5 after the stress application. Heat stress resulted in immediate reductions in dark-adapted photosystem II (PSII) quantum yield (Fv/Fm), net assimilation rate (A), stomatal conductance to water vapor (gs), and enhancement of stress volatile emissions, including enhanced emissions of green leaf volatiles (GLV), mono- and sesquiterpenes, light weight oxygenated volatile organic compounds (LOC), geranyl-geranyl diphosphate pathway volatiles (GGDP), saturated aldehydes, and benzenoids, with partial recovery by day 5. Changes in stress-induced volatiles were always less in leaves inoculated with B. linens RS16. However, net assimilation rate was enhanced by bacterial inoculation only in the 37 °C treatment and overall reduction of isoprene emissions was observed in bacterially-treated leaves. Principal component analysis (PCA), correlation analysis and partial least squares discriminant analysis (PLS-DA) indicated that different stress applications influenced specific volatile organic compounds. In addition, changes in the expression analysis of heat shock protein 70 gene (DnaK) gene in B. linens RS16 upon exposure to higher temperatures further indicated that B. linens RS16 has developed its own heat resistance mechanism to survive under higher temperature regimes. Taken together, this study demonstrates that foliar application of ACC deaminase containing PGPB can ameliorate heat stress effects in realistic biological settings.

Foliage inoculation by Burkholderia vietnamiensis CBMB40 antagonizes methyl jasmonate-mediated stress in Eucalyptus grandis.

Methyl jasmonate (MeJA) is widely used as a model chemical to study hypersensitive responses to biotic stress impacts in plants. Elevated levels of methyl jasmonate induce jasmonate-dependent defense responses, associated with a decline in primary metabolism and enhancement of secondary metabolism of plants. However, there is no information of how stress resistance of plants, and accordingly the sensitivity to exogenous MeJA can be decreased by endophytic plant growth promoting rhizobacteria (PGPR) harboring ACC (1-aminocyclopropane-1-carboxylate) deaminase. In this study, we estimated stress alleviating potential of endophytic PGPR against MeJA-induced plant perturbations through assessing photosynthetic traits and stress volatile emissions. We used mild (5 mM) to severe (20 mM) MeJA and endophytic plant growth promoting rhizobacteria Burkholderia vietnamiensis CBMB40 and studied how MeJA and B. vietnamiensis treatments influenced temporal changes in photosynthetic characteristics and stress volatile emissions. Separate application of MeJA markedly decreased photosynthetic characteristics and increased lipoxygenase pathway (LOX) volatiles, volatile isoprenoids, saturated aldehydes, lightweight oxygenated compounds (LOC), geranyl-geranyl diphosphate pathway (GGDP) volatiles, and benzenoids. However, MeJA-treated leaves inoculated by endophytic bacteria B. vietnamiensis had substantially increased photosynthetic characteristics and decreased emissions of LOX, volatile isoprenoids and other stress volatiles compared with non-inoculated MeJA treatments, especially at later stages of recovery. In addition, analysis of leaf terpenoid contents demonstrated that several mono- and sesquiterpenes were de novo synthesized upon MeJA and B. vietnamiensis applications. This study demonstrates that foliar application of endophytic bacteria B. vietnamiensis can potentially enhance resistance to biotic stresses and contribute to the maintenance of the integrity of plant metabolic activity.

Lethal heat stress-dependent volatile emissions from tobacco leaves: what happens beyond the thermal edge?

Natural vegetation is predicted to suffer from extreme heat events as a result of global warming. In this study, we focused on the immediate response to heat stress. Photosynthesis and volatile emissions were measured in the leaves of tobacco (Nicotiana tabacum cv. Wisconsin 38) after exposure to heat shock treatments between 46 °C and 55 °C. Exposure to 46 °C decreased photosynthetic carbon assimilation rates (A) by >3-fold. Complete inhibition of A was observed at 49 °C, together with a simultaneous decrease in the maximum quantum efficiency of PSII, measured as the Fv/Fm ratio. A large increase in volatile emissions was observed at 52 °C. Heat stress resulted in only minor effects on the emission of monoterpenes, but volatiles associated with membrane damage such as propanal and (E)-2-hexenal+(Z)-3-hexenol were greatly increased. Heat induced changes in the levels of methanol and 2-ethylfuran that are indicative of modification of cell walls. In addition, the oxidation of metabolites in the volatile profiles was strongly enhanced, suggesting the acceleration of oxidative processes at high temperatures that are beyond the thermal tolerance limit.

