Prions: En route from structural models to structures

The prion hypothesis^1–3 states that the prion and non-prion form of a protein differ only in their 3D conformation and that different strains of a prion differ by their 3D structure.^4,5 Recent technical developments have enabled solid-state NMR to address the atomic-resolution structures of full-length prions, and a first comparative study of two of them, HET-s and Ure2p, in fibrillar form, has recently appeared as a pair of companion papers.^6,7 Interestingly, the two structures are rather different: HET-s features an exceedingly well-ordered prion domain and a partially disordered globular domain. Ure2p in contrast features a very well ordered globular domain with a conserved fold, and—most probably—a partially ordered prion domain.⁶ For HET-s, the structure of the prion domain is characterized at atomic-resolution. For Ure2p, structure determination is under way, but the highly resolved spectra clearly show that information at atomic resolution should be achievable.Key words: prion, NMR, solid-state NMR, MAS, structure, Ure2p, HET-sDespite the large interest in the basic mechanisms of fibril formation and prion propagation, little is known about the molecular structure of prions at atomic resolution and the mechanism of propagation. Prions with related properties to the ones responsible for mammalian diseases were also discovered in yeast and funghi^8,9 which provide convenient model system for their studies. Prion proteins described include the mammalian prion protein PrP, Ure2p,¹⁰ Rnq1p,¹¹ Sup35,¹² Swi1,¹³ and Cyc8,¹⁴ from bakers yeast (S. cervisiae) and HET-s from the filamentous fungus P. anserina. The soluble non-prion form of the proteins characterized in vitro is a globular protein with an unfolded, dynamically disordered N- or C-terminal tail.^15–18 In the prion form, the proteins form fibrillar aggregates, in which the tail adopts a different conformation and is thought to be the dominant structural element for fibril formation.Fibrills are difficult to structurally characterize at atomic resolution, as X-ray diffraction and liquid-state NMR cannot be applied because of the non-crystallinity and the mass of the fibrils. Solid-state NMR, in contrast, is nowadays well suited for this purpose. The size of the monomer, between 230 and 685 amino-acid residues for the prions of Figure 1, and therefore the number of resonances in the spectrum—that used to be large for structure determination—is now becoming tractable by this method.Open in a separate windowFigure 1Prions identified today and characterized as consisting of a prion domain (blue) and a globular domain (red).Prion proteins characterized so far were found to be usually constituted of two domains, namely the prion domain and the globular domain (see Fig. 1). This architecture suggests a divide-and-conquer approach to structure determination, in which the globular and prion domain are investigated separately. In isolation, the latter, or fragments thereof, were found to form β-sheet rich structures (e.g., Ure2p(1-89),^6,19 Rnq1p(153-405)²⁰ and HET-s(218-289)²¹). The same conclusion was reached by investigating Sup35(1-254).²² All these fragements have been characterized as amyloids, which we define in the sense that a significant part of the protein is involved in a cross-beta motif.²³ An atomic resolution structure however is available presently only for the HET-s prion domain, and was obtained from solid-state NMR²⁴ (vide infra). It contains mainly β-sheets, which form a triangular hydrophobic core. While this cross-beta structure can be classified as an amyloid, its triangular shape does deviate significantly from amyloid-like structures of smaller peptides.²³Regarding the globular domains, structures have been determined by x-ray crystallography (Ure2p^25,26 and HET-s²⁷), as well as NMR (mammal prions^15,28–30). All reveal a protein fold rich in α-helices, and dimeric structures for the Ure2 and HET-s proteins. The Ure2p fold resembles that of the β-class glutathione S-transferases (GST), but lacks GST activity.²⁵It is a central question for the structural biology of prions if the divide-and-conquer approach imposed by limitations in current structural approaches is valid. Or in other words: can the assembly of full-length prions simply be derived from the sum of the two folds observed for the isolated domains?     

MEK1/2 and p38-like MAP kinase successively mediate H2O2 signaling in Vicia guard cell

