Characterization of PR-10 genes from eight Betula species and detection of Bet v 1 isoforms in birch pollen

Background  

Bet v 1 is an important cause of hay fever in northern Europe. Bet v 1 isoforms from the European white birch (Betula pendula) have been investigated extensively, but the allergenic potency of other birch species is unknown. The presence of Bet v 1 and closely related PR-10 genes in the genome was established by amplification and sequencing of alleles from eight birch species that represent the four subgenera within the genus Betula. Q-TOF LC-MS^E was applied to identify which PR-10/Bet v 1 genes are actually expressed in pollen and to determine the relative abundances of individual isoforms in the pollen proteome.     

Early responses of bark and wood boring beetles to an outbreak of the hemlock looper Lambdina fiscellaria (Guenée) (Lepidoptera: Geometridae) in a boreal balsam fir forest of North America

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Comparative use of Mindarus abietinus (Homoptera: Aphididae) by two coccinellids (Coleoptera: Coccinellidae), the native Anatis mali and the exotic Harmonia axyridis, in a Christmas tree plantation

The exotic coccinellid Harmonia axyridis (Pallas) recently expanded its range into eastern Canada and elsewhere in North America. We hypothesized that this coccinellid should be less well adapted to the prey Mindarus abietinus Koch. on balsam fir trees than the native coccinellid Anatis mali (Say), which evolved in close association with aphids on conifers in North America. We compared, under field conditions, prey use by both species by collecting data on their synchrony with M. abietinus, their prey searching and predation behaviors, life stage distribution in fir canopy, and their overall reproductive success in this system. The seasonal life cycle of A. mali was better synchronized with that of M. abietinus compared with that of H. axyridis. In spring, A. mali adults appeared nearly 2 wk earlier on trees than H. axyridis and were active predators of the aphid fundatrices. A. mali oviposition thus began before the aphid population started to grow, and its larvae were most active during peak aphid colonies. Behavioral observations showed that both adults and larvae of the native A. mali searched for prey more actively than those of H. axyridis. Distribution of life stages also showed that eggs and pupae had different distributions on trees and that the adult-to-adult net reproductive rate of A. mali was three times higher than that of H. axyridis. Thus, the native A. mali was better adapted than H. axyridis to prey on M. abietinus, possibly because it evolved for a much longer period of time with this prey in conifer habitats.     

Kinetic metabolic modelling for the control of plant cells cytoplasmic phosphate

A previously developed kinetic metabolic model for plant metabolism was used in a context of identification and control of intracellular phosphate (Pi) dynamics. Experimental data from batch flask cultures of Eschscholtiza californica cells was used to calibrate the model parameters for the slow dynamics (growth, nutrition, anabolic pathways, etc.). Perturbation experiments were performed using a perfusion small-scale bioreactor monitored by in vivo³¹P NMR. Parameter identification for Pi metabolism was done by measuring the cells dynamic response to different inputs for extracellular Pi (two pulse-response experiments and a step-response experiment). The calibrated model can describe Pi translocation between the cellular pools (vacuole and cytoplasm). The effect of intracellular Pi management on ATP/ADP and phosphomonoesters concentrations is also described by the model. The calibrated model is then used to develop a control strategy on the cytoplasmic Pi pool. From the identification of the systems dynamics, a proportional-integral controller was designed and tuned. The closed-loop control was implemented in the small-scale NMR bioreactor and experimental results were in accordance with model predictions. Thus, the calibrated model is able to predict cellular behaviour for phosphate metabolism and it was demonstrated that it is possible to control the intracellular level of cytoplasmic Pi in plant cells.     

Biphasic ethylene production during the hypersensitive response in Arabidopsis: A window into defense priming mechanisms?

