Maritta Kunert

Effects of Feeding Spodoptera littoralis on Lima Bean Leaves: IV. Diurnal and Nocturnal Damage Differentially Initiate Plant Volatile Emission

Continuous mechanical damage initiates the rhythmic emission of volatiles in lima bean (Phaseolus lunatus) leaves; the emission resembles that induced by herbivore damage. The effect of diurnal versus nocturnal damage on the initiation of plant defense responses was investigated using MecWorm, a robotic device designed to reproduce tissue damage caused by herbivore attack. Lima bean leaves that were damaged by MecWorm during the photophase emitted maximal levels of β-ocimene and (Z)-3-hexenyl acetate in the late photophase. Leaves damaged during the dark phase responded with the nocturnal emission of (Z)-3-hexenyl acetate, but with only low amounts of β-ocimene; this emission was followed by an emission burst directly after the onset of light. In the presence of 13CO2, this light-dependent synthesis of β-ocimene resulted in incorporation of 75% to 85% of 13C, demonstrating that biosynthesis of β-ocimene is almost exclusively fueled by the photosynthetic fixation of CO2 along the plastidial 2-C-methyl-d-erythritol 4-P pathway. Jasmonic acid (JA) accumulated locally in direct response to the damage and led to immediate up-regulation of the P. lunatus β-ocimene synthase gene (PlOS) independent of the phase, that is, light or dark. Nocturnal damage caused significantly higher concentrations of JA (approximately 2–3 times) along with enhanced expression levels of PlOS. Transgenic Arabidopsis thaliana transformed with PlOS promoter∷β-glucuronidase fusion constructs confirmed expression of the enzyme at the wounded sites. In summary, damage-dependent JA levels directly control the expression level of PlOS, regardless of light or dark conditions, and photosynthesis is the major source for the early precursors of the 2-C-methyl-d-erythritol 4-P pathway.

Ultrafast sampling and analysis of plant volatiles by a hand-held miniaturised GC with pre-concentration unit: Kinetic and quantitative aspects of plant volatile production

A method has been developed for continuous monitoring of the emission of volatiles from biological sources with high time resolution. High time resolution (up to 3 min) is achieved by an automatically operated, miniaturised gas chromatographic system equipped with a pre-concentration unit (zNoseTM). After calibration, the zNoseTM provides reliable quantitative data for individual compounds and allows their emission kinetics to be followed over longer periods of time without supervision. While the results of the zNoseTM provide information about actual composition of aromas during sampling, the analysis of simultaneously trapped volatiles supplies information about the total amount of emitted compounds and permits further spectroscopic analyses (Isotope-Ratio-MS, GC-MS, etc.). Both methods, analysis by the zNoseTM and sampling on charcoal traps, lead to comparable results. The new analytical system has been successfully used to study the kinetics of volatile emission from elicitor-treated Lima bean leaves (Phaseolus lunatus), aphid-damaged paprika plants (Capsicum annuum), and to follow the volatile emission from blossoms of the cactus Rebutia fabrisii over several days. The instrument is portable and can be used in the field.

De novo biosynthesis versus sequestration: a network of transport systems supports in iridoid producing leaf beetle larvae both modes of defense.

In the larval chrysomelines the de novo synthesis of monoterpenoids (iridoids) is believed to represent the ancestral state in the evolution of chemical defenses. Here we demonstrate that the iridoid producing larvae of Plagiodera versicolora and Phratora laticollis have the potential to sequester precursors from food. In nature, iridoids may even have a dual origin, namely plant-derived and de novo produced. The ability to sequester plant-derived precursors was proved by (i) (13)C-labelling of the terpenoids in the food plant, (ii) by larval feeding on leaves impregnated with analogs and labelled putative precursors for iridoid biosynthesis; and (iii) by injection of the precursors into the hemolymph followed by mass spectroscopic analysis of their distribution in the hemolymph, defensive secretion, and faeces. The experimental findings support a network of transport systems which allows a broader range of glucosides to enter and to leave the hemocoel, while only the appropriate precursor, 8-hydroxygeraniol-8-O-beta-d-glucoside, is channelled to the reservoir and processed to iridoids. The dual system of de novo biosynthesis and sequestration of phytogenic precursors may have favoured the larvae to shift from one host plant to another without losing their defense.

Poplar extrafloral nectaries: two types, two strategies of indirect defenses against herbivores.

Many plant species grow extrafloral nectaries and produce nectar to attract carnivore arthropods as defenders against herbivores. Two nectary types that evolved with Populus trichocarpa (Ptr) and Populus tremula × Populus tremuloides (Ptt) were studied from their ecology down to the genes and molecules. Both nectary types strongly differ in morphology, nectar composition and mode of secretion, and defense strategy. In Ptt, nectaries represent constitutive organs with continuous merocrine nectar flow, nectary appearance, nectar production, and flow. In contrast, Ptr nectaries were found to be holocrine and inducible. Neither mechanical wounding nor the application of jasmonic acid, but infestation by sucking insects, induced Ptr nectar secretion. Thus, nectaries of Ptr and Ptt seem to answer the same threat by the use of different mechanisms.