As a second messenger, H₂O₂ generation and signal transduction is subtly controlled and involves various signal elements, among which are the members of MAP kinase family. The increasing evidences indicate that both MEK1/2 and p38-like MAP protein kinase mediate ABA-induced H₂O₂ signaling in plant cells. Here we analyze the mechanisms of similarity and difference between MEK1/2 and p38-like MAP protein kinase in mediating ABA-induced H₂O₂ generation, inhibition of inward K⁺ currents, and stomatal closure. These data suggest that activation of MEK1/2 is prior to p38-like protein kinase in Vicia guard cells.Key words: H₂O₂ signaling, ABA, p38-like MAP kinase, MEK1/2, guard cellAn increasing number of literatures elucidate that reactive oxygen species (ROS), especially H₂O₂, is essential to plant growth and development in response to stresses,^1–4 and involves activation of various signaling events, among which are the MAP kinase cascades.^1–3,5 Typically, activation of MEK1/2 mediates NADPH oxidase-dependent ROS generation in response to stresses,^4,6–8 and the facts that MEK1/2 inhibits the expression and activation of antioxidant enzymes reveal how PD98059, the specific inhibitor of MEK1/2, abolishes abscisic acid (ABA)-induced H₂O₂ generation.^6,8,9 It has been indicated that PD98059 does not to intervene on salicylic acid (SA)-stimulated H₂O₂ signaling regardless of SA mimicking ABA in regulating stomatal closure.^2,6,8,10 Generally, activation of MEK1/2 promotes ABA-induced stomatal closure by elevating H₂O₂ generation in conjunction with inactivating anti-oxidases.Moreover, activation of plant p38-like protein kinase, the putative counterpart of yeast or mammalian p38 MAP kinase, has been reported to participate in various stress responses and ROS signaling. It has been well documented that p38 MAP kinase is involved in stress-triggered ROS signaling in yeast or mammalian cells.^11–13 Similar to those of yeast and mammals, many studies showed the activation of p38-like protein kinase in response to stresses in various plants, including Arabidopsis thaliana,^14–16 Pisum sativum,¹⁷ Medicago sativa¹⁸ and tobacco.¹⁹ The specific p38 kinase inhibitor SB203580 was found to modulate physiological processes in plant tissues or cells, such as wheat root cells,²⁰ tobacco tissue²¹ and suspension-cultured Oryza sativa cells.²² Recently, we investigate how activation of p38-like MAP kinase is involved in ABA-induced H₂O₂ signaling in guard cells. Our results show that SB203580 blocks ABA-induced stomatal closure by inhibiting ABA-induced H₂O₂ generation and decreasing K⁺ influx across the plasma membrane of Vicia guard cells, contrasting greatly with its analog SB202474, which has no effect on these events.^23,24 This suggests that ABA integrate activation of p38-like MAP kinase and H₂O₂ signaling to regulate stomatal behavior. In conjunction with SB203580 mimicking PD98059 not to mediate SA-induced H₂O₂ signaling,^23,24 these results generally reveal that the activation of p38-like MAP kinase and MEK1/2 is similar in guard cells.On the other hand, activation of p38-like MAP kinase^23,24 is not always identical to that of MEK1/2^8,25 in ABA-induced H₂O₂ signaling of Vicia guard cells. For example, H₂O₂^- and ABA-induced stomatal closure was partially reversed by SB203580. The maximum inhibition of both regent-induced stomatal closure were observed at 2 h after treatment with SB203580, under which conditions the stomatal apertures were 89% and 70% of the control values, respectively. By contrast, when PD98059 was applied together with ABA or H₂O₂, the effects of both ABA- and H₂O₂-induced stomatal closure were completely abolished (Fig. 1). These data imply that the two members of MAP kinase family are efficient in H₂O₂-stimulated stomatal closure, but p38-like MAP kinase is less susceptive than MEK1/2 to ABA stimuli.Open in a separate windowFigure 1Effects of SB203580 and PD98059 on ABA- and H₂O₂-induced stomatal closure. The experimental procedure and data analysis are according to the previous publication.^8,23,24It has been reported that ABA or NaCl activate p38 MAP kinase in the chloronema cells of the moss Funaria hygrometrica in 2∼10 min.²⁶ Similar to this, SB203580 improves H₂O₂-inhibited inward K⁺ currents after 4 min and leads it to the control level (100%) during the following 8 min (Fig. 2). However, the activation of p38-like MAP kinase in response to ABA need more time, and only recovered to 75% of the control at 8 min of treatment (Fig. 2). These results suggest that control of H₂O₂ signaling is required for the various protein kinases including p38-like MAP kinase and MEK1/2 in guard cells,^1,2,8,23,24 and the ABA and H₂O₂ pathways diverge further downstream in their actions on the K⁺ channels and, thus, on stomatal control. Other differences in action between ABA and H₂O₂ are known. For example, Köhler et al. (2001) reported that H₂O₂ inhibited the K⁺ outward rectifier in guard cells shows that H₂O₂ does not mimic ABA action on guard cell ion channels as it acts on the K⁺ outward rectifier in a manner entirely contrary to that of ABA.²⁷Open in a separate windowFigure 2Effect of SB203580 on ABA- and H₂O₂-inhibited inward K⁺ currents. The experimental procedure and data analysis are according to the previous publication.²⁴ SB203580 directs ABA- and H₂O₂-inactivated inward K⁺ currents across plasma membrane of Vicia guard cells. Here the inward K⁺ currents value is stimulated by −190 mV voltage.Based on the similarity and difference between PD98059 and SB203580 in interceding ABA and H₂O₂ signaling, we speculate the possible mechanism is that the member of MAP kinase family specially regulate signal event in ABA-triggered ROS signaling network,^1–4 and the signaling model as follows (Fig. 3).Open in a separate windowFigure 3Schematic illustration of MAP kinase-mediated H₂O₂ signaling of guard cells. The arrows indicate activation. The line indicates enhancement and the bar denotes inhibition.     