The hypersensitive response (HR) is a cell death phenomenon associated with localized resistance to pathogens. Biphasic patterns in the generation of H₂O₂, salicylic acid and ethylene have been observed in tobacco during the early stages of the HR. These biphasic models reflect an initial elicitation by pathogen-associated molecular patterns followed by a second phase, induced by pathogen-encoded avirulence gene products. The first phase has been proposed to potentiate the second, to increase the efficacy of plant resistance to disease. This potentiation is comparable to the “priming” of plant defenses which is seen when plants display systemic resistance to disease. The events regulating the generation of the biphasic wave, or priming, remains obscure, however recently we demonstrated a key role for nitric oxide in this process in a HR occurring in tobacco. Here we use laser photoacoustic detection to demonstrate that biphasic ethylene production also occurs during a HR occurring in Arabidopsis. We suggest that ethylene emanation during the HR represents a ready means of visualising biphasic events during the HR and that exploiting the genomic resources offered by this model species will facilitate the development of a mechanistic understanding of potentiating/priming processes.Key words: hypersensitive response, biphasic patterns, potentiation, defense priming, ethylene, ArabidopsisThe Hypersensitive Response (HR) is a cell death process which occurs at the site of attempted pathogen attack and which has been associated with host resistance.¹ Much work on the regulation of the HR has indicated the importance of H₂O₂,² and NO.³ A feature of H₂O₂ generation during the HR is its biphasic pattern (Fig. 1A). The first rise reflects elicitation by pathogen-associated molecular patterns (PAMPs)⁴ and the second reflects the interaction between a pathogen-encoded avirulence (avr) gene product with a plant resistance (R) gene. A key aspect of the first rise is the initiation of salicylic acid (SA) synthesis which potentiates the second rise and hence the potency of plant defense and the HR.⁵Open in a separate windowFigure 1Patterns of defense signal generation during the Pseudomonas syringae pv. phaseolicola elicited-hypersensitive response in tobacco (Nicotiana tabacum). Generation of (A) H₂O₂ (●, Mur¹⁸); (B) nitric oxide (◇; Mur¹² (C) salicylic acid (SA, ■¹⁹) and (D) ethylene (○ Mur⁹) during a HR elicited by Pseudomonas syringae pv. phaseolicola (Psph) in tobacco cv. Samsun NN. In (A) a phase where SA acts to augment the second rise in H₂O₂—the potentiation phase—is highlighted. The potentiation phase is likely to be similar to defense “priming”.⁶ Methodological details are contained within the appropriate references. (E) A possible model for biphasic defense signal regulation during the Psph-elicited HR in tobacco. During an initial phase NO and H₂O₂ act to initiate SA biosynthesis, where SA and NO act to initiate a “H₂O₂ biphasic switch”. This could initially suppress both SA and the H₂O₂ generation but subsequently acts to potentiate a second phase of H₂O₂ generation. This in turn increases SA biosynthesis which could act with NO to initiate the “C₂H₄ biphasic switch” to potentiate ethylene production. These (and other) signals contribute to initiation of the HR and SAR.This potentiation mechanism appears to be similar to defense priming; when whole plants display systemic resistance to disease as opposed to a localized resistance against pathogens. Priming can be initiated (the “primary stimulus”) following attack with a necrotizing pathogen (leading to “systemic acquired resistance”, SAR) or non-pathogenic rhizosphere bacteria (to confer “induced systemic resistance”, ISR). In the primed state a plant stimulates a range of plant defense genes, produces anti-microbial phytoalexins and deposits cell wall strengthening molecules, but only on imposition of a “secondary stimulus”.⁶ Such secondary stimuli include SA³ or PAMPs⁷ and is likely to be mechanistically similar to the potentiation step in the biphasic pattern of H₂O₂ generation (shaded in Fig. 1A). Accordingly, the two phases in the biphasic wave represent primary and secondary stimuli in priming.Highlighting a similarity between local HR-based events and priming, adds further impetus to efforts aiming to describe the underlying mechanism(s), however both phenomena remain poorly understood. Besides SA, both jasmonates and abscisic acid (ABA) have been shown to prime defenses as have a range of non-plant chemicals, with β-aminobutyric acid (BABA) being perhaps most widely used.^6,8 Mutants which fail to exhibit BABA-mediated potentiation were defective in either a cyclin-dependent kinase-like protein, a polyphosphoinositide phosphatase or an ABA biosynthetic enzyme.⁸We have recently investigated biphasic ethylene production during the HR in tobacco elicited by the nonhost HR-eliciting bacterial pathogen Pseudomonas syringae pv. phaseolicola.