A Duo of Potassium-Responsive Histidine Kinases Govern the Multicellular Destiny of Bacillus subtilis

ABSTRACT Multicellular biofilm formation and surface motility are bacterial behaviors considered mutually exclusive. However, the basic decision to move over or stay attached to a surface is poorly understood. Here, we discover that in Bacillus subtilis, the key root biofilm-controlling transcription factor Spo0A~Pi (phosphorylated Spo0A) governs the flagellum-independent mechanism of social sliding motility. A Spo0A-deficient strain was totally unable to slide and colonize plant roots, evidencing the important role that sliding might play in natural settings. Microarray experiments plus subsequent genetic characterization showed that the machineries of sliding and biofilm formation share the same main components (i.e., surfactin, the hydrophobin BslA, exopolysaccharide, and de novo-formed fatty acids). Sliding proficiency was transduced by the Spo0A-phosphorelay histidine kinases KinB and KinC. We discovered that potassium, a previously known inhibitor of KinC-dependent biofilm formation, is the specific sliding-activating signal through a thus-far-unnoticed cytosolic domain of KinB, which resembles the selectivity filter sequence of potassium channels. The differential expression of the Spo0A~Pi reporter abrB gene and the different levels of the constitutively active form of Spo0A, Sad67, in Δspo0A cells grown in optimized media that simultaneously stimulate motile and sessile behaviors uncover the spatiotemporal response of KinB and KinC to potassium and the gradual increase in Spo0A~Pi that orchestrates the sequential activation of sliding, followed by sessile biofilm formation and finally sporulation in the same population. Overall, these results provide insights into how multicellular behaviors formerly believed to be antagonistic are coordinately activated in benefit of the bacterium and its interaction with the host. IMPORTANCE Alternation between motile and sessile behaviors is central to bacterial adaptation, survival, and colonization. However, how is the collective decision to move over or stay attached to a surface controlled? Here, we use the model plant-beneficial bacterium Bacillus subtilis to answer this question. Remarkably, we discover that sessile biofilm formation and social sliding motility share the same structural components and the Spo0A regulatory network via sensor kinases, KinB and KinC. Potassium, an inhibitor of KinC-dependent biofilm formation, triggers sliding via a potassium-perceiving cytosolic domain of KinB that resembles the selectivity filter of potassium channels. The spatiotemporal response of these kinases to variable potassium levels and the gradual increase in Spo0A~Pi levels that orchestrates the activation of sliding before biofilm formation shed light on how multicellular behaviors formerly believed to be antagonistic work together to benefit the population fitness. Alternation between motile and sessile behaviors is central to bacterial adaptation, survival, and colonization. However, how is the collective decision to move over or stay attached to a surface controlled? Here, we use the model plant-beneficial bacterium Bacillus subtilis to answer this question. Remarkably, we discover that sessile biofilm formation and social sliding motility share the same structural components and the Spo0A regulatory network via sensor kinases, KinB and KinC. Potassium, an inhibitor of KinC-dependent biofilm formation, triggers sliding via a potassium-perceiving cytosolic domain of KinB that resembles the selectivity filter of potassium channels. The spatiotemporal response of these kinases to variable potassium levels and the gradual increase in Spo0A~Pi levels that orchestrates the activation of sliding before biofilm formation shed light on how multicellular behaviors formerly believed to be antagonistic work together to benefit the population fitness.

Precise RNAi-mediated silencing of metabolically active proteins in the defence secretions of juvenile leaf beetles

Allomones are widely used by insects to impede predation. Frequently these chemical stimuli are released from specialized glands. The larvae of Chrysomelina leaf beetles produce allomones in gland reservoirs into which the required precursors and also the enzymes are secreted from attached gland cells. Hence, the reservoirs can be considered as closed bio-reactors for producing defensive secretions. We used RNA interference (RNAi) to analyse in vivo functions of proteins in biosynthetic pathways occurring in insect secretions. After a salicyl alcohol oxidase was silenced in juveniles of the poplar leaf beetles, Chrysomela populi, the precursor salicyl alcohol increased to 98 per cent, while salicyl aldehyde was reduced to 2 per cent within 5 days. By analogy, we have silenced a novel protein annotated as a member of the juvenile hormone-binding protein superfamily in the juvenile defensive glands of the related mustard leaf beetle, Phaedon cochleariae. The protein is associated with the cyclization of 8-oxogeranial to iridoids (methylcyclopentanoid monoterpenes) in the larval exudates made clear by the accumulation of the acylic precursor 5 days after RNAi triggering. A similar cyclization reaction produces the secologanin part of indole alkaloids in plants.