Why have chloroplasts developed a unique motility system?

Organelle movement in plants is dependent on actin filaments with most of the organelles being transported along the actin cables by class XI myosins. Although chloroplast movement is also actin filament-dependent, a potential role of myosin motors in this process is poorly understood. Interestingly, chloroplasts can move in any direction and change the direction within short time periods, suggesting that chloroplasts use the newly formed actin filaments rather than preexisting actin cables. Furthermore, the data on myosin gene knockouts and knockdowns in Arabidopsis and tobacco do not support myosins'' XI role in chloroplast movement. Our recent studies revealed that chloroplast movement and positioning are mediated by the short actin filaments localized at chloroplast periphery (cp-actin filaments) rather than cytoplasmic actin cables. The accumulation of cp-actin filaments depends on kinesin-like proteins, KAC1 and KAC2, as well as on a chloroplast outer membrane protein CHUP1. We propose that plants evolved a myosin XI-independent mechanism of the actin-based chloroplast movement that is distinct from the mechanism used by other organelles.Key words: actin, Arabidopsis, blue light, kinesin, myosin, organelle movement, phototropinOrganelle movement and positioning are pivotal aspects of the intracellular dynamics in most eukaryotes. Although plants are sessile organisms, their organelles are quickly repositioned in response to fluctuating environmental conditions and certain endogenous signals. By and large, plant organelle movements and positioning are dependent on actin filaments, although microtubules play certain accessory roles in organelle dynamics.^1,2 Actin inhibitors effectively retard the movements of mitochondria,^3–6 peroxisomes,^5,7–11 Golgi stacks,^12,13 endoplasmic reticulum (ER),^14,15 and nuclei.^16–18 These organelles are co-aligned and associated with actin filaments.^{5,7,8,10–12,15,18} Recent progress in this field started to reveal the molecular motility system responsible for the organelle transport in plants.¹⁹Chloroplast movement is among the most fascinating models of organelle movement in plants because it is precisely controlled by ambient light conditions.^20,21 Weak light induces chloroplast accumulation response so that chloroplasts can capture photosynthetic light efficiently (Fig. 1A). Strong light induces chloroplast avoidance response to escape from photodamage (Fig. 1B).²² The blue light-induced chloroplast movement is mediated by the blue light receptor phototropin (phot). In some cryptogam plants, the red light-induced chloroplast movement is regulated by a chimeric phytochrome/phototropin photoreceptor neochrome.^23–25 In a model plant Arabidopsis, phot1 and phot2 function redundantly to regulate the accumulation response,²⁶ whereas phot2 alone is essential for the avoidance response.^27,28 Several additional factors regulating chloroplast movement were identified by analyses of Arabidopsis mutants deficient in chloroplast photorelocation.^29–32 In particular, identification of CHUP1 (chloroplast unusual positioning 1) revealed the connection between chloroplasts and actin filaments at the molecular level.²⁹ CHUP1 is a chloroplast outer membrane protein capable of interacting with F-actin, G-actin and profilin in vitro.^29,33,34 The chup1 mutant plants are defective in both the chloroplast movement and chloroplast anchorage to the plasma membrane,^22,29,33 suggesting that CHUP1 plays an important role in linking chloroplasts to the plasma membrane through the actin filaments. However, how chloroplasts move using the actin filaments and whether chloroplast movement utilizes the actin-based motility system similar to other organelle movements remained to be determined.Open in a separate windowFigure 1Schematic distribution patterns of chloroplasts in a palisade cell under different light conditions, weak (A) and strong (B) lights. Shown as a side view of mid-part of the cell and a top view with three different levels (i.e., top, middle and bottom of the cell). The cell was irradiated from the leaf surface shown as arrows. Weak light induces chloroplast accumulation response (A) and strong light induces the avoidance response (B).Here, we review the recent findings pointing to existence of a novel actin-based mechanisms for chloroplast movement and discuss the differences between the mechanism responsible for movement of chloroplasts and other organelles.     