⁹ As with H₂O₂ generation, this pattern reflected PAMP-and AVR-dependent elicitation events and included a SA-mediated potentiation stage. Crucially, we also showed that NO was a vital component in the SA-potentiation mechanism. When this finding is integrated with our other measurements of defense signal generation in the same host-pathogen system the complexity in the signaling network is revealed (Fig. 1). NO generation (Fig. 1B) appeared to be coincident with the first rise in H₂O₂ (Fig. 1A) which initiated SA biosynthesis^10,11 and together would contribute to the first small, but transient, rise in that hormone (Fig. 1C). In line with established models⁵ this momentary rise in SA coincides with the potentiation phase (shaded in Fig. 1A) required to augment the second rise in ROS. However, ethylene production seems to be correlated poorly with the patterns of NO, H₂O₂ and SA (Fig. 1D). Nevertheless, biphasic ethylene production was found to reflect PAMP and AVR-dependent recognition and included a SA-mediated potentiation step.⁹ Hence, ethylene production could be used as a post-hoc indicator of the potentiation mechanism. Therefore, our discovery that the second wave of ethylene production—a “biphasic switch”—is influenced by NO acting with SA could also be relevant to the H₂O₂ generation. Significantly, the second phases in both H₂O₂ and ethylene production occur exactly where SA and NO production coincides; in the case of H₂O₂ generation 2–4 h post challenge and with ethylene 6 h onwards (Fig. 1E).Thus, ethylene production represents a readily assayable marker to indicate perturbations in the underlying biphasic and possible priming mechanisms. As we have demonstrated, laser photoacoustic detection (LAPD) is a powerful on-line approach to determine in planta ethylene production in tobacco^9,12 but any mechanistic investigations would be greatly facilitated if the genetic resources offered by the model species Arabidopsis could be exploited.To address this, Arabidopsis Col-0 rosettes were vacuum infiltrated with either Pseudomonas syringae pv. tomato (Pst) avrRpm1 (HR-eliciting), the virulent Pst strain and the non-HR eliciting and non-virulent Pst hrpA strain. Ethylene production was monitored by LAPD (Fig. 2A). Significantly, Pst avrRpm1 initiated a biphasic pattern of ethylene production whose kinetics were very similar to that seen in tobacco (compare Figs. 2A with ​with1D).1D). Inoculations with Pst and Pst hrpA only displayed the first PAMP-dependent rise in ethylene production. Thus, these data establish that Arabidopsis can be used to investigate biphasic switch mechanism(s) in ethylene production during the HR and possibly defense priming. When considering such mechanisms, it is relevant to highlight the work of Foschi et al.¹³ who observed that biphasic activation of a monomeric G protein to cause phase-specific activation of different kinase cascades. Interestingly, ethylene has been noted to initiate biphasic activation of G proteins and kinases in Arabidopsis, although differing in kinetics to the phases seen during the HR.¹⁴ Further, plant defense priming has been associated with the increased accumulation of MAP kinase protein.⁶Open in a separate windowFigure 2Ethylene in the Pseudomonas syringae pv. tomato elicited-hypersensitive response in Arabidopsis thaliana. (A) Ethylene production from 5 week old short day (8 h light 100 µmol.m².sec⁻¹) grown Arabidopsis rosette leaves which were vacuum infiltrated with bacterial suspensions (2 × 10⁶ colony forming units.ml⁻¹) of Pseudomonas syringae pv. tomato (Pst) strains detected using laser photoacoustic detection (LAPD). Experimental details of the ethylene detection by LAPD are detailed in Mur et al.⁹ The intercellular spaces in leaves were infiltrated with the HR-eliciting strain Pst avrRpm1, (■), the virulent strain Pst (△) or the non-virulent and non-HR eliciting derivative, Pst hrpA (◇). (B) The appearance of Arabidopsis Col-0 and etr1-1 leaves at various h following injection with 2 × 10⁶ c.f.u.mL⁻¹ with of Pst avrRpm1. (C) Explants (1 cm diameter discs) from Arabidopsis leaf areas infiltrated with suspensions of Pst avrRpm1 were placed in a 1.5 cm diameter well, bathed in 1 mL de-ionized H₂O. Changes in the conductivity of the bathing solution, as an indicator of electrolyte leakage from either wild type Col-0 (◆), mutants which were compromised in ethylene signaling; etr1-1 (□), ein2-2 (▲) or which overproduced ethylene; eto2-1 (●) were measured using a conductivity meter. Methodological details are set out in Mur et al.⁹A further point requires consideration; the role of ethylene as a direct contributor to plant defense.¹⁵ The contribution of ethylene to the HR has been disputed,¹⁶ but in tobacco we have observed that altered ethylene production influenced the formation of a P. syringae pv. phaseolicola elicited HR.⁹ In Arabidopsis, cell death in the ethylene receptor mutant etr1-1 following inoculation with Pst avrRpm1 is delayed compared to wild type (Fig. 2B). When electrolyte leakage was used to quantify Pst avrRpm1 cell death, both etr1-1 and the ethylene insensitive signaling mutant ein2-1 exhibited slower death than wild-type but in the ethylene overproducing mutant eto2, cell death was augmented (Fig. 2C). These data indicate that ethylene influences the kinetics of the HR.Taking these data together we suggest that the complexity of signal interaction during the HR or in SAR/ISR could be further dissected by combining the genetic resources of Arabidopsis with measurements of ethylene production using such sensitive approaches as LAPD.     