Ozone and Wounding Stresses Differently Alter the Temporal Variation in Formylated Phloroglucinols in Eucalyptus globulus Leaves.

Formylated phloroglucinol compounds (FPCs) are a class of plant specialized metabolite present in the Myrtaceae family, especially in the genus Eucalyptus. FPCs are widely investigated due to their herbivore deterrence properties and various bioactivities of pharmaceutical relevance. Despite the increasing number of studies elucidating new FPCs structures and bioactivity, little is known about the role of those compounds in planta, and the effects of environmental stresses on FPC concentration. Ozone (O3) and wounding are key stress factors regularly confronted by plants. In this study, we investigated how O3, wounding, and their combination affected individual and total FPC foliar concentration of the economically important species Eucalyptus globulus. Six individual FPCs, including five macrocarpals and one sideroxylonal, showed different response patterns to the single and combined stresses. Total macrocarpals only increased under single O3 treatment, whereas total sideroxylonals only increased in response to wounding treatment, suggesting different physiological roles played by the two groups of FPCs predominantly existing in E. globulus foliage. Total FPCs increased significantly under individual wounding and O3 treatments but not under the combined treatment. A principal component analysis indicated that all different treatments had unique FPC fingerprints. Total phenolic contents increased in all O3 and wounding treatments, and a marginally positive correlation was found between total FPCs and total phenolic contents. We suggest that, depending on the concentration and composition, FPCs play multiple physiological roles in planta, including serving as antioxidants to scavenge the reactive oxygen species brought about by O3 and wounding stresses.

Methylobacterium oryzae CBMB20 influences photosynthetic traits, volatile emission and ethylene metabolism in Oryza sativa genotypes grown in salt stress conditions.

MAIN CONCLUSION: Inoculation of endophytic Methylobacterium oryzae CBMB20 in salt-stressed rice plants improves photosynthesis and reduces stress volatile emissions due to mellowing of ethylene-dependent responses and activating vacuolar H+-ATPase. The objective of this study was to analyze the impact of ACC (1-aminocyclopropane-1-carboxylate) deaminase-producing Methylobacterium oryzae CBMB20 in acclimation of plant to salt stress by controlling photosynthetic characteristics and volatile emission in salt-sensitive (IR29) and moderately salt-resistant (FL478) rice (Oryza sativa L.) cultivars. Saline levels of 50 mM and 100 mM NaCl with and without bacteria inoculation were applied, and the temporal changes in stress response and salinity resistance were assessed by monitoring photosynthetic characteristics, ACC accumulation, ACC oxidase activity (ACO), vacuolar H+ ATPase activity, and volatile organic compound (VOC) emissions. Salt stress considerably reduced photosynthetic rate, stomatal conductance, PSII efficiency and vacuolar H+ ATPase activity, but it increased ACC accumulation, ACO activity, green leaf volatiles, mono- and sesquiterpenes, and other stress volatiles. These responses were enhanced with increasing salt stress and time. However, rice cultivars treated with CBMB20 showed improved plant vacuolar H+ ATPase activity, photosynthetic characteristics and decreased ACC accumulation, ACO activity and VOC emission. The bacteria-dependent changes were greater in the IR29 cultivar. These results indicate that decreasing photosynthesis and vacuolar H+ ATPase activity rates and increasing VOC emission rates in response to high-salinity stress were effectively mitigated by M. oryzae CBMB20 inoculation.

Inoculation of Brevibacterium linens RS16 in Oryza sativa genotypes enhanced salinity resistance: Impacts on photosynthetic traits and foliar volatile emissions.