Mannan synthase activity in the CSLD family

Cellulose Synthase Like (CSL) proteins are a group of plant glycosyltransferases that are predicted to synthesize β-1,4-linked polysaccharide backbones. CSLC, CSLF and CSLH families have been confirmed to synthesize xyloglucan and mixed linkage β-glucan, while CSLA family proteins have been shown to synthesize mannans. The polysaccharide products of the five remaining CSL families have not been determined. Five CSLD genes have been identified in Arabidopsis thaliana and a role in cell wall biosynthesis has been demonstrated by reverse genetics. We have extended past research by producing a series of double and triple Arabidopsis mutants and gathered evidence that CSLD2, CSLD3 and CSLD5 are involved in mannan synthesis and that their products are necessary for the transition between early developmental stages in Arabidopsis. Moreover, our data revealed a complex interaction between the three glycosyltransferases and brought new evidence regarding the formation of non-cellulosic polysaccharides through multimeric complexes.Key words: mannan, mannose, plant cell wall, glycosyltransferase, cellulose synthase like, CSL, biosynthesis, hemicelluloseThe plant cell wall is mainly composed of polysaccharides, which are often grouped into cellulose, hemicelluloses and pectin. Since the discovery of the first cellulose synthase (CESA) genes in cotton fibers,¹ the synthesis of cellulose has been extensively studied.² In contrast, the glycosyltransferases responsible for synthesizing hemicelluloses and pectin are still largely unidentified.^3,4,5 The CESA genes are members of a superfamily that includes genes with a high sequence similarity with CESA genes and are named Cellulose Synthase Like (CSL).⁶ The CSL genes have themselves been grouped into nine families designated CSLA, -B, -C, -D, -E, -F, -G, -H and -J (Figure 1A).^5,6 Mannan and glucomannan synthase activity has been demonstrated in the CSLA family,^7,8,9 while members of the CSLC family have been implicated in synthesis of the xyloglucan backbone.¹⁰ CSLF and CSLH, which are found only in grasses, are involved in synthesis of mixed linkage glucan.^11,12 The function of the remaining CSL families has not been determined. We have reported our research on the CSLD family in a recent publication.¹³ Of all the CSL families, CSLD possesses the most ancient intron/exon structure and is the most similar to the CESA family.⁶ CSLD genes are found in all sequenced genomes of terrestrial plants including Physcomitrella and Selaginella suggesting a highly conserved function throughout the plant kingdom (Figure 1A). Five genes (CSLD1 to CSLD5) and one apparent pseudogene (CSLD6) have been identified in Arabidopsis thaliana.¹⁴ Bernal et al.^14,15 studied knock-out mutants of the individual genes and presented evidence for a role in cell wall biosynthesis for each Arabidopsis CSLD. To elucidate the activity of the CSLD proteins and obtain further understanding of their biological role, we generated double mutants csld2/csld3, csld2/csld5, csld3/csld5 and the triple mutant csld2/csld3/csld5. Immunochemical, biochemical and complementation assays brought evidence that CSLD5 or CSLD2 in concomitance with CSLD3 act as mannan synthases.Open in a separate windowFigure 1(A) Schematic representation of the CESA superfamily phylogeny. The inset on the right is a detailed phylogenetic tree of CSLDs from Selaginella moellendorffii, Arabidopsis thaliana and Oryza sativa. The figure is modified from Ulvskov and Scheller.⁵ (B) Comparison of csld2, csld3, csld5 with Col-0 20 days after germination. The inflorescences of csld2 and csld3 were similar to Col-0 whereas csld5 had a delayed growth. Scale bar: 1 cm. (C) Col-0 and csld2/csld3/csld5 (triple mutant, TM) 40 days after germination. After 40 days, the triple mutant was barely developed and, as shown in the magnified inset, displayed purple coloration indicating accumulation of anthocyanins, a typical stress response. Scale bar: 2 mm.     