Beetles Do It Differently : Two Stereodivergent Cyclisation Modes in Iridoid-Producing Leaf-Beetle Larvae

Larvae of the Chrysomelina species Phaedon cochleariae, Hydrothassa marginella, Phratora vulgatissima, Gastrophysa viridula, Gastrophysa atrocyanea, Gastrophysa cyanea and Gastrophysa polygoni produce the iridoid chrysomelidial (1) to defend themselves against predators. Feeding experiments with a deuterated precursor ([2H5]8‐hydroxygeraniol 9) and in vitro isotope exchange experiments with defensive secretion in 2H2O revealed differences in the cyclisation of the ultimate precursor 8‐oxogeranial (8) to 1, between members of the genus Gastrophysa and all other species. In P. cochleariae, H. marginella and P. vulgatissima 1 is most likely produced by a Rauhut–Currier‐type cyclisation via a “transoid dienamine”, with loss of a single deuterium atom from C(4) of the precursor. In contrast, members of the genus Gastrophysa cyclise 8 via a “cisoid dienamine” intermediate, with exchange of all three deuterium atoms from the methyl group at C(3). To study whether the different cyclisation modes influence the stereochemistry of 1, the absolute configuration of 1 of the larvae was determined by GC‐MS on a chiral column. In accordance with literature (J. Meinwald, T. H. Jones, J. Am. Chem. Soc. 1978, 100, 1883 and N. Shimizu, R. Yakumaru, T. Sakata, S. Shimano, Y. Kuwahara, J. Chem. Ecol. 2012, 38, 29), we found (5S,8S)‐chrysomelidial (1) in H. marginella and P. vulgatissima, but P. cochleariae and all investigated members of the genus Gastrophysa synthesise (5R,8R)‐chrysomelidial (1).

Glucose and Glucose Esters in the Larval Secretion of Chrysomela Lapponica; Selectivity of the Glucoside Import System from Host Plant Leaves

Larvae of Chrysomela lapponica (Coleoptera: Chrysomelidae) sequester characteristic O-glucosides from the leaves of their food plants, namely Betula and/or Salix The present study focuses on birch-feeding larvae of C. lapponica from the Altai region in East Kazakhstan. As in other sequestering leaf beetle larvae, the compounds are transported intact via different membrane barriers into the defensive system, followed by glucoside cleavage and subsequent transformations of the plant-derived aglycones. Unlike previous studies with model compounds, we studied the sequestration of phytogenic precursors by analyzing the complex pattern of glucosides present in food plant Betula rotundifolia (39 compounds) and compared this composition with the aglycones present as butyrate esters in the defensive secretion. In addition to the analytic approach, the insect’s ability, to transport individual glucosides was tested by using hydrolysis-resistant thioglucoside analogs, applied onto the leaf surface. The test compounds reach the defensive system intact and without intermediate transformation. No significant difference of the transport capacity and selectivity was observed between larvae of birch-feeding population from Kazakhstan, and previous results for larvae of birch-feeding population from the Czech Republic or willow-feeding populations. Overall, the transport of the phytogenic glucosides is highly selective and highly efficient, since only minor compounds of the spectrum of phytogenic glucoside precursors contribute to the limited number of aglycones utilized in the defensive secretion. Interestingly, salicortin 44 and tremulacin 60 were found in the leaves, but no aldehyde or esters of salicylalcohol. Surprisingly, we observed large amounts of free glucose, together with small amounts of 6-O-butyrate esters of glucose (27a/b and 28a/b).

Musty‐Earthy Scent in Cactus Flowers: Characteristics of Floral Scent Production in Dehydrogeosmin‐Producing Cacti

The musty‐earthy‐smelling sesquiterpenoid dehydrogeosmin (DHG) was found several years ago as a novel compound in flowers of some cactus species, which called attention to the biology of floral scents of the Cactaceae. Our studies showed that the odor bouquet of DHG‐producing cactus flowers is dominated by isoprenoids. All floral volatiles were produced by the perianth during the exclusively diurnal anthesis. Volatile emissions were diurnal, as was the daily opening schedule of flowers. Within one species, the odor composition of cultivated specimens varied strongly, whereas little variation was found among individuals of a natural population of the same species. However, field studies revealed that DHG is not produced by all specimens in natural populations. Our results on the timing and location of odor emission support the hypothesis that this unusual volatile may play a role in pollinator‐plant interactions.

Volatile Sampling from Biological Sources by the Closed-Loop-Stripping Technique

The closed-loop-stripping technique with absorber traps (e.g., charcoal, porous polymers) can be used to collect volatiles from different biological sources, for example, plants and insects. One notable advantage of the method is the high signal-to-noise ratio that can be achieved by continuous sampling of the volatiles from a producing source within a closed environment. The equipment required for the technique is inexpensive and can be easily adapted to the demands of the analyzed subject. Moreover, the system can be operated in the field since only air and a battery are needed. The volatiles can be recovered from the trap by thermodesorption or by elution with solvents. Solvent elution has the advantage that unknown compounds can be subjected to chemical transformations facilitating structure elucidation. Only very low amounts of solvents (20-40 μL) are needed to elute the microcarbon traps (1.5-5 mg). Single traps efficiently absorb volatiles up to the lower microgram range. Higher concentrations may result in insufficient trapping. In this protocol, we describe the collection of volatiles released from lima bean leaves (Phaseolus lunatus) after damage by feeding larvae of Spodoptera littoralis. The enclosed volume of air containing the emitted volatiles is circulated through miniature charcoal traps for 4 h or more. For analysis, the traps are eluted with dichloromethane.