Recruitment of closely linked genes for divergent functions: the seed storage protein (Glu-3) and powdery mildew (Pm3) genes in wheat (Triticum aestivum L.)

Wheat seed storage protein gene loci (Glu-3) and powdery mildew resistance gene loci (Pm3 and Pm3-like) are closely linked on the short arms of homoeologous group 1 chromosomes. To study the structural organization of the Glu-3/Pm3 loci, three bacterial artificial chromosome clones were sequenced from the A, B, and D genomes of hexaploid wheat. The A and B genome clones contained a Glu-3 adjacent to a Pm3-like gene organized in a conserved Glu-3/SFR159/Pm3-like structure. The D genome clone contained clusters of resistance gene analogs but no Pm3. Its similarity to the A and B genome was limited to the Glu-3/SFR159 region. Comparison of the B genome PM3-like deduced amino acid sequence with known PM3 functional isotypes reinforced the hypothesis of allelic evolution via block exchange by gene conversion/recombination. The advent of glutenin genes and the formation of the Glu-3/SFR159/Pm3 locus occurred after divergence of wheat from rice and Brachypodium. Comparison of the A genome homologous sequences permitted an estimate of time of divergence of ~0.3 million years ago. The B genome sequences were not colinear indicating that they could either be paralogs or represent different B genome progenitors. Analysis of the 11 complete retrotransposons indicated a time of divergence ranging from 0.29 to 5.62 million years ago, consistent with their complex nested structure.     

A novel, noninvasive transdermal fluid sampling methodology: IGF-I measurement following exercise

This study tested the hypothesis that transdermal fluid (TDF) provides a more sensitive and accurate measure of exercise-induced increases in insulin-like growth factor-I (IGF-I) than serum, and that these increases are detectable proximal, but not distal, to the exercising muscle. A novel, noninvasive methodology was used to collect TDF, followed by sampling of total IGF-I (tIGF-I) and free IGF-I (fIGF-I) in TDF and serum following an acute bout of exercise. Experiment 1: eight men (23 ± 3 yrs, 79 ± 7 kg) underwent two conditions (resting and 60 min of cycling exercise at 60% Vo(2)(peak)) in which serum and forearm TDF were collected for comparison. There were no significant changes in tIGF-I or fIGF-I in TDF obtained from the forearm or from serum following exercise (P > 0.05); however, the proportion of fIGF-I to tIGF-I in TDF was approximately fourfold greater than that of serum (P ≤ 0.05). These data suggest that changes in TDF IGF-I are not evident when TDF is sampled distal from the working tissue. To determine whether exercise-induced increases in local IGF-I could be detected when TDF was sampled directly over the active muscle group, we performed a second experiment. Experiment 2: fourteen subjects (22 ± 4 yr, 68 ± 11 kg) underwent an acute plyometric exercise condition consisting of 10 sets of 10 plyometric jumps with 2-min rest between sets. We observed a significant increase in TDF tIGF-I following exercise (P ≤ 0.05) but no change in serum tIGF-I (P > 0.05). Overall, these data suggest that TDF may provide a noninvasive means of monitoring acute exercise-induced changes in local IGF-I when sampled in proximity to exercising muscles. Moreover, our finding that the proportion of free to tIGF-I was greater in TDF than in serum suggests that changes in local IGF-I may be captured more readily using this system.