Nonhost resistance to Magnaporthe oryzae in Arabidopsis thaliana

Rice blast, caused by Magnaporthe oryzae, is a devastating disease of rice (Oryza sativa). The mechanisms involved in resistance of rice to blast have been studied extensively and the rice—M. oryzae pathosystem has become a model for plant—microbe interaction studies. However, the mechanisms involved in nonhost resistance (NHR) of other plants to rice blast are still poorly understood. We have recently demonstrated that AGB1 and PMR5 contribute to PEN2-mediated preinvasion resistance to M. oryzae in Arabidopsis thaliana, suggesting a complex genetic network regulating the resistance. To determine whether other defense factors: RAR1, SGT1 and NHO1, affected the A. thaliana-M. oryzae interactions, double mutants were generated between pen2 and these defense-related mutants. All these double mutants exhibited a level of penetration resistance similar to that of the pen2 mutant, suggesting that none of these mutants significantly compromised resistance to M. oryzae in a pen2 background.Key words: nonhost resistance, PEN2, RAR1, SGT1, NHO1Plants face microbial attacks and have evolved innate immunity systems to defend against these threats. The initial step of the immunity signaling pathway is recognition of intra- or extracellular pathogen-derived molecules. Externally oriented transmembrane-type proteins containing leucine-rich repeat (LRR) domains detect extracellular molecules, whereas cytoplasmic sensors possess nucleotide-binding (NB) and LRR domains (NLR).^1,2 The LRR domain serves as a pattern-recognition receptor to detect pathogen-derived molecules or host proteins that are targeted by pathogen peptides that have entered the cell, effectors.³ NLR-type sensors are the substrates of a structurally and functionally conserved chaperone complex that consists of HEAT SHOCK PROTEIN 90 (HSP90) and its cochaperone SUPPRESSOR OF THE G2 ALLELE OF SKP1 (SGT1). REQUIRED FOR MLA12 RESISTANCE 1 (RAR1) regulated the HSP90-SGT1 complex, resulting in the stabilization of NLR proteins. Thus, SGT1 and RAR1 are required for the function of multiple and distinct R genes that encode NLR immune sensors in plants.⁴ Experiments in RAR1-silenced transgenic rice lines showed that RAR1 is not essential for Pib, which encodes an NLR against rice blast fungus.⁵ In contrast, basal resistance to normally virulent races of rice blast fungus or bacterial blight is significantly reduced in RAR1-silenced lines. This result is consistent with earlier reports that RAR1 is involved in basal resistance to virulent Pseudomonas bacteria in Arabidopsis or blast fungus in barley.^6,7 The requirement of SGT1 for immunity in plants is shown mostly by transient silencing of a number of NLR proteins.^8,9 In addition, SGT1 is also required for immune responses triggered by non-NLR-type sensors.¹⁰ This requirement indicates that either SGT1 function is not limited to the NLR sensors, or some unknown SGT1-dependent NLR proteins also operate downstream of non NLR-type sensors. Furthermore, SGT1 is involved in nonhost resistance, indicating that SGT1 may be a general factor of disease resistance.¹⁰ An Arabidopsis mutant, nho1 (nonhost resistance 1), has been isolated on which Pseudomonas syringae pv. phaseolicola grows and causes disease symptoms.^11,12 It is significant that this mutant is also compromised in R-gene-mediated resistance to P. syringae.¹¹ Although NHO1 is the flagellin-induced glycerol kinase, whose exact function in NHR remains elusive.^12,13 A possible explanation might be that altered plant glycerol pools either directly or indirectly affect nutrient availability for P. syringae. NHO1 is also required for resistance to the fungal pathogen Botrytis cinerea, indicating that NHO1 is not limited to bacterial resistance.¹² However, these contributions to NHR to M. oryzae in A. thaliana have not been understood.To determine whether these factors were necessary for the resistance to M. oryzae in A. thaliana, the following A. thaliana mutants were inoculated with M. oryzae and monitored by microscopy: rar1-21;¹⁴ edm1-1;¹⁵ nho1-1,¹¹ (all Col-0 background). All these mutants exhibited a level of penetration resistance similar to that of the wild-type plants (data not shown), suggesting that none of these mutants significantly compromised resistance to M. oryzae. We have recently shown that among the penetration (pen) mutants, only the pen2,¹⁶ mutant allowed increased penetration into epidermal cells by M. oryzae.¹⁷ Thus, double mutants were generated between pen2 and these mutants to determine whether these factors were necessary for the resistance to M. oryzae in a pen2 background: pen2 rar1-21; pen2 edm1-1; pen2 nho1-1. All these double mutants exhibited a level of penetration resistance similar to that of the pen2 mutant (Fig. 1), suggesting that none of these mutants significantly compromised resistance to M. oryzae in a pen2 background. This might indicate that NHR against M. oryzae may not be conferred by RAR1- and SGT1-dependent NLR immune sensors. Alternatively, since there has been no report that RAR1 is required for any known transmembrane sensors, such as FLS2, EFR or Xa21, RAR1- and SGT1-independent transmembrane-type immune sensors may be required for NHR against M. oryzae. Future studies will be required to reveal the genetic and mechanistic requirements for NHR in A. thaliana-M. oryzae interactions.Open in a separate windowFigure 1Double mutant analysis to evaluate the role of the defense related genes on resistance to Magnaporthe oryzae in Arabidopsis thaliana. The frequency of M. oryzae penetration on double mutants at 3 days post-inoculation was expressed as a percentage of total appressoria. Data were collected from six independent plants per line. A minimum of 100 infection sites was inspected per leaf. Results represent mean ± standard error of three independent experiments.     