The emission of volatiles in response to salt stress in rice cultivars has not been studied much to date. Studies addressing the regulation of stress induced volatile emission by halotolerant plant growth promoting bacteria containing ACC (1-aminocyclopropane-1-carboxylate) deaminase are also limited. The objective of the present study was to investigate the salt alleviation potential of bacteria by regulating photosynthetic characteristics and volatile emissions in rice cultivars, and to compare the effects of the bacteria inoculation and salt responses between two rice genotypes. The interactive effects of soil salinity (0, 50, and 100 mM NaCl) and inoculation with Brevibacterium linens RS16 on ACC accumulation, ACC oxidase activity, carbon assimilation and stress volatile emissions after stress application were studied in the moderately salt resistant (FL478) and the salt-sensitive (IR29) rice (Oryza sativa L.) cultivars. It was observed that salt stress reduced foliage photosynthetic rate, but induced foliage ACC accumulation, foliage ACC oxidase activity, and the emissions of all the major classes of volatile organic compounds (VOCs) including the lipoxygenase pathway volatiles, light-weight oxygenated volatiles, long-chained saturated aldehydes, benzenoids, geranylgeranyl diphosphate pathway products, and mono- and sesquiterpenes. All these characteristics scaled up quantitatively with increasing salt stress. The effects of salt stress were more pronounced in the salt-sensitive genotype IR29 compared to the moderately salt resistant FL478 genotype. However, the bacterial inoculation significantly enhanced photosynthesis, and decreased ACC accumulation and the ACC oxidase activity, and VOC emissions both in control and salt-treated plants. Taken together, these results suggested that the ACC deaminase-containing Brevibacterium linens RS16 reduces the temporal regulation of VOC emissions and increases the plant physiological activity by reducing the availability of ethylene precursor ACC and the ACC oxidase activity under salt stress.

Differential regulation of volatile emission from Eucalyptus globulus leaves upon single and combined ozone and wounding treatments through recovery and relationships with ozone uptake.

Both ozone and wounding constitute two key abiotic stress factors, but their interactive effects on plant constitutive and stress-elicited volatile (VOC) emissions are poorly understood. Furthermore, the information on time-dependent modifications in VOC release during recovery from a combined stress is very limited. We studied the modifications in photosynthetic characteristics and constitutive and stress-induced volatile emissions in response to single and combined applications of acute ozone (4, 5, and 6 ppm) and wounding treatments through recovery (0.5-75 h) in a constitutive isoprene and mono- and sesquiterpene emitter Eucalyptus globulus. Overall, the photosynthetic characteristics were surprisingly resistant to all ozone and wounding treatments. Constitutive isoprene emissions were strongly upregulated by ozone and combined ozone and wounding treatments and remained high through recovery phase, but wounding applied alone reduced isoprene emission. All stress treatments enhanced emissions of lipoxygenase pathway volatiles (LOX), mono- and sesquiterpenes, saturated aldehydes (C7-C10), benzenoids, and geranylgeranyl diphosphate (GGDP) pathway volatiles. Once elicited, GGDP volatile, saturated aldehyde and benzenoid emissions remained high through the recovery period. In contrast, LOX emissions, and total mono- and sesquiterpene emissions decreased through recovery period. However, secondary rises in total sesquiterpene emissions at 75 h and in total monoterpenes at 25-50 h were observed. Overall, acute ozone and wounding treatments synergistically altered gas exchange characteristics and stress volatile emissions. Through the treatments and recovery period, stomatal ozone uptake rate and volatile emission rates were poorly correlated, reflecting possible ozone-scavenging effect of volatiles and thus, reduction of effective ozone dose and elicitation of induced defense by the acute ozone concentrations applied. These results underscore the important role of interactive stresses on both constitutive and induced volatile emission responses.

Ozone-triggered surface uptake and stress volatile emissions in Nicotiana tabacum 'Wisconsin'.

Ozone is a strong oxidant and a key stress elicitor. The immediate and longer term impacts of ozone are poorly understood in species with emission of both de novo synthesized and stored volatiles, such a tobacco (Nicotiana tabacum), which has terpene-containing glandular trichomes on the leaf surface. In this study, we exposed N. tabacum 'Wisconsin' leaves to acute ozone doses of 0 (control), 400, 600, 800, and 1000 ppb for 30 min and studied the effects of ozone exposure on ozone uptake, gas-exchange characteristics, and emissions of lipoxygenase pathway volatiles, monoterpenes, and sesquiterpenes. Foliage emissions of lipoxygenase pathway volatiles were quantitatively related to the severity of ozone exposure, but the stress dose vs. emission relationship was weaker for terpenoids. Analysis of leaf terpene content and composition indicated that several monoterpenes and sesquiterpenes were not stored in leaves and were synthesized de novo upon ozone exposure. The highest degree of elicitation for each compound was observed immediately after ozone treatment and it declined considerably during recovery. Leaf ozone uptake was dominated by non-stomatal deposition, and the emissions of total lipoxygenase pathway volatiles and mono- and sesquiterpenes were positively correlated with non-stomatal ozone deposition. Overall, this study demonstrates remarkably high ozone resistance of the studied tobacco cultivar and indicates that ozone's effects on volatile emissions primarily reflect modifications in the release of stored volatiles and reaction of ozone with the leaf surface structure.