Evolutionary origin of rhizobium Nod factor signaling

For over two decades now, it is known that the nodule symbiosis between legume plants and nitrogen fixing rhizobium bacteria is set in motion by the bacterial signal molecule named nodulation (Nod) factor.¹ Upon Nod factor perception a signaling cascade is activated that is also essential for endomycorrhizal symbiosis (Fig. 1). This suggests that rhizobium co-opted the evolutionary far more ancient mycorrhizal signaling pathway in order to establish an endosymbiotic interaction with legumes.² As arbuscular mycorrhizal fungi of the Glomeromycota phylum can establish a symbiosis with the vast majority of land plants, it is most probable that this signaling cascade is wide spread in the plant kingdom.³ However, Nod factor perception generally is considered to be unique to legumes. Two recent breakthroughs on the evolutionary origin of rhizobium Nod factor signaling demonstrate that this is not the case.^4,5 The purification of Nod factor-like molecules excreted by the mycorrhizal fungus Glomus intraradices and the role of the LysM-type Nod factor receptor PaNFP in the non-legume Parasponia andersonii provide novel understanding on the evolution of rhizobial Nod factor signaling.Open in a separate windowFigure 1Schematic representation of the genetically dissected symbiosis signaling pathway. In legumes rhizobium Nod factors and mycorrhizal Myc factors are perceived by distinct receptor complexes. In case of Nod factors these are the LysM-RK type receptors MtLYK3/LjNFR1 and MtNFP/LjNFR5, whereas Myc factors remain to be elucidated. In Parasponia PaNFP fulfils a dual function and acts in both symbioses. The subsequent common signaling pathway consists of several components including a plasma membrane localized LRR-type receptor (MtDMI2/LjSymRK), a cation channel in the nuclear envelope (MtDMI1/LjCASTOR/LjPOLLUX) and subunits of the nuclear pore (NUP85, NUP133), and a nuclear localized complex of calcium calmodulin dependent kinase (CCaMK) and interactor protein MtIPD3/LjCYCLOPS. Downstream of CCaMK the rhizobium and mycorrhiza induced responses bifurcate.Key words: parasponia, legumes, rhizobium, mycorrhizae, Nod factor     

Antennal responses of an oligolectic bee and its cleptoparasite to plant volatiles

Cleptoparasitic or cuckoo bees lay their eggs in nests of other bees, and the parasitic larvae feed the food that had been provided for the host larvae. Nothing is known about the specific signals used by the cuckoo bees for host nest finding, but previous studies have shown that olfactory cues originating from the host bee alone, or the host bee and the larval provision are essential. Here, I compared by using gas chromatography coupled to electroantennographic detection (GC-EAD) the antennal responses of the oligolectic oil-bee Macropis fulvipes and their cleptoparasite, Epeoloides coecutiens, to dynamic headspace scent samples of Lysimachia punctata, a pollen and oil host of Macropis. Both bee species respond to some scent compounds emitted by L. punctata, and two compounds, which were also found in scent samples collected from a Macropis nest entrance, elicited clear signals in the antennae of both species. These compounds may not only play a role for host plant detection by Macropis, but also for host nest detection by Epeoloides. I hypothesise that oligolectic bees and their cleptoparasites use the same compounds for host plant and host nest detection, respectively.Key words: Macropis fulvipes, Epeoloides coecutiens, Lysimachia punctata, oligolectic oil-bee, floral scent, dynamic headspace, GC-EAD, cuckoo bee, host nest findingBees are the most important animal pollinators worldwide, and guarantee sexual reproduction of many plant species.^1,2 This is especially true for female bees, which collect pollen and mostly nectar for their larvae and frequently visit flowers. For finding and detection of suitable flowers, bees are known to use, besides optical cues,^3,4 especially olfactory signals.^5–8 However, c. 20% of bees do not collect pollen for their larvae by their own, but enter nests of host bees and lay eggs into the broodcells.^1,9 The parasitic larvae subsequently feed the food that had been provided for the host larvae. These so called cuckoo or cleptoparasitic bees can be generalistic, indicating that they use species of several other bee groups as host, whereas others can be highly specialized, laying eggs in cells of only few host species.¹ Until now little is known about the cues used by the cuckoo bees for finding host nests. Nevertheless, Cane¹⁰ and Schindler¹¹ demonstrated that parasitic Nomada bees use primarily visual cues of the nest entrance holes for finding possible nests, and olfactory cues for detection of suitable host nests. The chemical cues used by the cleptoparasites originate from the host bee^10,11 and also pollen,¹⁰ the main larval provision. In most bee species, pollen is mixed together with nectar as larval provision, and both floral resources are known to emit volatiles.^12,13 It is unknown, whether cuckoo bees in search for host nests also use volatiles originating from nectar. While the odours of the host bee used as signal by the cleptoparasites, e.g., cuticiular hydrocarbons and glandular secretions, are often species-specific,¹⁴ the chemical cues from the larval provision may just indicate the presence of pollen in the nest without more specifity. As a consequence cuckoo bees could use species-specific host odours to detect nests of a suitable host, and odours released from the larval provision could indicate to them that broodcells are foraged. However, especially those cuckoo bees with oligolectic hosts foraging pollen only on few closely related plant species,¹ may also use the olfactory signals from host broodcell supplies as more specific cue for host nest detection. Thus the same signal from certain flowers may be used for different informations: for the host bee for host plant and for the cuckoo bee for host nest detection.In this concern I tested oligolectic Macropis (Melittidae, Melittinae) and its specific cuckoo bee, Epeoloides (Apidae, Apinae) by using gas chromatography coupled to electroantennographic detection (GC-EAD) on floral scent of Lysimachia (Myrsinaceae). Macropis is highly specialized on Lysimachia, because it is not only collecting pollen from plants of this genus, but also floral oil. Both floral products are the only provision for the larvae.^1,15 Recently, we have shown that the oil bee Macropis is strongly attracted to floral scent of its oil host Lysimachia though the compounds used for host plant finding are still unknown.⁷ Macropis is the only host of Epeoloides, and larvae of this cleptoparasite only feed on the Lysimachia pollen-oil mixture provided for the larvae of Macropis. Worldwide, there are only 2 species of this genus, one in North America and the other in Europe/Asia.^1,16,17 I hypothesized that both bee species respond to specific Lysimachia compounds, which may be used for host plant as well as host nest detection.The measurements with M. fulvipes (F.) and E. coecutiens (F.) antennae demonstrate that both bees, host as well as cuckoo bee, respond to some scent compounds emitted by inflorescences of Lysimachia punctata L. (Fig. 1), a plant being an important pollen and oil source for M. fulvipes. Macropis responded to much more Lysimachia compounds compared to the cuckoo bee, however, two compounds elicited clear signals in the antennae of both bee species: the benzenoid 1-hydroxy-1-phenyl-2-propanone, and the fatty acid derivative 2-tridecanone. Interestingly, both compounds are also emitted from the floral oil of this plant,⁷ and both compounds were also detected in scent samples collected by dynamic headspace in the entrance of a Macropis nest (Dötterl, unpublished data). Therefore, an Epeoloides female being in search for a host nest can detect volatiles emitted from the provision of the host bee at the entrance of a bee nest, and may use these specific compounds for detection of a Macropis nest provisioned with Lysimachia pollen and oil.Open in a separate windowFigure 1Coupled gas chromatographic and electroantennographic detection of a Lysimachia punctata headspace scent sample using antennae of a female oligolectic Macropis fulvipes and a female cleptoparasitic Epeoloides coecutiens bee. (1) 1-hydroxy-1-phenyl-2-propanone, (2) 2-tridecanone.Present results show that an oligolectic oil-bee as well as its cleptoparasite detects volatiles originating from the host plant of the pollen collecting bee, and that oligolectic bees as well as their cuckoo bees may use the same specific signals for host plant and host nest finding, respectively. Biotests are now needed to test this hypothesis.     