Glandular trichomes as a barrier against atmospheric oxidative stress: Relationships with ozone uptake, leaf damage, and emission of LOX products across a diverse set of species.

There is a spectacular variability in trichome types and densities and trichome metabolites across species, but the functional implications of this variability in protecting from atmospheric oxidative stresses remain poorly understood. The aim of this study was to evaluate the possible protective role of glandular and non-glandular trichomes against ozone stress. We investigated the interspecific variation in types and density of trichomes and how these traits were associated with elevated ozone impacts on visible leaf damage, net assimilation rate, stomatal conductance, chlorophyll fluorescence, and emissions of lipoxygenase pathway products in 24 species with widely varying trichome characteristics and taxonomy. Both peltate and capitate glandular trichomes played a critical role in reducing leaf ozone uptake, but no impact of non-glandular trichomes was observed. Across species, the visible ozone damage varied 10.1-fold, reduction in net assimilation rate 3.3-fold, and release of lipoxygenase compounds 14.4-fold, and species with lower glandular trichome density were more sensitive to ozone stress and more vulnerable to ozone damage compared to species with high glandular trichome density. These results demonstrate that leaf surface glandular trichomes constitute a major factor in reducing ozone toxicity and function as a chemical barrier that neutralizes the ozone before it enters the leaf.

Temporal regulation of terpene synthase gene expression in Eucalyptus globulus leaves upon ozone and wounding stresses: relationships with stomatal ozone uptake and emission responses.

Ozone and wounding are key abiotic factors but, their interactive effects on temporal changes in terpene synthase gene expression and emission responses are poorly understood. Here, we applied combined acute ozone and wounding stresses to the constitutive isoprenoid-emitter Eucalyptus globulus and studied how isoprene, 1,8-cineole, and isoledene synthase genes were regulated, and how the gene expression was associated with temporal changes in photosynthetic characteristics, product emission rates, and stomatal ozone uptake through recovery phase. Photosynthetic characteristics and emission rate of isoprene, 1,8-cineole, and isoledene were synergistically altered, while three TPS gene expressions were antagonistically altered by combined stress applications. A time-delay analysis indicated that the best correspondences between gene expression and product emission rates were observed for 0 h time-shift for wounding and 0-2 h time-shifts for separate ozone, and combined ozone and wounding treatments. The best correspondence between ozone uptake and gene expression was observed for 0-4 h time-shifts for separate ozone and combined ozone and wounding treatments. Overall, this study demonstrated that expression profiles of isoprene, the monoterpene 1,8-cineole, and the sesquiterpene isoledene synthase genes differentially influenced their corresponding product emissions for separate and combined ozone and wounding treatments through recovery.

Mono- and sesquiterpene release from tomato (Solanum lycopersicum) leaves upon mild and severe heat stress and through recovery: from gene expression to emission responses.

Plants frequently experience heat ramps of various severities, but how and to what degree plant metabolic activity recovers from mild and severe heat stress is poorly understood. In this study, we exposed the constitutive terpene emitter, Solanum. lycopersicum leaves to mild (37 and 41 °C), moderate (46 °C) and severe (49 °C) heat ramps of 5 min. and monitored foliage photosynthetic activity, lipoxygenase pathway volatile (LOX), and mono- and sesquiterpene emissions and expression of two terpene synthase genes, ß-phellandrene synthase and (E)-ß-caryophyllene/α-humulene synthase, through a 24 h recovery period upon return to pre-stress conditions. Leaf monoterpene emissions were dominated by ß-phellandrene and sesquiterpene emissions by (E)-ß-caryophyllene, and thus, these two terpene synthase genes were representative for the two volatile terpene classes. Photosynthetic characteristics partly recovered under moderate heat stress, and very limited recovery was observed under severe stress. All stress treatments resulted in elicitation of LOX emissions that declined during recovery. Enhanced mono- and sesquiterpene emissions were observed immediately after the heat treatment, but the emissions decreased even to below the control treatment during recovery between 2-10 h, and raised again by 24 h. The expression of ß-phellandrene and (E)-ß-caryophyllene synthase genes decreased between 2-10 h after heat stress, and recovered to pre-stress level in mild heat stress treatment by 24 h. Overall, this study demonstrates a highly sensitive heat response of terpenoid synthesis that is mainly controlled by gene level responses under mild stress, while severe stress leads to non-recoverable declines in foliage physiological and gene expression activities.