Duration of Priming of Two Indirect Plant Defenses

When plants are infested by herbivores, they emit herbivore-induced plant volatiles (HIPVs) that attract carnivorous natural enemies of herbivores. Furthermore, there are increasing evidences that defenses of intact plants against herbivores are primed when exposed to HIPVs. We previously reported that lima bean leaf volatiles induced by the herbivorous mites Tetranychus urticae primed two T. urtiae-induced indirect defenses in neighboring conspecific plants: HIPV emission and extrafloral nectar (EFN) secretion. An intriguing unanswered question is whether the durations of these two defenses are the same. Here, we show that the durations of the two defenses were the same for up to two days after the initiation of T. urticae damage. The two induced primed defense would act as a battery of defense in exposed plants.Key Words: herbivore-induced plant volatiles, indirect, defense, induced response, plant-plant interaction, primingWhen infested by herbivores, plants defend themselves indirectly by emitting herbivore-induced plant volatiles (HIPVs). One of the ecological functions of HIPVs is to attract carnivorous natural enemies of the herbivores.^1,2 Recently, it was reported that the emission of HIPVs primed defenses against herbivores in neighboring intact plants.^3–7 Thus, HIPVs also mediate interactions between infested and intact plants.⁸ The enhanced defense in response to HIPVs in intact plants is called ‘priming’, which has been studied intensively in plant-pathogen interactions,⁹ but not so in plant-insect interactions.We previously reported that exposure to HIPVs emitted from lima bean leaves infested by Tetranychus urticae primed HIPV production in detached intact conspecific leaves.³ We also reported that exposure to HIPVs, produced in response to T. urticae damage,⁴ primed the induced production of extrafloral nectar (EFN; an alternative food source for predators^10,11 in lima bean plants. An intriguing question is whether the two primed defenses work as a battery against T. urticae. To answer this, we examined the duration of primed HIPV production by lima bean plants using the same experimental set-up as our previous study of EFN priming by conspecific plants.⁴For exposure of plants to HIPVs, we used a 60 × 60 × 60 cm cage with two 30 × 30 cm windows on opposite sides of the cage.¹² As odor sources, we used eight plants that had been infested with 60 adult T. urticae females per plant for 1 day. Eight uninfested plants were used as control odor sources. Two uninfested plants were placed in a cage with the odor source plants and exposed to either HIPVs or uninfested plant volatiles (UPVs) for 10 days in a climate-controlled room (25 ± 2°C, 60–70% RH, 16:8; L:D).A Y-tube olfactometer¹³ was used to examine the response of the predators to HIPVs. Adult female P. persimilis were randomly selected from a colony and individually positioned at the beginning of the iron wire. When test mites reached the end of one arm of the olfactometer, their choice was recorded. We tested the olfactory responses of the predator toward (1) plants infested by T. urticae for two days after exposure to UPVs vs. plants infested by T. urticae for two days after exposure to HIPVs, and (2) plants infested by T. urticae for four days after exposure to UPVs vs. plants infested by T. urticae for four days after exposure to HIPVs.HIPV-exposed plants attracted more predators than UPV-exposed plants in a Y-tube olfactometer when infested by T. urticae for two days (Fig. 1A). By contrast, the predators did not distinguish between HIPV- and UPV-exposed plants when infested by T. urticae for four days (Fig. 1B). Our previous study showed that HIPV-exposed plants secreted significantly larger amounts of EFN secretion than UPV-exposed plants infested by T. urticae for two days under the same experimental condition as in this study.⁴ However, the difference was not significant when they were infested for four days.⁴Open in a separate windowFigure 1The olfactory response of P. persimilis females to volatiles from the odor-exposed plants, as determined in a Y-tube olfactometer: (A) plants infested by T. urticae for two days after exposure to UPVs (UPV-exposed—T. urticae 2d) vs. plants infested by T. urticae for two days after exposure to HIPVs (HIPV-exposed—T. urticae 2d), and (B) plants infested by T. urticae for four days after exposure to UPVs (UPV-exposed—T. urticae 4d) vs. plants infested by T. urticae for four days after the exposure to HIPVs (HIPV-exposed—T. urticae 4d). Asterisks beside each bar indicate a significant difference between the first trifoliate leaves and the primary leaves. Asterisks beside a bar indicate a significant difference (binomial test: p < 0.001).Lima bean plants increase the amount of endogenous jasmonic acid after exposure to HIPVs.¹⁴ Jasmonic acid, an important plant hormone regulating a defense signaling pathway against herbivores and pathogens,^15,16 is reported to be involved in the induction of both volatile emission^17,18 and EFN secretion¹⁹ in response to T. urticae damage in lima bean plants. The increase of endogenous jasmonic acid in HIPV-exposed plants may partly explain the simultaneous priming of the two defenses.In this study, we showed that the durations of priming of two indirect defenses were roughly the same for up to two days. Priming of these two indirect defenses would thus be a battery of defense at the outset of T. urticae damage. Further study is necessarily to test whether the primed battery of induced defense increases the fitness of the exposed plants.